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Vol. 55, Issue 1, 195-227, March 2003
,Centre National de la Recherche Scientifique UMR 7131, Hôpital Broussais, Bâtiment René Leriche, Paris, France (C.B.); Unit for Experimental Asthma and Allergy, The National Institute of Environmental Medicine, Karolinska Institutet, Stockholm, Sweden (S.-E. D.); Harvard Medical School, Brigham and Woman's Hospital, Boston, Massachusetts (J.M.D.); Department of Pharmacology, Merck & Co., West Point, Pennsylvania (J.F.E.); GlaxoSmithKline, King of Prussia, Pennsylvania (D.W.P.H.); Division Molecular Pharmacology, Pharmacological Sciences, Milan, Italy (S.N.); Center for Experimental Therapeutics and Reperfusion Injury, Department of Anesthesia Research Laboratory, Brigham and Woman's Hospital/Harvard Medical School, Boston, Massachusetts (C.N.S.); Department of Biochemistry and Molecular Biology, Faculty of Medicine, University of Tokyo, Tokyo, Japan (T.S., T.Y.)
Abstract
I. Introduction
II. General Considerations
A. Biochemical Pathways
B. Cellular Origins
C. Nomenclature for Leukotriene Receptors
III. Molecular Database for Leukotriene Receptors
A. Molecular and Structural Aspects of Dihydro-Leukotriene Receptors
1. BLT1.
2. BLT2.
3. Phenotypes Involving BLT Receptors.
B. Molecular and Structural Aspects of Cysteinyl-Leukotriene Receptors
1. CysLT1.
2. CysLT2.
C. Lipoxin Receptors
1. Molecular and Structural Aspects of Lipoxin Receptors.
D. Receptors and Cellular Signals
1. BLT.
2. CysLT.
3. Lipoxins.
E. Summary
IV. Properties and Significance of Leukotriene Receptors
A. BLT Functional and Radioligand Binding Studies
B. Cysteinyl-Leukotriene Functional Studies
1. Airway Smooth Muscle Contraction.
2. Vascular Smooth Muscle Contraction.
3. Vascular Smooth Muscle Relaxation.
4. Cardiovascular Effects.
5. Diverse Effects of Cysteinyl-Leukotrienes.
C. CysLT Radioligand Binding Studies
1. [3H]LTC4 Binding Sites.
2. [3H]LTD4 Binding Sites.
D. Evidence for Additional CysLT Receptor Subtypes
E. Clinical Studies Involving CysLT Receptors
F. Summary
G. Lipoxins Receptors
1. Functional and Radioligand Binding Studies.
2. Summary.
V. General Conclusions
References
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Abstract |
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The leukotrienes and lipoxins are biologically active metabolites derived from arachidonic acid. Their diverse and potent actions are associated with specific receptors. Recent molecular techniques have established the nucleotide and amino acid sequences and confirmed the evidence that suggested the existence of different G-protein-coupled receptors for these lipid mediators. The nomenclature for these receptors has now been established for the leukotrienes. BLT receptors are activated by leukotriene B4 and related hydroxyacids and this class of receptors can be subdivided into BLT1 and BLT2. The cysteinyl-leukotrienes (LT) activate another group called CysLT receptors, which are referred to as CysLT1 and CysLT2. A provisional nomenclature for the lipoxin receptor has also been proposed. LXA4 and LXB4 activate the ALX receptor and LXB4 may also activate another putative receptor. However this latter receptor has not been cloned. The aim of this review is to provide the molecular evidence as well as the properties and significance of the leukotriene and lipoxin receptors, which has lead to the present nomenclature.
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I. Introduction |
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Feldberg, Kellaway, and coworkers (Feldberg and Kellaway, 1938
;
Feldberg et al., 1938; Kellaway and Trethewie, 1940) observed that
perfusion of guinea pig lungs with antigen induced the release of a
material named "slow reaction smooth muscle-stimulating substance (SRS1)" that
caused a contraction of the isolated guinea pig ileum bioassay tissue.
These observations were confirmed by several workers (Schild et al.,
1951; Brocklehurst, 1960) who demonstrated that SRS (renamed
slow-reacting substance of anaphylaxis or SRS-A) was also released from
the human lung following antigen challenge. Sweatman and Collier (1968)
reported that SRS-A constricted human airways and the compound FPL
55712 (Augstein et al., 1973) was shown to inhibit SRS-A-induced
contractions in the guinea pig ileum assay. These observations provoked
an intense interest in elucidating the biochemical nature of this
entity. Initial attempts to characterize this substance revealed that
the factor was a low-molecular weight derivative of arachidonic acid
(Orange et al., 1973; Bach et al., 1977; Jakschik et al., 1977)
containing sulfur (Orange et al., 1973, Parker et al., 1979). SRS-A was
identified subsequently to be a family of lipid mediators known as
leukotrienes, a name derived from their cell source (leukocytes) and
their conjugated double bonds (triene) structure (Borgeat et al., 1976;
Borgeat and Samuelsson, 1979a,b,c; Murphy et al., 1979; Corey et al., 1980; Lewis et al., 1980; Morris et al., 1980; Rokach et al., 1980).
Leukotriene B4 (LTB4) was
the first of the leukotrienes to be isolated (Borgeat et al., 1976).
The elucidation of the structures and synthetic pathways for the
leukotrienes lead to a considerable amount of research on these
arachidonic acid metabolites (Fig. 1).
This work involved comprehensive assessments of the biological profiles
of both the cysteinyl-leukotrienes (cys-LTs:
LTC4, LTD4, and
LTE4) as well as dihydroxy-leukotriene
(LTB4) and, more recently, the lipoxins. Lipoxins
(LX), an acronym for eicosanoids, which are often generated during the
transcellular metabolism of arachidonic acid via the sequential actions
of the 15- and 5- or 5- and 12-lipoxygenase enzymatic pathways (Serhan
et al., 1984; Samuelsson et al., 1987). When the synthetic ligands were
made available many studies documented a myriad of actions for these
lipid mediators (Table 1) providing pertinent evidence for their possible patho-physiological roles in
inflammatory diseases, in particular asthma. During the last 20 years
significant efforts involving diverse chemical strategies have been
directed toward the identification and development of receptor
antagonists. These compounds have facilitated the identification and
characterization of distinct receptors, which are activated by either
the dihydroxy- or cysteinyl-leukotrienes.
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II. General Considerations |
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A. Biochemical Pathways
The leukotrienes are formed via activation of the 5-lipoxygenase
enzyme (5-LO) in collaboration with the "5-lipoxygenase activating protein" (FLAP). A prerequisite for this enzymatic reaction is the
hydrolyzation of arachidonic acid from membrane phospholipids by
phospholipase A2. The principal 5-LO products of
arachidonic acid metabolism are LTC4 and
LTB4 as well as 5-hydroxyeicosatetranoic acid
(5-HETE). In addition, eicosanoids that are formed by pathways that
involve the dual lipoxygenation of arachidonic acid by either 15- and
5-LO or 5- and 12-LO are referred to as lipoxins (Serhan et al., 1984;
Samuelsson et al., 1987). The transcellular metabolism of intermediates
such as LTA4 and 15(S)-HETE is
associated with LX formation (Serhan, 1994). LX and their carbon
15-epimer-LXs (aspirin-triggered lipoxins; ASA-15-epi-LX) are bioactive
and structurally distinct from other eicosanoids in that
they carry a conjugated tetraene system and are present in biological
matrix in two main forms that are positional isomers, namely, lipoxin A4
(5S,6R,15S-trihydroxyeicosa-7E,9E,11Z,13E-tetraenoic
acid) and lipoxin B4
(5S,14R,15S-trihydroxyeicosa-6E,8Z,10E,12E-tetraenoic acid; Serhan, 1997; Fig. 1). The aspirin-triggered form carry their C15
alcohol in the R configuration, which is inserted by COX-2
following aspirin treatment (denoted ASA-15-epi-LX). These metabolites are often produced during cell-to-cell interactions, and the principal targets appear to be platelets and leukocytes. During
these cellular interactions, platelets convert neutrophil derived
LTA4 to 5,6,-epoxytetraene through the action of
platelet 12-LO. However, under these conditions the term 12-LO is a
misnomer since this enzymatic activity was originally based on an
interaction with arachidonic acid. This enzyme functions as a 15-LO (LX
synthase) when the substrate is LTA4. Thus in an
inflammatory condition LTA4 serves as a pivotal
intermediate for both leukotriene and lipoxin formation.
B. Cellular Origins
The leukotrienes are formed in different cell types as well as via
transcellular metabolism involving multiple cells such as neutrophil
and platelets and vascular cells (Feinmark and Cannon, 1986;
Maclouf and Murphy, 1988; Sala et al., 1993). Human eosinophils and
neutrophils synthesize both LTC4 and
LTB4, respectively (Bray et al., 1980;
Ford-Hutchinson et al., 1980). Monocytes and macrophages also
synthesize both LTB4 and the cys-LTs (Samuelsson,
1983). LTC4 is metabolized to
LTD4 and LTE4 by the cells
in which this mediator is formed. In addition, the cys-LTs can be
transformed into 6-trans-LTB4 by
hypochlorous acid, which is generated during the respiratory burst in
leukocytes (Henderson et al., 1982; Lee et al., 1983).
LTB4 is also metabolized in the cells which
produce this metabolite, by a unique membrane bound cytochrome P450
enzyme. LTB4 is metabolized to
20-hydroxy-LTB4 (Hansson et al., 1981; Shak and
Goldstein, 1985; Soberman et al., 1985). There is also evidence
for a reductase dehydrogenase in polymorphonuclear leukocytes (PMN)
that appears to be specific for LTB4 (Powell et
al., 1989).
C. Nomenclature for Leukotriene Receptors
The previous IUPHAR publication (Coleman et al., 1995) introduced
two main classes of leukotriene receptors. One based on the biological
activities of leukotriene B4 and related
hydroxyacids, referred to as BLT receptors, and a second class
identified by the cysteinyl-leukotrienes (cys-LTs). The different
profiles of biological activity for these two classes of metabolites
were the initial basis for these categories and were supported by
structure-activity data obtained in studies with a variety of compounds
that selectively antagonized the different ligands. Activation of the
BLT receptors initially was shown to produce potent chemotactic
activities on leukocytes whereas the latter class (CysLT receptors)
stimulated smooth muscle as well as other cells. However, the
structures of the leukotriene receptors have recently been deduced from
the nucleotide sequences of the cDNAs and the encoding proteins are now
known for human, mouse, and rat. These data have permitted the IUPHAR
committee to establish the nomenclature for the leukotriene receptors,
and this is presented in Table 2. The
phylogenic tree for the different eicosanoid and bioactive lipid
G-protein-coupled receptors (GPCR) is illustrated in Fig.
2 and shows the molecular families with
the relationship between leukotrienes and lipoxins as well as other
proteins with seven transmembrane helices.
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The lipoxins, are chemically and functionally different from the
leukotrienes (Fig. 1). Although LXA4 and
LXB4 are similar in structure, these mediators
display biological activities that are quite distinct.
LXA4 interactions with neutrophils involves binding sites that are not recognized by LXB4
(Nigam et al., 1990; Fiore et al., 1992). LXB4 is
a potent agonist for stimulating proliferation and differentiation of
granulocyte-monocyte colonies from human mononuclear cells (Popov et
al., 1989), increasing the S-phase in the cell cycle and enhancing
nuclear protein kinase C activity (Beckman et al., 1992) actions, which
have not been reported for LXA4. However,
LXB4 has also been shown to share actions with
LXA4, such as, both selectively stimulate human
peripheral blood monocytes (Maddox and Serhan, 1996) and enhance
growth of myeloid progenitor cells (Stenke et al., 1991). Furthermore,
LXA4 does not activate BLT (Fiore et al., 1992)
but activates FPRL-1 receptors (Chiang et al., 2000; Resnati et al.,
2002; Perretti et al., 2002). These investigators have shown that ALX
and FPRL-1 are the same receptor and that LXA4 is
the natural and most potent ligand. In addition, Takano et al. (1997)
have identified the amino acid sequence for the receptor associated
with the LXA4 responses. In line with the IUPHAR
nomenclature directives, this committee recommends that ALX be used to
designate the receptor that has been cloned and is activated by the
native ligand LXA4 (Table 2).
LXB4-induced responses, although different from
those of LXA4, have not to date provided
sufficient evidence to specify another receptor. Since this receptor
has not been cloned, the LXB4 response is
associated with activation of a putative receptor.
The aim of this review is to present the evidence that led to the leukotriene nomenclature. To this end, information not only from the molecular database but also derived from the properties and significance of leukotriene receptors will be presented. Furthermore, the above nomenclature for the LX receptors is recommended as the framework for this evolving area of receptor research.
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III. Molecular Database for Leukotriene Receptors |
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A. Molecular and Structural Aspects of Dihydro-Leukotriene
Receptors
1. BLT1.
The cloning and characterization of the
BLT1 receptor was achieved by cDNA subtraction
using human leukemic cells HL-60, which were differentiated into
granulocyte-like cells (Yokomizo et al., 1997). The
BLT1 receptor was identified as a putative seven
transmembrane domain receptor with 352 amino acids. This receptor had
been initially misidentified as a purinergic receptor, P2Y7 (Akbar et
al., 1996). BLT1 shares low homology to P2Y
receptors and belongs to a family of receptors for chemoattractants
including complement receptors and a recently identified novel
prostaglandin D2 receptor, CRTH2 (Hirai et al.,
2001). The homology between the BLT1 receptor for mouse and humans is presented in Fig. 3.
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Fig. 3.
The sequence alignment of BLT1 and
BLT2 from human and mouse receptors. The amino acid
sequences were aligned using ClustalW and converted using Boxshade
3.21. The putative transmembrane domains of hBLT1 predicted
by Kyte-Doolittle hydrophobicity analysis are overlined and labeled as
I-VII. Consensus matches are boxed and shaded with darker shading for
identities and light shading for conservative substitutions. The amino
acid identity between human and mouse BLT1 was 78.6%
whereas BLT2 was 92.7%. The mouse sequence data are
available from Swiss-Prot under accession numbers (mBLT1:
no entry presently available) and (mBLT2: Q9JJL9).
reported that the BLT1 gene
consists of three exons located in a region of 5 kbp on chromosome 14. Whereas the open reading frame (ORF) for BLT1 is
in the last exon (exon 3), the basal promoter activity is found 100-bp
upstream from the transcriptional initiation site. This report and the
data of Yokomizo et al. (2000) demonstrated that there was no TATA or
CAT element near the transcription site. However, they did observe the
presence of an Sp-1 site at 
50 bp, which played a major role in the
basal transcription of BLT1. Since the promoter region of BLT1 is rich in GC sequences and
methylated in nonleukocyte cells but nonmethylated in leukocyte cells
expressing BLT1, Kato et al. (2000) have
suggested that the methylation state may, in part, be responsible for
transcription of the BLT1 gene.
COS-7 membranes transfected with human BLT1
exhibited [3H]LTB4
binding with a Kd of 0.15 nM,
comparable to the Kd of 0.14 nM for
the differentiated HL-60 cells (Fiore et al., 1993; Yokomizo et al.,
1997; Table 3). Various eicosanoids also
competed for the [3H]LTB4
binding to COS-7 membranes expressing human BLT1
and the potency ranking (Ki values)
were: LTB4 (0.38 nM) > 20-hydroxy-LTB4 (7.6 nM) = 12-oxo-LTB4 (7.6 nM) > 12R-HETE
(30 nM) > 20-COOH-LTB4 (190 nM). This
profile agrees with the previously characterized LTB4 binding sites in human granulocytes. The
structures and potencies of several antagonist for these receptors in
Chinese hamster ovary (CHO) cells expressing human BLT (CHO-BLT) are
presented in Table 4.
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; Huang et al., 1998). In addition,
BLT1 expression is inducible in activated
macrophages (Toda et al., 1999) and eosinophils treated with IL-5
(Huang et al., 1998), suggesting an association of
BLT1 with various inflammatory diseases.
BLT1-transgenic mice showed enhanced PMN
responses in the lung following ischemia-reperfusion and increased
expression of 5-lipoxygenase in peritonitis (Chiang et al., 1999).
Recently, two lines of BLT1-deficient mice have
been reported, and a reduction in leukocytes migration was demonstrated
(Haribabu et al., 2000; Tager et al., 2000).
2. BLT2.
During the analysis of transcriptional
regulation of human BLT1 gene (Kato et al.,
2000), a putative ORF for a novel GPCR with structural similarity to
BLT1 was identified (Yokomizo et al., 2000). This
novel receptor was also found in a human genome sequence database,
reported to act as a low-affinity receptor activated by
LTB4 (Kamohara et al., 2000; Tryselius et al.,
2000; Wang et al., 2000) and subsequently referred to as the
BLT2 receptor (Yokomizo et al., 2001b). The gene
structure for BLT2 has also been established
(Yokomizo et al., 2001b). Of considerable interest is that the promoter
region (Fig. 4) of human
BLT1 overlaps BLT2 ORF
(Kato et al., 2000). This represents the "promoter in ORF", as has
been reported in prokaryotes but the biological significance of this
rare gene structure is presently not clear. However, there is
sufficient evidence that BLT1 and
BLT2 form a gene cluster both in human
(chromosome 14 q11.2-q12;) and mouse (Yokomizo et al., 2000)
chromosomes suggesting that these receptors may be generated by gene
duplication.
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), and
a Kd value of 0.17 nM in COS-7 cells
(Wang et al., 2000). Although there are differences in the reported
Kd values (Table 3 and Table 4),
generally BLT2 is considered as a low-affinity
receptor since the pEC50 value of
LTB4 required for adenylyl cyclase inhibition and
the concentrations required for LTB4-dependent
chemotaxis were higher than those for BLT1 receptor activaton (Kamohara et al., 2000; Yokomizo et al., 2000). The
tissue distribution of BLT2 assessed by different
groups using Northern blots are varied. Using the ORF probe of
BLT2 (Kamohara et al., 2000; Yokomizo et al.,
2000) the highest expression was found in spleen, followed by
leukocytes and ovary. Other groups reported that
BLT2 is expressed highest in liver and intestine (Tryselius et al., 2000; Wang et al., 2000) using the 5'-UTR probes. The abundance of low-affinity binding sites for
LTB4 in spleen membrane supports the data showing
BLT2 expression in spleen (Showell et al.,
1998). Of interest, the rank order of potency of several eicosanoids
for BLT2 activation was
LTB4 > 12-epi-LTB4 > 12S-HETE > 12S-HPETE > 12R-HETE > 20-hydroxy-LTB4,
which is quite different from BLT1 (Yokomizo et
al., 2001c). Currently, there are no data on specific in vivo functions
for BLT2 receptors, however, lymphocytes have
been suggested to be the target, since BLT2 is
highly expressed in splenic T-cells (Yokomizo et al., 2001a).
3. Phenotypes Involving BLT Receptors.
Investigations with
transgenic mice expressing the human BLT1
receptor on leukocytes (Chiang et al., 1999) as well as targeted gene
disruption of the BLT1 receptor in knockout mice
(BLT/) indicate that an apparent phenotypic difference (Haribabu et
al., 2000; Tager et al., 2000) from wild type littermates is not
observed unless the animals are subject to experimental disease or
injury, which are known to stress the effector immune system (vide infra).
/ mice (Haribabu et al., 2000; Tager
et al., 2000) leukocytes exhibited a normal response to C5a and
platelet-activating factor (PAF), however, there was a selective loss
of responsiveness to exogenous LTB4. Tager et al.
(2000) also reported that adhesion to the endothelium in response to
LTB4 was diminished in leukocytes from
BLT1
/ animals. These
findings are in line with earlier observations with
LTB4 in the hamster cheek pouch (Raud et al.,
1991) and suggest that the BLT receptor is pivotal for the
LTB4-induced leukocyte activation.
Unfortunately, there are only a limited number of investigations
examining the relative input of LTB4 in
chemotaxis during inflammation in vivo in genetically modified animals.
Two groups (Haribabu et al., 2000; Tager et al., 2000) have provided
evidence that peritoneal inflammation is suppressed in these deficient mice. Interestingly, Haribabu et al. (2000) described a loss of this
protection at 72 h between / and +/+ animals, whereas Tager and colleagues (2000) observed an increase in protection between /
and +/+ with time intervals greater than 50 h. These latter investigators also reported a marked diminution in the number of
eosinophils, which accounted for virtually all of the changes in
cellular influx. In contrast, Haribabu et al. (2000) noted reductions
in both PMNs and macrophage populations. The basis for these
differences is presently not known, but may be related to the different
stimuli (zymosan versus thioglycollate) used to induce the inflammatory
cellular infiltration in vivo.
Expression of the human BLT1 receptor in
leukocytes has also been carried out using the CD11b promoter (Chiang
et al., 1999). BLT receptor-transgenic mice displayed enhanced
leukocyte responsiveness in acute dermal inflammation, with leukocyte
trafficking to remote organs (as in secondary organ reperfusion
injury), or in leukocyte recruitment following a peritoneal challenge.
These results are consistent with the interpretation that
LTB4 is associated with an excessive activation
of leukocytes and the 5-LO pathway provoking leukocyte-mediated injury.
Expression of the human BLT1 receptor in vivo
lead to an up-regulation of 5-LO expression and leukotriene biosynthesis (Chiang et al., 1999). These results suggest that receptor
expression may amplify proinflammatory circuits in vivo.
B. Molecular and Structural Aspects of Cysteinyl-Leukotriene
Receptors
1. CysLT1.
The cloning and characterization of
the human CysLT1 receptor
(hCysLT1) was achieved by two groups under the
general program of identifying cognate ligands for orphan GPCRs, a
process which has been termed "ligand fishing" (Lynch et al., 1999;
Sarau et al., 1999). The hCysLT1 receptor was
identified as a 337-amino acid putative seven transmembrane domain
receptor, termed either HG55 (Lynch et al., 1999) or HMTMF81 (Sarau et
al., 1999) (Fig. 5). The former
investigators demonstrated that LTD4 produced
activation of a calcium-activated chloride channel in Xenopus
laevis oocytes expressing the cRNA for HG55 but not in control
cells or oocytes expressing other GPCRs. This
LTD4-induced stimulation of oocytes was blocked
by the selective CysLT1 receptor antagonist
MK-571 (Lynch et al., 1999) (Table 5).
Similar results were obtained using the X. laevis
melanophore signaling assay and in mammalian monkey kidney COS-7 cells
expressing the HG55 (hCysLT1) receptor (Lynch et
al., 1999).
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Fig. 5.
Comparison of amino acid sequences of the human
CysLT1 and CysLT2 receptors. A
G-protein-coupled receptor snake diagram depiction of the amino acid
sequences of the human CysLT1 and CysLT2
receptors. The amino acid identities between the hCysLT1
and hCysLT2 receptors is 37.3%.
TABLE 5
CysLT1-selective and nonselective antagonists and
structures
) and has 31% amino acid identity to the
P2Y purinoceptor (Sarau et al., 1999).
Full Scatchard analyses of
[3H]LTD4 binding to the
HG55 (hCysLT1) COS-7 membranes demonstrated a
Kd of 0.3 nM and
Bmax of 50 fmol/mg of protein (Lynch
et al., 1999). In the structure-affinity relationships, the
IC50 for the agonists were
LTD4 (0.9 nM), LTC4 (350 nM), and LTE4 (200 nM). The affinities of
antagonists (Table 5) were similar to that demonstrated functionally in
other systems for the CysLT1 receptor as well as
in binding experiments on human lung or U937 cells (Frey et al., 1993;
Lynch et al., 1999). Likewise the HMTMF81
(hCysLT1) receptor expressed in HEK-293 cells
responded selectively to the cys-LTs with rank order of potency
(pEC50 value) were LTD4
(2.5 nM) > LTC4 (24 nM) > LTE4 (240 nM) (Sarau et al., 1999). In these
functional studies LTE4 was shown to be a partial agonist of the HMTMF18 (hCysLT1) receptor
expressed in HEK-293 cells (Sarau et al., 1999). This is similar to
evidence derived from the human bronchus (Labat et al., 1992), sheep
trachea (Mong et al., 1988), and U937 cells (Saussy et al., 1989).
[3H]LTD4 binding and
LTD4-induced calcium mobilization in HEK-293 cells expressing the HMTMF18 (hCysLT1) receptor
were potently inhibited by the structurally distinct CysLT receptor
antagonists and the rank order of potency in this system was
pranlukast = zafirlukast > montelukast > pobilukast
(Sarau et al., 1999). LTD4-induced calcium
mobilization in HEK-293 cells expressing the HMTMF18
(hCysLT1) receptor was not affected by pertussis
toxin and appeared to result from the release of intracellular calcium
stores (Sarau et al., 1999). In X. laevis melanophore
signaling system, the HG55 (hCysLT1) receptor was
shown not to couple via G
i (Lynch et al.,
1999).
Northern analyses of human tissues showed that the
hCysLT1 mRNA was detected as an approximately
2.8-kb species and was expressed in spleen, peripheral blood
leukocytes, less strongly in lung, bronchus small intestine, colon,
skeletal muscle, pancreas, placenta, and weakly in the prostate, heart,
brain, liver, kidney, and adipose tissues (Lynch et al., 1999; Sarau et
al., 1999). Further work is necessary to confirm whether expression in
some of these tissues may be attributable to expression of the receptor
on interstitial myeloid cells. The receptor was also detected in U937
cells and HL-60 cells, with an increase observed in dimethyl
sulfoxide-differentiated HL-60 cells (Sarau et al., 1999). In
HL-60 cells differentiated with sodium butyrate to eosinophilic-like
cells, interleukin-5 treatment resulted in a 5-fold increase in the
expression of the hCysLT1 (Thivierge et al.,
2000). Northern analysis in normal and asthmatic lungs suggested no
total differences in hCysLT1 mRNA expression,
although in situ studies in asthmatic tissues have not been reported
(Sarau et al., 1999). Using in situ hybridization, the
hCysLT1 mRNA was shown in lung smooth muscle
cells and interstitial macrophages, with little or no expression in
epithelial cells (Lynch et al., 1999). These in situ findings were
confirmed with immunohistochemical localization of the receptor protein
using a specific hCysLT1 antiserum (Figueroa et
al., 2001). In addition, the hCysLT1 mRNA and
protein have been detected recently in normal peripheral blood
eosinophils, subsets of monocytes and macrophages and in
pregranulocytic CD34+ cells (Figueroa et al., 2001). The hCysLT1 protein was not observed on CD4+ or CD8+
peripheral T cell populations (Figueroa et al., 2001). In situ and
immunohistochemical studies of the hCysLT1 in
diseased lung and other tissues are needed to determine whether the
expression of the hCysLT1 changes in asthma,
allergy, or other pulmonary and inflammatory diseases.
Two isoforms of the mouse CysLT1 receptor
(mCysLT1) have been cloned from a mouse lung cDNA
library (Maekawa et al., 2001; Martin et al., 2001; Mollerup et al.,
2001). A short isoform cDNA containing two exons encodes a polypeptide
of 339 amino acids with 87.3% amino acid identity to the
hCysLT1 (Lynch et al., 1999; Sarau et al., 1999,
Maekawa et al., 2001). A long isoform has two additional exons and an
in-frame upstream start codon resulting in a 13-amino acid extension at
the N terminus of the receptor (Maekawa et al., 2001). Northern blot
analysis of the mCysLT1 detecting both isoforms
of the receptor showed expression of a 3.5-kb transcript in the lung
and skin, whereas reverse transcriptase polymerase chain reaction
showed wide expression of the long isoform, with strongest expression
in the lung and skin. Unlike the hCysLT1, neither
form of the mouse receptor is expressed in the spleen. In addition, the
long rather than the short isoform of the mCysLT1 receptor is the closer counterpart to the human receptor, and that is
expressed more abundantly in the mouse tissues. The
mCysLT1 receptor maps to the X chromosome at band
XD. LTD4-induced intracellular calcium
mobilization in CHO cells stably expressing either isoform of
mCysLT1 was blocked by the
CysLT1 receptor antagonist MK-571 (Maekawa et
al., 2001). The rank order agonist functional potency for calcium
mobilization or the potency for competition of
LTD4 binding to the recombinant mouse receptor
was similar to that described above for the human receptor, except that
LTC4 was relatively less potent for the mouse
than for hCysLT1 (Lynch et al., 1999; Sarau et
al., 1999; Maekawa et al., 2001).
2. CysLT2.
The cloning and characterization of
the CysLT2 receptor
(hCysLT2) was initially reported by Heise et al.
(2000) (Fig. 5). This publication confirmed the previous
pharmacological characterization of a human
CysLT2 receptor in different tissues, based upon
the relative potencies of the cys-LT agonists and the lack of
sensitivity of the responses to classical CysLT1
receptor antagonists, and the antagonist activity of the partial
agonist BAY u9773 (Labat et al., 1992; Tudhope et al., 1994; Heise et
al., 2000). Subsequent to this initial publication, the Takeda group
published an article confirming the identification of the
hCysLT2 (Takasaki et al., 2000), and then a third
report by the Nothacker et al. (2000), on the characteristics of the
hCysLT2 was published, which revealed similar
distribution and functional data to the previous publications but with
more details on the partial agonist activity of BAY u9773 (Nothacker et
al., 2000). Recently, the cloned mCysLT2 has also been reported (Hui et al., 2001).
). Using FASTA, the hCysLT2 was shown to have 37.5%
amino acid identity with the hCysLT1 over a
315-amino acid overlap and the extreme carboxyl termini of these
proteins have little homology (Heise et al., 2000). The
hCysLT2 receptor was also identified from the Helix Research Institute database (Takasaki et al., 2000). The human
chromosomal localization of the hCysLT2 in 13q14,
a region that has been identified as a polygenic atopic linkage (Kimura et al., 1999).
The cRNA for the hCysLT2 was expressed in
X. laevis oocytes and either LTD4 or
LTC4 produced a calcium-dependent chloride flux
in these cells that was not blocked by the selective
CysLT1 receptor antagonist, MK-571, but was
inhibited by the dual
CysLT1/CysLT2 receptor
antagonist BAY u9773 (Heise et al., 2000). In addition, LTD4 and LTC4 elicited a
concentration-dependent activation of calcium flux in HEK-293 cells
coexpressing the aequorin bioluminescent protein and the recombinant
hCysLT2 (Heise et al., 2000). This activation was
blocked by BAY u9773 but not significantly by the CysLT1 antagonists MK-571, montelukast,
zafirlukast, or pranlukast (Heise et al., 2000). Experiments involving
radiolabeled LTD4 binding to COS-7 cell membranes
expressing the hCysLT2 demonstrated high-affinity
competition by LTD4 and
LTC4, with weaker competition by
LTE4 and no effect by LTB4
(Heise et al., 2000). Similar studies using radiolabeled
LTD4 binding to COS-7 cell membranes expressing the hCysLT2, showed that the dual antagonist BAY
u9773 fully competed for the binding, whereas
CysLT1 antagonists showed no or minimal competition (Heise et al., 2000). Furthermore, Kamohara et al. (2001)
have shown by in situ hybridization the presence of
hCysLT2 mRNA in human atrium, ventricle, and at
intermediate coronary arteries with little detection of
hCysLT1. These investigators also reported that
human coronary smooth muscle cells stimulated with
LTC4 caused an increase in calcium mobilization.
Thus the existence of a CysLT2 functional
receptor in the human heart has been documented.
Northern blot analyses of hCysLT2 expression
showed high expression in the human heart, adrenals, peripheral blood
leukocytes, placenta, spleen, and lymph nodes with weaker expression in
the brain (Heise et al., 2000; Nothacker et al., 2000; Takasaki et al.,
2000). A reverse transcription polymerase chain reaction comparison of
the CysLT receptors showed negligible hCysLT1
expression but high expression of hCysLT2, in the
heart and eosinophils (Mita et al., 2001), whereas only
hCysLT1 was expressed in tracheal tissue
(Takasaki et al., 2000). Both receptors were highly expressed in spleen
(Heise et al., 2000; Nothacker et al., 2000; Takasaki et al., 2000). In
situ hybridization analyses in the human lung demonstrated
hCysLT2 mRNA strongly expressed on interstitial
macrophages and weak expression on smooth muscle cells (Heise et al.,
2000). In human peripheral blood monocytes, about 30% of cells
expressed the hCysLT2, whereas greater than 90%
of purified human eosinophils expressed the receptor (Heise et al.,
2000). In the adrenal gland, in situ hybridization for the
hCysLT2 demonstrated localization to the
chromaffin-containing adrenal medulla cells (Heise et al., 2000). In
the heart, the hCysLT2 was expressed in Purkinje
fiber conducting cells (Heise et al., 2000).
The recent molecular cloning, expression and characterization of the
hCysLT1 and hCysLT2
receptors (Table 2) represents a significant milestone in the history
of CysLT research and is predicted to lead to a renaissance in interest
in this area of biology and therapeutics. The findings have confirmed
much of the earlier pharmacological characterization of the two
receptors, which were based solely on the functional profiling of the
effects of agonists and antagonists in different cellular and tissue
systems. However, there are some unexpected findings with regard to
genomic structure and tissue expression, as well as the relatively low homology (38%) between the two receptors. The cloning of the receptors allows the generation of tools to investigate in more detail the regulation of CysLT1 and
CysLT2 receptor expression, the roles of these
CysLT receptors in normal and diseased states, and their potential as
new therapeutic targets. The ultimate goals will be the identification
of potent and selective CysLT2 receptor antagonists and combined
CysLT1/CysLT2 receptor
antagonists and their clinical evaluation in the diverse diseases in
which the cys-LTs have been implicated as significant
pathophysiological mediators.
C. Lipoxin Receptors
Of the nonprostanoid eicosanoid GPCRs, the
LXA4 receptor (ALX) was the first recognized at
the molecular level (Fiore et al., 1993, 1994). In addition, ALX was
initially identified as the only inhibitory or anti-inflammatory
receptor that acts via an agonist role as a "stop signal" (Fiore et
al., 1994; Serhan, 1994, 1997; Takano et al., 1997). This action
appears to be a unique flexibility of GPCR that functions within the
immune system. Since LXA4 shares some structural
features with LTC4 and LTD4
as well as prostaglandins, LXA4 competed for
CysLT1 receptors identified on isolated human
vascular endothelial cells (Gronert et al., 2001) and mesangial cells
(McMahon et al., 2000) and antagonized either
LTC4- or LTD4-induced
bronchoconstriction in humans (Christie et al., 1992) and animals (Badr
et al., 1989; Gronert et al., 2001). In addition, lipoxin
B4 has also been reported to activate another
receptor. The present nomenclature for the lipoxin receptors is
therefore based on the cloned receptor sequence as well as the
observation that LXA4 is the natural and most
potent ligand. In contrast, the putative receptor activated by
LXB4 has not been cloned. ALX activation has been
reported to generate intracellular stop signals (Serhan et al.,
1994; Levy et al., 1997, 1999) and thereby promote resolution of inflammation.
1. Molecular and Structural Aspects of Lipoxin
Receptors.
Based on the finding that functional ALX are inducible
in promyelocytic lineages (HL-60 cells) (Fiore et al., 1993), several putative receptor cDNAs cloned earlier from myeloid lineages and designated orphans were screened for their ability to bind and signal
in response to LXA4 (Fiore et al., 1994). When
transfected into CHO cells, one of the orphans (previously denoted as
pINF114 or a formyl peptide receptor-like-1 (FPRL-1), displayed both
specific [3H]LXA4 binding
with high affinity (Kd of 1.7 nM) and
demonstrated ligand selectivity when compared with
LXB4, LTB4,
LTD4, and prostaglandin E2
(Fiore et al., 1994). LXB4 did not act via the
ALX receptor and interacted with a specific receptor present on human
leukocytes (Maddox and Serhan, 1996). In transfected CHO cells,
LXA4 activated both GTPase and released
arachidonic acid from membrane phospholipids, indicating that this cDNA
encodes a functional receptor for ALX in myeloid cells. A mouse ALX
receptor cDNA was also identified and cloned from a spleen cDNA
library. This receptor expressed in CHO cells displayed specific
[3H]LXA4 binding, and
LXA4 initiated GTPase activity (Takano et al.,
1997).
) or FPRH1 (Bao et al., 1992) was
designated by different research groups. Similarly this receptor was
also known as FPR2 (Ye et al., 1992) or RFP (receptor related to FPR)
(Perez et al., 1992). ALX was also cloned by Nomura et al. (1993) from
human monocyte cDNA library and was designated as an orphan receptor
(HM63). Both human and mouse ALX cDNA contain an open reading frame of
1051 nucleotides that encode a protein of 351 amino acids. Northern
blot analysis demonstrated that ALX mRNA is ~1.4 kb in both human and
mouse (Takano et al., 1997). Deduced amino acid sequences indicated
that ALX belongs to the GPCR superfamily characterized by seven
putative transmembrane segments with N termini on the extracellular
side of the membrane and C terminus on the intracellular side.
The overall homology between human and mouse ALX is 76% in nucleotide
sequence and 73% in deduced amino acid (Takano et al., 1997). A high
homology was present in the sixth transmembrane segment and the second
intracellular loop, a finding that suggests an essential role for these
regions in ligand recognition and signaling. The molecular evolution,
as assessed from computer-based sequence analyses indicates that ALX
(Fig. 2) is not related to prostanoid receptors and belongs to a
rapidly growing cluster of chemoattractive peptide and chemokine
receptors, exemplified by fMLP, C5a, and IL-8 receptors (Toh et al.,
1995). The BLT1 receptor cloned from human HL-60
cells (Yokomizo et al., 1997) and mouse eosinophils (Huang et al.,
1998) shares an overall homology of ~30% with ALX in deduced amino
acid sequences.
Human and mouse ALX cDNA, when transfected into CHO cells, displayed
specific binding to
[3H]LXA4, with a
Kd of 1.7 nM (Fiore et al., 1994) and
1.5 nM (Takano et al., 1997), respectively. Human ALX-transfected CHO
cells were also tested for binding with other eicosanoids, including
LXB4, LTD4,
LTB4, and prostaglandin E2.
Only LTD4 shows competition with
[3H]LXA4 binding, with a
Ki value of 80 nM (Fiore et al.,
1994). Moreover, LXA4 does not compete with
radiolabeled LTB4 binding to either recombinant
human BLT1 or isolated human peripheral blood
neutrophils (Fiore et al., 1992; Chiang et al., 2000). Hence, the
ability of LXA4 and
15-epi-LXA4 to block
LTB4-induced responses in vivo and in isolated
PMNs may not be via direct interactions at either
BLT1 or BLT2. Although ALX
shares ~70% homology with FPR, ALX binds
[3H]fMLP with only low affinity
(Kd ~5 µM) and therefore is
selective for LXA4 by 3 orders of magnitude
(Fiore and Serhan, 1995).
Preparation of radiolabeled
[11,12-3H]LXA4 (Brezinski
and Serhan, 1991) enabled the direct characterization of specific
LXA4 binding sites present on PMNs (Fiore et al.,
1992). Intact PMN demonstrated specific and reversible
[11,12-3H]LXA4 binding
(Kd ~0.5 nM and
Bmax ~1,830 sites/PMN) that are modulated by guanosine stable analogs. These
LXA4-specific binding sites are inducible in
HL-60 cells exposed to differentiating agents (e.g., retinoic acid,
dimethyl sulfoxide, and phorbol 12-myristate 13-acetate) and confer
LXA4-induced phospholipase D activation in
these cells (Fiore et al., 1993). The various ligands that activate the
ALX receptor are presented in Table 6.
These results provided further evidence that LXA4
interacts with specific membrane-associated receptors on human
leukocytes. These binding data have corroborated with the functional
results for LXA4. In contrast, although
functional studies (Maddox et al., 1998) have indicated the existence
of a receptor activated by LXB4, this receptor
has not been cloned and is presently referred to as a putative
receptor. In addition, the receptor binding investigations with
LXB4 must await the synthesis of a suitable
LXB4 radiolabel with high specificity. Such an
analog has proven to be quite difficult to achieve.
|
). In humans, ALX mRNA is also abundant in PMNs, as
well as in spleen, lung, placenta, and liver (Fiore et al., 1994;
Takano et al., 1997). In human enterocytes, ALX is inducible by IL-13
and interferon present in crypt and brush-border colonic epithelial
cells (Gronert et al., 1998). The location of this receptor provides
additional evidence that ALX is not associated with bacterial
chemotaxis as observed for N-formyl peptide FPR-signaling
since the gastrointestinal tract always has a very high level of
bacteria present.
D. Receptors and Cellular Signals
Whereas the cascades of cellular events subsequent to GPCR
activation have been the subject of many investigations, the exact signal transduction mechanisms for either the leukotrienes or the
lipoxins have not been completely elucidated. Generally, agonist interactions with GPCRs involve activation of heterotrimeric G-proteins associated with a group of conventional cellular events. However, effectors for GPCRs that are independent of G-proteins are also known
to exist (Hall et al., 1999). G-proteins, composed of -, 
-, and
-subunits each encoded by a different gene, appear often to be cell
specific. Upon ligand-receptor activation, the G- and
G-subunits stimulate a variety of intracellular molecular systems. Furthermore, G-protein activation leads to increases in
intracellular Ca2+ and modifications in a number
of membrane ion channels.
The cellular responses to ligand activation of GPCRs can also be
up-regulated through priming of cells and down-regulated by
desensitization. Two types of desensitization have been described, one
that results from phosphorylation of the agonist-occupied receptor by
G-protein-coupled receptor kinases. These phosphorylated receptors are
associated with the arrestin family of proteins. A second type of rapid
desensitization (loss of response) following phosphorylation by either
second messenger-activated kinases (protein kinase A, protein kinase C)
or inhibition of phospholipase C, which are activated by different
receptors or signaling processes. Generally, this second type of
desensitization does not require agonist-receptor occupancy. In
addition, Didsbury et al. (1991) also demonstrated "cross-receptor
desensitization", a phenomenon that has been reported for the
chemoattractant family receptors. Presently, an exploration of these
latter mechanisms associated with the actions of leukotrienes and
lipoxins at the molecular level has received little attention.
1. BLT.
Investigations involving the intracellular signaling
of BLT receptor activation have been performed in peripheral leukocytes specifically granulocytes. One of the problems involved in such studies
is that these cells have a limited life span (24 h) making drug and
transfection studies difficult. These limitations have caused several
investigators to use either CHO cells expressing human BLT receptors
(Yokomizo et al., 1997) or to perform reconstitutional studies with the
heterotrimeric GTP-binding proteins (Miki et al., 1990; Igarashi et
al., 1999). Although high-affinity binding of
LTB4 (BLT1 receptor) is
found essentially in leukocytes and macrophages, the G-proteins
associated with the functions in these cells has not been clearly
established. Furthermore, the intracellular signaling pathways for BLT
may depend on the G-proteins expressed in the different cells. For
example, most of the LTB4-dependent signals in
granulocytes appear to be mediated by Gi-like
G-proteins, (granulocytes express abundant Gi
proteins, mainly Gi2), whereas in the nervous
system Gi1 and Go are
mainly present (Simon et al., 1991). In several cell types,
LTB4 signals via
Gi-proteins are inhibited by pretreatment of
pertussis toxin (PTX). However, LTB4-induced
calcium mobilization in CHO-BLT1 was not affected by PTX, suggesting the coupling with Gq-like
molecules in these latter cells. Chemotaxis and inhibition of adenylyl
cyclase by LTB4 were completely PTX-sensitive in
CHO-BLT1 cells. The coupling of
BLT1 with various G-subunits was examined by
cotransfection studies using COS-7 cells, and
BLT1-mediated phospholipase C activation was
shown to be mediated by Gi6- and
G-subunits released from Gi (Gaudreau et
al., 1998). When expressed heterologously in CHO, HeLa, and COS-7
cells, BLT2 activation led to the inhibition of
adenylyl cyclase and an increase in calcium. However,
BLT2 activation was less potent in mobilizing
calcium than BLT1 receptor activation (Yokomizo
et al., 2000). BLT2 was also shown to mediate LTB4-dependent chemotaxis through
Gi-like G-proteins (Kamohara et al., 2000;
Yokomizo et al., 2000). Recently, Woo et al. (2002) have suggested that
LTB4 stimulation of the Rac-extracellular signal-regulated kinase cascade associated with the generation of
reactive oxygen species-mediated chemotaxis in Rat-2 cells was via
activation of the BLT2 receptor. This suggestion,
although not conclusive, was supported by the observations that
BLT1 expression has not been detected in Rat-2
fibroblasts whereas BLT2 was expressed. Furthermore, the LTB4 stimulation of reactive
oxygen species was observed at high concentrations (0.3-1 µM), which
are within the range for BLT2 activation and are
2 orders of magnitude higher than that observed for activation of
BLT1. In addition, this
LTB4 stimulation was blocked by ZK 158252. In an
attempt to understand the mechanisms involved in BLT receptor
desensitization, Gaudreau et al. (2002) have reported some initial
molecular evidence. These investigators showed that the cytoplasmic
tail of BLT1 receptor was intimately involved in
the regulation of desensitization and that the amino acid threonine
(Thr308) was implicated in the GPCR-specific
kinase phosphorylation associated with this phenomenon. This study
therefore provides pertinent leads for understanding those structural
elements associated with BLT1 receptor regulation.
; Kuhns
et al., 1998). While activation of the LTB4
receptor does stimulate intracellular Ca2+
mobilization (Powell et al., 1996), this Ca2+
appears not to be essential for chemotaxis and plays a role only in
degranulation and other responses of human neutrophils (Serhan et al.,
1982; Luscinskas et al., 1990; Rola-Pleszczynski et al., 1993). Such
studies indicate that the unraveling of the intracellular events
associated with the BLT receptor activation with the second messenger
cascades are only beginning to be understood.
2. CysLT.
Unfortunately, most studies concerning the CysLT
receptors have involved only LTD4 activation of
CysLT1 receptors. There is little information
available concerning G-protein and Ca2+
mobilization when the CysLT2 receptor is
activated. Initial studies (Kuehl et al., 1984; Crooke et al., 1989,
1990; Watanabe et al., 1990) demonstrated that
LTD4 activation of the
CysLT1 receptor lead to G-protein activation and
the release of several second intracellular messengers, namely,
diacylglycerol, inositol phosphates, and Ca2+,
events which were followed by activation of protein kinase C (PKC) and
accompanied by the mobilization of Ca2+ derived
from both intracellular and extracellular stores. Clark et al. (1985)
demonstrated that LTD4 activation of
CysLT1 receptors also led to the release of
arachidonic acid via stimulation of phospholipase
A2, which was associated with an enhanced
transciption of phospholipase A2 activating
protein. Expression of this latter protein was controlled by activation
of topoisomerase I, which in turn was regulated by PKC (Mattern et al.,
1991).
; Oliva et al., 1994), or
Ca2+ release without influx (Bouchelouche et al.,
1990) and in many cell types can induce both Ca2+
influx and release (Mong et al., 1984b, 1987a, 1988; Sjolander et al.,
1990). In human airways, Gorenne et al. (1998) demonstrated that
LTD4-induced contractions may mobilize
Ca2+ via a receptor-operated channel. Recently,
Accomazzo et al. (2001) provided evidence that
LTD4 contractions in human airways did not elicit
a marked elevation in
[Ca2+]i but rather
activated Ca2+ via a
Ca2+-independent isoform of protein kinase C
(PKC
). These observations supported an initial report that PKC
activation may be associated with increased force development at
constant [Ca2+]i (Masuo
et al., 1994). In addition, PKC was shown to be necessary for the
generation of the LTD4-induced
Ca2+ signal in intestinal epithelial cells.
Together these results suggest that the Ca2+
signaling for LTD4 contractions in human airways
may involve several intracellular pathways. Unfortunately, the other
ligands (LTC4 and LTE4)
have not been examined in these studies. Interestingly, Sjolander et
al. (1990) proposed that LTD4 and
LTE4 activated different receptors associated
with different intracellular signals. However, these observations have
not been pursued. The initial observations of Gronroos et al. (1995,
1996, 1998) have recently received support from several investigators
(Hoshino et al., 1998; Thodeti et al., 2000; Massoumi and Sjolander,
2001; Paruchuri et al., 2002) and provided evidence that
LTD4 activation of an epithelial cell line
(THP-1) led to an association of a protein other than the
heterotrimeric G-proteins and this mobilization was necessary for the
mobilization of calcium. Although these investigators observed that
LTD4 activation of THP-1 cells altered mitogen-activated protein kinase via protein kinase C and Raf-1, an
effect which was PTX-insensitive, they also reported that the chemotactic response of these cells was PTX-sensitive. These
observations suggest that diverse pathways at the G-protein level may
also exist when LTD4 interacts with the receptor.
However, these investigators did not evaluate different receptor
antagonists to determine which receptor was associated with these
different pathways.
3. Lipoxins.
The cytoplasmic signaling cascade of the ALX
receptor is also highly specific and selective for different cell
types. In human PMNs, LXA4 stimulates rapid lipid
remodeling and release of arachidonic acid via a PTX-sensitive
G-protein (Nigam et al., 1990) and blocked intracellular generation of
inositol 1,4,5-trisphosphate (Grandordy et al., 1990) as well as
Ca2+ mobilization (Lee et al., 1989). In
contrast, in human monocytes and THP-1 cells,
LXA4 triggers intracellular calcium release
(Romano et al., 1996; Maddox et al., 1997), suggesting a different
intracellular signaling pathway than in PMNs despite identical receptor
sequences. In addition, distinct signaling in monocyte and PMNs was
further supported by different responses to LXA4
in these cell types. LXA4 modulates
mitogen-activated protein kinase activities in mesangial cells in a
PTX-insensitive manner (McMahon et al., 2000), suggesting the presence
of an additional ALX receptor subtype and/or signaling pathway for ALX.
Since the ALX receptor has been shown to switch recognition and
function with certain chemotactic peptides, the G-proteins and
intracellular pathways involved may prove to be a difficult but
fascinating area to explore. One of the problems presently confronting
investigators in this area of research is the availability of the
ligands. Studies on G-protein and intracellular messengers are
presently limited (Kang et al., 2000), since stable analogs for
LXA4 and LXB4 have only
recently become available.
E. Summary
Within the last few years, a considerable effort at the molecular
level has been undertaken to identify the leukotriene receptors. However, data involving chimeric constructs of the leukotriene receptors have only recently been reported (Gaudreau et al., 2002). In
contrast, there are several observations that warrant further investigation. For example, the mouse CysLT1
cloned receptor is activated by all three native ligands and
antagonized by MK-571. However, the ligand profile for the
mCysLT1 is quite different from that observed in
the human CysLT1 receptor, since the mouse CysLT1 receptor exhibited little response to
LTC4. An explanation for this difference is not
readily apparent. In addition, MK-571 potentiated
Ca2+ mobilization in CHO cells transfected with
mCysLT1 long isoform cDNA (Maekawa et al., 2001).
The exact reason for this specific effect has not been explored.
Recently, Ogasawara et al. (2002) reported different pharmacological
properties of the CysLT2 receptor between human
and mouse, and they also showed that mCysLT1 and mCysLT2 tissue expression was different in some
strains of mice. These data suggest that receptor distributions may be
different within and between species.
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IV. Properties and Significance of Leukotriene Receptors |
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Well before the cloning of the leukotriene receptors, a considerable amount of indirect evidence had already suggested their existence. To date, the molecular database has confirmed the pharmacological database. However, a more thorough appreciation of ligand-receptor interactions under both physiological and pathophysiological conditions can only be achieved by an assessment of both these databases. Therefore, an effort has been made in this section to provide the evidence that reported the properties and significance of leukotriene receptors and to highlight those observations that suggested other receptor subtypes may exist.
A. BLT Functional and Radioligand Binding Studies
As early as 1974, certain monohydroxy eicosanoids, including the
platelet arachidonate lipoxygenase product (12-HETE), were shown to be
neutrophil chemoattractants (Turner et al., 1975) as well as other
monohydroxy products (5-HETE, 12-HETE) (Goetzl and Pickett, 1980;
Powell et al., 1996). In addition, certain derivatives of
LTB4, namely, 12-oxo-LTB4
and 20-hydroxy-LTB4 were reported to induce
chemotaxis (Yokomizo et al., 1993, 1996). However, each required higher
concentrations than LTB4 to induce chemotaxis in
experiments when cells were placed in isolated Boyden-type chambers
(Ford-Hutchinson et al., 1980). Furthermore, radioligand binding
studies (Goldman and Goetzl, 1982; Snyder et al., 1984; Goldman et al.,
1985; Gorman et al., 1985; Falcone and Aharony, 1990; Maghni et al.,
1991) provided more support for the existence and distribution of
specific receptors for LTB4. The differences in
relative potencies between LTB4 and the
mono-HETEs (LTB4 
HETE) also provided evidence
and facilitated the identification and classification of the cognate
receptor. However, the leukocyte surface receptors on neutrophils
involved in chemotaxis by which other mono-HETEs activate these cells
(Powell et al., 1996; Falgueyret and Riendeau, 2000) have not been identified.
B. Cysteinyl-Leukotriene Functional Studies
1. Airway Smooth Muscle Contraction.
Drazen et al.
(1980) reported that LTC4 and
LTD4 were equipotent in guinea pig tracheal
smooth muscle preparations whereas LTD4 was a
more selective peripheral airway agonist, since
LTD4 exhibited a 100-fold greater potency than
LTC4 in the lung parenchymal strip suggesting the
presence of two CysLT receptors. The heterogeneity of leukotriene
receptors received further support from Krell et al. (1983) who showed
that the potency of LTD4 in the guinea pig lung
parenchymal tissues (pEC50, 9.2) was
significantly different from that observed in the tracheal preparations
(pEC50, 7.8). In addition, Fleisch et al. (1982)
demonstrated that the potency of LTD4 was
different in tissue preparations derived from various organs. These
investigators reported that in the guinea pig ileum LTD4 had a pEC50 of 9.2, whereas in the trachea from this species the
pEC50 was 8.5. However, when contractile studies
were performed on human airways, results showed that
LTC4, LTD4, and
LTE4 were equipotent as contractile agonists
(Buckner et al., 1990; Labat et al., 1992) with
pEC50 values of approximately 10 nM, that is, about one-thousandth of the pEC50 values for
histamine in the same tissues (Dahlén et al., 1980). However,
LTE4 was markedly less active than
LTC4 and LTD4 in these
tissues (Buckner et al., 1986). A comparison of the cys-LT agonist
potencies between different tissues illustrates that considerable
variation exists in functional assays (pEC50
values 11 to 7) (Jones et al., 1984; Hand and Schwalm, 1987; Gardiner
et al., 1990; Gieske et al., 1990; Norman et al., 1990; DeLegge et al.,
1993). Based solely upon agonist potencies, one is unable to surmise
whether either a single or several receptors are present in these
preparations. This requires additional information from structural,
operational, and molecular biological studies. However, the data
suggest a marked difference between LTE4 and the
other cys-LTs. Interestingly, human vascular preparations exhibited
exquisite sensitivity to these mediators (Allen et al., 1994). reported a
pKB value for FPL 55712 of 6.9 in the
lung parenchymal tissues when LTD4 was used as
the agonist with a Schild plot slope of 0.72; similar results were
demonstrated by Krell et al. (1981). Furthermore, these latter
investigators showed that the potency of FPL 55712 (pKB) for antagonism of
LTD4-induced contraction in the parenchyma decreased with increasing concentrations of this antagonist, suggesting that in this tissue either factors may be released which were not
blocked by FPL 55712 or the profile of antagonism of this compound was
not competitive. In the guinea pig trachea, Krell et al.
(1981) calculated a pKB (6.4) for FPL
55712 for inhibition of LTD4 contraction. When
these preparations were contracted with LTC4, the
pKB for FPL 55712 was similar to that
obtained against LTD4. In contrast, when tracheal
tissues were treated with indomethacin the
pKB obtained against
LTC4 was 5.7. Thus, in the trachea, while FPL
55712 antagonized both LTD4-induced and
LTC4-induced contractions, the
pKB values differed (6.4 and 5.7, respectively). In parenchymal tissues, contractions to
LTD4, but not those to
LTC4, were inhibited. Fleisch et al. (1982)
reported their results with FPL 55712 against LTD4 contractions in different tissues and
demonstrated pKB values of 6.1 and 6.3 in parenchyma and trachea whereas in the ileum the value was 7.1; the
pA2 values were calculated to be 7.3 for the ileum, 6.5 for the trachea, and 6.0 for the parenchyma. These data also indicated that the dissociation constants for FPL 55712 derived from the trachea and parenchyma were markedly different from
that obtained in the ileum. Additional experiments with FPL 55712 supported the notion that different leukotriene receptors existed,
since FPL 55712 failed to antagonize the LTC4
contractions in the lung parenchymal strips and exhibited a
pKB of 9.2 against LTD4-induced contractions in the guinea pig
trachea. Since FPL 55712 was reported to have effects other than
leukotriene antagonism, including PDE inhibition (Welton et al., 1981),
the development of other specific compounds was required to establish
more definitive evidence for the presence of at least two receptors.
Many compounds with much greater potency
(Ki values of about 0.1-10 nM;
pA2 values or
pKB values of 9.5-7.5) and markedly
improved selectivity compared with FPL 55712 have been
pharmacologically identified and comprehensively characterized (Jones
et al., 1983; Buckner et al., 1986; Tucker and Weichman, 1986; Hay et
al., 1987; Krell et al., 1987, 1990; Muccitelli et al., 1987; Jones et
al., 1989, 1995; Torphy et al., 1989; Labat et al., 1992; Gardiner et
al., 1994). Collectively, the results with these chemically distinct
antagonists (Table 5) provided pertinent pharmacological support for
the presence of two receptors in the various tissue preparations.
2. Vascular Smooth Muscle Contraction.
Hanna et al. (1981)
reported that cys-LTs contracted not only isolated human airways but
also human pulmonary veins and noted that the maximum responses on
human pulmonary arteries were small (Schellenberg and Foster, 1984;
Bourdillat et al., 1987). Berkowitz et al. (1984) studied vascular
preparations from several species (rat, rabbit, and guinea pig) and
observed small contractions in guinea pig pulmonary veins, the inferior
vena cava and jugular vein; the effects of LTC4
were not examined. In the rabbit renal vein,
LTC4- and LTD4-induced
contractions (Kito et al., 1981), although only small or no contractile
responses, were observed in pulmonary and portal veins. Gleason et al.
(1983) also reported that LTD4-induced
contractions in the guinea pig-isolated pulmonary artery were small.
Together these observations suggested that a number of vascular
preparations exhibited little or no contractile response to the
cys-LTs. Whether this lack of contraction was due to the absence of
receptors or to factors that are released and mask the response has not
been systematically studied (Bäck et., 2002). However, in human
vascular preparations, Allen et al. (1992) reported a pronounced
constriction by LTC4 and
LTD4 in human-isolated saphenous veins and the
agonists were equipotent.
showed that FPL 55712 and ONO-RS-411 blocked the
LTD4 contractions in the guinea pig basilar
artery demonstrating a single specific receptor. However, in human
pulmonary veins, unlike those in human airways, the contractions
induced by cys-LTs were not affected by the
CysLT1 receptor-selective antagonists MK-571 and
ICI 198615 (Labat et al., 1992; Gorenne et al., 1996). These results
suggested the presence of distinct CysLT receptors on the human
pulmonary veins compared with either human bronchus or trachea (Jones
et al., 1982; Buckner et al., 1986). In contrast, Rinkema et al. (1993)
showed that the LTD4 contractions in the guinea
pig inferior vena cava were blocked by tomelukast and WY 48252. However, the contractions induced by LTC4 were
blocked in a biphasic fashion by these two antagonists, that is, the
low concentrations of LTC4 were not affected by
the antagonists suggesting two CysLT receptors. Therefore, in
some species, such as the guinea pig, vascular smooth muscle may
contain either one or two CysLT receptors associated with the
contractions. Whether the CysLT receptors in the human pulmonary veins,
which are resistant to these CysLT1 receptor
antagonists, are the same as the receptors in the guinea pig vena cava,
which are activated by LTC4, remains to be
established. Recently, Bäck et al. (2000b) have reported that
contractions induced by LTC4 in porcine pulmonary arteries were resistant to MK-571 (CysLT1
antagonist) as well as BAY u9773 (dual antagonist,
CysLT1/CysLT2), suggesting
that another receptor may be associated with this tissue response.
3. Vascular Smooth Muscle Relaxation.
Secrest et al. (1985)
reported that, in canine renal arteries where tone had been induced by
a contractile agonist, LTD4 provoked relaxations.
Similar results were obtained with LTC4 and
LTD4 in guinea pig pulmonary artery (Sakuma et
al., 1987; Sakuma and Levi, 1988) and isolated human saphenous veins
(Allen et al., 1992), and LTD4 in isolated human
pulmonary arteries and veins (Ortiz et al., 1995).
LTD4 relaxed canine renal blood vessels and was
approximately 100-fold more potent on veins than arteries. Whether or
not this latter difference is due to receptor density or
receptor-effector coupling is presently unknown.
LTC4 was not examined in these tissues. In canine
splanchnic venous capacitance vessels similar relaxations for
LTC4 and LTD4 have been
observed. Furthermore, in canine coronary arteries both
LTC4 and LTD4 relaxed the
tissues with the latter agonist being 10 times more potent. The data
reported indicate that the agonist potencies for relaxation of human
vascular preparations (pEC50 values,
approximately, 11-10) (Allen et al., 1992; Ortiz et al., 1995) are
markedly different from the potencies required to produce contractions
in the same tissue (pEC50 values, approximately,
9-7) (Schellenberg and Foster, 1984; Bourdillat et al., 1987; Allen et
al., 1992; Labat et al., 1992; Ortiz et al., 1995; Stanke-Labesque et
al., 2000). Such data suggest that either the receptors on the
endothelium associated with the relaxation are different from the
receptors responsible for the contraction or the G-protein second
messengers may be coupled more efficiently. In addition,
CysLT1 receptor antagonism of contractions in
preparations from the human lung (Table
7) suggest that the receptor present in
vascular smooth muscle may be different from that in the human airways.
Interestingly, the LTD4-induced relaxation of
isolated human pulmonary arteries and veins has also been reported to
be resistant to CysLT1 antagonists, MK-571 and
ICI 198,615 (Ortiz et al., 1995). The question remains as to whether or
not the receptor present on the endothelium and associated with the
relaxation is the same as that present on the vascular smooth muscle
and responsible for the contraction.
|
; Secrest and Chapnick, 1988)
reported that the endothelium-dependent relaxations were produced by
activation of a specific receptor since the response was attenuated by
FPL 55712. Sakuma and Levi (1988) observed that the relaxations induced
by LTC4 and LTD4 in the
isolated guinea pig thoracic aorta were antagonized with similar
potency by the CysLT1 receptor antagonist, ICI
198,615, suggesting that both agonists activate a single receptor
(CysLT1). This antagonism was also observed in
the guinea pig pulmonary artery. Therefore, on the endothelium of
guinea pig arterial preparations, a single receptor was present (Sakuma
and Levi, 1988), and activation was associated with relaxation. In
contrast, this does not appear to be the case in the canine renal
arteries and veins (Pawloski and Chapwick, 1993a; Pawloski and
Chapwick, 1993b) or in the human pulmonary arteries and veins (Ortiz et
al., 1995). In the latter study, the endothelium present on the human
pulmonary veins contains two CysLT receptors: a
CysLT2-type responsible for relaxation and a
CysLT1 receptor associated with the release of a
contractile factor (Ortiz et al., 1995).
4. Cardiovascular Effects.
Subsequent to the early
investigations (Levi and Burke, 1980; Levi et al., 1980), which
reported that partially purified SRS markedly reduced coronary blood
flow, a number of other investigators have explored the cardiovascular
effects of the cys-LTs in a variety of different animal models. The
early studies have shown that the cys-LTs are potent vasoconstrictors
of the coronary vasculature in both isolated perfused hearts (Woodman
and Dusting, 1982; Letts and Piper, 1983; Letts et al., 1983) and in a
number of species in vivo (Michelassi et al., 1982; Panzenbeck and
Kaley, 1983; Fiedler et al., 1984, 1987; Leffler et al., 1984; Fiedler
and Abram, 1987; Kopia et al., 1987; Michelassi et al., 1987) including human (Marone et al., 1988). In sheep and pigs, cys-LTs induced not
only coronary vasoconstriction but also ischemia and impaired left
ventriclar function (Michelassi et al., 1982; Ezra et al., 1983;
Fiedler et al., 1985). In isolated perfused guinea pig heart preparations (Terashita et al., 1982; Letts and Piper, 1983; Roth et
al., 1985), LTC4 and LTD4
caused a reduction in myocardial contractility concommitant with the
vasoconstriction. In perfused rat hearts (Bittl et al., 1985),
LTD4 caused not only a reduction in flow but also
reduced the spontaneous heart rate to a greater extent than
contractility, suggesting an action on conductivity in this species
(Feuerstein et al., 1981; Zukowska-Grojec et al., 1982; Zukowska-Grojec
et al., 1984; Tomoike et al., 1987).
also showed that FPL 55712 antagonized the
cardiodepressant effects of the cys-LTs suggesting that these effects
were via activation of a specific receptor and noted that the potency
of FPL 55712 was greater against ventricular contraction induced by
LTD4 compared with those of
LTC4, suggesting that there may be two receptors
present in the guinea pig heart. These investigators also reported that
in the human heart, the negative inotropic effect of cys-LTs was
similar to that of the guinea pig and the rank order potency
(LTD4 > LTC4
>LTE4) was the same for both species. In other
studies using isolated guinea pig cardiac muscle preparations, the data
have been contradictory. Letts and Piper (1983) reported no myocardial
effects whereas other investigators (Falcone et al., 1991a,b) have
demonstrated positive inotropic affects.
The use of potent and selective CysLT1 receptor
antagonists provided an alternate approach for establishing the effects
of cys-LTs in the perfused heart preparations. McLeod and Piper (1991) reported a differential antagonistic effect against
LTC4 and LTD4. ICI 198,615 selectively antagonized LTD4-induced increases in coronary vascular resistance with no significant effect against LTC4. Pobilukast antagonized both
LTC4 and LTD4 responses
whereas MK-571 was without effect on cardiac vascular resistance.
However, ICI 198,615 antagonized both LTC4- and
LTD4-induced ventricular contractility. These
data suggested that the reduction in contractility by
LTC4 may be more sensitive to the actions of the
antagonists when compared with their action against the
vasoconstrictory effect of LTC4. The failure of
MK-571 to antagonize the coronary vasoconstriction was also observed
against ventricular contractility induced by LTC4
and LTD4. In contrast, Falcone et al. (1990)
showed specific binding of [3H]ICI 198,615 to
guinea pig cardiac ventricular membranes, suggesting the existence of
CysLT1 receptors in this tissue, whereas Egan et
al. (1989) demonstrated that selective antagonism of the leukotriene responses did not reduce myocardial effects. Although the data from
animal studies remain controversial, the results derived from the
guinea pig indicate that the antagonist profile in the guinea pig heart
and lung preparations were not similar and suggested that the receptors
in cardiac vessels may be different from those in the airways of this species.
Marone et al. (1988) reported the effects of cys-LTs following
intracoronary injection in man. These investigators showed that low
doses of exogenous LTD4 and
LTC4 induced immediate changes of cardiovascular
function consisting of a fall in blood pressure, a rise in heart rate,
and sympathoadrenergic activation. These initial alterations were
followed by a marked vasoconstriction of small resistance coronary
vessels. In another study, Albazzaz et al. (1989) assessed the
cardiopulmonary effects of leukotriene C4
inhalation in nonasthmatic and asthmatic patients. Although the mean
cardiac output decreased without changes in blood pressure and heart
rate in both subjects, there was difficulty in separating the cardiac
output modifications from the relative contribution of the pulmonary
vasoconstriction, bronchoconstriction, and reduced cardiac output,
which followed the challenge. The use of the potent and selective
CysLT1 receptor antagonists for such in vivo
studies in man may provide an alternative for understanding the role of these mediators in cardiovascular disease.
5. Diverse Effects of Cysteinyl-Leukotrienes.
Human umbilical
vein endothelial cells (HUVECs) have been reported to produce (Sjostrom
et al., 2001) as well as respond to all cys-LTs (Datta et al., 1995) by
releasing von Willebrand Factor as well as an increased expression of
P-selectin The effects were blocked by the CysLT1
receptor antagonist, pobilukast. These investigators demonstrated a
concomitant surface expression of P-selectin and release of
high-molecular weight von Willebrand Factor by the cys-LTs due to
stimulation of exocytosis of the Weibel-Palade body pools rather than
promoting the constitutive release from endothelial cells. In contrast,
Pedersen et al. (1997) showed that the P-selectin secretion induced by
LTD4 and LTC4 was not inhibited by three different CysLT1 receptor
antagonists (zafirlukast, SB 205,312 and pobilukast). However, in this
latter study the pool of P-selectin monitored by their assay was not
described. The difference between the effects of the
CysLT1 antagonists may depend on which pool of
P-selectin was assayed rather than a subset of receptors.
,b; Evans et
al., 1989; Obata et al., 1992; Bochnowicz and Underwood, 1995); the
mechanism may involve contraction of endothelial cells resulting in
gaps in the endothelium of venules (Joris et al., 1987). This
influence of the cys-LTs, which appeared to involve both direct and
indirect pathways, was inhibited by either FPL 55712 (Woodward et al.,
1983a) or pranlukast (Nakagawa et al., 1992; Bochnowicz and Underwood,
1995) indicating that a CysLT1 receptor was involved.
There is increasing evidence that the cys-LTs may also influence
inflammatory cell function since they enhance the recruitment of
eosinophils. For example, inhaled LTE4 (Laitinen
et al., 1993) or LTD4 selectively increased
airway eosinophil numbers when assessed in bronchoalveolar lavage from
humans (Smith et al., 1993). Similar results were obtained with
LTC4 or LTD4 in guinea pigs
(Foster and Chan, 1991; Underwood et al., 1996), with single
administration of inhaled LTD4 elicited a marked
increase in eosinophils, which was maintained for at least 4 weeks
(Underwood et al., 1996). The receptor responsible for this phenomenon
of the cys-LTs appears to be CysLT1 receptor
(Foster and Chan, 1991; Underwood et al., 1996) and, at least in guinea
pigs, appeared to involve IL-5 (Underwood et al., 1996), a key cytokine
in eosinophil proliferation, activation, and survival. Pobilukast
inhibited LTD4-induced chemotaxis of peripheral
blood eosinophils from nonasthmatic volunteers (Spada et al., 1994).
Furthermore, the CysLT1 receptor antagonists
pranlukast, MK-571, ICI 198,615 inhibited antigen-induced eosinophil
influx in rats (Salmon et al., 1999), guinea pigs (Foster and Chan,
1991), and cynomolgus monkeys (Turner et al., 1994).
The effects of the cys-LTs on mucus secretion have been explored only
to a limited degree. Initial studies involved the incorporation of
radiolabels such as [3H]glucosamine into
secretory cells (Shelhamer et al., 1980; Coles et al., 1983; Kim et
al., 1989, 1993). Although the cys-LTs were shown to increase release
of radiolabeled material from the secretory elements of the human
bronchus, there is little information on the effects of selective
antagonists (Lundgren et al., 1990). LTC4 or
LTD4 induced an increase in mucus secretion from
cultured human airway mucosal explants, an effect which was antagonized by FPL 55712 (Marom et al., 1982; Coles et al., 1983). Pobilukast inhibited the aerosolized LTD4-induced increase
in epithelial mucus secretion in guinea pig airways in vitro (Hoffstein
et al., 1990), suggesting that the effects of the cys-LTs on mucus
secretion are CysLT1 receptor-mediated. Labat et
al. (1999) have reported that challenge of human bronchial preparations
with LTD4 failed to elicit increased amounts of
the MUC5AC gene product, suggesting that there may or may not be
alterations in the secretory activity of human airways depending upon
which secretory product is monitored.
Panettieri et al. (1998) reported that although
LTD4 alone had no significant effect on DNA
synthesis in human cultured airway smooth muscle cells, there was a
marked potentiation in the airway smooth muscle proliferation induced
by the classical mitogens, epidermal growth factor, or thrombin. This
effect of LTD4 was inhibited by either pranlukast
or pobilukast, but not zafirlukast, suggesting activation of a CysLT
receptor other than CysLT1 (Panettieri et al.,
1998). Evidence for an influence of the cys-LTs on airway smooth muscle
proliferation is derived from the results of two in vivo studies in
rats following chronic antigen challenge where airway smooth muscle
proliferation and hyperreactivity (Wang et al., 1993) or eosinophil
infiltration (Salmon et al., 1999) were attenuated by the
CysLT1 receptor antagonists, MK-571 (Wang et al.,
1993) or pranlukast (Salmon et al., 1999).
There is also evidence in guinea pigs that the cys-LTs may influence
lung function by modulating the afferent nervous system, specifically
enhancing activity of capsaicin-sensitive sensory C fibers (Stewart et
al., 1984; Undem and Weinreich, 1993; Undem et al., 1993). For example,
LTD4 enhanced the release of substance P
(Bloomquist and Kream, 1990) and potentiated tachykinin-mediated, nerve-induced responses (Ellis and Undem, 1991) in guinea pig trachea.
Vagal nerve-induced contraction and microvascular permeability in
guinea pig airways are inhibited by CysLT1
receptor antagonists (Ellis and Undem, 1991), indicating that the
effects of the cys-LTs on tachykinin-containing sensory C fibers are
mediated via the CysLT1 receptor in guinea pig airways.
There are a number of other observations concerning the cys-LTs that
require further investigation. Few studies have been performed to
examine these mediators in angiogenesis (Tsopanoglou et al., 1994)
where pobilukast has been reported to block responses to
LTD4, albeit with less potency than those to
LTC4. The effects of these mediators on liver
metabolism has been overlooked but an investigation has demonstrated
that both LTD4 and LTC4 are equipotent in altering glucose and lactate output, and this
modification is blocked by CGP 35949, a CysLT1
antagonist (Iwai and Jungermann, 1988, 1989). In addition,
investigations on the actions of the cys-LTs on renal function
(Chapnick, 1984; Filep et al., 1985; Piper et al., 1985; Petric and
Ford-Hutchinson, 1995), the pulmonary circulation (Smedegard et al.,
1982; Kadowitz and Hyman, 1984; Jones and Masson, 1985; Malik et al.,
1985; Garcia et al., 1987; Ohtaka et al., 1987; Schreiber et al.,
1987), systemic circulation (Pfeffer et al., 1983), and on the
microcirculation (Dahlén et al., 1981; Bisgaard et al., 1982,
1985, 1986; Soter et al., 1983; Chan and Ford-Hutchinson, 1985; Hua et
al., 1985) to established specific CysLT receptors have not been pursued.
C. CysLT Radioligand Binding Studies
The presence of heterogeneous CysLT receptors was also supported
by data from experiments using classical radioligand binding techniques. Cheng et al. (1985) provided evidence that the CysLT receptors were widely distributed in different organs, tissues, and
species. Furthermore, autoradiographic studies with the labeled agonists have also demonstrated the presence of CysLT receptors in lung
(Carstairs et al., 1988), endothelial cells (Chau et al., 1986), uterus
(Chegini and Rao, 1988b), corpus lutea (Chegini and Rao, 1988a), as
well as brain (Goffinet, 1986). In addition, a number of smooth muscle
or transformed cells in culture were also shown to possess these
receptors (Krilis et al., 1983; Mong et al., 1988; Frey et al., 1993).
The radioligand binding studies have not only demonstrated the presence
of specific sites for [3H]LTC4,
[3H]LTD4, and
[3H]LTE4 binding but also
have shown that there is often a preferential binding for one ligand
and not the other (Mong et al., 1984a, 1985; Cheng et al., 1985). These
latter observations suggested that certain tissues may contain a single
class of receptor responsible for the physiological actions of cys-LTs.
Although a considerable amount of evidence demonstrated that membrane
preparations from a number of tissues bind all the radiolabeled
agonists, there are data suggesting the existence of at least two
sites: one associated with
[3H]LTC4 and one that
binds [3H]LTD4 and
[3H]LTE4 (Cheng and
Townley, 1984b).
However, three major points became apparent from the radioligand
binding studies that need to be highlighted. First, interpretation of
the results of [3H]LTC4
binding assays were complicated by the rapid metabolic transformation
of LTC4 to LTD4 if this was
not controlled by enzymatic inhibition. Inhibitors of this metabolism
must frequently be incorporated in
[3H]LTC4 binding studies.
In addition, recent binding studies (Ravasi et al., 2000) have also
suggested the necessity to include S-decyl-glutathione (a
high-affinity ligand for nonreceptor LTC4 binding
sites) in the membrane preparations. Second, there was often no
correlation between the
[3H]LTC4 binding studies
and the functional studies (contraction) as in the case of either the
guinea pig ileum (Norman et al., 1990), guinea pig lung strip (Mong et
al., 1985; Norman et al., 1987), or guinea pig uterus (Weichman and
Tucker, 1982; Levinson, 1984). In contrast, a correlation was observed
between the antagonist effects in the
[3H]LTD4 binding studies
and the ability of the antagonists to block LTD4-induced contractions (Fig.
6). Furthermore, in the rat lung the
radioligand binding data provided evidence for the presence of two
sites (Pong et al., 1983; Metters et al., 1991) whereas the functional
data (contraction) suggested a single receptor was associated with the
contractions with LTC4 as the full agonist (Norman et al., 1994; Gardiner et al., 1994). Third, the number of
[3H]LTC4 binding sites in
most tissues was in excess of 10 pmol/mg of protein whereas the number
of binding sites for
[3H]LTD4 was markedly
lower. These observations suggested that
[3H]LTC4 may bind to
other cellular entities, such as glutathione S-transferase
(GST) as has been reported by several investigators (Sun et al., 1987;
Metters et al., 1994; Bannenberg et al., 1999). In addition,
LTC4 may also bind to specific exporters in human eosinophils (Lam et al., 1992), to ATP-dependent carriers in rat liver
(Ishikawa et al., 1990; Keppler et al., 1992), and to
multidrug-resistant protein 1 (Qian et al., 2001). The GST data (Sun et
al., 1987) supported the original observations of Mong et al. (1985)
who demonstrated that the glutathione conjugates (SK&F 103,050 and SK&F
103,024), which were devoid of contractile activity in the guinea pig
lung and trachea, exhibited a high affinity to the specific site
labeled by [3H]LTC4.
Furthermore, these glutathione conjugates did not antagonize the
LTC4-induced contractions in the guinea pig
trachea. These observations suggest
[3H]LTC4 binding to GST
may mask the LTC4 receptors present in the preparations and thereby explain the reason for the lower affinity for
LTC4 binding sites when compared with the
functional data. However, the evidence reported by several groups in
functional studies have shown that in guinea pig tracheal preparations
the LTC4-induced contractions, in the presence of
inhibitors of -glutamyl transpeptidase (Snyder and Krell, 1984;
Charette and Jones, 1987), are resistant to the classical
CysLT1 antagonists suggesting a distinct receptor
for LTC4.
|
1. [3H]LTC4 Binding Sites.
Despite
the [3H]LTC4 binding to
nonreceptor proteins, which is now well established,
[3H]LTC4 binding has been
demonstrated in the central nervous system (Schalling et al., 1986),
brain capillaries (Mayhan et al., 1986; Black et al., 1987),
endothelial cell membranes (Muller et al., 1987), as well as human
erythrocytes (Ghiglieri-Bertez et al., 1986) and leukocytes
(Baud et al., 1987b). Unfortunately, the significance of this
[3H]LTC4 binding is
presently unknown. Cheng et al. (1985) showed that membrane
preparations from the guinea pig uterus did not bind
[3H]LTD4, data that were
also confirmed in kinetic binding experiments (Mong et al., 1985). The
[3H]LTC4 binding
Kd value (10 nM) in the membrane
fractions in the uterus (Levinson, 1984) was similar to that reported
for binding to intact cultured smooth muscle cells from the hamster vas
deferens (Krilis et al., 1983) but slightly lower than that reported
for the guinea pig and rat lung membranes (Hogaboom et al., 1983: Pong
et al., 1983). Similar concentrations of LTC4
competed for [3H]LTC4
binding in the guinea pig ileal muscle (Nicosia et al., 1984). Together
these data are consistent with the notion that a specific high-affinity
receptor was present in the uterus. Further studies have been performed
using brain homogenates, which contain the highest activity of
[3H]LTC4 binding sites,
and the potency order of the cys-LTs for inhibition of the brain
[3H]LTC4 binding is
identical to their inhibitory order in the uterine homogenate. In
addition, these studies have demonstrated that the relative ability of
LTC4, LTD4, and
LTE4 to inhibit
[3H]LTC4 binding in the
brain and uterine membrane fraction was the same as their ability to
reduce binding in the smooth muscle cell and lung homogenates (Krilis
et al., 1983, 1984; Pong et al., 1983; Cheng et al., 1985). In
contrast, the IC50 of LTC4 to inhibit brain [3H]LTC4
binding differs from that value obtained in the uterine homogenate.
This suggests a difference between the dissociation constant
(Kd) of brain and uterine
[3H]LTC4 binding sites.
Data from initial studies are comparable with these results and have
shown that the Kd of lung
[3H]LTC4 binding
apparently differed from that demonstrated in the smooth muscle cell
(Krilis et al., 1983; Pong et al., 1983). Whether this difference is
due to the existence of high- and low-affinity LTC4 receptor sites (Krilis et al., 1983) awaits
further studies. Interestingly, Cheng and Townley (1984a) also
demonstrated that LTC4 was more potent than
LTD4 in the ability to produce uterine contractions. These results would suggest that in the guinea pig uterine preparations
[3H]LTC4 binds to a
receptor that may be responsible for cys-LT-induced uterine contraction
in this species.
2. [3H]LTD4 Binding Sites.
The
initial data from radioligand binding in a variety of membrane
preparations demonstrated the presence of
[3H]LTD4 binding sites.
There was also a correlation between the tissue distribution and the
binding sites for
[3H]LTD4 and
[3H]LTE4 (Cheng et al.,
1985). In addition, the apparent affinity constants for agonists and
antagonists (Mong et al., 1985) suggested that
[3H]LTD4 and
[3H]LTE4 bound to the
same receptor in guinea pig lung preparations. Aharony et al. (1989)
provided the pertinent evidence that this was indeed the case in the
guinea pig lung. However, these investigators demonstrated that not
only was the rank order inhibition potency similar when obtained
against either ligand but also the absolute potency for
LTC4, LTD4, and
LTE4 was higher against
[3H]LTE4 than that
observed with [3H]LTD4.
These results suggested a discriminative binding of
[3H]LTE4 toward a subset
of high-affinity LTD4 receptors. Although ICI
198,615 exhibited equal potency for inhibiting both
[3H]LTE4 and
[3H]LTD4, there were
subtle differences; namely, LTD4 was
significantly less potent than ICI 198,615 for inhibiting
[3H]LTD4 binding whereas
no such difference was observed against [3H]LTE4. Furthermore,
the rank order antagonist potency against [3H]LTD4 was ICI 198,615 analog > ICI 198,615 > zafirlukast whereas against
[3H]LTE4 the antagonists
were equipotent. These investigators also reported that there was a
higher sensitivity to inhibition by sodium ions and GTP analogs for
[3H]LTE4 binding compared
with [3H]LTD4. These data
provided evidence for the existence of heterogeneous LTD4 receptors. This suggestion has received some
support from Shirley and Cheng (1991) who demonstrated that either
LTD4 (Ki = 0.49 nM) or ICI 198,615 (Ki = 6.89 nM) interacted with a
single homogeneous
[3H]LTD4 binding site,
whereas the competitive binding results of either
LTC4 (in the presence of AT-125) or
LTE4 exhibited high- and low-affinity
[3H]LTD4 binding sites.
, 1992), the binding sites for
the same radioligand in one tissue may also be different from that
observed in another preparation. In guinea pig lung membranes,
[3H]LTD4 exhibits a
Kd of ~0.43 nM whereas in the
membranes obtained from the guinea pig myocardium the
Kd is 3.4 nM. In the latter study,
Hogaboom et al. (1985a,b) reported that the myocardial [3H]LTD4 binding sites
were neither modulated by guanine nucleotides nor divalent cations and
the sulfhydryl-reducing reagents altered the maximum number of sites
without any effect on binding affinity. Unfortunately the significance
of these myocardium sites has not been explored.
In a study involving [3H]ICI 198,615, Aharony
et al. (1988) reported that a CysLT receptor (which corresponds to the
current CysLT1 receptor) was coupled to a
G-protein and exhibited high- and low-affinity states. In addition,
Metters and Zamboni (1993) identified the CysLT1
receptor as a single polypeptide with a molecular mass of 45 kDa using
direct photoaffinity labeling. These investigators demonstrated that
this protein was inhibited by both agonist and antagonists with
potencies (IC50) similar to those reported in
radioligand binding studies. Furthermore, both cations and nucleotide
analogs modulated the photolabeled protein. This latter characteristic
was analogous to [3H]LTD4
specific binding in the guinea pig lung membranes.
D. Evidence for Additional CysLT Receptor Subtypes
The reported functional data suggest that other CysLT receptors
may exist. Initial reports have shown that the
LTC4 contractions of the guinea pig trachea are
resistant to the classical CysLT1 antagonists
(Snyder and Krell, 1984). Furthermore, in the guinea pig lung
preparation, MK-571 exhibited a low affinity for the cys-LTs
(pKB 5.8) and BAY u9773 did not block
the cys-LT contractions (Gardiner et al., 1993; Sakata and Bäck,
2002), data which do not fit the classical CysLT1
profile. One interpretation is that there may be a
CysLT1 receptor subtype. In addition, Tudhope et al. (1994) observed a residual contraction in the guinea pig lung strip
subsequent to CysLT1/CysLT2
antagonism by BAY u9773. Recently, Bäck et al. (2000a,b) have
reported that the contractions to LTC4 in porcine
and human pulmonary arteries were not antagonized by either MK-571 or
BAY u9773, data which further suggest the presence of another CysLT
receptor responsible for the LTC4-induced contractions. These latter observations have received support from the
work of Northacker et al. (2000) who demonstrated that BAY u9773 was a
selective CysLT2 agonist. Although BAY u9773
contracted human pulmonary veins (Labat et al., 1992) in porcine and
human pulmonary arterial preparations, this compound did not modify basal tone. These data suggest that the CysLT receptor associated with
the contractions of human pulmonary arteries may be different from
those receptors characterized as CysLT1 and
CysLT2. These observations have been extended by
Walch et al. (2002) who suggested a "novel CysLT receptor" based on
the data in the human pulmonary artery (Table 7).
Panettieri et al. (1998) demonstrated an enhanced
LTD4 proliferation of human smooth muscle cells
in the presence of epidermal growth factor. These investigators
suggested that the CysLT1 receptor associated
with proliferation may be different from the
CysLT1 receptor activated in human airways
responsible for contraction. This interpretation was based on the low
affinity of the well known CysLT1 antagonists,
pranlukast and pobilukast, which blocked the effect of
LTD4 in this assay. In addition, the authors also reported that zafilukast did not work at a high concentration. Together
these data do not suggest that based only on zafirlukast the receptor
is different but that all of the antagonists were less active at
concentrations that caused a significant shift of
LTD4 contractions in the human airways. However,
the contractile response at CysLT1 receptors in
human airways was also induced by LTC4 and
LTE4, the proliferative response should therefore also be induced by these ligands as well. Until such data are presented, the results are preliminary and not conclusive for the
existence of another receptor subtype.
Using molecular techniques, Ogasawara et al. (2002) demonstrated
different pharmacological characteristics of
mCysLT2 and hCysLT2. These
investigators reported that paranlukast, which inhibited
hCysLT1, antagonized
mCysLT2 responses as determined by
Ca2+ mobilization and receptor-induced promoter
activation. Furthermore, the distribution of mRNA expression of
mCysLT1 and mCysLT2 in tissues between different strains of mice was also different These results provide further evidence that other CysLT receptor subtypes may
exist as has previously been suggested (Mellor et al., 2001). Indeed
the up-regulation of the CysLT1 receptor by
different cytokines (Mellor et al., 2001; Thivierge et al., 2000,
2001) raises the further question as to whether or not the
"induced receptor" is similar to the "constitutive receptor".
Recently, results generated from radioligand binding studies in human
lung membrane preparations suggested that
[3H]LTC4 binding (Capra
et al., 1998) may be associated with a specific LTC4 receptor different from the classical
CysLT1 and CysLT2 receptors (Ravasi et al., 2000, 2002). These latter investigators demonstrated that the LTC4 contractions of human lung
parenchyma were via a classical CysLT1 receptor
activation whereas the
[3H]LTC4 binding
(high-affinity site) was insensitive to GTP. Furthermore, the
CysLT1 receptor antagonists, zafirlukast and
iralukast, did not interact with this site.
Although, the above results suggest that other receptor subtypes may be present in different preparations, this hypothesis is based solely on indirect pharmacological assessment and should be interpreted cautiously. Extension of the current CysLT receptor classification requires additional investigation, namely, structural, operational, and molecular biological information.
E. Clinical Studies Involving CysLT Receptors
Most molecules that have been identified as antagonists of the
CysLT1 receptor in isolated guinea pig tissue
antagonize the effects of LTC4 and
LTD4 to an equal extent (Buckner et al., 1986, 1990) in human isolated bronchial tissues. These data suggest that in
healthy human medium-sized airways CysLT1 is the
only leukotriene receptor responsible for the contractile response. These data have also received some support from Civelli et al. (1987)
who demonstrated that the number of
[3H]LTC4 binding sites in
membrane preparations derived from human segmental bronchi at different
levels of the respiratory tract were similar even though there was
large interindividual variation. Furthermore, these investigators
reported that [3H]LTC4
may actually label LTD4 sites as well, since
LTD4 bound with higher affinity to the lower
capacity class site. Although certainly not conclusive, these latter
observations suggest that this uniform distribution of sites may
reflect the functional receptors reported by other investigators
(Buckner et al., 1986; Labat et al., 1992).
Inhalation of aerosols generated from solutions of cysteinyl
leukotrienes results in airway obstruction manifested as decreased specific airway conductance (SGaw) or
as decreased flow rates (measured from partial or full expiratory
flow-volume curves) (Drazen, 1986); LTC4 and
LTD4 are nearly equipotent contractile agonists
in intact humans. Nebulizer concentrations on the order of 10 µM are
required to reduce the maximal expiratory flow rate (measured from a
partial flow-volume curve,
V30-P) by 30% in
nonasthmatic subjects. Approximately 30-fold greater concentrations of
LTE4 are required for an equivalent physiological
effect in nonasthmatic subjects. Comparable nebulizer concentrations
are required for reduction of a similar magnitude in the
SGaw. Compared with a reference
agonist in nonasthmatic subjects (with
V30-P or
SGaw as the index of airway
obstruction), LTC4 and LTD4
are approximately 2000 times more potent than histamine, whereas
LTE4 is 30 to 100 times more potent than
histamine as a bronchoconstrictor agonist (Weiss et al., 1982; Griffin
et al., 1983; Barnes et al., 1984; Smith et al., 1985; Adelroth et al.,
1986; Greenberger et al., 1986; Kern et al., 1986; Davidson et al.,
1987). Complete dose-response curves show that the plateau response to
LTD4 is greater than that to methacholine (Bel et
al., 1989). Since no such difference is evident with dose-response
curves for 2- to 4-mm isolated bronchi, these findings are consistent
with the hypothesis that airways from physically distinct loci (i.e.,
small versus large airways) respond to both stimuli
(LTD4 and methacholine) in normal human subjects.
Patients with asthma also exhibit bronchoconstrictor responses when
they inhale aerosols generated from solutions of cys-LTs (Griffin et
al., 1983; Adelroth et al., 1986; Davidson et al., 1987; Pichurko et
al., 1989; Smith et al., 1993); the aerosol generator concentrations of
LTC4, LTD4, and
LTE4 required to decrease the
V30-P by approximately 30%
are about 30- to 100-fold less than is required in normal human
subjects. The bronchoconstrictor responses of asthmatic subjects to
these leukotrienes are all manifest within 3 to 5 min after aerosol
inhalation; the duration of the effect is related to the severity of
the bronchospasm but is on the order of 20 to 30 min when the decrement
in the V30-P is 30%.
Thus the predominant biological effect of administration of leukotrienes by inhalation is broncoconstriction mediated by the CysLT1 receptor. There may be other effects when leukotrienes are administered systemically but at present there are inadequate data to make a statement.
F. Summary
The development of selective antagonist for specific CysLT cell surface receptors was undertaken to harness the detrimental aspects of the inflammatory process specifically in asthma. This research has lead to compounds with clinical benefits in asthmatic patients and the milestones in the sequence of events which have led to this therapy are presented in Table 8.
|
Therefore, based on the CysLT1 antagonists, certain therapeutic goals have been achieved. However, the evidence to suggest that the metabolites of the 5-lipoxygenase enzymatic pathway activate other CysLT receptors, which may have further implications in pathophysiological conditions, needs to be addressed. Unfortunately, there are no selective antagonists for the CysLT2 receptor. Recent molecular studies have shown that this receptor is expressed in several human tissues (heart, brain, peripheral blood leukocytes) but an exploration of their role in these tissues remains markedly compromised until selective pharmacological compounds become available.
Interestingly, there is some evidence from vascular studies that a
CysLT receptor may be induced during pathological conditions, such as
atherosclerosis (Allen et al., 1993, 1998) since cys-LT-induced contractions were only observed in tissues derived from patients with
this disease. In addition, the potency of the cys-LTs are markedly
altered during aging (Duncan and Douglas, 1985). Whether these data
reflect modification in receptor number, affinity, or second messenger
pathways has never been pursued. Recently, Mellor et al. (2001) have
reported that the CysLT1 receptor may function as
a UDP pyrimidinergic receptor. These latter results indicate that the
eicosanoid GPCRs may exhibit a dual receptor role. UDP acting with the
CysLT1 receptor is analogous in some respects to
the peptides that can act at the ALX. These observations provide future
perspectives for studies in receptor classification.
G. Lipoxins Receptors
1. Functional and Radioligand Binding Studies.
Although
LXs are ancient molecular structures conserved and produced by fish as
well as frog blood cells (Rowley et al., 1994), the potential
physiologic roles of LX and related compounds have not been widely
tested outside of the context of inflammation and the immune system.
The principal actions reported for LX focus on leukocytes, in
particular neutrophils, monocytes, and eosinophils in vitro and in
vivo, suggesting that these cells are the major targets. The potency of
action of LX on human leukocytes is in the nanomolar to subnanomolar
range. Experiments with LXA4 were initially
performed on guinea pig lung strips to establish structure-activity relationships. These early reports indicated stereospecificity, that
is, the 5S,6R-orientation of the two hydroxyl
groups positioned immediately adjacent to the carboxylic end of the
conjugated tetraene was essential for the contractile activity in the
submicromolar range. These stereospecific requirements suggested the
presence of specific LXA4 recognition sites.
Although this evidence was essential in establishing the biological
activities evoked by LXA4, the use of isolated
lung strip as a target tissue model for endogenous LX actions in view
of recent findings may be misleading. These compounds, in particular
LXA4, act in the subnanomolar ranges to initiate
protective actions. In this regard, LXA4
possesses human leukocyte-selective actions that implicate lipoxins as
endogenous stop signals or chalones (Serhan, 1994, 1997). In addition,
LXA4 stimulates rapid lipid remodeling and
release of arachidonic acid in human PMN, which is sensitive to PTX
treatment (Grandordy et al., 1990; Nigam et al., 1990). These results
point to the involvement of a GPCR and indicate that
LXA4 stimulates selective functions of human
leukocytes (PMN and monocytes) that are markedly different from the leukotrienes.; Fiore et al., 1992; McMahon et al., 2000; Gronert et al., 2001) and block LTD4 binding to mesangial cells (Badr
et al., 1989) and isolated HUVECs (Takano et al., 1997). HUVECs
specifically bind
[3H]LXA4 with a
Kd of 11 nM, which can be inhibited by
LTD4 and the CysLT1
receptor antagonist, pobilukast (Fiore et al., 1993). This receptor is
inducible in HUVECs and identical to the CysLT1 (Gronert et al., 2001). Therefore, LXA4 interacts
with at least two classes of cell surface receptors: one specific for
LXA4 on leukocytes, the other shared by
LTD4 that is present on HUVEC and mesangial
cells, namely, CysLT1.
). LXA4 was also reported recently to
bind to the Ah receptor, which is a ligand-activated transcription factor (Schaldach et al., 1999). The finding that
LXA4 blocks either PAF- or fMLP-stimulated
eosinophil chemotaxis (Soyombo et al., 1994) suggests that functional
ALX is also present on eosinophils.
ALX activation on human PMNs evokes inhibition of
LTB4- and fMLP-stimulated PMN adhesion (by
down-regulating CD11/CD18), chemotaxis and transmigration as well as
degranulation (Serhan, 1997). In human enterocytes (T84), LX analogs
inhibit TNF-induced IL-8 release (Gronert et al., 1998) as well as
pathogen-induced IL-8 secretion at the mRNA level (Gewirtz et al.,
1998). In addition, LXA4 analogs inhibit rat
leukocyte rolling and adherence by attenuating P-selectin expression in
mesenteric microvasculature (Scalia et al., 1997). When applied to
mouse ears, LX analogs markedly reduced LTB4-induced PMN infiltration and vascular
permeability (Takano et al., 1998). Moreover,
LXA4 inhibits PMN recruitment to inflamed glomeruli in vivo (Papayianni et al., 1995), further supporting the
anti-inflammatory actions of LX to activate "stop signaling" via
the ALX receptor.
To further investigate ALX receptors and their function in
inflammation, analogs based on the native structures of
LXA4 and LXB4 were designed
to resist rapid inactivation (Serhan et al., 1995; Takano et al.,
1998). Methyl groups were placed on carbon-15 and carbon-5 of
LXA4 and LXB4 structures,
respectively, to block dehydrogenation (Serhan et al., 1995; Clish et
al., 2000).
15(R,S)-methyl-LXA4 is a
racemic stable analog of both LXA4 and the
aspirin-triggered 15-epi-LXA4. Additional analogs
of LXA4 were synthesized with a phenoxy group
bonded to carbon-16 and replacing the 
-end of the molecule. This
design permits 16-phenoxy-LXA4 to resist
potential -oxidation and to be protected from dehydrogenation and
potential -oxidation in vivo by the stearic hindrance of the bulky
aromatic ring. The actions of LX analogs were tested in vitro and in
vivo and proved to be potent LX mimetics (Clish et al., 1999, 2000). Furthermore, the topical application of LX and 15-epi-LX analogs in a
mouse ear model of acute inflammation demonstrated that these analogs
are potent inhibitors of LTB4- and phorbol
12-myristate 13-acetate-initiated neutrophil-recruitment as well as
PMN-mediated vascular injury (Takano et al., 1997, 1998). These LX
mimetics proved to be more potent as topical agents than
LTB4 receptor antagonists within the range of the
clinically used anti-inflammatory steroid dexamethasone (Takano et al.,
1998). In addition, in a rat model of endothelial dysfunction
characterized by reduced nitric oxide release and up-regulation of
adhesion molecules such as P-selectin, superfusion with LX analogs
dramatically inhibited leukocyte rolling (Scalia et al., 1997). Thus,
these analogs of LX and 15-epi-LX serve as potent topically active
agents that inhibit PMN recruitment and PMN-mediated changes in
vascular permeability.
In addition to LXA4 and aspirin-triggered
LXA4 analogs, ALX is activated by and competed
with a series of rogue synthetic peptides that are also chemotactic
(Chiang et al., 2000). CHO cells with Gq/o
expressing ALX can chemotax in response to either the synthetic peptide
MMK-1 or 15-epi-LXA4 analog. CHO cell chemotaxis to 15-epi-LXA4 was not only ligand-dependent but
also Gq/o-dependent in that without
Gq/o neither LXA4 nor
15-epi-LXA4 or their analogs could stimulate
chemotaxis. Several synthetic nonphysiological peptides interact with
ALX-related receptors such as FPRL2 (Christophe et al., 2001). The
bioactive LX and 15-epi-LX analogs, as well as select small peptides,
each competed for specific
[3H]LXA4 binding with
recombinant human ALX, and the N-glycosylation of the
receptor proved essential for peptide but not
LXA4 recognition. Several chimeric receptors
(Chiang et al., 2000) were constructed from receptors with opposing
functions, namely ALX and BLT1. Results with
these chimeric receptors also showed that the seven-transmembrane segment and adjacent regions of ALX are essential for
LXA4 recognition, and additional regions of ALX
are required for high-affinity binding of the peptide ligands. These
initial findings indicated that a single seven-transmembrane receptor
can switch recognition as well as function with certain chemotactic
peptides from stimulatory to inhibitory with aspirin-triggered lipoxins
(ATL) and LX (lipid ligands). This evidence also raises the possibility
that ALX activation by LX or ATL can protect the host from potentially
deleterious PMN-induced responses associated with innate immunity and
direct effector responses to sites of tissue injury by recognition of mitochondrial-derived peptide protein fragments. Hence, activation of
ALX by LXA4, aspirin-triggered
15-epi-LXA4 or their analogs can dampen
inflammation by evoking a stop signal as well as blocking either
LTC4 or LTD4 responses at
CysLT1 receptors (Gronert et al., 2001). The in
vivo combination of these sites of LXA4 action (ALX and CysLT1) favor resolution of inflammation
and protection from acute PMN-mediated tissue injury.
2. Summary.
The ability to inhibit the expression of
pro-inflammatory messengers, regulate trafficking, and sequestration of
leukocytes as well as antagonize the CysLT1
receptor, illustrate that LX and ATL by activating specific receptors
have clearly established roles in a variety of vascular-related
inflammatory phenomenon. Of considerable interest is the ability of the
stable aspirin-triggered lipoxin analogs (ATLa) to antagonize the
LTD4 activation of a CysLT1
receptor in the mouse ear model (Gronert et al., 2001). Previous
investigators (Ortiz et al., 1995) have reported that LTD4, via CysLT1 receptor
activation, released a contractile factor from endothelial cells in
human pulmonary vessels. This factor masked the functional relaxations
associated with nitric oxide. Whether or not the
CysLT1 receptor antagonized by ATLa in the murine
vascular model is identical to that present on the endothelium of the
human pulmonary vessels remains to be determined. However, lipoxin
activation of an ALX receptor also inhibits
LTD4-induced mesangial cell proliferation
(McMahon et al., 2000). These investigators demonstrated that two
receptor subtypes were present in these cells, namely, an ALX receptor
and an ALX/CysLT shared receptor. Recently, McMahon et al. (2002) have
extended these observations to include further cross-talk between
receptors, since leukotrienes and lipoxins also interact with PDGF
receptor in mesangial cell proliferation. These data provide
evidence of the complex mechanisms involved in mediator-receptor
activation associated with cellular proliferation.
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V. General Conclusions |
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TopPreviousNextReferences
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The classification of the receptors that are activated by
leukotrienes and lipoxins was initially derived from data obtained in
functional investigations. The pharmacological studies provided substantial evidence for the existence of specific and distinct receptors that were activated by these lipid mediators and are now well
supported by the recombinant systems. However, the molecular cellular
mechanisms for the contractions of vascular smooth muscle by
LTC4 and LTD4, which are
resistant to the classical CysLT1 antagonists has
yet to be elucidated. Since the observations of Labat et al. (1992), no
known selective antagonists has been reported. Without such compounds
the identification of CysLT receptors is severely compromised and our
nomenclature for the receptors associated with vascular inflammation
will be difficult to postulate. In fact, the CysLT receptor on the
human pulmonary endothelium is also resistant to the classical CysLT
antagonists (Ortiz et al., 1995). Presently, there is no way of
identifying the receptor associated with the relaxations induced by
LTD4 in human pulmonary vessels from the receptor
responsible for the contractions induced by this same ligand.
In addition to these areas of research, future work in splice variants of the different receptors may lead to a better understanding of the cellular signal transduction pathways subsequent to activation by the ligand. Presently, little information is available based on this approach.
The use of transgenic animals have clearly demonstrated that BLT1 plays a role in the recruitment of eosinophils to the inflamed site as well as a gender-associated effect related to mouse survival in an anaphylactic model. These results strongly suggest that BLT1 receptors are intimately linked with acute inflammation in vivo. One of the most promising aspects of knockout mice is the uncovering of new receptors or the possible roles for receptor subtypes. The role of CysLT receptors in these knockout animals remains to be elucidated
The recent evidence provided by Ogasawara et al. (2002) concerning the
pharmacological differences between hCysLT2 and
mCysLT2 together with CysLT receptor distribution
in different mouse strains suggests that the choice of the animal model
and the extrapolation to human inflammation warrants considerable
reflection. Furthermore, in human airways activation of the
CysLT1 receptor is responsible for the
bronchoconstriction both in normal and asthmatic subjects. However,
there is no evidence for airway constriction in mice (Martin et al.,
1988; Richter and Sirois, 2000), and the mCysLT1 receptor has only been implicated in constriction of the microvascular smooth muscles leading to increased permeability and extravasation. Although the genomic organization of the mCysLT1
has been reported by Maekawa et al. (2001), there is a need for a
similar exploration of the human CysLT1 receptor.
The relative potencies of LTC4,
LTD4, and LTE4 in a number
of functional studies demonstrated that LTE4 is
generally less potent and markedly less efficient in activating the
CysLT receptors. The receptor studies involving cells transfected with
specific CysLT receptors have confirmed this observation. However,
there are now very few investigations that are based on the use of
LTE4. This is unfortunate, since there are
several reports suggesting that this ligand may activate another
receptor (Snyder and Krell, 1984; Mong et al., 1985; Sjolander et al.,
1990; Sakata and Bäck, 2002) or may not activate the known
classical CysLT receptors (Walch et al., 2002). Although these
observations are only suggestive, care should be taken so as not to
overlook these pertinent published data, which may offer interesting
leads to our understanding the leukotriene receptors.
Young (1989) postulated that a hydrophobic pocket existed at the
receptor for the tetraene chain (the unstable triene moiety found in
LTC4 or LTD4). An earlier
report (Snyder and Bernstein, 1987) had demonstrated that several
leukotriene analogs, where the heptyl
(C7H15) was positioned
either para, meta, or ortho on the phenyl ring altered the
pharmacological potency profile of the analog. Whether or not the use
of stable but pharmacologically active analogs of the cys-LTs would
provide clearer results for the presence of two separate CysLT
receptors in functional studies based on agonist potency has received
little attention. Such tools may be of some benefit in stereochemical
investigations for the identification of receptorial proteins.
Interestingly, the functional identification and classification of the
ALX receptor has been systematically evaluated using the LX stable analogs.
The identification and classification of the CysLT receptors has been based essentially on the ability of antagonists to block or not to block a physiological response. Although this classification has proven to be reliable at the functional and more recently at the molecular levels, future studies need to evaluate not only several antagonists but also the different ligands, especially when the functional profiles of the antagonists do not appear to match what has previously been reported.
Based on investigations involving ALX and BLT receptors, there are
several phenomenon that need to be highlighted. "Receptor induction" as seen in animal models of vascular inflammation (mouse ear model) (Gronert et al., 2001) has now been widely accepted. These
data demonstrated that receptor message is markedly increased in
tissues or cells where a pro-inflammatory agent has been used as a
primer. In addition, other receptor subtypes may be induced, as in the
case of the human mast cell, where IL-4 induced a receptor with a
pharmacological profile different from the classical CysLT receptors
(Mellor et al., 2001). These data warrant further attention since
inducible receptors may not be identical to constitutive receptors
(receptors that are present under normal conditions). In addition, the
G-protein second messenger systems must equally be explored. Along
similar lines, there is an intriguing observation that at least one
receptor, namely, CysLT1 may act as a
pyrimidinergic (UDP) receptor (Mellor et al., 2001) suggesting that
GPCRs may be quite versatile at the ligand-G-protein interface.
Although the significance of these latter findings remains to be
established, both observations provide fruitful areas for future work.
Furthermore, the observations that LTB4 is
associated with an excessive activation of leukocytes as well as the
5-LO pathway suggests a role for BLT receptor "tone" and indicates
that receptor expression amplifies pro-inflammatory circuits in vivo.
In the last few years there has been some controversy as to whether or
not the BLT receptor may act as a coreceptor for macrophage-trophic human immunodeficiency virus (HIV) of the type 1 strains. Owman et al.
(1998) reported that this receptor mediated entry of primary HIV
isolates into CD14 cells. However, results from transfected cells
apparently raise some question as to such a role for the BLT1 receptor (Martin et al., 1999). Whether or
not BLT1 antagonists can be used to prevent HIV
infection warrants perhaps further pursuit.
Although there is considerable optimism about these recent developments in lipid mediator receptor classification, only by further work in this area, as well as in vascular inflammation models, will a potential for therapeutic exploitation be realized.
| |
Footnotes |
|---|
During the preparation of this manuscript, Professor Nicosia passed
away following a long illness. Her efforts in the organization of this
committee and her scientific work on the leukotriene receptors over the
past years will be missed.
Address correspondence to: Charles Brink (Chairman), Researcher (CNRS CR-1), CNRS UMR 7131, Hôpital Broussais, Bâtiment René Leriche, 96, rue Didot, 75014 Paris, France. E-mail: charlesbrink{at}hotmail.com
DOI: 10.1124/pr.55.1.8
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Abbreviations |
|---|
SRS, slow reaction smooth muscle-stimulating substance; SRS-A, slow reacting substance of anaphylaxis; LT, leukotriene; LX, lipoxin; LO, lipoxygenase; HETE, hydroxyeicosatetraenoic acid; PMN, polymorphonuclear leukocyte; cys-LT, cysteinyl-leukotriene; IUPHAR, International Union of Pharmacology; GPCR, G-protein-coupled receptor; kbp, kilobase pair(s); ORF, open reading frame; bp, base pair(s); CHO, Chinese hamster ovary; IL, interleukin; UTR, untranslated region; HPETE, hydroperoxyeicosatetraenoic acid; PAF, platelet-activating factor; HEK, human embryonic kidney; FPR, formyl peptide receptor; fMLP, formyl-methionyl-leucyl-phenylalanine; PTX, pertussis toxin; PKC, protein kinase C; HUVEC, human umbilical vein endothelial cell; GST, glutathione S-transferase; ATL, aspirin-triggered lipoxin; HIV, human immunodeficiency virus.
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-initiated neutrophil responses and trafficking: regulators of a cytokine-chemokine axis.
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Copyright © 2003 by The American Society for Pharmacology and Experimental Therapeutics
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 H. Ge, J. Weiszmann, J. D. Reagan, J. Gupte, H. Baribault, T. Gyuris, J.-L. Chen, H. Tian, and Y. Li Elucidation of signaling and functional activities of an orphan GPCR, GPR81 J. Lipid Res., April 1, 2008; 49(4): 797 - 803. [Abstract] [Full Text] [PDF]
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M. Arita, T. Ohira, Y.-P. Sun, S. Elangovan, N. Chiang, and C. N. Serhan Resolvin E1 Selectively Interacts with Leukotriene B4 Receptor BLT1 and ChemR23 to Regulate Inflammation J. Immunol., March 15, 2007; 178(6): 3912 - 3917. [Abstract] [Full Text] [PDF]
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J.-L. Gao, A. Guillabert, J. Hu, Y. Le, E. Urizar, E. Seligman, K. J. Fang, X. Yuan, V. Imbault, D. Communi, et al. F2L, a Peptide Derived from Heme-Binding Protein, Chemoattracts Mouse Neutrophils by Specifically Activating Fpr2, the Low-Affinity N-Formylpeptide Receptor J. Immunol., February 1, 2007; 178(3): 1450 - 1456. [Abstract] [Full Text] [PDF]
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K. Wada, M. Arita, A. Nakajima, K. Katayama, C. Kudo, Y. Kamisaki, and C. N. Serhan Leukotriene B4 and lipoxin A4 are regulatory signals for neural stem cell proliferation and differentiation FASEB J, September 1, 2006; 20(11): 1785 - 1792. [Abstract] [Full Text] [PDF]
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M. P. Abbracchio, G. Burnstock, J.-M. Boeynaems, E. A. Barnard, J. L. Boyer, C. Kennedy, G. E. Knight, M. Fumagalli, C. Gachet, K. A. Jacobson, et al. International Union of Pharmacology LVIII: Update on the P2Y G Protein-Coupled Nucleotide Receptors: From Molecular Mechanisms and Pathophysiology to Therapy Pharmacol. Rev., September 1, 2006; 58(3): 281 - 341. [Abstract] [Full Text] [PDF]
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N. Chiang, C. N. Serhan, S.-E. Dahlen, J. M. Drazen, D. W. P. Hay, G. E. Rovati, T. Shimizu, T. Yokomizo, and C. Brink The Lipoxin Receptor ALX: Potent Ligand-Specific and Stereoselective Actions in Vivo Pharmacol. Rev., September 1, 2006; 58(3): 463 - 487. [Abstract] [Full Text] [PDF]
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K. A. Lundeen, B. Sun, L. Karlsson, and A. M. Fourie Leukotriene B4 Receptors BLT1 and BLT2: Expression and Function in Human and Murine Mast Cells. J. Immunol., September 1, 2006; 177(5): 3439 - 3447. [Abstract] [Full Text] [PDF]
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C.-W. Lee, R. Rivera, S. Gardell, A. E. Dubin, and J. Chun GPR92 as a New G12/13- and Gq-coupled Lysophosphatidic Acid Receptor That Increases cAMP, LPA5 J. Biol. Chem., August 18, 2006; 281(33): 23589 - 23597. [Abstract] [Full Text] [PDF]
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M. Arita, S. F. Oh, T. Chonan, S. Hong, S. Elangovan, Y.-P. Sun, J. Uddin, N. A. Petasis, and C. N. Serhan Metabolic Inactivation of Resolvin E1 and Stabilization of Its Anti-inflammatory Actions J. Biol. Chem., August 11, 2006; 281(32): 22847 - 22854. [Abstract] [Full Text] [PDF]
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 B. Uzonyi, K. Lotzer, S. Jahn, C. Kramer, M. Hildner, E. Bretschneider, D. Radke, M. Beer, R. Vollandt, J. F. Evans, et al. Cysteinyl leukotriene 2 receptor and protease-activated receptor 1 activate strongly correlated early genes in human endothelial cells PNAS, April 18, 2006; 103(16): 6326 - 6331. [Abstract] [Full Text] [PDF]
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 T. D. Mickleborough, M. R. Lindley, A. A. Ionescu, and A. D. Fly Protective Effect of Fish Oil Supplementation on Exercise-Induced Bronchoconstriction in Asthma Chest, January 1, 2006; 129(1): 39 - 49. [Abstract] [Full Text] [PDF]
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 V. Capra, S. Ravasi, M. R. Accomazzo, S. Citro, M. Grimoldi, M. P. Abbracchio, and G. E. Rovati CysLT1 receptor is a target for extracellular nucleotide-induced heterologous desensitization: a possible feedback mechanism in inflammation J. Cell Sci., December 1, 2005; 118(23): 5625 - 5636. [Abstract] [Full Text] [PDF]
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M. Back, D.-x. Bu, R. Branstrom, Y. Sheikine, Z.-Q. Yan, and G. K. Hansson Leukotriene B4 signaling through NF-{kappa}B-dependent BLT1 receptors on vascular smooth muscle cells in atherosclerosis and intimal hyperplasia PNAS, November 29, 2005; 102(48): 17501 - 17506. [Abstract] [Full Text] [PDF]
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 P Gyllfors, M Kumlin, S-E Dahlen, F Gaber, P-O Ehrs, and B Dahlen Relation between bronchial responsiveness to inhaled leukotriene D4 and markers of leukotriene biosynthesis Thorax, November 1, 2005; 60(11): 902 - 908. [Abstract] [Full Text] [PDF]
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 N. Wettschureck and S. Offermanns Mammalian G Proteins and Their Cell Type Specific Functions Physiol Rev, October 1, 2005; 85(4): 1159 - 1204. [Abstract] [Full Text] [PDF]
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K. Terawaki, T. Yokomizo, T. Nagase, A. Toda, M. Taniguchi, K. Hashizume, T. Yagi, and T. Shimizu Absence of Leukotriene B4 Receptor 1 Confers Resistance to Airway Hyperresponsiveness and Th2-Type Immune Responses J. Immunol., October 1, 2005; 175(7): 4217 - 4225. [Abstract] [Full Text] [PDF]
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T. Okuno, T. Yokomizo, T. Hori, M. Miyano, and T. Shimizu Leukotriene B4 Receptor and the Function of Its Helix 8 J. Biol. Chem., September 16, 2005; 280(37): 32049 - 32052. [Abstract] [Full Text] [PDF]
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 A. Casas, F. P. Gomez, B. Dahlen, J. Roca, J. A. Barbera, S-E. Dahlen, and R. Rodriguez-Roisin Leukotriene D4-induced hypoxaemia in asthma is mediated by the cys-leukotriene1 receptor Eur. Respir. J., September 1, 2005; 26(3): 442 - 448. [Abstract] [Full Text] [PDF]
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I. Prinz, C. Gregoire, H. Mollenkopf, E. Aguado, Y. Wang, M. Malissen, S. H.E. Kaufmann, and B. Malissen The Type 1 Cysteinyl Leukotriene Receptor Triggers Calcium Influx and Chemotaxis in Mouse {alpha}{beta}- and {gamma}{delta} Effector T Cells J. Immunol., July 15, 2005; 175(2): 713 - 719. [Abstract] [Full Text] [PDF]
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Y. Iizuka, T. Yokomizo, K. Terawaki, M. Komine, K. Tamaki, and T. Shimizu Characterization of a Mouse Second Leukotriene B4 Receptor, mBLT2: BLT2-DEPENDENT ERK ACTIVATION AND CELL MIGRATION OF PRIMARY MOUSE KERATINOCYTES J. Biol. Chem., July 1, 2005; 280(26): 24816 - 24823. [Abstract] [Full Text] [PDF]
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S. Paruchuri, O. Broom, K. Dib, and A. Sjolander The Pro-inflammatory Mediator Leukotriene D4 Induces Phosphatidylinositol 3-Kinase and Rac-dependent Migration of Intestinal Epithelial Cells J. Biol. Chem., April 8, 2005; 280(14): 13538 - 13544. [Abstract] [Full Text] [PDF]
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V. R. Jala, W.-H. Shao, and B. Haribabu Phosphorylation-independent {beta}-Arrestin Translocation and Internalization of Leukotriene B4 Receptors J. Biol. Chem., February 11, 2005; 280(6): 4880 - 4887. [Abstract] [Full Text] [PDF]
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 Y. Hui, Y. Cheng, I. Smalera, W. Jian, L. Goldhahn, G. A. FitzGerald, and C. D. Funk Directed Vascular Expression of Human Cysteinyl Leukotriene 2 Receptor Modulates Endothelial Permeability and Systemic Blood Pressure Circulation, November 23, 2004; 110(21): 3360 - 3366. [Abstract] [Full Text] [PDF]
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T. C. Beller, A. Maekawa, D. S. Friend, K. F. Austen, and Y. Kanaoka Targeted Gene Disruption Reveals the Role of the Cysteinyl Leukotriene 2 Receptor in Increased Vascular Permeability and in Bleomycin-induced Pulmonary Fibrosis in Mice J. Biol. Chem., October 29, 2004; 279(44): 46129 - 46134. [Abstract] [Full Text] [PDF]
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N. Murakami, T. Yokomizo, T. Okuno, and T. Shimizu G2A Is a Proton-sensing G-protein-coupled Receptor Antagonized by Lysophosphatidylcholine J. Biol. Chem., October 8, 2004; 279(41): 42484 - 42491. [Abstract] [Full Text] [PDF]
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 C. Lefevre, B. Bouadjar, A. Karaduman, F. Jobard, S. Saker, M. Ozguc, M. Lathrop, J.-F. Prud'homme, and J. Fischer Mutations in ichthyin a new gene on chromosome 5q33 in a new form of autosomal recessive congenital ichthyosis Hum. Mol. Genet., October 1, 2004; 13(20): 2473 - 2482. [Abstract] [Full Text] [PDF]
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M. J. Paul-Clark, T. van Cao, N. Moradi-Bidhendi, D. Cooper, and D. W. Gilroy 15-epi-lipoxin A4-mediated Induction of Nitric Oxide Explains How Aspirin Inhibits Acute Inflammation J. Exp. Med., July 6, 2004; 200(1): 69 - 78. [Abstract] [Full Text] [PDF]
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 M. Back, H. Qiu, J. Z. Haeggstrom, and K. Sakata Leukotriene B4 is an indirectly acting vasoconstrictor in guinea pig aorta via an inducible type of BLT receptor Am J Physiol Heart Circ Physiol, July 1, 2004; 287(1): H419 - H424. [Abstract] [Full Text] [PDF]
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D.-S. Im Discovery of new G protein-coupled receptors for lipid mediators J. Lipid Res., March 1, 2004; 45(3): 410 - 418. [Abstract] [Full Text] [PDF]
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C. Brink, S.-E. Dahlen, J. Drazen, J. F. Evans, D. W. P. Hay, G. E. Rovati, C. N. Serhan, T. Shimizu, and T. Yokomizo International Union of Pharmacology XLIV. Nomenclature for the Oxoeicosanoid Receptor Pharmacol. Rev., March 1, 2004; 56(1): 149 - 157. [Abstract] [Full Text] [PDF]
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 B. McMahon and C. Godson Lipoxins: endogenous regulators of inflammation Am J Physiol Renal Physiol, February 1, 2004; 286(2): F189 - F201. [Abstract] [Full Text] [PDF]
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S. Paruchuri and A. Sjolander Leukotriene D4 Mediates Survival and Proliferation via Separate but Parallel Pathways in the Human Intestinal Epithelial Cell Line Int 407 J. Biol. Chem., November 14, 2003; 278(46): 45577 - 45585. [Abstract] [Full Text] [PDF]
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T. Okuno, H. Ago, K. Terawaki, M. Miyano, T. Shimizu, and T. Yokomizo Helix 8 of the Leukotriene B4 Receptor Is Required for the Conformational Change to the Low Affinity State after G-protein Activation J. Biol. Chem., October 17, 2003; 278(42): 41500 - 41509. [Abstract] [Full Text] [PDF]
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 E. Sundstrom, L. Lastbom, A. Ryrfeldt, and S.-E. Dahlen Interactions among Three Classes of Mediators Explain Antigen-Induced Bronchoconstriction in the Isolated Perfused and Ventilated Guinea Pig Lung J. Pharmacol. Exp. Ther., October 1, 2003; 307(1): 408 - 418. [Abstract] [Full Text] [PDF]
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K. Masuda, H. Itoh, T. Sakihama, C. Akiyama, K. Takahashi, R. Fukuda, T. Yokomizo, T. Shimizu, T. Kodama, and T. Hamakubo A Combinatorial G Protein-coupled Receptor Reconstitution System on Budded Baculovirus: EVIDENCE FOR G{alpha}i AND G{alpha}o COUPLING TO A HUMAN LEUKOTRIENE B4 RECEPTOR J. Biol. Chem., June 27, 2003; 278(27): 24552 - 24562. [Abstract] [Full Text] [PDF]
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S. Hong, K. Gronert, P. R. Devchand, R.-L. Moussignac, and C. N. Serhan Novel Docosatrienes and 17S-Resolvins Generated from Docosahexaenoic Acid in Murine Brain, Human Blood, and Glial Cells. AUTACOIDS IN ANTI-INFLAMMATION J. Biol. Chem., April 18, 2003; 278(17): 14677 - 14687. [Abstract] [Full Text] [PDF]
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