|
|
||||||||
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
| |
Abstract |
|---|
|
|
|---|
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.
| |
I. Introduction |
|---|
|
|
|---|
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.
|
|
| |
II. General Considerations |
|---|
|
|
|---|
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.
|
|
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.
| |
III. Molecular Database for Leukotriene Receptors |
|---|
|
|
|---|
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.

View larger version (69K):
[in a new window]
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).
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)
|
|
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.
|
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
/
animals. These
findings are in line with earlier observations with
LTB4 in the hamster cheek pouch (Raud et al.,
1991
/
and +/+ animals, whereas Tager and colleagues (2000)
/
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)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
).

View larger version (39K):
[in a new window]
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
i (Lynch et al.,
19992. 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
).
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
).
|
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 G
i
proteins, mainly G
i2), 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 G
i6- and
G
-subunits released from G
i (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.
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
).
). These observations supported an initial report that PKC
activation may be associated with increased force development at
constant [Ca2+]i (Masuo
et al., 1994
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)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, an