Foreword

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A literature review on kinin pharmacology that appeared in this journal in 1980 was based on a systematic effort to define two receptor types for bradykinin- (BK ^b) related peptides, and proposed a receptor nomenclature (B₁, B₂) (Regoli and Barabé, 1980). Since then, that historical paper has been frequently cited and the nomenclature, widely adopted. The physiologically prominent B₂ receptor (B₂R) subtype has certainly been the subject of more intensive efforts in drug development, structure-function studies, and physiological investigations. However, the B₁ receptor (B₁R), activated by a class of kinin metabolites (des-Arg⁹-BK and Lys-des-Arg⁹-BK), is now a defined molecular entity (fig. 1) and its characterization, including its rapid up-regulation in tissue injury, has emerged as an important area of investigation within the study of the kallikrein-kinin system. Indeed, a major interest in the B₁R is its inducible character, an unusual feature for a G-protein coupled receptor.

View larger version (12K): [in this window] [in a new window]   Fig. 1.   Primary structure and putative domains of the human B1R. The amino acid sequence (one letter code) starts at the upper left, an extracellular domain. Asterisks represent putative sites of N-glycosylation. A putative disulfide bond between Cys residues of the second and third extracellular domain is also represented. Residues in bold are identical in all four species for which the B1R has been sequenced (human, rabbit, mouse, rat). The underlined C-terminal sequence, a part of a sequence extension found only in the human molecule, has been the immunogen for the production of anti-B1R antibodies. Numbered residues are referred to in the description of mutant or chimeric constructions (see text). Data from Hess et al. (1996), GenBank file no. U66107, MacNeil et al. (1995), and Menke et al. (1994). Sequence variants for human B1R are reported: R146 (Bastian et al., 1997), R246, and S259 (GenBank file no. U22346).

We will present the findings on the kinin B₁R with emphasis on contemporary molecular research and, when possible, on the human molecules, cells, tissues, and subjects. We do not aim at an exhaustive or historical coverage of early work on B₁Rs, which was essentially based on functional approaches and structure-activity investigations of peptide analogs; the reader is referred to earlier reviews for coverage of these aspects (Marceau, 1995; Marceau and Regoli, 1991; Regoli and Barabé, 1980). Finally, the field of B₁R research cannot be isolated from pharmacological and molecular studies of the BK B₂Rs or from other components of the kallikrein-kinin system. A comparative and parallel coverage will be used in several sections of the present text.

	I. Introduction: The B1 Receptors in the Kallikrein-Kinin System

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Knowledge concerning the molecular elements of the kallikrein-kinin system is being constantly refined (table 1; reviewed by Kaplan et al., 1997; Margolius, 1995). A single human kininogen gene codes for the hepatic production of both high (HMW) and low molecular weight (LMW) kininogen via alternative splicing. Kininogens are multi-domain proteins that include the BK sequence, but also domains capable of binding Ca²⁺, binding to the cell surface or inhibiting the cysteine proteinases. Kallikreins are the enzymes that cleave kininogens and liberate kinins (defined as BK-related peptides). Plasma prekallikrein is cofactor of coagulation that is involved in a reciprocal activation reaction with the Hageman factor in the contact system. Plasma kallikrein releases BK (the nonapeptide H-Arg-Pro-Pro-Gly-Phe-Ser-Pro-Phe-Arg-OH) from HMW-kininogen (Margolius et al., 1995). There is also evidence that HMW-kininogen adsorbed on extracellular (M) proteins of Streptococcus pyogenes can release BK in the presence of prekallikrein, thus extending the physiopathological applications of the contact system to sepsis (Ben Nasr et al., 1997).

Tissue kallikreins are represented in humans by three related enzymes (hK1 to hK3). Tissue kallikrein (hK1) is widely expressed in glandular and duct cells, as well as in neutrophils (Wu et al., 1993), cultured vascular smooth muscle cells (SMCs), colonic goblet cells, and renal distal tubules and collecting ducts (Chen et al., 1995a, 1995b; Nolly et al., 1993; Oza et al., 1990). Tissue kallikreins are synthesized as zymogens and little is known of their production, storage, or release. Pure tissue kallikreins are not equally capable of generating biologically active kinins: hK1, the classical tissue kallikrein, is highly effective, whereas hK3 (also called prostate-specific antigen) is inactive, hK2 being of intermediate potency (Deperthes et al., 1997). The exclusive prostatic localization of hK2 and hK3 is currently being challenged, as they are found in other cell types (e.g., a human breast cancer line expresses hK2; Hsieh et al., 1997). Although hK1 is preformed in the human kidney, further synthesis of this protease occurs in this organ after trauma or immunopathological insult (Cumming et al., 1994). Human tissue kallikrein preferentially releases the decapeptide Lys-BK (kallidin) from kininogens, as found in the nasal secretions of subjects with allergic or viral rhinitis (Nacleiro et al., 1988; Proud et al., 1983).

The metabolism of kinins will be briefly summarized here, because B₁Rs are selectively and exclusively stimulated by one class of kinin metabolites (fig. 2). BK exhibits a remarkably short half-life in the blood plasma in vitro (10 to 50 sec, depending on species; Décarie et al., 1996b) or in vivo. The great susceptibility of these peptides to hydrolysis accounts for this rapid disposal. BK and Lys-BK are predominantly metabolized in the circulation by the angiotensin I converting enzyme (ACE; EC 4.4.15.1; kininase II; Erdös, 1990). ACE removes the C-terminal dipeptide from BK or Lys-BK, which leads to their complete inactivation (K_m of about 1 µM for this reaction). ACE is predominantly a surface enzyme located on the luminal membrane of endothelial cells, an observation which explains the extensive pulmonary inactivation of kinins. The proximal nephron epithelium is also rich in ACE (Erdös, 1990). ACE eventually cleaves further its primary metabolite, BK_1-7, into shorter fragments (e.g., BK_1-5).

View larger version (10K): [in this window] [in a new window]   Fig. 2.   Proposed cleavage sites for Lys-BK and Lys-des-Arg9-BK by the kininases. Abbreviations: AmM, aminopeptidase M; AmP, aminopeptidase P; NEP, neutral endopeptidase 24.11; ACE, angiotensin I converting enzyme; CPN, carboxypeptidase N; CPM, carboxypeptidase M.

An aminopeptidase P (aminoacyl proline aminopeptidase; EC 3.4.11.9) which cleaves the Arg¹-Pro² bond, may also contribute to the pulmonary inactivation of BK (Ryan et al., 1994; Ward, 1991). Another form of membrane-bound peptidase, the "neutral" endopeptidase (E.C. 3.4.24.11), cleaves two bonds in BK (Gly⁴-Phe⁵ and Pro⁷-Phe⁸; Zolfaghari et al., 1989). Endopeptidase 24.11 is notably located at a high concentration in the proximal nephron epithelium and is also present on the membrane of some leukocytes, but barely detectable in the vasculature or plasma (Erdös, 1990; Ward, 1991).

Kininase I activity is of particular importance, because it is believed to generate the physiological agonists of B₁Rs, the des-Arg⁹-kinins, from native kinins. This activity is composed of the arginine carboxypeptidases, carboxypeptidase N from plasma (EC 3.4.17.3), and carboxypeptidase M, predominantly membrane-bound and widely distributed, including in the microvasculature (table 1; Erdös, 1990; Ward, 1991). These are enzymes of rather low affinity (K_m 20 to 50 µM for BK and carboxypeptidase N) and broad specificity, digesting several other peptides generated by trypsin-like enzymes, such as the anaphylatoxins derived from complement. The relative role of arginine carboxypeptidases (kininase I) in the metabolism of kinins is discussed below.

The metabolism of des-Arg⁹-kinins differs from that of native kinins in several important points. First, des-Arg⁹-BK does not react with arginine carboxypeptidases, as it is devoid of the corresponding C-terminal residue (Drapeau et al., 1991a). Second, ACE cleaves a C-terminal tripeptide from B₁R agonists, yielding BK_1-5, but at a slower rate, and with much less affinity (K_m 130 to 240 µM) than the removal of a C-terminal dipeptide from BK (Drapeau et al., 1991a). The C-terminal position of Phe⁸ in des-Arg⁹-BK may protect the Pro⁷-Phe⁸ bond from the endopeptidase 24.11; this requires confirmation.

Aminopeptidase M (EC 3.4.11.2), present in plasma, hydrolyzes Lys-BK into BK, and Lys-des-Arg⁹-BK into des-Arg⁹-BK (Proud et al., 1987; Sheik and Kaplan, 1989). This reaction is pharmacologically neutral for the B₂R agonists BK and Lys-BK, because these peptides exhibit similar potencies. However, it represents a relative inactivation for the B₁R agonists, as des-Arg⁹-BK is an agonist of lesser affinity for the B₁Rs than Lys-des-Arg⁹-BK in some species (see below).

The conversion effectiveness of native kinin sequences (BK, Lys-BK) into their respective des-Arg⁹ metabolites is a question of considerable interest for the physiological role of B₁Rs, because the other competing metabolic pathways lead to fragments inactive on both B₁ and B₂Rs (Regoli and Barabé, 1980; see also below). Novel analytical techniques for BK- and des-Arg⁹-BK-like immunoreactive peptides have shown that ACE accounts for more than 75% of the hydrolysis of exogenous synthetic BK (500 nM) in the plasma of humans, rabbits, or rats (Décarie et al., 1996b), and more than 40% in cardiac membrane preparations derived from the same species (Blais et al., 1997b). The formation of des-Arg⁹-BK is minor in these species (0.9 to 3.4% of BK converted), much less than previous estimates based on high concentrations of exogenous BK as a substrate (Marceau et al., 1981; Proud et al., 1987). Blocking the competing ACE pathway with enalaprilat improved the conversion in all species (Blais et al., 1997b; Décarie et al., 1996b). Thus, when considering the relative contribution of several kininases, the mere existence of measurable des-Arg⁹-kinins in vivo could be questioned. However, in the plasma or cardiac membrane preparations of the four cited species, the half-life of exogenous des-Arg⁹-BK was 4- to 12-fold longer than that of BK under the same experimental conditions (Blais et al., 1997b; Décarie et al., 1996b). If this is an accurate representation of the situation in vivo, it may explain why the in vivo concentration of immunoreactive des-Arg⁹-BK is consistently higher than that of immunoreactive BK. Simply put, des-Arg⁹-BK, although not effectively produced, has a much greater capacity to accumulate than BK.

Kinins are very difficult to measure accurately, in part because the sampled blood contains all the necessary components to generate and destroy these peptides in vitro. For instance, the contact of glass in test tubes activates the plasma kallikrein. Current methods for measuring kinin concentrations rely on antibody-based techniques (radioimmunoassays, enzyme immunoassays) preceded by complex extraction procedures (Décarie et al., 1994; Odya et al., 1983; Raymond et al., 1995). In venous blood sampled from normal humans, the average concentration of immunoreactive des-Arg⁹-BK (204 pg/ml) is higher than that of BK (67 pg/ml) (Odya et al., 1983). These values are not significantly changed inpatients with essential hypertension or under ACE blockade with enalapril (Odya et al., 1983). The urine of these individuals also contains both BK and des-Arg⁹-BK immunoreactivities that do not differ between groups. The authors point out that venous blood or urine measurements may not reflect the status of the kinins at the tissue level, because of metabolism by multiple pathways. In the arterial blood of anesthetized rabbits, captopril treatment increases the BK immunoreactive concentration, but does not influence the concentration of des-Arg⁹-BK. The latter could be selectively and importantly increased by treatment with bacterial lipopolysaccharide (LPS), and even more so in animals pretreated with captopril (Raymond et al., 1995). Species-dependent differences in the metabolism and the rate of activation of the kallikrein-kinin system may be important parameters which determine the relative concentrations of BK and des-Arg⁹-BK. Local concentrations of kinins may generally be of greater interest, as the kinins are autacoids rather than hormones. Interestingly, both BK and des-Arg⁹-BK immunoreactivities increase in inflammatory lesions caused by carrageenan in the rat (Burch and DeHaas, 1990; Décarie et al., 1996a).

The discussion above is centered on the C-terminal structure of kinins, so important for receptor subtype selectivity (see below). However, various limitations are apparent in the analytical approach for des-Arg⁹-kinins. The antibodies efficient to discriminate the C-terminal structure do not differentiate between kinin homologues that differ in their N-terminus (e.g., BK, Lys-BK, Ile-Ser-BK; Décarie et al., 1994; Raymond et al., 1995). This represents a major limitation of our analytical capabilities, as Lys-des-Arg⁹-BK is the most potent B₁R agonist in humans, des-Arg⁹-BK being relatively ineffective (see below, Section II.A.). Combining the antibody-based method with chromatographic separation of homolog peptides may be helpful to discriminate among them (Décarie et al., 1994), although with a probable loss of sensitivity. An unambiguous analytical method for measuring Lys-des-Arg⁹-BK still has to be invented. An additional problem is that the Pro (3) residue in either BK or des-Arg⁹-BK may rather be hydroxyproline in a sizable proportion of the kinin molecules, caused by a partial posttranslational modification of high molecular weight kininogen in the human species (Schlüter et al., 1997). Although this substitution does not adversely affect receptor affinity (Regoli et al., 1996), its effect on metabolism or antigenicity is unknown. Finally, an inflammation-induced increase of immunoreactive des-Arg⁹-BK, as in the case of acute edema produced by carrageenan in the rat paw, does not necessarily predict an important role for B₁Rs, as these receptors are apparently absent in this acute model that dissociates the agonist from the response (Décarie et al., 1996a).

	II. Seven Criteria to Classify Kinin Receptors into the B1 and B2 Subtypes

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Kinins, positively charged peptides, influence tissues and cells by stimulating membrane receptors. Two types of receptors are currently completely defined in several species using a series of seven criteria, detailed in this chapter, although very significant species-specific pharmacological differences are documented. The first three criteria are pharmacological as defined by Schild (1973). The other ones are of more biochemical and molecular nature. Binding (table 2) and biochemical experiments based on cloned receptor subtypes have been valued in our coverage, because they offer the ultimate proofs regarding the behavior of definite molecular entities.

In vitro smooth muscle preparations assume a particular importance in the history of kinin receptor classification. The first hint of the receptor heterogeneity was the atypical order of agonist potency found for BK and its fragment on the isolated rabbit aorta: the fragment without the C-terminal arginine residue, des-Arg⁹-BK, was more potent than the intact nonapeptide (Regoli et al., 1977). A few other preparations are worth mentioning at this point, as they are useful to compare potency orders obtained in binding assays with cloned receptors in each species: comprehensive data are available for the contractility mediated by B₁Rs in the human umbilical vein (Gobeil et al., 1996b), the longitudinal muscle of the rat ileum (Meini et al., 1996), and the mouse stomach (Allogho et al., 1995). These preparations were further used to determine the potency and intrinsic activity of antagonists and the postisolation regulation of B₁Rs (see below). Of the preparations mentioned above, the rabbit aorta and the rat ileum respond to kinins only via B₁R, whereas the human umbilical vein and mouse stomach possess both receptor subtypes, a situation that complicates pharmacological analyses.

The potency order of agonists, as established in separate binding assays (table 2), confirms that the removal of the C-terminal arginine is essential for high affinity on the B₁R and detrimental for affinity toward the B₂R. This holds true for Lys-BK (kallidin) and Lys-des-Arg⁹-BK (des-Arg¹⁰-kallidin), which have higher affinities for the human and rabbit B₁Rs compared with BK and des-Arg⁹-BK, respectively (table 2). Met-Lys-BK, resulting from a different cleavage of the kininogen sequence, does not significantly differ from Lys-BK, being a relatively selective B₂R agonist in the human and rabbit (table 2). The only natural kinin sequence with a subnanomolar affinity for human and rabbit B₁Rs is Lys-des-Arg⁹-BK, a fact of particular significance that may suggest that the B₁R belongs to the "tissue" kallikrein-kinin system, jointly with tissue kallikrein that generates Lys-BK (kallidin) from low molecular weight kininogen. This does not invalidate the experimental use of des-Arg⁹-BK in certain animal species as a tool of high specificity for B₁Rs; however, this peptide has a low absolute affinity for the human B₁R. This is reflected by the low relative potency of functional responses induced by des-Arg⁹-BK as compared with those elicited by Lys-des-Arg⁹-BK, in the human umbilical vein assay (Gobeil et al., 1997), or in cells expressing recombinant human B₁Rs (MacNeil et al., 1997).

Limited functional data based on porcine renal vein contractility show that this B₁R, not yet cloned, is also preferentially stimulated by Lys-des-Arg⁹-BK (pD₂ of 8.4, versus 7.2 for des-Arg⁹-BK; Rizzi et al., 1997). By contrast, Lys-des-Arg⁹-BK has no decisive advantage over des-Arg⁹-BK in small rodents such as mouse (table 2) or rat. The sequenced kininogen genes in small rodents differ from the human ones, as the amino acids preceding the BK sequence are arginine, or Ile-Ser in T-kininogen, not lysine (Hess et al., 1996; Takano et al., 1997). In view of this recent discovery the precise sequence released by tissue kallikreins in rats and mice becomes an analytical issue of interest. Only BK has been isolated from rat urine and other experimental systems based on rat tissue kallikrein, but not Arg-BK, Ile-Ser-BK or Lys-BK (Hagiwara et al., 1995). A form of coevolution of the B₁R with the kininogen genes may have occurred in rodents to maintain the B₁R functional by conferring a subnanomolar affinity to des-Arg⁹-BK, which is reflected by the high potency of this peptide (superior or equal to Lys-des-Arg⁹-BK in functional assays based on rat or mouse smooth muscle contractility; Allogho et al., 1995; Meini et al., 1996; or cultured rat SMCs, where des-Arg⁹-BK exerts its effect with an EC₅₀ of about 300 pM; Dixon and Dennis, 1997).

Thus, the B₁R is obviously specialized across species to respond to different kinin metabolites, either des-Arg⁹-BK or Lys-des-Arg⁹-BK, generated by arginine carboxypeptidases, such as carboxypeptidase N and M. No other kinin fragment seems to retain pharmacological activity. For instance, des-Arg¹-BK or des(Phe⁸, Arg⁹)-BK (the primary metabolite generated by ACE) do not retain significant activity on either known receptor type (Regoli and Barabé, 1980). The presence of arginine carboxypeptidases in functional systems frequently distorts the potency estimates for BK or Lys-BK on the B₁R, as these sequences are transformed into their respective des-Arg metabolites (discussed by Marceau and Regoli, 1991). Even a binding assay based on the cloned mouse B₁R seems to be prone to this error, as the apparent potency of BK in a competition with the ligand [³H]Lys-des-Arg⁹-BK is decreased by a factor of 5 in the presence of the arginine carboxypeptidase inhibitor MERGETPA (Pesquero et al., 1996). This was not the case for des-Arg⁹-BK. Thus, intact BK and Lys-BK may exhibit a very high selectivity toward B₂R.

Most published binding studies to human or animal B₁Rs have used the agonist ligand [³H]Lys-des-Arg⁹-BK (table 2). [¹²⁵I]Tyr-Gly-Lys-Aca-Lys-des-Arg⁹-BK is an alternative high affinity B₁R ligand which can be also used (Levesque et al., 1995a). This agonist incorporates a N-terminal extension which essentially acts as a spacer between iodotyrosine and the Lys-des-Arg⁹-BK sequence.

A synthetic agonist modeled on Lys-des-Arg⁹-BK incorporates modifications to improve resistance to metabolism: Sar-[D-Phe⁸]des-Arg⁹-BK is a high affinity B₁ receptor agonist of high selectivity that is completely resistant to blood aminopeptidase, kininases I and II (angiotensin I converting enzyme) and kidney neutral endopeptidase (Drapeau et al., 1991b, 1993). In a binding assay to rabbit B₁ receptors, this analog was 7-fold more potent than des-Arg⁹-BK, but 11-fold less potent than Lys-des-Arg⁹-BK (Levesque et al., 1995a). It is very potent in inducing contraction of isolated rabbit aorta (fig. 3D). Contractility data from the human umbilical veins also suggest an intermediate potency between des-Arg⁹-BK and Lys-des-Arg⁹-BK in the human B₁R (Gobeil et al., 1997). However, Sar-[D-Phe⁸]des-Arg⁹-BK is equipotent to Lys-des-Arg⁹-BK as an hypotensive agent in LPS-pretreated rabbits (Drapeau et al., 1991b) and the hypotensive episodes are prolonged (fig. 3C). The analog is inert in rabbits not treated with LPS, caused by the lack of cardiovascular B₁Rs in these animals (see below). The hypotensive effect of Sar-[D-Phe⁸]des-Arg⁹-BK is antagonized by Lys-[Leu⁸]des-Arg⁹-BK, a B₁R antagonist, and its analogues, but not by a B₂R antagonist Hoe 140 (Drapeau et al., 1993). A formal pharmacokinetic approach showed that Sar-[D-Phe⁸]des-Arg⁹-BK has a longer half-life than Lys-des-Arg⁹-BK in the rabbit circulation (Audet et al., 1997), probably accounting for the decisive advantage of the modified analog in vivo. Sar-[D-Phe⁸]des-Arg⁹-BK is also more potent than des-Arg⁹-BK as an algesic agent in a rat model of chronic inflammation (Davis and Perkins, 1994a), and may be superior to any natural sequence for demonstrating the LPS induction of hypotensive responses mediated by B₁Rs in this species (Nicolau et al., 1996). Sar-[D-Phe⁸]des-Arg⁹-BK appears to be a valid tool across different species to demonstrate the presence of a B₁R population. The replacement of a central tetrapeptide (Pro²-Phe⁵) in Lys-des-Arg⁹-BK by alkyl spacers, optimally 12-aminododecanoic acid, is another reported approach to modify a natural B₁R agonist (Tancredi et al., 1997). In this series of compounds, the best one retained 10% of Lys-des-Arg⁹-BK potency in the rat ileum contractility assay.

View larger version (26K): [in this window] [in a new window]   Fig. 3.   The rabbit treated with lipopolysaccharide (LPS), an animal model for the study of the regulation of the B1R. (A) Hypotensive effect of BK, Lys-BK and their des-Arg9-BK fragments in anesthetized rabbits. The endotoxin-treated animals (dotted lines) were injected with 10 µg of E. coli LPS 5 h before anesthesia. Peptides were injected intra-arterially as boluses. Abcissa: dose of peptide (ng per 1.5 to 2 kg animal) ; ordinate: fall of the mean arterial pressure, mmHg. Points are means of 5 to 18 determinations; the asterisk indicates value significantly changed by the LPS treatment. Reprinted in a modified form from Marceau and Regoli (1991), p. 40, by permission from Marcel Dekker, Inc. (B) B1R mRNA concentration in rabbit hearts relative to those of a housekeeping gene, GAPDH. Animals were untreated (controls) or injected with 25 µg/kg LPS 1 or 3 h before they were killed. Values are means ± SEM of triplicate determinations (with permission from Marceau et al., 1997, and the NRC Research Press). (C) Hemodynamic changes induced by an intraarterial bolus injection of the B1R agonist Sar-[D-Phe8]des-Arg9-BK (750 ng/kg) in anesthetized rabbits previously treated with LPS (30 µg/kg i.v. 5 h before anesthesia). CO, cardiac output reading derived from an electromagnetic flowmeter placed on the thoracic aorta; MABP, mean arterial blood pressure; TRP, calculated total peripheral resistance. The asterisk indicates value significantly different from baseline. Modified, from Audet et al. (1997), with permission from ASPET. (D) Initial responsiveness of aortic rings derived from control (saline vehicle i.v.) or LPS-treated rabbits (25 µg/kg, i.v. 8 h before they were killed). The cumulative concentration-effect curve of the B1R agonist Sar-[D-Phe8]des-Arg9-BK was established after a short in vitro incubation (45 min) to minimize the influence of isolation and tissue incubation on the responses. Values (means ± SEM of eight determinations from four animals in each group) are expressed as a percent of an internal contractile standard, the maximal effect of phenylephrine, established in each tissue. The maximal effects of the kinin differed significantly between groups (J.-F. Larrivée, D. Bachvarov, and F. Marceau, unpublished).

The development of kinin receptor antagonists has been pursued for more than two decades (Stewart, 1995). Selective antagonists have been crucial to define kinin receptor subtypes. The prototype of the kinin B₁R antagonists was [Leu⁸]des-Arg⁹-BK (Regoli et al., 1977). The B₁ nomenclature was latter applied to the rabbit aortic preparation in which the receptors were initially defined by both a typical order of agonist potency and by these antagonists. However, it became clear that these antagonists were not active in the most common contractile bioassays for BK (e.g., rat uterus, guinea pig ileum) or in vivo (hypotension) (Regoli and Barabé, 1980). The B₂R was not well defined up to Schild's criteria until 1985, when the first generation of antagonists based on [D-Phe⁷]BK were produced (Vavrek and Stewart, 1985). Since then, more efforts have been invested into the development of B₂R antagonists, relative to B₁R antagonists. Hoe 140 (icatibant; D-Arg[Hyp³, Thi⁵, D-Tic⁷, Oic⁸]-BK; abbreviations for unusual residues defined in footnote h to table 2, previous page; Hock et al., 1991) is a representative of the "second generation" B₂R antagonists, as it has several hours duration of action in animal models. Hoe 140 also exhibits a very high affinity (table 2) and is a competitive antagonist in most species with the noticeable exception of the rabbit (nonequilibrium insurmountable antagonism; Bachvarov et al., 1995). The effect of Hoe 140 on the human B₁R has probably been underestimated in the binding study quoted in table 2, because other functional or binding experiments rather indicate a K_i around 100 to 400 nM (Aramori et al., 1997; Bastian et al., 1997; Gobeil et al., 1996b; Zuzack et al., 1996). However, in practical terms, this peptide still exhibits a high selectivity toward B₂Rs. Hoe 140 has been used in many studies, particularly to assess the role of kinins in various models of experimental pathology. NPC 17731 is a B₂R antagonist that is chemically and pharmacologically similar to Hoe 140 (table 2). The first nonpeptide kinin antagonists, the phosphonium WIN 64338 and the quinoline FR 173657, are selective B₂R antagonists (Aramori et al., 1997; Sawutz et al., 1994; table 2). The latter compound is orally bioavailable, an important step toward the clinical evaluation of such antagonists.

Thus far, nonpeptide B₁R antagonists have not been yet reported. Structure-activity relationships for early peptide antagonists of the B₁R are discussed elsewhere (Regoli and Barabé, 1980). In summary, the substitution of Phe⁸ in des-Arg⁹-BK by a residue with an aliphatic (Ala, Ile, Leu, D-Leu, norleucine) or saturated cyclic hydrocarbon chain (cyclohexylalanine) produced antagonists of variable potency. Early experiments have also shown that substituting Pro⁷ (e.g., with Ala or Gly) can produce partial agonists or antagonists (Regoli and Barabé, 1980). Substitutions at this position were introduced in some of the more recent B₁R antagonists. The N-terminal extension of [Leu⁸]-des-Arg⁹-BK by a Lys residue confers a higher affinity in the species which have a similar relationships for agonists: the rabbit and human (table 2). Thus, Lys-[Leu⁸]des-Arg⁹-BK is the optimal B₁R antagonist based on natural amino acids. This family of peptides exhibits a competitive behavior and an almost ideal selectivity for the B₁R subtype (table 2). A tritiated form of Lys-[Leu⁸]des-Arg⁹-BK has been used as a ligand for the human B₁R in a recent study (Bastian et al., 1997), and binding competition data were very similar to those obtained with the agonist ligand [³H]Lys-des-Arg⁹-BK. Thus, the antagonist activity of [Leu⁸]des-Arg⁹-BK and its homologues is a reasonable basis for the pharmacological definition of the kinin B₁R.

Improving the stability and selectivity of B₁R antagonists is critically important for physiopathological investigations and therapeutic applications. Peptides generally suffer from many disadvantages as potential therapeutic agents, such as metabolic instability, poor oral absorption, rapid elimination, short duration of action and, in some cases, partial agonist activity. A metabolically stable peptide B₁R antagonist, Ac-Lys-[MeAla⁶, Leu⁸]des-Arg⁹-BK, has been elaborated by successive modifications of the high affinity prototype, Lys-[Leu⁸]des-Arg⁹-BK, with the main purpose of documenting the metabolic pathways of this class of agents (Drapeau et al., 1993). The N-methyl-alanine residue in position 6 removes recognition sites for ACE and neutral endopeptidase (fig. 2). Thus, although the novel antagonist peptide was not very potent (pA₂ of 6.5 in the rabbit aorta; K_i of 670 nM on cloned human B₁R, table 2), it showed resistance to ACE, neutral endopeptidase and also aminopeptidases, and exhibited an increased duration of action (15-30 min) as compared with the reference compound, against cardiovascular responses induced by an exogenous B₁R receptor agonist in LPS-pretreated rabbits. Ac-Lys[D-Nal⁷, Ile⁸]des-Arg⁹-BK (also referred to as R-715) has been designed with a similar rationale, and proved to be partially resistant to ACE, although retaining a high affinity for the human and rabbit B₁R (pA₂ of 8.5 in the rabbit aorta; Gobeil et al., 1996a; table 2).

Recently, metabolically stable B₁ and mixed B₁ + B₂ antagonists of high affinity and stability have been synthesized. B9858 [Lys-Lys-[Hyp³, Igl⁵, D-Igl⁷, Oic⁸]des-Arg⁹-BK] belongs to this series and has some selectivity for B₁Rs (table 2; Gera et al., 1996; Gobeil et al., 1997). This peptide retains the Lys⁰ residue, a favorable feature for binding to human B₁Rs (table 2). In binding assays, B9858 is more potent on the human B₁Rs than on its murine homologues: it is functionally highly active as a competitive antagonist against Lys-des-Arg⁹-BK-induced contractility of the human umbilical vein (pA₂ 9.2) and in the rabbit aorta (pA₂ 8.4; Gobeil et al., 1997). In the anesthetized dog, representing a hemodynamic system coexpressing B₁ and B₂Rs (see below, Section III.C.), B9858 is reported to be 20 times more potent than Lys-[Leu⁸]des-Arg⁹-BK in antagonizing des-Arg⁹-BK-induced hypotension, and the antagonist effect lasts for more than 4 h (versus about 15 min for Lys-[Leu⁸]des-Arg⁹-BK; Stewart et al., 1996).

Optimal peptide B₂R antagonists retain both Arg¹ and Arg⁹ residues, and B₁R agonists or antagonists typically lack Arg⁹. However, some novel antagonists with backbones constrained by nonnatural residues are somewhat more promiscuous in their selectivity. The des-Arg⁹ fragment of Hoe 140 has been studied by some investigators (table 2). This peptide may not be a metabolite of Hoe 140. It exhibits increased affinity toward the B₁R in all species studied, but retains fairly high residual antagonistic effects on B₂Rs in functional in vitro (Rhaleb et al., 1992) or in vivo studies (rat blood pressure, Lagneux and Ribuot, 1997). Similarly, NPC 18565 is the des-Arg⁹ fragment of NPC 17731, but exhibits a better selectivity and affinity for the human B₁R than Hoe 140 des-Arg (table 2). However, recently produced antagonists retaining Arg⁹, such as B9430 (D-Arg-[Hyp³, Igl⁵, D-Igl⁷, Oic⁸ ]BK) represent combined B₁ and B₂R antagonists, although the corresponding des-Arg⁹ fragment has a large selectivity toward B₁Rs (Burkard et al., 1996). B9430 has been deliberately exploited as a dual kinin receptor antagonist in the blood pressure assay of the dog where it can abolish, at certain doses, hypotensive responses to either BK or des-Arg⁹-BK without affecting the effects of several other unrelated agonists (Stewart et al., 1996). The development of this compound not only demonstrates that a "polypharmaceutic" approach covering both receptor types is possible, but also that the structures of the B₁ and B₂R molecules are sufficiently similar to be antagonized by a single drug, a fact not appreciated until recently. Conformational analyses of B-9430 and B-9858 have been performed (Sejbal et al., 1997). The peptides exhibit no observable secondary structure in aqueous solution. However, some common and divergent elements emerge under certain experimental conditions, such as a type II -turn involving residues 2 to 5 common to both B₁ and B₂ antagonists. Such efforts may lead to the discovery of a universal "pharmacophore" for antagonizing kinin receptors.

Partial agonists are often discovered upon development of antagonist drugs. For example [Leu⁸]des-Arg⁹-BK, the prototype B₁R antagonist, may exhibit fairly high partial agonist behavior in some species, especially in the rats and mice. This has practical implications, because one of the most exciting emerging therapeutic applications of B₁R antagonists, analgesia, is commonly based on models involving these species (see below, Section IV.C.). The molecular structure of both the drug and the receptor are critically important for the partial agonist behavior of certain antagonists, as shown in a study involving the cloned human or murine B₁Rs transiently expressed in a cell line that constitutively produces the calcium sensitive photoprotein aequorin (MacNeil et al., 1997). [Leu⁸]des-Arg⁹-BK or Lys-[Leu⁸]des-Arg⁹-BK increased intracellular calcium concentrations in cells expressing the murine receptor (about 40% of the maximal activity of the full agonist des-Arg⁹-BK), whereas B9858 and R-715 were not active in this respect. None of these peptides acted as agonists on cloned human B₁Rs. Functional studies based on mouse or rat isolated tissues that express B₁Rs have confirmed that R-715 and des-Arg-Hoe 140 possess no or very low intrinsic stimulant activities in these species (Meini et al., 1996; Teater and Cuthbert, 1997; Allogho et al., 1995). However, partial agonist behavior of [Leu⁸]des-Arg⁹-BK or Lys-[Leu⁸]des-Arg⁹-BK was observed in some preparations (e.g., in the mouse isolated stomach, the rat isolated colon; Allogho et al., 1995; Teater and Cuthbert, 1997). This does not necessarily indicate the existence of B₁R subtypes that could be differentiated using a partial agonist behavior of some antagonists. If a pharmacological effect requires the stimulation of a very large proportion of the receptors on each cell, ligands with reduced efficacy are more likely to behave as antagonists (discussed by Leslie, 1987). This model may be applied quite well to the interaction of [Leu⁸]des-Arg⁹-BK on rat B₁Rs, as the maximum contractile (agonist) effect of [Leu⁸]des-Arg⁹-BK, relative to that of des-Arg⁹-BK, increases as a function of incubation time in the rat longitudinal ileum (i.e., the intrinsic activity is variable and increases; Meini et al., 1996). This is one of the smooth muscle preparations that exhibit in vitro up-regulation of B₁Rs upon isolation (see below, Section III.A.).

A partial agonist behavior may explain why the analgesic effect of [Leu⁸]des-Arg⁹-BK in rats and mice may be limited to a relatively narrow window of dosage (around 30 nmol/kg), as higher doses may exacerbate pain perception (Rupniak et al., 1997). No existing peptide antagonist seems ideal in term of affinity or selectivity to fully characterize the analgesic potential of B₁R blockade in small rodents (Rupniak et al., 1997).

Cross-desensitization is a valid criterion to distinguish substances acting on the same receptors (Schild, 1973). This implicitly refers to adaptation mechanisms present in, or closely associated with the receptor molecules. Thus, a "fading" of a response caused by the depletion of intracellular stores of an ion or some other desensitizing mechanisms distal to the receptors cannot be linked to receptor selectivity and would not qualify in exploiting this approach for receptor classification. In rat mesangial cells, a line that coexpresses both B₁ and B₂Rs, it has been possible to down-regulate a functional response (acute increase of [Ca²⁺]_i) to BK without depressing that to des-Arg⁹-BK, and vice versa (Bascands et al., 1993). An inositol trisphosphate formation or calcium response to des-Arg⁹-BK has been demonstrated upon BK desensitization in bovine endothelial cells or rabbit mesenteric artery SMCs, but the inverse was not readily demonstrable, because B₁R-mediated responses were not as tachyphylactic (Mathis et al., 1996; Smith et al., 1995;).

The two types of kinin receptors differ significantly in regard to receptor-mediated ligand internalization: exposure of cloned human B₂R to BK results in a rapid receptor-mediated ligand internalization and the sequestration of both the receptor and the G protein subunit in caveolae, accompanied by a profound loss of surface receptor binding (Austin et al., 1997; de Weerd and Leeb-Lundberg, 1997). In contrast, the activation of the cloned B₁R is not associated with ligand-induced receptor internalization at 37°C (Austin et al., 1997), and exhibits much less desensitization (further discussed below, Section III.D.). Experiments using truncated or chimeric human B₁ and B₂Rs in which the cytoplasmic carboxy terminus is either missing, or exchanged between the two receptor subtypes have indicated that the cytoplasmic carboxy terminus of the human B₂R contains sequences responsible for the receptor-mediated ligand internalization and receptor sequestration, which are not present in the C-terminal cytoplasmic domain of the B₁R subtype (Faussner et al., 1996), a domain of low interspecies conservation (fig. 1). Replacement from Cys³²⁴ of the cytoplasmic carboxy terminus of the B₂R with that of the B₁R has greatly reduced ligand-receptor complex internalization, whereas replacement from Cys³³⁰ of the cytoplasmic carboxy terminus of the B₁R (see fig. 1) with the B₂R counterpart has lead to a striking increase (~80% within 10 min) of internalization. Another study has confirmed the important role of the carboxy tail in B₂R internalization, identifying also other structural determinants (Prado et al., 1997). Thus, the consistent differences in signaling persistence between B₁ and B₂Rs appear to have a definite molecular basis and are of great potential interest to understand the relative importance of kinin receptor types in physiopathology. Whether other molecular mechanisms of desensitization are functionally important for kinin receptors (temporary loss of coupling of the G proteins, transcriptional suppression) is yet to be determined.

The structural features of the B₁R defined upon the expression cloning of the receptor cDNA confirmed its belonging to the superfamily of G protein-coupled receptors, exhibiting a typical 7 transmembrane domain architecture (fig. 1). Recently, it has been shown that CHO cells stably transfected with human B₁Rs cause Gq/11 and, to a lesser extent, Gi_1,2 to bind GTP upon stimulation with Lys-des-Arg⁹-BK (Austin et al., 1997). Thus, the identity and relative importance of G protein subtypes linked to the B₁R are similar to the ones coupled to B₂R (e.g., see de Weerd and Leeb-Lundberg, 1997). Both B₁ and B₂ receptors are primarily linked to polyphosphoinositide phospholipase C (PLC) activation. Specifically, activation of the naturally regulated B₁R is linked to increased phosphatidylinositol turnover in all systems studied up to now (reviewed by Marceau, 1995; see also Smith et al., 1995; Butt et al., 1995; see application to rabbit aortic SMCs, fig. 4A). Human B₁Rs stably expressed in Chinese hamster ovary (CHO) or 293 cells increase the turnover of inositol-phosphates when stimulated with Lys-des-Arg⁹-BK (Austin et al., 1997; Bastian et al., 1997). This effect was not prevented by pertussis toxin treatment in CHO cells, suggesting that the Gq/11 protein may be the more important than Gi_1,2 for coupling the B₁R to PLC (-isoform; Austin et al., 1997).

View larger version (46K): [in this window] [in a new window]   Fig. 4.   The rabbit cultured arterial smooth muscle cell (SMC), a model for the regulated cellular effects of the B1R. Middle: immunofluorescence pattern for -actin in rabbit aortic SMCs, passage 1. (A) Inositol phosphates (IP) extracted from rabbit aortic SMCs 5 min after exposure to des-Arg9-BK (1.7 µM) and pretreated or not with human recombinant IL-1 (5 ng/ml, 20 h before challenge). The asterisk indicates value significantly different from the corresponding control; the dagger indicates value significantly different from the corresponding one without IL-1 treatment. (Modified from Levesque et al., 1993, with permission.) (B) The B1R Bmax is significantly (asterisk) increased in these cells by the 16- to 20-h IL-1 treatment in a proportion very similar to the effect of the cytokine on IP hydrolysis. Nevertheless, the baseline population of B1Rs is important under the culture conditions employed (Levesque et al., 1995a). (C) Persistent [Ca2+]i signaling in a single representative SMC derived from the rabbit mesenteric artery and exposed to increasing concentrations of des-Arg9-BK (DBK). The signal (Fura-2 fluorescence) is composed of oscillating peaks, of an increase of the baseline, or both, depending on the individual cell and the agonist concentration. From Mathis et al. (1996), with permission of the authors, editor, and ASPET. (D) Effects of kinins on [3H]-thymidine incorporation into rabbit aortic SMCs in cells preteated with IL-1 (0.25 ng/ml, 24 h) and diclofenac (500 nM, 4 days). Both types of treatments are required to record a significant (asterisk) incorporation. Modified from Levesque et al. (1995b), with permission. Figures 4A and 4D were used with permission from the British Journal of Pharmacology; 109:1254-1262,1993; and 116:1673-1679,1995, respectively.

Calcium signaling was instrumental in the expression cloning of the B₁R (Menke et al., 1994). A transient increase in [Ca²⁺]_i (largely dependent on intracellular stores) is conceivably mediated by inositol 1,4,5-trisphosphate, one of the direct products of PLC, and this has been observed in response to stimulation of naturally expressed B₁Rs in animal or human cells (Bascands et al., 1993; Bkaily et al., 1997; Marsh and Hill, 1994; Mathis et al., 1996; Smith et al., 1995; fig. 4C) or cloned human or murine B₁R expressed in heterologous systems (Austin et al., 1997; Bastian et al., 1997; MacNeil et al., 1997). The sustained or oscillating phase of intracellular calcium increase is relatively more dependent on calcium influx by largely uncharacterized mechanisms (Bascands et al., 1993; partial blockade by nickel: Mathis et al., 1996; Smith et al., 1995). There is preliminary evidence for the activation of multiple types of calcium channels by the B₁R agonists in vascular SMCs (L, T, and R types, the latter being relatively more important in the sustained phase, Bkaily et al., 1997).

The moderate stimulatory effect of B₁R agonists on DNA synthesis in rabbit vascular SMCs (fig. 4D) is apparently dependent on another product of PLC, diacylglycerol, that activates a protein kinase C (PKC) (Levesque et al., 1995b). The SMC contractility mediated by B₁Rs (in the rabbit aorta) probably involves the cooperation of PKC and Ca²⁺ of both intra- and extracellular sources (Levesque et al., 1993). Filamin translocation from the membrane to the cytosol is another example of a Ca²⁺-dependent response elicited by either B₁ or B₂R stimulation in cultured bovine ECs (Wang et al., 1997). In this case, PKC action attenuates the response by limiting changes in calcium concentrations.

Thus, the identity of second-messengers is of limited value for the classification of the kinin receptors caused by common G protein subtypes and downstream elements. The most interesting differences observed so far may rather relate to the temporal patterns in systems that naturally coexpress both receptors. In bovine endothelial cells, rat mesangial cells, and rabbit mesenteric SMCs, the rise in [Ca²⁺]_i is more persistent and less tachyphylactic if elicited by B₁R stimulation, as opposed to B₂ activation, (Bascands et al., 1993; Mathis et al., 1996; Smith et al., 1995). Similarly, the effect of the B₁R stimulation on PLC activation is similarly more persistent than that the B₂R, in separate transfected CHO cell lines expressing analogous densities of each receptor type (Austin et al., 1997). In addition, these comparable cell lines were used to show that B₁Rs mediate much less receptor-ligand complex internalization than B₂Rs (Austin et al., 1997), a fact that could explain the persistence of B₁R signaling. Structural determinants for B₂R internalization not shared by the B₁R, are discussed above (Section II.C.) and may be the molecular basis of these temporal differences.

The kinin receptors might be involved in other transduction mechanisms, different from those described above. For instance, both B₁ and B₂Rs are equally capable of suppressing platelet-derived growth factor- (PDGF) induced DNA synthesis in rat arterial SMCs (Dixon and Dennis, 1997). The mechanism of this suppression is still obscure, as the effects were not dependent on PLC, prostaglandins (PGs), or cyclic adenosine 3',5'-monophosphate (AMP), or correlated with the extent of PLC activation (the B₁R being relatively ineffective in this respect; Dixon and Dennis, 1997).

Secondarily released mediators formed by Ca²⁺ dependent enzymes, such as endothelial nitric oxide synthase and cytosolic phospholipase A₂, may account for the production of nitric oxide (Drummond and Cocks, 1995a; Pruneau et al., 1996) and eicosanoids (Levesque et al., 1995b) mediated by B₁Rs. The secondary mediators are also shared with B₂Rs in many experimental systems, and are of considerable importance for the in vivo pharmacology of B₁Rs (see below, Section IV.). NO and PGs act in an autocrine or paracrine manner, thus extending the signaling mechanisms that kinins may activate in receptive tissues (notably to cyclic guanosine 3',5'-monophosphate and cyclic AMP).

The two kinin receptor subtypes have been further defined on molecular basis after the recent isolations and characterizations of their genes. A BK receptor was first cloned from rat uterus, using a Xenopus oocyte expression assay and shown to have the pharmacological profile of a B₂R subtype (McEachern et al., 1991). Subsequently, the human B₂R has been cloned from the fibroblast cell line CCD-16Lu by PCR and cDNA screening (Hess et al., 1992), its genomic organization studied (Kammerer et al., 1995; Ma et al., 1994) and its localization mapped to chromosome 14q32 (Ma et al., 1994; Powell et al., 1993). The murine and rabbit B₂R genes were also characterized (Bachvarov, et al. 1995; McIntyre et al., 1993). The deduced amino acid sequences of the B₂Rs from the four species studies have shown extensive similarity (80 to 84%), consistent with being orthologs of the same gene.

The B₁R became a defined molecular entity since the cloning and sequencing of the corresponding human cDNA. That the human embryonic cell line IMR-90 expresses B₁Rs has been known for some time, and receptor stimulation leads to metabolic effects such as collagen and DNA synthesis (Goldstein and Wall, 1984). Menke et al. (1994) observed that these cells bind [³H]Lys-des-Arg⁹-BK and that the binding is vigorously up-regulated by pretreating the cells with IL-1, consistent with current regulation studies (see below, Section III.). These stimulated cells were an enriched source for the isolation of mRNA further used for the expression cloning of the human B₁R cDNA. Different molecular weight mRNA fractions were consecutively injected in X. laevis oocytes and the photoprotein aequorin was used as an indicator of the ability of the B₁R agonist Lys-des-Arg⁹-BK to mediate Ca²⁺ mobilization in the injected oocytes. A 1307-bp clone that included a 1059 nucleotide open reading frame encoding a 353-amino acid protein was thus isolated and proposed to encode the B₁R (Menke et al., 1994; predicted primary structure, fig. 1). This discovery was followed by the full analysis of the cDNA of the B₁R (1.4 kb), considerably smaller than that coding for B₂R (over 4 kb), and by the determination of the corresponding genomic organization and chromosomal localization (14q32 between markers D14S265 and D14S267) (Bachvarov et al. 1996, 1998b; Chai et al., 1996; Yang and Polgar, 1996; fig. 5). The rabbit and mouse homologues of the B₁R gene were also isolated and characterized (MacNeil et al., 1995; Pesquero et al., 1996) as there is 68 to 78% homology between the B₁Rs of the three species studied. A rat nucleotide sequence with a similar level of homology is proposed to encode the rat B₁R (GenBank sequence U66107), but its pharmacological profile is not reported yet. The cloned human B₁R also exhibits the seven transmembrane structure typical for G protein coupled receptors, although its amino acid sequence identity with the B₂R type of BK receptor is only about 36%. Nevertheless, they are the closest relatives based on the degree of similarity, followed by angiotensin receptors.

View larger version (17K): [in this window] [in a new window]   Fig. 5.   Schematic structure of the human B1 receptor gene (Bachvarov et al., 1996; Yang and Polgar, 1996). The hatched region represents the uninterrupted coding sequence. Intron 1 length has been estimated to be 6 to 7 kb. Motifs of functional significance are indicated (start and stop codons, the polyadenylation signal, the TATA box in the 5' flanking sequence, an important NF-B-like site). Two polymorphic sites that can be studied by restriction length polymorphism are indicated (AciI, TaqI). Some of the fragments tested for promoter activity in constructions involving a reporter gene are indicated by numbered lines terminated with an arrowhead pointing to the right (sense orientation), or the left (antisense orientation) : (1) a 1.9-kb fragment 5' of the coding sequence and spanning exon 2 (Chai et al., 1996); (2) a 0.87-kb fragment in the same region; (3 and 4) 0.45-kb fragments 5' of and partially overlapping exon 1; (5) a 0.92-kb fragment starting in intron 1 and spanning exon 2 (Yang and Polgar, 1996); (6) a 0.14-kb gene fragment (111/+34 relative to the transcription initiation site) containing a consensus TATA box (Ni et al., 1998); (7) a 226-nt gene fragment (139/+86); (8) the most active promoter fragment tested (735/+86); a postulated negative control region in the core promoter is located 5' relative to fragment 8 (D. R. Bachvarov, R. Drouin, M. Angers, J.-F. Larrivée, M. Bachvarova, and F. Marceau, in preparation).

The predicted intracellular carboxy tail of the human B₁R is longer than that of their rabbit, mouse or rat counterparts (fig. 1). Perhaps significantly, the only effective antiserum to human B₁R to date has been derived from a rabbit immunized with the 16-mer C-terminal peptide (underlined in fig. 1; Hess et al., 1996). These antibodies detected human recombinant B₁Rs expressed under the control of a viral promoter in transfected COS cells (immunofluorescence, Western blot; Hess et al., 1996). Four immunoreactive bands were identified in the Western blot experiments, corresponding to 39, 31, 25, and 20 kDa. Because the predicted molecular weight of the human B₁R without carbohydrate is 40.4 kDa, it is quite possible that all four bands correspond to proteolytic cleavage fragments. All the published immunohistochemistry of the B₁R (see Section IV.) can be traced back to this antiserum.

Few studies of the structure-function relationship have been performed on the B₁R, and they are always comparative to the B₂R, for which a putative binding pocket involving transmembrane (TM) domain VI and the adjacent extracellular residues has been characterized (discussed by Leeb et al., 1997). The similarity between the two receptor subtypes is significantly higher in these regions. Leeb et al. (1997) have constructed chimeric human B₁ and B₂Rs in which TM-VI domains have been exchanged and have shown that TM-VI in both receptor subtypes are important for the ability of these receptors to discriminate between their subtype-selective agonists. Substitution of B₁ TM-VI into the B₂R dramatically reduced the affinity of the B₂R-selective agonist BK. This affinity was fully restored when two residues, Tyr²⁵⁹ and Ala²⁶³ near the extracellular surface of the chimeric B₁R-TM-VI (see fig. 1) were replaced with the corresponding residues in the wild-type B₂R, which are Phe²⁵⁹ and Thr²⁶³. Conversely, substitution of the B₂ TM-VI into the B₁R reduced the high affinity binding of the B₁-selective agonist Lys-des-Arg⁹-BK. Interestingly, the latter chimera retained a functional response ([Ca²⁺]_i increase) to a high concentration of Lys-des-Arg⁹-BK and to Lys-BK, the latter peptide confirming its status as a B₁ and B₂R agonist. TM-VI is probably less important for ligand docking in the B₁R than in the B₂R. Recent experiments have focused on TM-III exchange between the two kinin receptors, as this domain is presumably facing the TM-VI docking domain (Fathy et al., 1997; L. M. F. Leeb-Lundberg, personal communication). Introduction of B₁R TM-III into the B₂R sequences decreased the affinity of the B₂R selective agonist BK, whereas the substitution of B₂R TM-III into the B₁R decreased the affinity of the selective ligand Lys-des-Arg⁹-BK. High affinity for BK was restored when Lys¹¹⁸ in B₁R TM-III (numbering as in B₁R) was replaced with the corresponding wild-type B₂R residue (Ser¹¹¹). These observations and complementary analyses suggest that Lys¹¹⁸ in the B₁R structure repels the positive charge of the ligand C-terminal Arg⁹, thus providing a possible explanation for the fundamental feature of the B₁R pharmacological profile: selectivity for des-Arg⁹-BK homologues. These types of experiments further show the surprising structural compatibility of the two types of human kinin receptors, already suggested by the recent development of promiscuous antagonist ligands (see above, Section II.B.).

There is no genetic support for the existence of B₁ or B₂R subtypes in any given species, although fairly large differences of pharmacological profiles exist from one species to another. Experiments based on cloned and highly homologous receptors expressed in heterologous systems show conclusively that these pharmacological discrepancies are species differences (table 2).

Perhaps it can be postulated that the ultimate form of receptor antagonism is the targeted disruption ("knockout") of receptor genes by homologous recombination. In mammalian species this technology, based on manipulations of embryonic stem cells, is currently restricted to the mouse. The B₂R knockout mice fail to respond to BK (smooth muscle, afferent nerve stimulation, etc.), indicating that no other B₂R subtype or variant responsive to that peptide exists in this species (Borkowski et al., 1995). These animals develop normally and may not exhibit important hemodynamic changes. However, if overloaded with dietary NaCl, they develop severe hypertension with end organ damage (Alfie et al., 1996). The relative importance of renal deficit in B₂R knockout mice is an important novel aspect of the kallikrein-kinin system, further exemplified by a polymorphism in the human B₁R gene (see below, Section III.C.). There is ample evidence that cytokine or postinjury regulation of the B₁R gene expression is not disrupted in B₂R knockout mice (Asonganyi et al., 1996; Cuthbert et al., 1996; Rupniak et al., 1997; Seabrook et al., 1997; see Section IV.). Such results are important, because both kinin receptor genes lie close to each other on human chromosome 14. This clustering may not represent a functionally coordinated locus comparable to those of globins or serum albumin homologues, for instance.

The B₁R knockout mice have been produced quite recently (Bader et al., 1997; J. B. Pesquero, personal communication). These animals develop normally, are normotensive, but fail to functionally respond to des-Arg⁹-BK (e.g., contractility of the mouse isolated stomach). Further, basal and LPS-induced expression of B₁R mRNA is absent (RT-PCR, reverse transcriptase polymerase chain reaction). Further experiments will be needed to exploit this murine strain in different physiopathological conditions. In this context, the creation of a double B₁/B₂R knockout strain is extremely intriguing.

The transcriptional activity of the receptor genes is regulated by both tissue-specific and physiological factors. This may be relevant for receptor classification, and refers to the promoter function of distinct genes. Although there is some evidence that B₂R expression is transcriptionally regulated in cultured cells (e.g., modest positive regulation by cyclic AMP, PDGF and IL-1; Bathon et al., 1992; Dixon, 1994; Dixon et al., 1996; Schmidlin et al., 1997; suppression by tumor necrosis factor-; Sawutz et al., 1992), these receptors are constitutively expressed in a wide variety of tissues. One of the few systematic efforts to document B₂R regulation concerns a sexual dimorphism of B₂R expression in several organs of the rat, the females expressing more of the corresponding mRNA (detected using RT-PCR; Madeddu et al., 1997). Ovariectomy reduced the hypotensive effect of BK and estrogen treatment restored the hemodynamic effect of the kinin; however, hemodynamic responses to some agonists for other receptors followed a similar pattern (Madeddu et al., 1997). Based on hemodynamic responses to des-Arg⁹-BK, the B₁Rs were not regulated by estrogen in these studies. In some chronic inflammation models, B₂R function may be depressed (several examples will be given in Section IV.), but it is not known whether this is caused by transcriptional repression or ligand-mediated down-regulation. In contrast, a large body of evidence shows that the B₁R is generally absent from normal tissues and animals (with some exceptions), but is rapidly induced after certain types of injuries in many species (Marceau, 1995). Molecular and genetic techniques have recently confirmed this receptor up-regulation. The regulatory mechanisms of gene expression are a recent focus of the research on B₁Rs, which is covered in detail in the next section.

	III. Immunological and Molecular Analysis of B1 Receptor Regulation by Tissue Injury

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Smooth muscle contractility measured using organ bath methodology has been historically important to discover and characterize the B₁Rs. The distinct pharmacological profile of the kinin B₁R was initially derived from the analysis of kinin-induced contractility of the rabbit aorta (Regoli et al., 1977). On this preparation, exogenous kinins exert a contractile effect that develops from an initial null level but increases in magnitude as a function of the in vitro incubation time (Bouthillier et al., 1987). In this sense, the rabbit isolated aorta is a model for many other smooth muscle preparations derived from normal animals and used to illustrate the phenomenon of postisolation B₁R induction. Many such systems from various animal species are reviewed elsewhere (Marceau, 1995). To cite just a few recently documented examples, the rat portal and pig renal veins also demonstrate this phenomenon based on contractility (Campos and Calixto, 1994; Rizzi et al., 1997). It is now clear that nonmuscle cellular elements can also exhibit the postisolation induction of B₁R: the vascular endothelial and colonic epithelial cells acquire such a responsiveness in vitro from a null or low initial level in the bovine or porcine isolated coronary artery and rat isolated colon, respectively (Drummond and Cox, 1995a; Pruneau et al., 1996; Teater and Cuthbert, 1997). In such preparations, the de novo synthesis of B₁R is supported by the selectivity of the regulated behavior; similar responses mediated by other receptor types (including B₂Rs in some preparations) are much more stable as a function of time. Characteristically, the use of metabolic inhibitors also support the postisolation B₁R induction. Inhibition of RNA synthesis with actinomycin D, of protein synthesis with cycloheximide, anisomycin or puromycin, or of protein maturation with brefeldin A or tunicamycin, specifically prevent the development of a responsiveness to exogenous B₁R agonists without displaying toxic effects on other types of responses, or an acute inhibitory effect on responses to kinin that had been allowed to develop without metabolic inhibitors (Deblois et al., 1991; Drummond and Cox, 1995a; Pruneau et al., 1996; Teater and Cuthbert, 1997). Brefeldin A is a selective inhibitor of the translocation of newly synthesized proteins from the endoplasmic reticulum to the Golgi apparatus. A membrane receptor with transmembrane domain(s) would follow such a relatively slow maturation process and accordingly, the drug completely and selectively prevented des-Arg⁹-BK-induced contraction in the rabbit isolated aorta (Audet et al., 1994). The B₁R contains up to three putative but conserved N-glycosylation sites (fig. 1), which may be of importance for receptor assembly, stability or function. This is supported by the partial inhibitory effect of tunicamycin on the development of responses to des-Arg⁹-BK in rings of rabbit aorta (Audet et al., 1994). The biochemical effects of some of the metabolic inhibitors have been validated in assays involving the incorporation of [³⁵S]methionine (Deblois et al., 1991) or [³H]leucine into the protein fraction and of [³H]mannose into the glycoprotein fraction of rabbit aortic tissue (Audet et al., 1994).

The isolation-induced B₁R induction also occurs in human tissue preparations. Rings of human coronary arteries develop in vitro as a function of time the capacity to respond to des-Arg⁹-BK resulting in an endothelium-dependent relaxation that is competitively antagonized by [Leu⁸]des-Arg⁹-BK, but not by Hoe 140 (Drummond and Cocks, 1995b). The human isolated ileum and umbilical vein develop responsiveness to B₁R agonists as a function of time, as evidenced using a full range of agonists and antagonists for kinin receptors, and this increase of responsiveness was prevented by treatment with a protein synthesis inhibitor (Gobeil et al., 1996b; Sardi et al., 1997; Zuzack et al., 1996). In all these systems, B₂Rs were present, but in a preformed and stable manner, and mediated qualitatively similar effects.

The rabbit aortic contractility has also been exploited to show that cytokines, corticosteroids and drugs inhibitory for mitogen-activated protein (MAP) kinases influence the up-regulation of B₁Rs. Some inflammatory cytokines and growth factors increase the rate of sensitization to B₁R agonists in rabbit aortic rings: IL-1, IL-2, epidermal growth factor (EGF), and oncostatin M have a similar effect on the rabbit aorta (Bouthillier et al., 1987; Deblois et al., 1988, 1991; Levesque et al., 1995b), and interferon- on the bovine isolated mesenteric artery (De Kimpe et al., 1994). The most potent stimulants increase both the maximal effects and the apparent potencies of B₁R agonists. However, the induction of B₁R-mediated responses is still prevented by cycloheximide in cytokine-stimulated aortic rings (Audet et al., 1994). Glucocorticoids (dexamethasone and others), but not an estrogen, suppressed extensively the up-regulation of the B₁R-mediated responses, either under the spontaneous or cytokine-stimulated conditions (Deblois et al., 1988). This also applies to the isolation-induced up-regulation of B₁R in the human umbilical vein (Sardi et al., 1997). These findings are suggestive for a gene regulated by the cytokine network and immunopathology. However, the cytokines regulating the process are very diverse as far as their receptor structure and signal transduction system pathways are involved. It was recently observed that freshly isolated rabbit aortic rings from normal animals show high MAP kinase activities (p38, ERK and SAPK/JNK pathways) relative to untreated cultured cells of various types, and these activities were further up-regulated by exogenous IL-1 (SAPK/JNK) or EGF (ERK) (Larrivée et al., 1998). An inhibitor of the p38 MAP kinase, SB 203580, selectively inhibited by ~75% the spontaneous sensitization to des-Arg⁹-BK over 6 h. SB 203580 also significantly reduced the development of the response to des-Arg⁹-BK as stimulated by IL-1 or EGF (by about 50% at 6 h). Both the spontaneous and IL-1-stimulated up-regulation of responsiveness to des-Arg⁹-BK were strongly inhibited by the MEK1 inhibitor PD 98059 (by about 40%); however, the drug did not affect des-Arg⁹-BK-induced responses in tissues treated with EGF. There is no specific inhibitor of the SAPK/JNK MAP kinase pathway to date, thus it cannot be excluded that this pathway also regulates B₁R expression. The protein kinase inhibitors failed to inhibit protein synthesis or to acutely block the contractile effect of des-Arg⁹-BK, suggesting that they do not influence B₁R transduction mechanisms. Thus, protein kinase inhibitors reveal the role of cell injury-controlled MAP kinase pathways, and singularly of the p38 pathway, in the induction of B₁R (Larrivée et al., 1998). These pathways conceivably may serve as mediators between cytokine receptor activation or direct cell injury (hyper- or hypoosmolarity, nutrient deprivation, oxidative stress or hypoxia) and the activation of the multiple transcription factors. The isolation-induced paradigm of B₁R induction has provided useful insights into the implicated immunological/pathological mechanisms and has indicated a minimal time (about 1 to 2 h) for the functional appearance of the response. The use of metabolic inhibitors, introduced in isolated organ pharmacology to study B₁R regulation, has revealed the reactivity of such preparations and has subsequently been important for the analysis of other systems (e.g., the cytokine induction of NO synthase or arachidonate metabolism in isolated blood vessels; Beasley et al., 1991; Petitclerc et al., 1994).

An in vivo paradigm for B₁R induction which has been particularly studied is the induction of cardiovascular responses to the corresponding agonists by a treatment with bacterial LPS in rabbits and other animals (see below Sections IV.A.). The evidence for transcriptional regulation of the receptor and its immunological basis will be reviewed here. Rabbits receiving intravenous injections with a sublethal dose (10 to 40 µg) of LPS exhibit dose-related hypotensive responses to exogenous intraarterial des-Arg⁹-BK (Regoli et al., 1981) or Lys-des-Arg⁹-BK (Drapeau et al., 1991b) after 5 h (see also fig. 3). By contrast, control animals do not respond significantly to these agents (fig. 3A). LPS pretreatment does not modify the basal blood pressure or the hypotensive responses to BK (mediated by B₂Rs) or to other agonists, and is not effective if given immediately before hemodynamic assessment, indicating a minimal lag time, probably necessary for protein synthesis and maturation. Tissues removed from LPS-pretreated animals (e.g., vascular strips, fig. 3D) exhibit functional responses to B₁R agonists within the first hour of in vitro incubation, suggesting that the response was acquired in vivo before death. Des-Arg⁹-BK is a vasodilator of the coronary arteries only in hearts removed from LPS-pretreated animals and perfused in vitro (Langendorff technique; Regoli et al., 1981). The B₁R mRNA up-regulation in heart tissue accordingly precedes this responsiveness in LPS-treated animals (Marceau et al., 1997; fig. 3B). Northern blot experiments performed on poly(A⁺) RNA derived from freshly isolated aortic smooth muscle of rabbits pretreated or not with LPS produced results virtually identical with those presented in figure 3B (J. F. Hess, D. R. Bachvarov, and F. Marceau, unpublished data). This model suggests that the whole cardiovascular system of the rabbit can be sensitized to B₁R agonists without modifying the preexisting population of B₂Rs or of receptors for other agents. The transcriptional activation of the B₁R gene is supported in this model by the kinetics of the corresponding mRNA in rabbit tissues after LPS injection, preceding the functional responses. LPS induction of cardiovascular responses has been observed in other species (see Section IV.A.) and supportive molecular evidence of de novo formation of B₁Rs has been generated. Autoradiography of [³H]Lys-des-Arg⁹-BK (6 nM) bound to frozen sections of porcine pulmonary arteries and thoracic aortas has revealed that the specific binding signal associated with the media SMCs is increased at least 3-fold by a pretreatment with LPS (Schremmer-Danniger et al., 1996). RT-PCR has been used to study the expression of the B₁R in the mouse (Pesquero et al., 1996). This highly sensitive and nonlinear amplification technique revealed B₁R transcripts in several organs of the normal mouse, but these signals were markedly increased by LPS treatment, notably in the heart, lung, and liver.

LPS, a complex polymer usually in the form of high molecular weight aggregates, is derived from Gram-negative bacteria and is capable of recruiting numerous host effector systems and modifying gene expression in various cell types. Notably, LPS is an inducer of cytokine synthesis in leukocytes and other cell types. An alternate cytokine inducer and pyrogen, the muramyl-dipeptide, is a synthetic LMW activator of cytokine production and can substitute for LPS to induce cardiovascular sensitivity to des-Arg⁹-BK in rabbits (Bouthillier et al., 1987). Intravenously injected human recombinant IL-1 is also active in this respect (Deblois et al., 1991). Bridging the gap between the in vivo model and the molecular receptor populations, binding assays of radiolabeled B₁R agonists to rabbit cultured vascular SMCs have confirmed that IL-1, EGF, and LPS up-regulate the B₁R population (B_max), without influencing receptor affinity (Galizzi et al., 1994; Levesque et al., 1995a; Schneck et al., 1994). In these studies, a relatively high background of B₁Rs in the cells may be a culture-induced artifact, perhaps derived from MAP kinase stimulation by serum in the medium or by stress. Nevertheless, we have observed that exogenous IL-1 stimulates B₁R B_max by a factor very similar to the potentiating effect of IL-1 on phospholipase C by des-Arg⁹-BK in rabbit aortic SMCs (fig. 4A and B). Other studies, based on different culture conditions, have demonstrated a more dynamic regulation of the receptor population by cytokines and LPS. The rabbit B₁R cloning by subtraction hybridization took advantage of the presence of the B₁R in a cDNA library constructed from cultured aortic SMCs, from which a library derived from fresh aortic tissue was subtracted, thus enriching the receptor cDNA (MacNeil et al., 1995).

Another important in vivo paradigm for B₁R up-regulation is the mediation of hyperalgesia in various models of persistent or chronic inflammation based on the rat or mouse (see below, Section IV.C.). The hyperalgesia models in rats, based on various primary irritants or noxious stimuli, can also reveal the B₁R-mediated loss of mechanical or thermal tolerance when the rats receive injections of recombinant cytokines such as IL-1, IL-2, IL-8 (Davis and Perkins, 1994b), or nerve growth factor (Rueff et al., 1996). The natural IL-1 receptor antagonist, IRA, prevented the hyperalgesia produced by intraarticular injection of IL-1, IL-2, or IL-8, suggesting that the effect of some cytokines may be ultimately linked to the autocrine/paracrine production of IL-1 (Davis and Perkins, 1994b). Isolated and cultured mouse superior cervical ganglia expressed B₁R (detected as a functional response, i.e., depolarization after exposure to Lys-des-Arg⁹-BK in the presence of captopril) only in tissues treated in vitro with IL-1 or IL-8, but not leukemia inhibitory factor (Seabrook et al., 1997). The same report showed B₁R transcripts in neural tissue by in situ hybridization, although the functional correlates were not developed.

In summary, immunological analysis of B₁R up-regulation in vivo and in vitro indicates that inflammatory cytokines play an important role in transcriptional activation of the corresponding gene, with a prominent place for IL-1. However, an obligatory role of IL-1 in B₁R expression under all experimental conditions is not proven. For instance exogenous IRA, a competitive antagonist of IL-1 or - at the level of IL-1 membrane receptors, failed to inhibit the spontaneous sensitization to des-Arg⁹-BK as a function of time in rabbit aortic rings, although IRA was effective to suppress the potentiating effect of exogenous IL-1 on this process (Petitclerc et al., 1992). More direct forms of cellular stress (isolation of tissues and incubation in nutrient-poor physiological media; long term cell culture), typically applied in the absence of leukocytes, may bypass the need for cytokines and their receptors, by activating downstream stress-sensitive MAP kinases (Larrivée et al., 1998). In addition, biochemical responses to B₁R agonists that are distant from the receptors, such as PGI₂ synthesis (Galizzi et al., 1994) or DNA synthesis (fig. 4D), may be amplified to a greater extent by IL-1 or other cytokines than expected on the basis of B₁R B_max changes, possibly caused by various postreceptor interactions between the kinin and cytokine signaling pathways. Similarly, an immediate synergistic effect of EGF with des-Arg⁹-BK-induced contractile effect is observed in the rabbit isolated aorta, and this is not likely to be mediated by a B₁R up-regulation (Deblois et al., 1992), although long-term induction of B₁Rs by EGF has been shown (Schneck et al., 1994). The molecular basis of an EGF receptor-B₁R interaction is not determined yet.

The inducible behavior of the B₁R, whose expression is restricted to immunopathology, is a substantially documented concept, but important exceptions may exist. In addition to the significant species-dependent differences in ligand affinities, major discrepancies in the regulatory function of the B₁R may exist at least in the dog and the cat. Hypotension, natriuresis and renal vasodilator responses were observed in normal dogs injected with des-Arg⁹-BK (Lortie et al., 1992; Nakhostine et al., 1993). Some tissues isolated from the dog also exhibit an immediate and stable response to B₁R agonists (reviewed by Marceau, 1995). Complex hemodynamic effects are also produced by des-Arg⁹-BK in the feline pulmonary circulation (DeWitt et al., 1994). In each case, the B₂R coexisted and the identity of each receptor type was also validated with the use of appropriate antagonists. It is tempting to speculate that a difference in the B₁R gene promoter of the order Carnivora can explain the constitutive expression of cardiovascular B₁Rs. The guinea pig gallbladder is a smooth muscle preparation which acquires a contractile response to des-Arg⁹-BK in a time- and protein synthesis-dependent manner (Cabrini and Calixto, 1997). However, this response is antagonized by Hoe 140, not by [Leu⁸]des-Arg⁹-BK, prompting these authors to conclude that the response to des-Arg⁹-BK is mediated by inducible B₂Rs. Molecular biology approaches are needed to clarify whether the canine and feline constitutive B₁Rs are the real species homologues of the genetically defined B₁Rs, whether the guinea pig receptors responsive to des-Arg⁹-BK are B₂Rs, and whether immunopathology can regulate the expression of all these receptors.

In such animal species as the pig and rats, some reports suggest a minimal but measurable hemodynamic responsiveness to exogenous des-Arg⁹-BK (Siebeck et al., 1989; Madeddu et al., 1997), although it is clear that treatments with bacterial products can up-regulate these responses (Lagneux and Ribuot, 1997; Siebeck et al., 1989; Tokumasu et al., 1995). Whether a low population of B₁Rs plays a physiological role in normal animals, at least in some organs, is not a trivial issue. Stomach strips from apparently normal mice exhibit an immediate and constant contractile response to B₁R agonists (Allogho et al., 1995), and exogenous des-Arg⁹-BK is inflammatory when injected into the pleural space of naive mice, but not when injected into the paw (Vianna and Calixto, 1998). Further, the B₁R antagonist [Leu⁸]des-Arg⁹-BK reduces the glomerular filtration rate and the urine concentration in normal rats infused with saline (Fenoy and Roman, 1992). Whether tissue-specific constitutive B₁Rs can be invoked in these examples could not be excluded. However, subclinical infectious disease in "control" animals is another possible explanation for a "baseline" B₁R population. For example, in pigs, spontaneous infections are common and this can predict the level of cardiovascular response to des-Arg⁹-BK (Siebeck et al., 1996). A systematic effort will be needed to clarify these issues.

The body of evidence summarized above shows that the B₁R is a strongly regulated gene. The analysis of the promoter region of the human B₁R gene has been initiated using constructions involving a reporter gene (luciferase or chloramphenicol acetyl transferase) transfected in the human cell lines (IMR-90, HEK-293 or HepG2) or rat aortic SMCs (Chai et al., 1996; Ni et al., 1998; Yang and Polgar, 1996; D. R. Bachvarov, R. Drouin, M. Angers, J.-F. Larrivée, M. Bachvarova, and F. Marceau, in preparation). DNA sequencing and analysis had revealed several potential regulatory sites in the noncoding parts of the genes, in fact too many to make useful predictions. Some of the gene fragments tested for promoter activity are indicated in fig. 5. An initial study showed a weak promoter activity in the 5' region of the coding sequence, amounting to a maximal of about 10% of that of a positive control, the Rous sarcoma virus promoter (maximal activity in the 1.9-kb fragment indicated as 1 in fig. 5; Chai et al., 1996). This fragment spans exon 2, a fact that was not known at this time. Recent experiments showed that this fragment does not confer regulatory effects to IL-1 (Ni et al., 1998). Knowledge of the 3-exon structure prompted Yang and Polgar (1996) to cover more systematically potential regulatory domains (fragments 2 to 5, fig. 5). Although fragment 2 promoted some expression of the reporter gene in SV40-transformed IMR-90 cells, fragment 3 (located 5' to exon 1) was about 10-fold more active. Fragment 4, the antisense counterpart of fragment 3, as well as fragment 5, covering a part of exon 1 and exon 2, were essentially inactive. This comparative study established the predominant promoter activity of the region located 5' relative to exon 1.

A 2.6-kb fragment localized upstream from the first intron of the receptor gene has been recently studied for promoter activity (Ni et al., 1998). Different fragments of this region, cloned in front of a reporter gene (firefly luciferase), exhibited promoter activity in several cell lines, but a regulated behavior only in some lines. Successive deletions indicated that a 0.14-kb 5' flanking fragment (6 in fig. 5) was sufficient for transcriptional activity and inducibility by IL-1, TNF- and LPS and suppression by dexamethasone and by a putative anti-oxidant inhibitor of nuclear factor B (NF-B) (Ni et al., 1998). An NF-B-like binding sequence at 64 to 55 relative to the major transcription initiation has been found; its mutagenesis abolished most of the regulatory effects. Another group studied the 4.2-kb fragment upstream of exon 1 (D. R. Bachvarov, R. Drouin, M. Angers, J.-F. Larrivée, M. Bachvarova, and F. Marceau, in preparation). The promoter activity of this zone was addressed using chloramphenicol acetyl transferase reporter gene constructs. Transient transfection of reporter gene constructs in IMR-90 cells indicated that a 226-nt gene fragment (139 to +86 relative to the transcription initiation site; fragment 7 in fig. 5) containing a consensus TATA box, was sufficient to direct transcription in the cell line IMR-90, a cell line spontaneously expressing the B₁R. The corresponding antisense fragment was not active. Promoter activity was not observed in four constructs lacking the TATA box which is located 21 to 28 bp upstream of the major transcription initiation site, indicating its functional importance. Promoter activity was not importantly modulated by IL-1 or dexamethasone in IMR-90, by contrast to the findings of Ni et al. (1998) in SMCs. The presence of IL-1 responsive element(s) in the region studied was indirectly shown by transient transfections using the human embryonal kidney cell line HEK-293, which expresses the IL-1 receptor type I gene only at a very low level. Cotransfection experiments, using a construct expressing the human IL-1 receptor type I gene together with promoter constructs, doubled the promoter activity of fragments as short as the 226-nt fragment 7, confirming the presence of IL-1-responsive motif(s) in the region studied. Further experiments in IMR-90 cells based on several constructions of various length identified a positive and a negative control regions, localized upstream from the TATA box (fig. 5). The most active fragment, indicated as 8 in fig. 5, exhibited about two-thirds of the activity of a cytomegalovirus promoter in transfected IMR-90 cells, and spanned a polymorphic site of potential importance (see below, Section III.E.). A negative control region was located upstream to the maximally active promoter fragment 8.

Thus, the promoter region of the human B₁R bears the characteristics of an eukaryotic inducible promoter with a functional TATA box, and contains additional positive and negative control elements. Evidence of tissuespecific and cytokine regulatory control was also obtained. At this time, it cannot be excluded that regulatory elements lie outside the studied regions, for instance in the large intron 1.

Induction of B₁R in IMR-90 cells by immunostimulants may involve a stabilization of the corresponding mRNA, in addition to transcriptional stimulation, suggesting a role for of a posttranscriptional regulator (Zhou et al., 1998). IL-1 doubled the mRNA half-life to 2 h; protein synthesis inhibitors were even more potent to increase B₁R mRNA stability in these cells (Zhou et al., 1998). Perhaps related to these findings, protein synthesis inhibitors applied as a "pulse" of 1 to 3 h on rabbit aortic rings were found to paradoxically increase the subsequent up-regulation of B₁R-mediated responses (Deblois et al., 1991).

Human genetic studies have shown that a transmissible low urinary kallikrein excretion is associated with a positive history of hypertension (Berry et al., 1989). Investigations on genetic polymorphisms of kinin receptors have been initiated. Of the described B₂R gene polymorphisms identified, only one has been shown to be of potential clinical significance: an exon 1 polymorphism in which alleles differ by a 9-bp deletion, designated () versus the complete sequence, designated (+) (Lung et al., 1997). The () allele, presumably more stable relative to the action of RNases, appears to confer a higher level of expression and is always present in the most symptomatic cases of C1 inhibitor deficiency (hereditary angioedema with angioedema crises, 21 patients examined). Thus, the B₂R () allele is proposed to modulate in a dominant manner the phenotype (penetrance) of the basic genetic defect in this disorder, the C1 inhibitor deficiency. The latter plasma component is an endogenous inhibitor of plasma kallikrein activity (table 1).

Minor variations in the coding sequence of the human B₁R have been reported (see fig. 1 legend); whether these are genetic polymorphisms or the result of methodological errors in DNA sequencing has not resolved. Based on the genomic structure of the human B₁R for kinins, the presence of possible allelic polymorphisms of this gene was investigated using both restriction fragment length polymorphism and single strand conformation polymorphism methods (Bachvarov et al., 1998b). It was reasoned that the protective effect of the kallikrein-kinin system on the kidney and cardiovascular system could be altered if the polymorphic alleles were functionally different, leading to decompensation and end organ damage with the less functional alleles, as is seen in an extreme model, i.e., the knockout mice for the B₂R gene submitted to a NaCl overload (Alfie et al., 1996). Two B₁R gene polymorphisms were found and the frequencies of the corresponding allele pairs were determined in healthy volunteers and inpatients with a history of end stage renal failure (Bachvarov et al., 1998b). An A¹⁰⁹⁸  G polymorphism has been identified in exon 3 in a minority of volunteer blood donors. This polymorphism is located 35 nucleotides downstream of the stop codon and 14 nucleotides upstream of the polyadenylation signal, and appeared to be clinically neutral. The A  G substitution introduces an additional TaqI restriction site (fig. 5). A second, and more frequent polymorphism consists of a single base substitution (G^-699  C) in a positive control region of the promoter (position relative to the major transcription initiation site; this generates an additional AciI restriction site, fig. 5). This polymorphism is significantly less frequent in the population of renal failure patients (33.3% prevalence in 102 healthy volunteers, versus 20.6% in 287 diseased individuals, P = 0.014) and determines in IMR-90 cells a significantly increased activity of the promoter function in constructions involving both versions of gene fragment 8 (fig. 5) cloned in front of a reporter gene. The altered prevalence of this allele was also found in several etiological subgroups of uremic patients. Thus, the polymorphism of the B₁R promoter may be a marker of prognostic significance for the preservation of renal function in diseased individuals. The hypothetical influence of the relative B₁R overexpression determined by the C allele could not be linked to a specific etiology, but may rather be related to nonspecific compensatory mechanism(s), as with resistance to ischemic damage, maintenance of glomerular filtration, etc. It is not currently known whether the alleles are associated with negative clinical aspects (e.g., amplified role of kinins as inflammatory and pain mediators).

	IV. Physiopathological Relevance of B1 Receptor Up-Regulation

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The conservation of the B₁R structure (fig. 1) and regulatory mechanisms in several mammalian species suggests that this system affords a selective advantage in stressful situation such as infection or cardiovascular or renal disease. Cytokine and MAP kinase regulation of this gene are likely to make the B₁R up-regulation a rather nonspecific tissue response to many intense stressful situations (e.g., 42°C heat shock applied to anesthetized rats, Lagneux and Ribuot, 1997). In experimental pathology, the contribution of the kinin B₁R has been very generally overlooked, until recently. Part of the problem may be related to the less-than-ideal properties of the available peptide B₁R antagonists (discussed above). However, typical of inflammatory mediators, the pharmacological blockade of the B₁R system in inappropriate forms of inflammation (e.g., autoimmune, allergic) is emerging as a useful therapeutic intervention, based on animal models. Practically nothing is known about the clinical pharmacology of the B₁R. Investigators have initiated the immunohistochemical or in situ hybridization detection of the B₁R in human tissue sections. Although these results are very interesting, their relevance to receptor population is not always clear, because most adult "normal" tissue donors are either clinically ill defined or objectionable (e.g., posttraumatic death that was probably preceded by an episode of cardiovascular shock; Raidoo et al., 1997).

The in vivo induction of B₁Rs in the rabbit by a LPS injection described above, is based on functional and molecular evidence (fig. 3; Section III.B.). As for the physiopathological implications, this model exhibits selectivity, as several other treatments recruiting immunological effectors failed to induce the state of responsiveness to the selective B₁R agonist des-Arg⁹-BK (e.g., in vivo activation of circulating neutrophils with N-formyl-Met-Leu-Phe; Bouthillier et al., 1987). The dominant vasoconstrictor effect of B₁R agonists in large conduction vessels (the rabbit aorta, the human umbilical vein, etc.), conveniently used for in vitro pharmacological studies (see above), may only be important in a limited number of cases; for instance, both B₁ and B₂Rs have been hypothetically involved in postpartum umbilical vessel closure (Abbas et al., 1998). Such vasoconstriction may not be predictive for the response of smaller muscular or resistance vessels, where vasorelaxation mediated by various secondary released autacoids (eicosanoids, nitric oxide) may overshadow contractile effects (as seen in the perfused rabbit heart, the rabbit isolated carotid and mesenteric arteries; Regoli et al., 1981; Churchill and Ward, 1987; Pruneau and Bélichard, 1993). The mechanism of the hypotension induced by the metabolically stable B₁ agonist Sar[D-Phe⁸]des-Arg⁹-BK has been studied in LPS-pretreated rabbits. The duration, but not the amplitude, of the hypotensive episodes were reduced by indomethacin (Drapeau et al., 1991b) or diclofenac (Audet et al., 1997), suggesting a modulating effect of secondarily released prostaglandins. The mechanism of the prolonged hypotension caused by the stable B₁R agonist, Sar-[D-Phe⁸]des-Arg⁹-BK, appears to be complex in LPS-pretreated rabbits: a fall of peripheral vascular resistance accounts for the early response, but the prolonged hypotension associated with persistent receptor stimulation is explained by a fall of cardiac output (Audet et al., 1997). Pharmacological evidence showed that PG-stimulated sympathetic nervous system activation follows B₁R activation in this system, comparable to the Lys-des-Arg⁹-BK-induced depolarization of IL-1-pretreated mouse sympathetic ganglia in vitro, where a prostanoid intermediate is suspected (Seabrook et al., 1997). As mentioned above, induction of cardiovascular responsiveness to des-Arg⁹-BK by bacterial endotoxin may also apply for the pig (Siebeck et al., 1989, 1996) and the rat (Lagneux and Ribuot, 1997; Nicolau et al., 1996; Tokumasu et al., 1995), but no detailed hemodynamic analysis is available in these species. Dog blood vessels express constitutive B₁Rs mediating hypotension when des-Arg⁹-BK is injected intravascularly. Vasodilation (such as in the coronary vasculature), fall of peripheral resistance and increase of cardiac output were observed during these episodes (Bélichard et al., 1996; Lamontagne et al. 1996). Bélichard et al., (1996) performed their experiments under ganglionic blockade with hexamethonium and, accordingly, did not observe manifestations of sympathetic activation. However, Lamontagne et al. (1996) monitored des-Arg⁹-BK-induced tachycardia that could be prevented by propranolol. These authors agree that at least part of the vasodilator effect of des-Arg⁹-BK is dependent on nitric oxide, as N^G-nitro-L-arginine inhibits the coronary vasodilator effect or part of the hypotension that follows intraarterial injection of the B₁R agonist; by comparison, prostanoids seem unimportant. The same two effector systems of hemodynamic B₁R-mediator response, NO-dependent vasodilation superimposed to vasoconstriction of sympathetic origin, are acting in a concurrent manner during the infusion of des-Arg⁹-BK into the cat pulmonary circulation (DeWitt et al., 1994).

Because either B₁ or B₂R activation in vivo results in hypotension, one can wonder about the role of kinins in septic shock, a cardiovascular condition that can be reproduced by injecting large doses of LPS (typically 500 µg/kg or more in rodents or rabbits). Low doses of LPS up-regulate B₁Rs, but do not necessarily produce much of the corresponding agonist(s), as infusions of B₁R antagonists do not change the baseline blood pressure (Drapeau et al., 1993). However, there is evidence for a massive consumption of kininogen and Hageman factor in rabbits injected with large doses of LPS (Erdös and Miwa, 1968). This situation is therefore a possible field of application for the kinin antagonists developed in the last decade. However, B₂R antagonists have been disappointing in treating septic shock in animals and humans, despite some favorable hemodynamic effects in the early phase of the pathology (Fein et al., 1997; Félétou et al., 1996). A proper evaluation of a very good B₁R antagonist has not been reported in this condition. An adverse effect of [Leu⁸]des-Arg⁹-BK, combined with a B₂R antagonist, has even been suggested in short term septicemia in the pig, but the effect of the B₁R antagonist alone has not been reported (Siebeck et al., 1996). It is likely that B₁R-mediated vasodilation and sympathetic system stimulation, as observed in several animal species, represents useful cardiovascular compensatory mechanisms during the initial phase of systemic sepsis, for instance by increasing tissue oxygen delivery and improving lung circulation (Siebeck et al., 1996). On this basis, a pharmacotherapeutic intervention with a B₁R agonist has been advocated in systemic sepsis (Siebeck et al., 1997). Decompensation in this condition is a relatively late event, in which excessive B₁R stimulation may also participate. This may be illustrated by the counterproductive drop of cardiac output in the presence of a persistent low peripheral resistance in LPS-pretreated, anesthetized rabbits injected with Sar-[D-Phe⁸]des-Arg⁹-BK (fig. 3C), and is possibly associated with a decreased venous return. The intense and prolonged fall of cardiac output was also observed when Sar-[D-Phe⁸]des-Arg⁹-BK was infused in this model (A. Audet, F. Rioux, and F. Marceau, unpublished data). Cardiovascular complications of sepsis may respond differently to drugs as a function of the duration of the pathology and of the species, and a systematic approach to this problem could help to clarify the potential of B₁R antagonists in this condition.

The expression of kinin receptors by cardiomyocytes and cardiac conduction tissue is further supported by a PG-independent negative chronotropic effect of BK mediated only by B₂Rs in the canine sinus node (Ribuot et al., 1993). Both B₁ and B₂R stimulation can prolong the action potential duration in a preparation of rat ventricular muscle (Gouin et al., 1996). The B₁R agonist Sar-[D-Phe⁸]des-Arg⁹-BK was used in this demonstration. Whether postisolation up-regulation of B₁R has occurred in the latter system is a possibility that has not been rigorously tested. A significant decrease in heart rate was measured during the prolonged hypotension induced by Sar-[D-Phe⁸]des-Arg⁹-BK in the rabbit (Audet et al., 1997), and it is not excluded that a direct effect of the peptide on the heart contributed to the observed drop of cardiac output.

ACE inhibitors have been very successful in clinical practice for treatment of hypertension and heart failure. The contribution of kinins and B₂Rs to the therapeutic and side effects of cardiovascular drugs which block ACE (such as captopril, enalapril, and others) has attracted much interest. These inhibitors not only repress the activation of angiotensin I into angiotensin II, but also have the capacity to potentiate endogenous kinins if they are formed in sufficient amounts during a pathological state (Linz et al., 1995; Marceau, 1997). However, the clinical evidence for this phenomenon is still limited. A privileged experimental approach to test this hypothesis in animals has been to combine the administration of a B₂R antagonist with that of an ACE inhibitor to observe a more or less complete antagonism of the therapeutic effect of the latter drug. Kinin contribution assessed in that manner varies from small to important, depending on the animal model of cardiovascular pathology (Marceau, 1997). The possibility that B₁Rs also participate in the tissue effects of ACE inhibitors has not been systematically tested. The B₁Rs are not up-regulated 3 to 20 h after large intravenous doses of enalaprilat, enalapril or captopril in normal rabbits (based on Northern blot detection of B₁R transcripts in the heart or on in vivo hemodynamic analysis; Marceau et al., 1997; Deblois et al., 1991). However, these drugs were not planned to be given to normal individuals, and the pathological context (e.g., end organ damage in hypertension) may locally modify the receptor population. It is of considerable interest that BK increases in the effluent of rat isolated hearts perfused with Krebs buffer during a reperfusion period that followed ischemia; in this case the des-Arg⁹-BK immunoreactivity increases only when ACE was concomitantly blocked using ramiprilat (Lamontagne et al., 1995). This suggests that the ischemic pathology and the pharmacotherapy of cardiovascular disease with ACE inhibitors can cooperate to produce B₁R agonists. In view of the possible regulation of B₁R population in such situation (see below), there is clearly an opportunity to assess the importance of the duality of receptor types for kinins. Virtually nothing is known about B₁R regulation in hypertension, cardiac failure and their complications. However, it is interesting to note that cardiac failure is associated with a high production of inflammatory cytokines in humans (Lommi et al., 1997).

Angioplasty applied to the rabbit carotid artery, a form of vascular lesion, is associated with both the proliferation of SMCs and the acquisition of a contractile response to the B₁R agonist des-Arg⁹-BK (Pruneau et al., 1994). Although no causal relationship is proven between the two findings, B₁R stimulation leads to DNA synthesis in cytokine treated rabbit aortic SMCs (fig. 4D). Only cells pretreated with cytokines, such as IL-1, EGF, or oncostatin M, exhibit this stimulatory effect of kinins (Levesque et al., 1995b). However, B₁R stimulation mediates the suppression of DNA synthesis in PDGF-stimulated rat mesenteric artery SMCs (Dixon and Dennis, 1997). It has been recently observed that the immunoreactivity of the human B₁R is highly increased in all cell types that compose atheromatous plaques in tissue section of large arteries (endothelial cells, foamy macrophages, infiltrating leukocytes, proliferating SMCs; Raidoo et al., 1997). Immunoreactive B₂Rs were also somewhat overexpressed in these lesions, but this staining was consistently inferior to that of the B₁Rs. Kinin receptor up-regulation may be determined by the inflammatory nature of atherosclerosis, but its precise role (contributing or protective) in the development of human ischemic disease is difficult to predict, because of the conflicting nature of the in vitro animal evidence.

Ischemia is a basic pathological process caused by a local circulatory deficit and there is ample evidence that it activates the kallikrein-kinin system (e.g., see Lamontagne et al., 1995). Several of the MAK kinases, more notably p38, are activated by experimental ischemia or ischemia/reperfusion in rat and dog organs (Yin et al., 1997). These signaling molecules may determine B₁R induction after tissue injury (discussed above, Section III.A.). Various experimental settings based on rat isolated cardiac tissue have been used to document indirectly ischemia-induced B₁R up-regulation by their capacity to modulate positively or negatively norepinephrine release or to preserve endothelium-dependent vasodilation (Bouchard et al., 1998; Chahine et al., 1993; Feng et al., 1997; Foucart et al., 1997). The understanding of the observed relatively rapid B₁R induction (30-50 min) would benefit from the use of metabolic inhibitors and from molecular approaches. Further indirect evidence of B₁R induction by ischemia in the rat is derived from a model of cerebral artery occlusion (Relton et al., 1997). In this system, administration of a B₂R antagonist reduced the cerebral infarct size under some experimental conditions, a finding disputed by other investigators (Campbell et al., 1997). However, the B₁R antagonist B9858 exerted no direct effect on the pathology, but this peptide, as well as Lys-[Leu⁸]des-Arg⁹-BK, significantly attenuated the beneficial effect of the B₂R antagonist (Relton et al., 1997). It was concluded that B₁ and B₂R have differential effects on ischemic brain insult, with a rather protective effect for the B₁Rs (Relton et al., 1997).

The investigators who first had access to synthetic BK have shown initially that this relatively simple peptide reproduced the cardinal signs of inflammation when injected into animal tissues, including inflammatory swelling (edema) (Elliot et al., 1960). Exudation of protein-rich fluid from the circulation is largely determined by vascular mechanisms involving the physical separation of endothelial cells, particularly at the level of postcapillary venules. Local vasodilation produced by kinins (inflammatory hyperemia) further facilitates exudation. Consistent with the regulatory patterns of kinin receptor subtypes, kinins stimulate B₂Rs when injected in normal tissues. For instance, an assessment of the acute edema produced by kinin injection into the rat paw, based on a full set of agonist and antagonist peptides, showed mediation of the edema formation by B₂Rs without a significant involvement of B₁Rs (Whalley et al., 1984). Thus, a specific B₂R antagonist, such as FR 167344, exerts some antiinflammatory activity in acute animal models associated with the activation of the kallikrein-kinin system (Asano et al., 1997).

However, systemic treatment with a relatively low dose of LPS sensitized in 24 h the rat paw to the inflammatory effect of exogenous des-Arg⁹-BK, with some loss in the effect of B₂R stimulation (Campos et al., 1996). Similarly, inflammation in more sophisticated immunopathological models in the rat also evidences B₁R up-regulation. A shift from B₂ to B₁R mediation for kinin-induced exudation has been observed during the course (1 to 5 days) of antigen-induced chronic arthritis in the affected joint (Cruwys et al., 1994). A full set of agonists (BK, des-Arg⁹-BK) and antagonists (Hoe 140, [Leu⁸]des-Arg⁹-BK) further supported this conclusion. Moreover, the latter B₁R antagonist became progressively more effective to reduce the basal inflammatory plasma extravasation in this model, although the relative anti-inflammatory efficacy of Hoe 140 declined, suggesting that endogenous B₁R agonist(s) were active at the site of the maturing lesion (Cruwys et al., 1994). Bacterial peptidoglycan-induced arthritis in the rat is also a form of immunological hypersensitivity; in this model, infusion of either a B₁ or a B₂R peptide antagonist reduced the inflamed paw volume and diameter, the combination of both types of antagonists being optimal 72 h after the sensitization (Blais et al., 1997a). Chronic infection with the attenuated mycobacterium strain BCG determines a persistent (10 weeks) state of sensitivity to exogenous des-Arg⁹-BK, as assessed by paw edema formation after local injection (Campos et al., 1997). The edema response to a B₂R agonist was not changed in this model. Interestingly, the repeated administration of the B₂R agonist [Tyr⁸]-BK in the rat paw sensitized the animal to des-Arg⁹-BK, while the edema caused by [Tyr⁸]-BK exhibited tachyphylaxis (Campos et al., 1995). This suggests that intense B₂R stimulation may trigger directly or indirectly B₁R up-regulation to amplify tissue responses to kinins during an inflammatory reaction.

Streptozotocin-injected mice eventually develop an insulin-dependent form of diabetes mellitus; [Leu⁸]des-Arg⁹-BK, but not Hoe 140, could prevent this if administered twice daily from day 3 though day 13 (Zuccolo et al., 1996). The inhibitor of tissue kallikrein, aprotinin, is also active in this respect. The major therapeutic end point was the glycemia at day 13, which failed to increase in animals treated with the B₁R antagonist. These fascinating results may be related to an anti-inflammatory effect of the B₁R antagonist, as the insulin deficiency is the complication of a chemically induced inflammation of the Langherans' islets. It would be desirable to further study several aspects of this model, including peptide doses that were high. Rabbit blood vessels have been shown to exhibit an immediate contractile response to des-Arg⁹-BK if isolated from animals submitted to inflammatory treatments (immune complex-induced arthritis, Farmer et al., 1991; or some surgical procedures as simple as a sham intervention; Davies and Hagen, 1994). As discussed above, the early response of isolated tissues to a B₁R agonist is suggestive for an in vivo B₁R formation in this animal (as in fig. 3D). Thus, an increasing body of evidence suggests that B₁Rs are up-regulated in various models of inflammation. However, this is generally an assumption that has not been substantiated using molecular approaches in most models.

Acute phase proteins are plasma components, usually of hepatic origin, whose concentration is modified by intense or systemic inflammation or sepsis. In rodents, a major acute phase reactant is T-kininogen, a kininogen homologue containing the Ile-Ser-BK sequence and corresponding to a gene lacking in humans. Carragenin-induced acute inflammation in the rat paw is a rapidly evolving paw edema partially prevented by administration of Hoe 140, not of a B₁R antagonist (Raymond et al., 1996). However, this form of local inflammation is followed by the production of T-kininogen (systemic inflammation) which is further increased by treatment with either Hoe 140 or Lys-[Leu⁸]des-Arg⁹-BK, suggesting a negative feedback between kinin receptor stimulation and kininogen production (Raymond et al., 1996). Precisely the same systemic inflammatory reaction has been observed in the course of peptidoglycan-induced arthritis in the rat (Blais et al., 1997a). In the latter model, the local content of immunoreactive BK and des-Arg⁹-BK increased 4- to 5-fold in the inflamed paw, but it is not clear whether peripherally formed kinins can reach an hepatic site of action via the circulation. An alternate, but not proven possibility, is the existence of an hepatic kallikrein-kinin system that would include both B₁ and B₂Rs and would be perhaps activated from a distant site by cytokines. More experiments need to be done to explain this type of observation.

Leukocytes are inflammatory and immunological effectors of prime importance, and their functions are diversified. There is no extensive published evidence of important effects of kinins on these cells, at least in humans. However, some incomplete or preliminary in vitro observations are intriguing, as they suggest that B₁Rs may be expressed by phagocytic cells and lymphocytes. Carl et al. (1996) observed that freshly isolated human neutrophils respond to BK or Lys-des-Arg⁹-BK by an elastase secretion mediated by B₂ and B₁Rs, respectively; thus, the Lys-des-Arg⁹-BK-induced protease secretion was capable of increasing the permeability of an endothelial layer maintained in vitro only in the presence of neutrophils. The second system includes circulating lymphocytes, which seem to exhibit in vitro a chemokinetic response to kinins via the stimulation of B₁Rs (McFadden and Vickers, 1989). These observations are of great potential interest if several experimental parameters familiar to leukocyte biologists can be controlled (e.g., purity of cell preparations, postisolation activation, subtyping of lymphocytes) and if rigorous pharmacological and genetic experiments confirm the kinin receptor expression in these cells. B₁R can contribute in vivo to leukocyte recruitment, but this appears to be through an indirect mechanism (Ahluwalia and Perretti, 1996). Polymorphonuclear leukocytes converge in 6 day-old air pouches under the skin of mice when further locally treated with murine IL-1 (4 h). The leukocyte accumulation is partially prevented by [Leu⁸]des-Arg⁹-BK, but not by Hoe 140. Conversely, des-Arg⁹-BK increased the migration into IL-1-treated pouches. The effect of the kinin on leukocyte accumulation was also inhibited by antagonists of the neuropeptides substance P and CGRP related peptide (Ahluwalia and Perretti, 1996), indicating that des-Arg⁹-BK-stimulated sensory nerve afferents probably released locally neuropeptides that, in turn, determined the chemotactic response. There is also strong pharmacological evidence that the inflammatory effect of des-Arg⁹-BK injected in the pleural space of mice is mediated by neurokinins and calcitonin-gene related peptide (Vianna and Calixto, 1998). Thus, B₁Rs may play a role in neurogenic inflammation. Stimulation of nervous afferents by kinins is examined in the next section.

BK is one of the few mediators of inflammation that directly stimulates afferent nerves. This is caused by the presence of B₂R on neural elements, notably in the sensory ganglions and dorsal layers of the spinal cord (Dray et al., 1988). The autonomic nervous system is also affected by BK. At least in the rat, BK produces part of its inflammatory effect and recruits the hypothalamic-pituitary-adrenal axis to release corticosteroids via the stimulation of sympathetic postganglion (Green et al., 1997). The expression of B₂R in the neurones is evidenced by several approaches, including systems based on cultured cells (Naruse et al., 1992), in which BK-induced depolarization is a distant effect from calcium signaling. Although exogenous kinins produce pain perception stimulating B₂Rs in the blister base of human skin (Whalley et al., 1987) or in rat tissues (Steranka et al., 1988), the analgesic potential of B₂R antagonists has proved to be limited to very acute inflammatory situations (see below). Several studies have now shown that B₁R antagonists surpass B₂R antagonists in preventing or reversing inflammatory hyperalgesia in various models based on the rat and the mouse. Perkins et al. (1993) first described the antinociceptive effect of [Leu⁸]des-Arg⁹-BK in rats submitted to Freund's adjuvant-induced hyperalgesia in the rat knee or ultraviolet ray-induced hyperalgesia in the rat paw. This peptide (1 to 10 nmol/kg intravenously) proved to be effective for about 2 to 3 h. The time course of these experimental pathologies revealed that the B₂R antagonist Hoe 140 was effective at the beginning, but that the B₁R ligand progressively became the best analgesic in a few days after the lesion initiation. These kinetics are consistent with the B₁R up-regulation by tissue injury; in addition, exogenous des-Arg⁹-BK exacerbated the pain. Later, it was found that IL-1, IL-2, IL-8, nerve growth factor, the neuropeptide substance P, the afferent nerve stimulant capsaicin and repeated injections of BK can all produce, after some hours-days of latency, inflammatory hyperalgesia that was favorably influenced by [Leu⁸]des-Arg⁹-BK in models involving the measurement of mechanical or thermal hyperalgesia (Davis and Perkins, 1994a,b, 1996; Khasar et al., 1995; Perkins and Kelly, 1993; Rueff et al., 1996; Sufka and Roach, 1996; Tonussi and Ferreira, 1997). Mice exhibited a [Leu⁸]des-Arg⁹-BK-induced reduction of pain induced by formalin injection (licking behavior) only in LPS-pretreated animals (Campos et al., 1995). Exogenous des-Arg⁹-BK also exacerbated formalin pain in LPS-treated mice. Related to the idea of latency in the induction of B₁Rs, a late phase of formalin-induced pain in the mouse may be partially dependent on B₁Rs. B₂R knockout mice exhibit this phase, which responds to [Leu⁸]des-Arg⁹-BK, and also the slow developing hyperalgesia to Freund's adjuvant (Rupniak et al., 1997). A sophisticated behavioral approach (place preference paradigm) shows that [Leu⁸]des-Arg⁹-BK is preferred against Hoe 140 as an analgesic by rats subjected to adjuvant-induced inflammation, and that the former peptide, unlike opioids, has no potential for abuse (no positive reinforcement) (Sufka and Roach, 1996). In models based on rats, exogenous des-Arg⁹-BK may further increase the hyperalgesia if it is not already at maximal level under the effect of the inflammatory stimulus. However, it became recently apparent that very low doses of the agonist may be analgesic through the release of endogenous opioid peptides (Davis and Perkins, 1997).

As mentioned above (Section III.B.), administration of the exogenous IL-1 receptor antagonist prevented not only the hyperalgesia caused by IL-1, but also that caused by IL-2, IL-8, capsaicin or substance P (Davis and Perkins, 1994b, 1996), suggesting that some of these factors are dependent on the local IL-1 production before triggering the hypothetical B₁R up-regulation. These observations do not necessarily mean that nerve terminals acquire B₁Rs in tissue injury context; the soma of these neurons can be located distantly from the localized inflammatory site. Pharmacological evidence suggests that B₁Rs may rather control PG production locally (Davis and Perkins, 1994b), and some PGs are well-known hyperalgesic substances, as recently shown by the high threshold of pain perception in knockout mice for the PGI₂ receptor (Murata et al., 1997). The analgesic profile of B₁R antagonists resembles that of nonsteroidal antiinflammatory drugs (Rupniak et al., 1997), which are agents known to inhibit the fatty acid cyclooxygenase necessary for PG production. Thus, a chain of mediators (a cytokine, a kinin, eicosanoids) may be involved in these reactions. Repeated attempts have generally failed to show that neural elements (e.g., dorsal root ganglion neurones) can acquire functional responses to B₁R agonists (Davis et al., 1996). According to this interpretation, peripheral B₁Rs located on nonneural cells (e.g., fibroblasts, endothelial cells, etc.) may produce PGs as secondarily mediators, which, in turn, would sensitize the nerve endings in the periphery. However, negative results are limited by the experimental conditions employed. Seabrook et al. (1997) showed that a mouse sympathetic ganglion treated in vitro with IL-1 could depolarize in response to Lys-des-Arg⁹-BK. In addition, mRNA transcripts coding for B₁R were detected in mouse sensory dorsal root ganglia, and somewhat overexpressed in tissues from B₂R^/ animals, though without detectable function. Cultured rat dorsal root ganglia could acquire binding sites corresponding to B₁Rs in addition to the preexisting B₂Rs, but this process was very slow, taking 2 days (Von Banchet et al., 1996). In this study, the experimental approach was rather unusual (inhibition of gold-labeled BK binding by a panel of peptides), but the cell type identity was unambiguous. Thus, neural expression of B₁Rs now appears to be possible.

Fever is another reaction to sepsis and inflammation in which a cytokine messenger is believed to affect a neural center with a PG intermediate. Could the kinins assume an intermediate position in this mediation chain, as in the inflammatory hyperalgesia? The answer seems to be positive: intravenously administered LPS causes a fever reaction in the rat which is initially inhibited by intracerebroventricular (i.c.v.) injection of Hoe 140, but, after 2.5 h, [Leu⁸]des-Arg⁹-BK is more effective (Coelho et al., 1997). LPS pretreatment 24 h before testing is no longer pyrogenic, but abrogates the pyrogenic effect of i.c.v. BK, and confers a pyrogenic effect to des-Arg⁹-BK administered by the same route (Coelho et al., 1997). It should be noted that previous studies involving i.c.v. injection of LPS in the rat failed to demonstrate B₁R induction (Walker et al., 1996). Nevertheless, the intermediate role of kinins was supported by the antipyretic effect of Hoe 140 in this study. The origin of the discrepancy is not clear, but may be related to different time frames, drug dosages and route of administration for LPS.

The importance of tissue kallikrein of renal origin is well documented and generally seen as nephroprotective, natriuretic, and diuretic (Majima and Katori, 1995; Margolius et al., 1995), a suggestion confirmed by the natriuretic deficit of B₂R^/ mice and the altered frequency of a B₁R gene polymorphism in patients with a history of end stage renal failure (see above, Section III.E.).

The use of selective B₁ and B₂R antagonists in volume-loaded rats suggests, that the natriuretic and vasodilator effect of endogenous kinins is mediated by B₂R, whereas B₁R blockade reduces the glomerular filtration rate (Fenoy and Roman, 1992). In normal dogs, natriuresis and renal vasodilator responses were observed after injection with the B₁R agonist des-Arg⁹-BK (Lortie et al., 1992). In this case, the B₂Rs coexist and mediate effects that are partially overlapping (vasodilation). The dog may be an exceptional species, as renal B₁Rs are preformed (see above). The status of kinin receptors in the human kidney is now beginning to be explored. In situ hybridization with a riboprobe specific for B₁R indicates that the B₁R is distributed in epithelial cells of the parietal layer of Bowman's capsule and the thin segment of Henle's loop; thus, the B₁R seems to be colocalized in the nephron with the B₂R, kallikrein, kallistatin, and LMW-kininogen (Wang et al., 1996). Tissue sections were derived from kidneys of adults with unknown clinical history, making impossible any statement about B₁R regulation. However, the B₁R mRNA was shown to be grossly overexpressed in cells forming a malignancy of the human kidney (Wang et al., 1996). Thus the B₁R has the potential to be present both in the kidney vasculature and in renal parenchymal cells, and probably to be regulated by inflammation and cytokines.

The relevance of B₁R up-regulation is more documented in the urinary tract. The urinary bladder isolated from the rat or the rabbit acquires a contractile response to B₁R agonists in a time- and protein-synthesis dependent process (Butt et al., 1995; Marceau et al., 1980; Roslan et al., 1995). More importantly, chemical inflammation of the bladder promotes the B₁R up-regulation in vivo in rats (Marceau et al., 1980; Roslan et al., 1995). Although the mechanisms of this induction are still unclear, it seems that inflammatory mediators as well as protein synthesis are involved. It is tempting to speculate that the hypercontractile state of the infected or inflamed bladder is dependent on this up-regulation. In the mucosa-free rabbit urinary bladder it has been shown that des-Arg⁹-BK can evoke contractions largely via activation of B₁Rs, which have similar properties, including time-dependent induction, to B₁Rs in the rabbit isolated blood vessels (Butt et al., 1995).

E. Others

1. Fibrosis. Fibrosis is a postinflammatory pathological process affecting the lung and many other tissues. There are no drug known to be clearly effective in controlling fibrosis in humans (Goldstein and Fine, 1995). Kinin B₁Rs are perhaps related to fibrosis and B₁R antagonists may be worth trying to prevent it. The regulation of B₁R expression by cytokines and chronic inflammation would make a role for these receptors plausible if the relevant cell types were permissive. Immunohistochemistry of transbronchial biopsies in several patients and in control subjects reveals that the human B₁R is densely expressed in the fibrotic zones of lungs of patients with sarcoidosis or progressive systemic sclerosis, but undertectable in control lungs (Nadar et al., 1996). The human embryonic fibroblasts IMR-90 exhibit mitotic and collagen synthesis responses to des-Arg⁹-BK (Goldstein and Wall, 1984). The original observations of Appleton et al. (1994) suggest that the B₁R agonist des-Arg⁹-BK is the strongest agonist to contract myofibroblasts in granulation tissue from the rat (relevance for retraction of scar tissue).

2. Gastroenterology. The intestinal SMCs at all levels contain B₂Rs that generally mediate contraction (Regoli and Barabé, 1980). However, the genetic program leading to the expression of B₁Rs after tissue injury is active in these tissues, as in isolated strips of human colon (Couture et al., 1981) or ileum (Zuzack et al., 1996). Thus, a responsiveness to des-Arg⁹-BK or Lys-des-Arg⁹-BK is acquired in vitro in a time- and protein synthesis-dependent process.

	V. Conclusions

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Although many receptor populations are regulated, the B₁R is a peculiar example of a G protein-coupled receptor that is inducible. The regulation process was partly characterized before the molecular definition of this receptor, because large changes in the pharmacological responses allowed indirect monitoring of the receptor induction. More recent molecular approaches have essentially confirmed that the B₁R gene is transcriptionally induced in tissue injury situations. This system remarkably integrates different humoral components of the inflammatory reactions, such as the cytokines, the blood proteolytic cascades and the secondary activation of eicosanoid synthesis. Use of kinin antagonists for both receptors in recent years supported such a sequential involvement of these mediators in phenomena such as inflammatory hyperalgesia, fever and, perhaps, hepatic regulation of acute phase reactants. This could be ultimately explained by the local coexpression of key components of the kallikrein-kinin systems, including kininogen, tissue kallikrein and both receptor types, by various cell types (vascular, renal cells, etc.) in responding organs.

Twenty years after the description of the B₁R pharmacological profile, it is now clear that some tissue effects of kinins cannot be antagonized with B₂R ligands such as Hoe 140 and FR 173657, especially in diseased individuals. The cells types, second-messengers and pharmacological effects under the control of B₁ or B₂Rs are largely identical, with some possible exceptions. The specific role of B₁R induction may be to amplify the tissue effects of kinins as a function of time via the synthesis of novel receptor molecules that are resistant to tachyphylaxis and stimulated by the abundant des-Arg⁹-metabolites of kinins. The therapeutic consequences of this are just beginning to be appreciated, with the recent demonstration of the better analgesic effect of the B₁R antagonists in chronic inflammatory pain, relative to the B₂R antagonists.

We have mentioned above several avenues for additional research on the B₁R. Notably, knowledge of the structure-function relationship of the receptor protein is relatively undeveloped. Most pathological models have not used molecular approaches suitable to describe the B₁Rs in terms of cell localization and gene regulation. The genetic definition of B₁ and B₂Rs would be helpful in additional animal species where the pharmacological profile (guinea pig) or the regulation (dog, cat) might be different, compared with more familiar species. Progress in the analytical biochemistry of kinins in health and disease, with special reference to Lys-des-Arg⁹-BK, is also needed. The physiopathological roles of B₁ and B₂Rs should be more widely investigated, notably in relation with leukocytes and ischemia. Finally, nonpeptide B₁R antagonists of great selectivity and clinical pharmacology studies on the kinin B₁R are needed to evaluate the clinical relevance of pharmacological interventions on the B₁R.

	VI. Addendum

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The B₁R receptor promotor was recently analyzed by transient DNA transfections of constructions in SV-40 transformed IMR-90 cells (Yang et al, 1998). This study confirms that the promotor functions in a cell type specific manner, with weak stimulatory effect of LPS, TNF- or phorbol ester on transcription, but with a significant effect of BK. Furthermore, a negative regulatory region (682 to 604) and an enhancer (548 to 448) were defined in a relatively distal portion of the promotor. The enhancer may bind AP-1 and other unidentified factors.

Chemical inflammation of the rat urinary bladder is associated with a decrease of micturition threshold when the bladder is progressively inflated; a viscero-visceral hyper-reflexia is the cause of this phenomenon. Kinin receptor antagonists were shown recently to inhibit this hyper-reflexia: Hoe 140 prevents or reverses it, whereas [Leu⁸]des-Arg⁹-BK only reverses the hyper-reflexia when administered at least 5 h after the induction of local inflammation (Jaggar et al, 1998). This suggests that endogenous kinins are mediators of the afferent part of the reflex, with a temporal shift of mediation from B₂ to B₁Rs.

Patients with inflammatory bowel disease were found to exhibit allele frequency alterations similar to but more profound than those observed in patients with renal failure (promotor B₁R polymorphism; Bachvarov et al, 1998a).