Kinin B₁ receptors: key G-protein-coupled receptors and their role in inflammatory and painful processes

Abstract

Kinins are a family of peptides implicated in several pathophysiological events. Most of their effects are likely mediated by the activation of two G-protein-coupled receptors: B₁ and B₂. Whereas B₂ receptors are constitutive entities, B₁ receptors behave as key inducible molecules that may be upregulated under some special circumstances. In this context, several recent reports have investigated the importance of B₁ receptor activation in certain disease models. Furthermore, research on B₁ receptors in the last years has been mainly focused in determining the mechanisms and pathways involved in the process of induction. This was essentially favoured by the advances obtained in molecular biology studies, as well as in the design of selective and stable peptide and nonpeptide kinin B₁ receptor antagonists. Likewise, development of kinin B₁ receptor knockout mice greatly helped to extend the evidence about the relevance of B₁ receptors during pathological states. In the present review, we attempted to remark the main advances achieved in the last 5 years about the participation of kinin B₁ receptors in painful and inflammatory disorders. We have also aimed to point out some groups of chronic diseases, such as diabetes, arthritis, cancer or neuropathic pain, in which the strategic development of nonpeptidic oral-available and selective B₁ receptor antagonists could have a potential relevant therapeutic interest.

Introduction

Kinins are a group of peptides involved in a series of pathophysiological processes. They are formed in plasma and tissues in response to infection, tissue trauma or inflammatory alterations. Once formed and released, kinins exert most of their biological effects by the activation of two G-protein-coupled receptors, denoted B₁ and B₂. B₂ receptors are distributed in a constitutive manner throughout the central and peripheral tissues and present higher affinity for bradykinin (BK) and Lys-BK peptides. On the other hand, B₁ receptors display high affinity for the metabolites des-Arg⁹-BK and Lys-des-Arg⁹-BK. It is worth noting that B₁ receptors are not usually expressed under physiological conditions, but may be quickly upregulated under several inflammatory stimuli (see Calixto et al., 2000; 2001).

Lewis (1964) demonstrated for the first time that BK was able to evoke all classical signals of inflammation, such as increase in vascular permeability, oedema formation and pain. Soon after, many works pointed out that both B₁ and B₂ receptors are involved in the onset and maintenance of inflammatory and nociceptive alterations (Regoli & Barabé, 1980; Proud & Kaplan, 1988; Ahluwalia & Perretti, 1999; Calixto et al., 2000; 2001; Couture et al., 2001). For various reasons, research on the involvement of B₂ receptors in nociceptive and inflammatory states has progressed more quickly than that on B₁ receptors. Curiously, the first B₁ receptor antagonist was developed almost 10 years before the first B₂ receptor antagonist, but the characterization of B₁ receptors was delayed, probably due to its complex ability to be induced. Thus, the role of B₂, but not B₁ receptors, in inflammatory and nociceptive processes was widely explored during the 1980s and 1990s. This was favoured by the systematic development of selective peptidic B₂ receptor antagonists by the pharmaceutical companies, as at this time, the industry believed that B₂ receptors were more attractive as pharmacological targets (in comparison to B₁ receptors) for the possible treatment of several pathological states (Regoli et al., 1998; Stewart et al., 1999; Bock & Longmore, 2000). In addition, cloning studies with B₂ receptors were initiated as early as 1991, whereas the B₁ receptor was first cloned only in 1994 (see details below). Other factors have also largely contributed to the delay of the studies on B₁ receptors. Firstly, B₂ receptor knockout mice were generated in 1995 (Borkowski et al., 1995), while the generation of B₁ receptor knockout mice was only achieved later in 2000 (Pesquero et al., 2000). In addition, the development of nonpeptidic selective B₂ receptor antagonists was achieved almost 10 years before the synthesis of the first nonpeptidic selective B₁ receptor antagonist (Table 1).

Table 1.

Selective kinin B₁ receptor antagonists

Name	Structure	Main characteristics	Reference
[Leu8]-des Arg9-BK	[Leu8]-des-Arg9-BK	Peptidic antagonist, first-generation, widely employed for studying the involvement of B1 receptors in inflammatory and nociceptive models. Mainly active in vivo when administered by topical or s.c. routes.	Regoli et al. (1977), Regoli & Barabé(1980)
Lys[Leu8]-des-Arg9-BK	Lys[Leu8]-des-Arg9-BK	Peptidic antagonist, first generation. Presents pharmacological actions similar to those described for [Leu8]-des-Arg9-BK. Has higher affinity for rabbit and human B1 receptors.	Regoli & Barabé (1980), Drapeau et al. (1991)
[des-Arg9]-HOE 140	Des-Arg9-𝒟-Arg [Hyp3,Thi5, 𝒟-Tic7,Oic8]	Peptidic antagonist, second generation. Presents increased potency and stability in relation to first generation antagonists.	Wirth et al. (1991)
[des-Arg9]-NPC 17731	𝒟-Arg-Arg-Pro-Hyp-Gly-Phe-Ser-𝒟Hype-(Transpropyl-Oic)	Peptidic antagonist, second generation. Only a Few pharmacological studies have been conducted with this antagonist.	Cabrini & Calixto (1997), Vianna & Calixto (1998)
R-715	AcLys[𝒟-βNal7,Ile8]des-Arg9-BK	Peptidic antagonist, third generation. Has less susceptibility to proteolytic degradation than second generation antagonists. Several recent works have employed this compound to evaluate the involvement of B1 receptors during pain and inflammation. Active when administered systemically by s.c., i.v. or i.p. routes.	Gobeil et al. (1996)
R-954	AcOrn[Oic2, (αMe)Phe5,𝒟-βNal7, Ile8] des-Arg9-BK	Peptidic antagonist, third generation. Used at the same doses and schedules employed for R-715	Gobeil et al. (1996), Neugebauer et al. (2002)
B-9858	LysLys[Hyp3,Igl5,𝒟-Igl7,Oic8] des-Arg9-BK	Peptidic antagonist, third generation. Extremely potent and long-lasting B1 receptor antagonist. Some reports indicate that this compound may present B2 receptor antagonistic properties.	Stewart et al. (1996), Rhaleb et al. (1992), Larrivee et al. (2000)
B-9958	Lys-Lys-Arg-Pro-Hyp-Gly-CpG-Ser-Dtic-CpG	Peptidic antagonist, fourth generation. A highly potent and long-acting B1 receptor antagonist, without any action on B2 receptors.	Stewart et al. (1996), Gobeil et al. (1997)
Benzodiazepine antagonist		Nonpeptidic B1 receptor antagonist. Presents in vivo efficacy against inflammatory pain when administered by the i.p. route.	Wood et al. (2003)
Dihydroquinoxalinone antagonist		Nonpeptidic antagonist. It presents higher affinity for human and rabbit B1 receptors, with low affinity for rat B1 receptor. In vivo, shows antinociceptive actions in a rabbit assay of hyperalgesia.	Su et al. (2003)
SSR240612	(2R)-2-[((3R)-3-(1,3-benzodioxol-5-yl)-3-{[(6-methoxy-2-naphtyl)sulfonyl]amino}propanoyl)amino]-3-(4-{[2R,6S)-2,6-dimethylpiperidinyl] methyl} phenyl)-N-isopropyl-N-methylpropanamide hydrochloride	Nonpeptidic B1 receptor antagonist. The first B1 receptor antagonist with proven efficacy when administered by oral route in several models of pain and inflammation.	Gougat et al. (2004)

The former relevant in vivo studies regarding the role of B₁ receptors in pain and inflammation were initiated only after B₁ receptor cloning studies. At the same time, certain pioneer works provided convincing evidence that B₁ receptors could be functionally upregulated in vivo following proinflammatory or trauma stimuli (Perkins et al., 1993; Perkins & Kelly, 1993; Davis & Perkins, 1994; Campos & Calixto, 1995; Khasar et al., 1995; Perkins et al., 1995; Campos et al., 1996). The great progress in molecular biology had also made it possible to demonstrate that both B₁ receptor mRNA and protein expression could be markedly enhanced under many stressful conditions. Nowadays, signalling pathways underlying B₁ receptor induction during inflammatory and/or nociceptive alterations are better understood, even though various points in the cascade of B₁ receptor modulation still remain elusive. In addition, a major effort toward the development of selective B₁ receptor antagonists has been undertaken, mainly by the pharmaceutical companies. Most of this effort entails the screening of large compound libraries in order to identify more potent and selective antagonists see (Table 1). In the present review, we will discuss the main advances obtained in the last 5 years on understanding the role of B₁ receptors in pain and inflammation, with special emphasis on some pathophysiological alterations where B₁ receptor induction and/or activation might be of pertinent clinical interest. We will also call attention to some mechanisms involved in the upregulation of B₁ receptors during inflammatory and painful conditions.

The kinin B₁ receptor as the special one

There are, so far, several reasons for describing kinin the B₁ receptor as a special entity in relation to the classical well-known G protein-coupled receptors. Many particular characteristics draw the attention of researchers to B₁ receptors. Without doubt, the most interesting feature regarding this receptor is its complex pattern of expression. As described above, B₁ receptors are normally absent, but may be highly and rapidly upregulated following inflammatory stimuli. This is an unusual characteristic of G protein-coupled receptors. In fact, the pattern of induction of B₁ receptors resembles more closely to tyrosine-kinase-linked receptors. Although some literature data has demonstrated that other G-protein-coupled receptors can be also upregulated – these include the platelet-activating factor (PAF), the neurokinin NK-1, the protease-activated receptors 1 and 4 and occasionally even the kinin B₂ receptor – none of them present the same profile and complexity of induction observed for B₁ receptors (Marceau, 1995; Donaldson et al., 1997; Calixto et al., 2000; 2001). Interestingly, kinin B₁ receptors seem to be upregulated under the same conditions described for the inducible proinflammatory enzymes cyclooxygenase-2 (COX-2) and the inducible nitric oxide synthase (iNOS). In this way, the stimuli (and also the cellular signalling pathways) shown to be capable of increasing the expression of B₁ receptors are the same as those recognized for stimulating the upregulation of COX-2 and iNOS (Calixto et al., 2000; 2001). Furthermore, transcriptional and post-transcriptional tools required for B₁ receptors expression seem to be the same necessary for regulating COX-2 and iNOS enzymes (Zhou et al., 1998; 1999; Dixon et al., 2000; Haddad et al., 2000; Lasa et al., 2001; Dixon, 2004). At this moment, it is not easy to explain why a G-protein-coupled receptor behaves as a typical inducible enzyme and this constitutes a very interesting field of research that will probably receive much attention. Despite its inducible profile, it is important to mention that in some tissues, especially at the central nervous system, kinin B₁ receptors can be found constitutively expressed, a feature that is shared with the enzyme COX-2.

Some recent reports have indicated the existence of a crosstalk between B₁ and B₂ receptors. Therefore, evidence has also shown that persistent stimulation of B₂ receptors might result in B₁ receptor upregulation (Campos & Calixto, 1995; Phagoo et al., 1999; 2000; Campos et al., 2001; Todorov et al., 2003; Rashid et al., 2004). It is worth noting that stimulation of B₂ receptors leads to transient increases in Ca²⁺ concentration and to the fast desensitization of these receptors. Regarding B₁ receptors, they do not seem to be susceptible to desensitization mechanisms and their stimulation results in sustained elevations of Ca²⁺ concentration (Faussner et al., 1999; Marceau et al., 2002). Recent studies conducted with fluorescent conjugated proteins have demonstrated that B₁ receptors, in contrast to B₂ receptors, normally do not internalize following agonist stimulation, but they seem to translocate and aggregate (by a caveolae-mediated mechanism) after agonist binding, probably to facilitate the amplification of B₁ receptor-mediated responses. In addition, other relevant points are likely to contribute to the extension of this idea: (1) in some cases, B₁ receptor upregulation appears to be secondary to the downregulation of B₂ receptors; (2) some transduction pathways activated by B₂ receptors are known to be capable of inducing B₁ receptors; and (3) stimulation with both B₁ and B₂ receptor agonists may lead to B₁ receptors induction (Campos et al., 1996; 2001; Hayashi et al., 1998; Modéer et al., 1998; Pan et al., 1998; Phagoo et al., 1999; 2000). Finally, it has been established that B₁ and B₂ receptor genes are both located at the same chromosome of humans (chromosome 14), rats (chromosome 6) and mice (chromosome 12) (Chai et al., 1996; Cayla et al., 2002). This feature might also contribute to facilitating a balanced regulation of B₁ and B₂ receptors.

Molecular characterization of B₁ receptor

In the present section, we have remarked on some of the most important findings concerning the molecular biology studies carried out on kinin B₁ receptors.

When, McEachern et al. (1991) cloned the B₂ receptor-encoding cDNA from rats, Southern blot analysis indicated that if a distinct gene was responsible for encoding B₁ receptor, it would not be highly homologous to the B₂-enconding gene. Later, Webb et al. (1994) presented important evidence suggesting that mRNA-encoding B₁ receptor was distinct in size and in coding sequence from that encoding B₂ receptor. At the same time, Menke et al. (1994) reported the first cloning expression of kinin B₁ receptor from the human embryonic lung fibroblast cDNA library. Afterwards, B₁ receptor has been cloned from rabbit (MacNeil et al., 1995), mouse (Pesquero et al., 1996), rat (Ni et al., 1998a), dog (Hess et al., 2001) and monkey (Hess et al., 2002). Differences in the amino-acid sequence of B₁ receptors from different species range between 69 and 97%. Greater degrees of homology are observed between mouse and rat (88%) and monkey and human (97%) receptors (Hess et al., 2002).

Pharmacological evidence has shown that human, rabbit, mouse and monkey receptors present higher affinity (until 1000 times) for Lys-des-Arg⁹-BK in comparison to des-Arg⁹-BK, whereas rat and mouse receptors present similar affinities for both B₁ receptor agonists. This difference is also observed for the B₁ receptor antagonists Lys-des-Arg⁹-[Leu⁸]-BK and des-Arg⁹-[Leu⁸]-BK (Regoli et al., 2001; Hess et al., 2002). Recent molecular studies have proposed that these differences in affinity rely on the heterogeneity of the extracellular domain IV of the B₁ receptor. Therefore, it has been proposed that N-terminal lysine preferentially binds to the extracellular domain IV in the human B₁ receptor (Fathy et al., 2000; Hess et al., 2002). From this, it is worth noting that B₁ receptor agonists and antagonists might present significant differences of affinity and potency depending on the species studied. This is mainly important for the development of antagonists and also for the preclinical studies: selective B₁ receptor antagonists with similar affinities for all species would be preferable.

Molecular biology studies have widely contributed for understanding the inducible characteristic of B₁ receptors. It has demonstrated that 5′-flanking region of B₁ receptor gene (located upstream from the initiation site) holds several putative transcriptional regulatory sequences, including the TATA box and positive and negative control elements, with all characteristics of a core promoter (Bachvarov et al., 1996; Yang & Polgar, 1996; Ni et al., 1998b; Schanstra et al., 1998; Angers et al., 2000). Ni et al. (1998a, b) and Schanstra et al. (1998) have reported an NF-κB-binding domain, although the precise position remains elusive. Ni et al. (1998b) have further suggested putative sequences for AP-1 and CREB regulation. These pieces of evidence have largely contributed to the recent advances obtained in the comprehension of the mechanisms and signalling pathways involved in the upregulation of B₁ receptors (see the next section of this review).

Other relevant molecular studies need to be mentioned. For instance, footprint analysis of the promoter region has revealed that it is possibly bound by several sequence-specific DNA binding proteins, like GATA-1, PEA3, AP-1, CAAT, Sp1, Pit-1a, Oct-1 and CREB (Angers et al., 2000). With the construction of a minigene (1.8 kb promoter plus exon 1, 1.5 kb intron 1, exon 2 and intron 2), it has been verified that motifs not included in the promoter sequence, such as 5′-UTR and intronic regions, are also required for induction of this gene (Yang et al., 2001b). The cotransfection of c-Jun with the minigene causes an increase of the promoter activity in a concentration-dependent manner, probably through interaction with multiple AP-1 sites. On the other hand, the tumour suppressor protein p53 has been demonstrated to suppress B₁ receptor promoter activity in a concentration-dependent manner (Yang et al., 2001a). Finally, it has been suggested that the 3′-untranslated region (3′-UTR) containing a polyadenylation signal in the B₁ receptor gene might be involved in the mRNA stability processes and its ultimate expression, since its absence directly reflects on the half-life of B₁ receptor mRNA (Zhou et al., 1999). These data further extends the notion that the kinin B₁ receptor represents an unusual protein-G-coupled receptor which can be modulated by the activation of highly specialized mechanisms.

Main mechanisms involved in the upregulation of B₁ receptors

The induction of B₁ receptors has been associated with the production of inflammatory mediators, stimulation of inflammatory cells, and, finally, activation of several intracellular signalling pathways. Understanding which intracellular signalling pathways mediate B₁ receptor induction is of high interest in the discovery of therapeutic targets. In this section, we have summarized the recent advances made on the mechanisms involved in B₁ receptor upregulation in several inflammatory models (Figure 1).

Figure 1.

Possible mechanisms underlying B₁ receptor upregulation. Stressful stimuli (such as infection and inflammation) are able to activate several signalling pathways including MAPKs, PKC and PI3-K, which in turn might modulate transcriptional factors (e.g. NF-κB, AP-1 and CREB), and are supposed to be crucial for the regulation of B₁ receptor expression. AP-1, activating protein-1; AKT/PKB, protein kinase B; CREB, c-AMP response element-binding; ERK, extracellular signal-regulated kinase; JNK, c-jun NH₂-terminal kinase; MAPK, mitogen-activated protein kinase; MEKK, mitogen-activated protein kinase kinase kinase; MEK, mitogen-activated protein kinase kinase; NF-κB, nuclear factor-κB; PI3-K, phosphatidylinositol-3 kinase; PLC, phospholipase C; PKC, protein kinase C; TLR, Toll-like receptors.

Primary studies concerning B₁ receptor expression have reported the importance of in vitro incubation time in the increase of functional responses to B₁ receptor agonists in many isolated organ preparations. These studies have also shown that the time-dependent B₁ receptor upregulation is sensitive to RNA and protein synthesis inhibitors, suggesting its dependence on de novo protein synthesis (see Regoli & Barabé, 1980; Marceau et al., 1998). Although the elucidation of the pivotal molecular mechanisms implicated in the regulation of these processes has only just begun, recent literature data has already shown that B₁ receptor upregulation can be modulated at different levels. The main pharmacological strategies applied to the study of the mechanisms involved in the upregulation of B₁ receptors are listed in Table 2 .

Table 2.

Some possible pathways for pharmacological modulation of B₁ receptor upregulation

Target	Pharmacological modulation
mRNA transcription	Actinomycin D
Translocation	Brefeldin A
Translation/ N-glycosylation	Cycloheximide
	Tunicamycin
Inhibition of ribonuclease	MAPK inhibitors
Interference with mRNA unstable regions (AUUUA)	Dexamethasonea
NF-κB activation	Inhibitors of IκB kinase (PDTC, BAY 117082)
	Proteasome activity inhibitors (TLCK, MG 132)
Cytokine modulation	Inhibitors of cytokines synthesis, antibodies, recombinant receptors
Protein kinase inhibitors	Blockers of TKs, PKC and MAPKs, etc.

For further details, refer to the following reviews: Marceau et al. (1998), Calixto et al. (2000; 2001), Couture et al. (2001).

^aDexamethasone is able to block B₁ receptor induction at several points.

The determination of distinct signalling pathways involved in the regulation of B₁ receptor expression has been accomplished by several approaches. Gene sequence analysis has revealed that the B₁ receptor promoter presents numerous sequence-specific motifs for different DNA binding proteins, which indicates that B₁ receptor expression could be modulated by several transcriptional factors (Bachvarov et al., 1996; Yang & Polgar, 1996; Ni et al., 1998b; Schanstra et al., 1998; Angers et al., 2000). In this context, there is now a considerable amount of experimental evidence indicating that NF-κB, a transcriptional factor that governs the expression of gene-encoding cytokines, chemokines, growth factors, cell adhesion molecules, and some acute phase proteins in health and various pathological states, plays a pivotal role in the regulation of B₁ receptor induction. It has been demonstrated that specific NF-κB blockers are able to prevent B₁ receptor upregulation both in vitro (Sardi et al., 2000; 2002; Medeiros et al., 2001; 2004; Phagoo et al., 2001; Sabourin et al., 2002) and in vivo (Campos et al., 1999; Fernandes et al., 2003; Passos et al., 2004). Indeed, studies conducted by Ni et al. (1998b) have revealed the existence of an NF-κB-like binding site on the human B₁ receptor promoter, which seems to be essential for the control of receptor transcription following exposure to certain inflammatory agents such as IL-1β, Tumour necrosis factor α (TNFα) or lipopolysaccharide (LPS). Furthermore, it has been demonstrated that in cultured human lung fibroblasts, B₁ receptor upregulation induced by IL-1β is modulated at the transcriptional level, in a process mediated by the activation of NF-κB (Schanstra et al., 1998). Recently, we have verified that tissue damage in the rat portal vein is also able to induce the expression of B₁ receptor, in a process involving IκBα degradation and the subsequent translocation of NF-κB into the nucleus with an increase in NF-κB DNA-binding activity (Medeiros et al., 2004).

Protein phosphorylation has also been reported to exert an important role in the regulation of many cellular processes, including gene expression and protein synthesis (Hunter, 1995). Thus, it has been suggested that the activation of some classes of protein kinases is essential for the induction of the B₁ receptor. In fact, Larriveé et al. (1998) were the first to show that both spontaneous and IL-1β-induced B₁ receptor upregulation in rabbit aorta are greatly sensitive to inhibitors of tyrosine kinase, extracellular signal-regulated kinase (ERK) or p38 MAPK pathway. In addition, it has been demonstrated that protein kinase C (PKC) activation also critically regulates the B₁ receptor mRNA expression in cultured human lung fibroblasts (Schanstra et al., 1998). Other studies have confirmed and extended these previous data. It has been shown that in vivo B₁ receptor upregulation induced by inflammatory cytokines IL-1β or TNFα involves the activation of PKC, tyrosine kinase and ERK (Campos et al., 1999). Moreover, PKC is also involved in the B₁ receptor upregulation in different isolated tissue preparations (Medeiros et al., 2001; Ueno et al., 2002). Of interest is the recent data demonstrating that p38 MAPK and c-Jun NH₂-terminal kinase (JNK) are critically involved in the regulation of B₁ receptor mRNA expression induced by vascular tissue trauma (Medeiros et al., 2004). Altogether, these data clearly demonstrates the importance of different classes of protein kinases in the modulation of B₁ receptor induction. However, the precise mechanisms through which this regulation occurs is currently a matter of debate. Recently, several studies have suggested that protein kinases may regulate the expression of certain inflammatory genes, acting at both transcriptional and post-transcriptional levels. In addition, the activation of protein kinases has been associated to the increased activation of different transcriptional factors and to the regulation of the half-lives of many inflammatory mediator mRNAs (e.g. COX-2, TNFα and IL-1β) (Dunn et al., 2002; Clark et al., 2003; Lahti et al., 2003; Yang et al., 2003). Following this, a recent study has suggested that B₁ receptor expression could also be regulated through the enhancement of mRNA stabilization (Zhou et al., 1999). This notion has been extended further by Haddad et al. (2000), who demonstrated that B₁ receptor induction in human lung fibroblasts (HEL 299), following incubation with IL-1β and TNFα, is largely dependent on post-transcriptional mechanisms, being this effect greatly sensitive to dexamethasone and p38 MAPK inhibitors. Therefore, it is tempting to suggest that the regulation of B₁ receptor might be controlled both transcriptionally and post-transcriptionally. Therefore, the activation of protein kinases could certainly be involved in the enhancement of both transcriptional factor activity and B₁ receptor mRNA stability. However, additional studies are still required to better understand and confirm this hypothesis.

The B₁ receptor and its role in development and maintenance of inflammatory diseases

Several pieces of evidence indicate that B₁ receptor induction plays a critical role in inflammatory pathological states. In the next sections, we have highlighted the main recent findings, which implicate B₁ receptors in inflammatory models. Data described herein is summarized in Table 3.

Table 3.

Inflammatory stimuli associated with B₁ receptors up-regulation

Stimuli	References
Infectious stimuli – LPS, polyinosininc polycytidylic acid, trypomastigotes from T. cruzi	McLean et al. (1999), Deblois & Horkick (2001); Wille et al. (2001), Ni et al. (2003), Bachar et al. (2004), Passos et al. (2004), Todorov et al. (2003)
Myocardial, intestinal and pancreatic ischemia–reperfusion injury	Mazenot et al. (2001), Lagneux et al. (2002), Kuebler et al. (2003), Souza et al. (2004)
Airway stimulation – challenge with OVA to sensitized animals; intrapleural injection of carrageenan; rhinitis	Huang et al. (1999), Christiansen et al. (2002), Hayashi et al. (2002), Eric et al. (2003), Landgraf et al. (2003)
Type I STZ-induced diabetes	Campos et al. (2001), Couture et al. (2001), Gabra & Sirois (2002), Abdouh et al. (2003), Vianna et al. (2003)
Some kinds of cancer – astrocytic tumour; prostatic cancer	Raidoo et al. (1999), Barki-Harrington et al. (2003), Taub et al. (2003)
Cystitis – induced by cyclophosphamide	Bélichard et al. (1999)
Injection of some phlogistic agents – zymosan, CFA, carrageenan and kaolin, PAF, the proinflammatory cytokines IL-1β and TNFα	Campos et al. (1999), Bélichard et al. (2000), Fernandes et al. (2003), Fox et al. (2003), Seegers et al. (2004)

B₁ receptors and infection

The effects of treatment with bacterial endotoxin on B₁ receptor upregulation (both functional and molecular) have been extensively reported (see Marceau et al., 1998; Calixto et al., 2000; 2001). Likewise, infection seems to constitute a classical signal known for its ability to induce B₁ receptors. A very interesting study conducted by DeBlois & Horlick (2001) suggested that the haemodynamic and inflammatory responses observed after systemic LPS treatment in green monkeys are largely mediated by B₁ activation. The authors reported that the selective B₁ receptor agonist des-Arg⁹-BK caused a marked fall in blood pressure, accompanied by an increase in heart rate only in monkeys treated with LPS. In the same way, des-Arg⁹-BK-induced skin oedema formation was observed solely in monkeys treated with LPS. The work of Deblois & Horlick (2001) was the first experimental evidence indicating the upregulation of B₁ receptors in non-human primates. In addition, it has been shown that gene deletion of B₁ receptors prevents the endotoxic shock in mice (Pesquero et al., 2000). Extending this idea, Ni et al. (2003) demonstrated that transgenic overexpression of B₁ receptors renders mice more susceptible to endotoxic shock and greatly potentializes the oedematogenic responses caused by the inflammatory agent carrageenan. Based on these results, it is tempting to speculate that the new generation of nonpeptidic B₁ receptor antagonists see (Table 1) could represent a valuable tool for the control/treatment of situations associated with disseminated infection, such as sepsis.

Studies conducted with rodents confirm and further extend the proposition that endotoxins classically induce B₁ receptors. For instance, B₁ receptors seem to exert an important role in vascular changes and hypotension induced by LPS from Escherichia coli in rats (McLean et al., 1999). LPS has also been found to markedly increase plasma extravasation induced by the selective B₁ receptor agonist des-Arg⁹-BK in the duodenum, ileum and trachea of rats (Wille et al., 2001). More recently, Passos et al. (2004) have demonstrated the occurrence of kinin B₁ receptor upregulation after the local administration of LPS into the rat paw, in a process involving activation of the transcriptional factor NF-κB, neutrophil migration, and release of inflammatory mediators (e.g. IL-1β, TNFα and PAF). Another recent and relevant publication has shown that incubation of tracheal organ culture for 1 or 4 days with the bacterial products LPS and polyinosininc polycytidylic acid enhances BK- and des-Arg⁹-BK-mediated contractile responses by mechanisms largely dependent on protein synthesis, MAPK activation and NF-κB activation (Bachar et al., 2004).

More recently, it has been widely demonstrated that LPS and other endotoxins induce several inflammatory alterations by binding to a family of receptors denoted toll-like receptors (TLR). In the case of LPS, it has been reported that their actions are mediated by the activation of TLR4 receptors and the consequent stimulation of NF-κB and several protein kinases, associated with the release of proinflammatory cytokines (e.g. IL-1β or TNFα) (for more details see Beg, 2002; Dobrovolskaia & Vogel, 2002; Akira et al., 2003). In this regard, all these signalling pathways have now been demonstrated to be involved in the process of B₁ receptor upregulation.

Another aspect which deserves attention is the recent evidence implicating the B₁ receptor as an adjuvant for Trypanosoma cruzi infection. Recent data have shown that intradermal injection of trypomastigotes into the mouse paw results in a long-lasting oedema formation: the first phase of oedematogenic response (3 h) is blocked by the B₂ receptor antagonist Hoe 140, whereas the late phase (24 h) can be prevented by treatment with the selective B₁ receptor antagonist B9858. Late-phase oedema has also been abolished in B₁ receptor knockout mice (Scharfstein et al., 2000; Todorov et al., 2003). These results have led authors to conclude that B₁ receptors are upregulated following local infection with T. cruzi and to point out the importance of B₁ receptors for the progress of Chagas disease. Also of interest are the results from the same group showing that B₁ receptors (as well as B₂ receptors) seem to facilitate the invasion of T. cruzi to the host cells. These conclusions are based on evidence indicating that both B₁ and B₂ receptor antagonists are effective in preventing the parasitic infection of Chinese Hamster ovary cells which coexpress B₁ and B₂ receptors (Todorov et al., 2003).

Ischaemia–reperfusion lesions

Literature data have suggested that B₁ receptors might exert a pivotal role in postischaemic situations. Thus, it has been shown that B₁ receptors can be induced in the endothelial cells of rabbits (Mazenot et al., 2001) or in the hearts of mice (Lagneux et al., 2002) submitted to myocardial ischaemia–reperfusion. In the rabbit model, the vascular effects evoked by ischemia–reperfusion can be significantly reduced by a selective B₁ receptor antagonist – [Leu⁸]-des-Arg⁹-BK (Mazenot et al., 2001). In mice, either pharmacological blocking with selective B₁ antagonist [Leu⁸]-des-Arg⁹-BK or gene deletion of B₁ receptors largely diminishes the infarction's extension, an effect that further reinforces the relevance of B₁ receptors under these conditions. Kuebler et al. (2003) have affirmed that B₁ receptors also seem relevant in the ischaemia–reperfusion of rat pancreas. The authors have demonstrated, by means of binding studies, that B₁ receptor expression is augmented by about 22-fold after pancreatic ischaemia–reperfusion injury. They have also shown that the B₁ receptor antagonist CP-0298 (alone or in combination with the B₂ antagonist CP-0597) significantly reduces the number of adherent leukocytes in postcapillary venules (Kuebler et al., 2003). Another recent contribution has demonstrated that intestinal ischaemia–reperfusion injury results in a striking increase of B₁ receptor expression in rats (Souza et al., 2004). In this model, B₁ receptor upregulation is probably secondary to the activation of B₂ receptors, as it can be significantly blocked by selective B₂ receptor antagonists. It should be noted that all the main inflammatory parameters such as, changes of vascular permeability, neutrophil migration and cytokines production, are completely abolished in B₁ receptor knockout mice.

Ischaemia–reperfusion lesions are associated with a marked migration of inflammatory cells, cytokine generation and activation of kinases and transcriptional factors (e.g. p38 MAPK and NF-κB, respectively) (Jones et al., 2003; Lien et al., 2003). It is noteworthy that all these signalling pathways have been correlated with the process of B₁ receptor upregulation. Taking into account these considerations, it is tempting to suggest that B₁ receptors modulation could be a very interesting target for the control and/or treatment of the alterations observed after ischaemia–reperfusion injury.

Inflammation of airways

Kinins seem to exert a central role in the control and maintenance of the inflammatory states of the airways. The relevance of B₂ receptors has been widely investigated, but only a few publications have remarked on the role of B₁ receptors in the airways. Thus, some recent reports have pointed out the critical relevance of kinin B₁ receptors in allergen-induced bronchial hyper-responsiveness. Huang et al. (1999) have demonstrated that challenge with ovalbumin (OVA) results in a marked increase of B₁ receptor mRNA expression in the lungs of previously sensitized rats. This increase peaks between 2 and 6 h and remains elevated up to 24 h following the challenge with OVA. Another recent report has shown that allergic lung inflammation in ovalbumin-sensitized mice is significantly diminished by the systemic treatment of animals with the selective B₁ receptor antagonist R-954 (Landgraf et al., 2003). The same effect of R-954 has been observed in immunocomplex-induced lung inflammation (Landgraf et al., 2004). These findings were extended and confirmed by Eric et al. (2003), who has demonstrated that treatment with B₁ receptor antagonists R-715 or R-954 significantly reduces lung eosinophilia evoked by OVA in sensitized mice. Importantly, allergen-evoked hyperalgesia in OVA-sensitized rats has been found to be markedly reduced by local treatment with [Leu⁸]-des-Arg⁹-BK (Lavich et al., 2003). In addition, B₁ receptor mRNA levels are consistently increased in carrageenan-induced pleurisy at 3 h, remaining elevated up to 5 h after challenge in rats (Hayashi et al., 2002). The same authors have also reported that administration of either of B₁ receptor antagonists [Leu⁸]-des-Arg⁹-BK or des-Arg⁹-D-Arg-[Hyp³, Thi⁵, D-Tic⁷, Oic⁸]-BK is able to prevent carrageenan-induced exudation. Furthermore, Pesquero et al. (2000) have reported that disruption of the B₁R gene in mice results in a marked reduction of the inflammatory responses induced by carrageenan in the pleurisy model in mice. Interestingly, the relevance of B₁ receptors in human airway inflammation has recently been reported by Christiansen et al. (2002). These authors have shown that nasal tissue samples from allergic rhinitis present a significantly higher B₁ receptor mRNA expression in comparison to tissue samples obtained from normal subjects.

Some in vitro studies conducted with cell lines from airways confirm the data obtained in vivo and reinforce the notion that B₁ receptors are critical for the pathological conditions affecting the airways. For instance, B₁ receptors are upregulated by the incubation of IL-1β and TNFα in human embryonic lung fibroblasts HEL 299 (Haddad et al., 2000). Likewise, Phagoo et al. (2001) have shown that stimulation of human lung fibroblasts with IL-1β or des-Arg⁹-bradykinin results in a marked upregulation of B₁ receptors. Moreover, in pulmonary A549 and human bronchial epithelial cells, treatment with the proinflammatory cytokines IL-1β or TNFα causes a remarkable enhancement of B₁R mRNA expression (Newton et al., 2002). Very recently, an interesting study conducted by Zhang et al. (2004) has demonstrated that the incubation of mouse tracheal segments (for at least 4 days) results in both functional and molecular upregulation of B₁ receptors, these responses being further enhanced by the continuous exposure to the proinflammatory cytokine TNFα. This work also demonstrates that the upregulation of B₁ receptors following TNFα exposure is largely dependent on the activation of MAPKs JNK and ERK 1/2.

Diabetes and B₁ receptor modulation

Type I diabetes constitutes an autoimmune disorder in which insulin production is affected by the destruction of pancreatic β-cells. It has been well documented that inflammatory alterations exert a critical role in the development of diabetes – a massive infiltration of inflammatory cells, the elevated production of proinflammatory cytokines (e.g. IL-1β TNFα) and the generation of free radicals are the main features related to the destruction of β-cells in the pancreas (see Couture et al., 2001; Hohmeier et al., 2003). The progress of the diabetes and the constant fluctuation of glucose levels are associated with changes in vascular structure (mainly related to the endothelium) and the production of vasoactive substances. In this context, the kinin system has often been implicated in the pathophysiology of diabetes (Garcia Leme et al., 1973; Couture et al., 2001). Moreover, the extent to which diabetes may influence the modulation of B₁ receptors has been the matter of several recent publications. Campos et al. (2001) have shown that the induction of diabetes with the nitrosamine streptozotocin (STZ) results in a long-lasting (8 weeks) functional upregulation of B₁ receptors in the rat paw, as indicated by a marked increase in the oedematogenic response induced by the selective B₁ receptor agonist des-Arg⁹-BK in diabetic rats. This effect is associated with a significant reduction of B₂ receptor-mediated paw oedema. Based on these results, it is possible to assume that during diabetes development, inducible B₁ receptors may be overexpressed, whereas constitutive B₂ receptors are downregulated. The functional induction of B₁ receptors has also been described in the pleural cavity of rats following STZ treatment (Vianna et al., 2003). In this model, both mononuclear and neutrophil influx in response to intrapleural injection of des-Arg⁹-BK has been found to be markedly enhanced in diabetic rats. In addition, autoradiographic B₁ receptors binding specific sites are significantly augmented in the lungs of STZ (4 days)-treated rats, as compared with control animals (Couture et al., 2001; Vianna et al., 2003). It has further been demonstrated that intrathecal injection of des-Arg⁹-BK results in a biphasic nociceptive response following acute (24 h) administration of STZ (Couture et al., 2001). The importance of B₁ receptors in diabetes has also been shown by a series of studies indicating that systemic treatment with the highly selective B₁ receptor antagonists R-715 and R-954 consistently prevents thermal hyperalgesia induced by STZ (Gabra & Sirois, 2002; 2003a, 2003b). Recently, Abdouh et al. (2003) have suggested a potential role for B₁ receptors in diabetes-associated retinopathies: a marked increase in autoradiographic B₁ receptor-specific binding sites, associated with the enhancement of des-Arg⁹-BK-induced vasodilatation, has been observed in the retinas of STZ (4–21 days)-treated rats. In this regard, it is possible to propose that selective B₁ receptor antagonists would constitute a very attractive strategy for the control of some of the symptoms associated with diabetes, namely inflammatory and neuropathic complications.

Kinin B₁ receptors and cancer

An increasing body of evidence has emerged indicating that kinins and their receptors appear to be involved in cancer (Mahabeer & Bhoola, 2000; Stewart, 2003). The known mitogenic properties of kinins and their ability to activate tyrosine and MAP-kinase cascades could explain, at least in part, the effects of kinins in tumour growth and migration (Mahabeer & Bhoola, 2000; Bhoola et al., 2001; Stewart, 2003). In addition, as cancer growth and metastasis are critically dependent on the activation of inflammatory pathways, it is possible to conclude that B₁ receptor upregulation could play an important role in this scenario. Most publications regarding the participation of kinin receptors in cancer indicate a prevalent role for B₂ receptors. Thus, immunohistochemical and autoradiographic studies have pointed out a marked distribution of B₂ receptors in several human and mouse tumour cells (Wang et al., 2001; Wu et al., 2002). Some studies conducted with MG63 human osteosarcoma cells and mice bearing sarcoma 180 cells have discarded a role for B₁ receptors in cancer (Wang et al., 2001; Ishihara et al., 2001; 2002). On the other hand, B₁ receptors immunoreactivity has been found to be markedly increased in astrocytes and endothelial cells, as well as in the stromal blood vessels from human astrocytic tumour biopsies (Raidoo et al., 1999). In addition, Barki-Harrington et al. (2003) have reported that both B₁ and B₂ receptors activation seem to be linked with the mitogenic signalling in androgen-insensitive prostate cancer PC3 cells. This evidence has been further extended by recent results showing that B₁ receptors are present in prostatic intraepithelial neoplasia and malignant lesions, but not in benign prostate tissues (Taub et al., 2003). These data permit us to suggest that kinin B₁ receptor antagonists might be potentially useful as adjuvant therapies in some types of cancer. The mechanisms underlying the precise role of B₁ receptors in cancer remain to be investigated and constitute a promising and growing area of research.

Participation of B₁ receptors in other inflammatory states

The modulation of B₁ receptors and their participation in other inflammatory alterations has been demonstrated by several additional contributions. Thus, in the model of cystitis induced by cyclophosphamide in rats, B₁ receptor selective agonists cause a marked contractile response of the urinary bladder, an effect that is not observed in naïve animals (Bélichard et al., 1999). Also, a consistent and progressive increase of B₁R mRNA expression in the urinary bladder for up to 48 h following cyclophosphamide treatment has been reported (Bélichard et al., 1999). These data suggest that B₁ receptor induction and activation could constitute an important event during the development of cystitis.

In the acute and severe synovitis induced by intra-articular injection of carrageenan and kaolin, a significant increase in B₁R mRNA expression has been observed in the rat knee synovia (Seegers et al., 2004). Sainz et al. (2004) have reported that in peptidoglycan–polysaccharide-induced arthritis in rats, the selective B₁ receptor antagonist B9858 causes a significant decrease in lymphocyte homing and leukocyte transmigration.

Kinin-induced rat paw oedema is a classical in vivo model of inflammation that has been widely used for studying B₁ receptor upregulation. In this model, B₂ receptor agonists induce a marked oedema formation in naïve animals, while the injection of selective B₁ agonists evokes only slight alterations in paw volume. However, systemic or local treatment with several distinct inflammatory stimuli evokes a considerable increase in B₁ receptor-mediated paw oedema. Of note is data showing that acute local treatment with the proinflammatory cytokines IL-1β and TNFα (15–120 min) results in a rapid- onset and time-dependent increase in rat paw oedema caused by the selective B₁ receptor agonists des-Arg⁹-BK and des-Arg¹⁰-kallidin (Campos et al., 1998). The effects of IL-1β and TNFα in the functional upregulation of B₁ receptors in the rat paw have been found to be modulated by the secondary production of inflammatory cytokines, by the activation of several protein kinases, and also by the transcriptional factor NF-κB (Campos et al., 1998; 1999). Participation of NF-κB activation in the upregulation of des-Arg⁹-BK-induced paw oedema has also been evidenced 7 days after the surgical removal of adrenal glands (Cabrini et al., 2001). Additional evidence has indicated that intraplantar injection of IL-1β induces a striking augmentation of B₁ receptor mRNA expression in the rat paw (Campos et al., 2002). In the same publication, it was proposed that B₁ receptor upregulation was directly linked to the activation of chemotatic mediators and neutrophil migration. Likewise, Fernandes et al. (2003) have recently shown that PAF, a known chemotatic agent, was capable of inducing both functional and molecular upregulation of B₁ receptors when administered locally (6 h prior) into the rat paw. The mechanisms involved in PAF effects are largely related to the stimulation of PAF receptors, NF-κB activation, and neutrophil migration. Similar findings have been described by Passos et al. (2004) following treatment (12 h) with LPS in the rat paw. Recently, Gougat et al. (2004) have demonstrated that des-Arg⁹-BK produced a marked oedema formation in mice locally pretreated with IL-1β (5 ng/paw, 40 min before). Interestingly, they report that des-Arg⁹-BK-induced mouse oedema was dose-dependently inhibited by the systemic treatment (by oral or i.p. routes) with the new nonpeptidic B₁ receptor antagonist SSR240612.

Participation of kinin B₁ receptors in painful processes

Pain may be defined as an unpleasant sensorial and emotional experience associated with actual or perceived tissue damage by a noxious (damaging) stimulus (Watkins & Maier, 2003). As the emotional component of pain is very difficult to measure, especially in experimental animals, the sensorial component of pain (nociception) is more commonly assessed in preclinical and clinical pain studies. BK is among the most potent endogenous algogen substances, and its role in nociceptive processes has been extensively reviewed (Dray & Perkins, 1997; Calixto et al., 2000; 2001; Couture et al., 2001). BK, acting at B₂ kinin receptors, is able to produce excitation and sensitization of the free endings of C- and Aδ-primary afferent fibres (nociceptors) leading to the production of overt nociception (stimuli-independent pain), hyperalgesia (exaggerated response to a painful stimulus) and allodynia (pain produced by a previously innocuous stimulus) in experimental animals and humans. The activation of B₂ receptors triggers several well-known signalling pathways to produce depolarization and lowering of the threshold of the nociceptors, including phospholipase C and A₂, protein kinase C, formation of COX and lypooxygenase metabolites from arachidonic acid, and activation of vanilloid receptors and tetrodotoxin-resistant sodium currents (Dray & Perkins, 1997; Premkumar & Ahern, 2000; Ferreira et al., 2004). On the other hand, the participation of kinin B₁ receptors in pain transmission is a relatively new area of research and is even less explored. B₁ receptors have generally been implicated in the modulation of the persistent and chronic inflammatory hyperalgesia induced by different agents, including cytokines (IL-1β, IL-2 and IL-8, but not TNFα and IL-6), bacterial components (LPS, CFA and Mycobacterium bovis bacillus Calmette–Guerin – BCG), irritants (carrageenan and capsaicin), ultra-violet irradiation, and substance P (Perkins & Kelly, 1993; Perkins et al., 1993; Davis & Perkins, 1994; Khasar et al., 1995; Rupniak et al., 1997; De Campos et al., 1998; Poole et al., 1999; Ganju et al., 2001; see for review: Dray & Perkins, 1997; Calixto et al., 2000; 2001). However, recent data regarding the constitutive expression of the B₁ receptor in sensory neurones and the participation of B₁ receptors in chronic models of inflammatory and neuropathic pain (especially using knockout mice and selective nonpeptide receptor antagonists) has dramatically improved our comprehension about the critical role of B₁ receptor activation during painful processes. Thus, in the next section, we will focus our attention on these novel ideas and we will try to demonstrate the potential of the B₁ receptor as a new and relevant target for the development of a new class of analgesic drugs.

Expression of B₁ receptors in pain-transmitting neurones

Pain is produced by the stimulation of small-diameter primary afferent fibres that innervate regions of the head and body and arise from cell bodies in trigeminal and dorsal root ganglia (DRG), respectively (Julius & Basbaum, 2001). In a first attempt to identify B₁ receptors in sensory neurons, Davis et al. (1996) were unable to detect specific any binding to [³H]-des-Arg¹⁰-kallidin rat DRG cultured for 7–8 days. Afterwards, expression of B₁ receptor mRNA was detected in freshly isolated mouse and rat DRG by means of RT–PCR (Seabrook et al., 1997; Levy & Zochodne, 2000; Yamaguchi-Sase et al., 2003). The constitutive expression of B₁ receptors in rat and mouse sensory neurones was further confirmed by the use of immunohistochemical staining (Ma et al., 2000; Wotherspoon & Winter, 2000). Furthermore, peripheral B₁-containing neurons were found in peripheral nerve terminals, such as those that innervate rat urinary bladder (Wotherspoon & Winter, 2000). B₁ receptor staining was also localized in rat, mouse and monkey trigeminal and DRG ganglia (Ma et al., 2000; Wotherspoon & Winter, 2000; Shughrue et al., 2003; Rashid et al., 2004). B₁ receptors were predominantly expressed by small diameter DRG neurones, colocalized with calcitonin gene-related peptide or isolectin B4 (Ma, 2001).

It has been well demonstrated that both B₁ receptor mRNA and protein are constitutively present in the spinal cord of rats, mice, monkeys and humans (Couture & Lindsey, 2000; Wotherspoon & Winter, 2000; Ma & Heavens, 2001; Shughrue et al., 2003). It is worth noting that B₁ receptor immunoreactivity has been identified in the superficial layers of the dorsal horn, confined mainly to the spinal terminals of primary afferent fibres (Couture & Lindsey, 2000; Wotherspoon & Winter, 2000; Ma & Heavens, 2001). Subsets of dorsal horn neurons project axons and transmit pain messages to higher brain structures related by the somatic, affective and autonomic responses to pain (Hunt & Mantyh, 2001). Of interest is the fact that basal B₁ receptor expression has been described in several structures related to pain transmission and modulation, including the somatosensory cortex and thalamus (Ongali et al., 2003; Shughrue et al., 2003). However, the function of B₁ receptors in these regions remains obscure.

Role of kinin B₁ receptor in acute and chronic painful processes

Much about the earlier studies on the role of B₁ receptors in physiological and pathological processes has been elucidated on the basis of functional studies or the use of selective receptor antagonists. However, because of the problems of selectivity and agonist activity, and the rapid degradation of some of the antagonists, advances in understanding the role played by B₁ receptors in most physiological and pathological processes has been hampered. The generation of the B₁ knockout mouse has made it possible to expand our current knowledge regarding the contribution of this receptor in nociceptive processes (Pesquero et al., 2000). B₁ receptor-deficient mice present hypoalgesia against the acute overt nociception induced by capsaicin or formalin and by high intensity heat stimuli (Pesquero et al., 2000). This evidence confirms and extends previous data that demonstrates the role of B₁ receptor in acute pain. For instance, intraplantar or systemic treatment with the selective B₁ receptor antagonist des-Arg⁹-Leu⁸-BK was found to be capable of reducing capsaicin, glutamate, and first-phase of formalin-induced pain (Shibata et al., 1989; Corrêa & Calixto, 1993; Sufka & Roach, 1996; Beirith et al., 2003; J.B. Calixto, unpublished results). Since the nociceptive behaviours of the former tests are very short (lasting up to 10 min), it seems improbable that B₁ receptor expression depends on de novo protein synthesis (see details above) and the involvement of constitutively expressed receptors is indicated. However, the peripheral injection of B₁ receptor agonists rarely induces nociception in naïve animals (Perkins & Kelly, 1994; Khasar et al., 1995; Ganju et al., 2001; Fox et al., 2003; Ferreira et al., 2004). Moreover, so far, there is no functional evidence showing that B₁ receptor agonists directly activate peripheral terminals or the cell bodies of sensory neurones (Dray et al., 1992; Davis et al., 1996; Seabrook et al., 1997; Brand et al., 2001). Thus, it has been suggested that B₁ receptors on the peripheral sensory nerve terminals need some stimulation to prime their nociceptive action (Ma, 2001). In fact, des-Arg⁹-BK is able to produce overt nociception when intraplantarly coadministered with formalin (Campos et al., 1995; De Campos et al., 1998). The mechanisms involved in this short-lasting functional induction of B₁ receptor remain elusive and require further study.

Studies carried out with B₁ receptor knockout mice have also confirmed the important role played by B₁ receptors in the development and maintenance of chronic pain. Chronic pain differs substantially from acute pain, not only in terms of the persistence, but also in relation to the maladaptive neuroplasticity described at various levels of the nervous system (Woolf & Mannion, 1999). Peripheral injection of CFA has been used as an experimental animal model of arthritis, causing persistent hyperalgesia and allodynia to mechanical and thermal stimuli that is developed as early as 2 h after its administration and persists for weeks. As observed in humans, this nociceptive behaviour takes place on both ipsilateral and contralateral sides of the injection, an effect which is mediated by local nociceptor sensitization and systemic neuronal (such as central sensitization) and immune (such as increase in cytokine serum level) mechanisms (Shenker et al., 2001). Gene deletion of the B₁ receptor reduces ipsilateral, and in particular contralateral, thermal hyperalgesia and mechanical allodynia induced by CFA (Ferreira et al., 2001). These findings confirm previous studies which have indicated that the B₁ receptor antagonists des-Arg⁹-Leu⁸-BK, des-Arg¹⁰-Hoe 140 and B9858 are capable of inhibiting CFA-induced nociception in rats, mice and rabbits, respectively (Perkins et al., 1993; Panesar et al., 1998; Mason et al., 2002). Moreover, intraplantar injection of des-Arg⁹-BK induces contralateral mechanical hyperalgesia and allodynia in rats pretreated with intraplantar CFA (Khasar et al., 1995; Fox et al., 2003). Interestingly, in the experiment of Fox et al. (2003), B₁ receptor-immunoreactivity was significantly increased 24 h after CFA administration in both ipsilateral and contralateral small DRG neurons. It is worth noting that the nonpeptidic B₁ receptor antagonist derived from dihydroquinoxalinone produces potent antinociceptive action in the model of CFA-induced hyperalgesia in rabbits (Su et al., 2003).

Painful neuropathies may result from nerve injury, chronic treatment with certain drugs and metabolic disorders (Woolf & Mannion, 1999). As the mechanisms underlying these syndromes are not fully understood, available therapy does not provide satisfactory pain relief and patients suffer from chronic intractable pain. Several studies have demonstrated the important role played by kinins and their receptors in neuropathic pain induction. Increased levels of B₁ receptor mRNA or protein have been found in dorsal root ganglion after sciatic nerve constriction injury in rats and mice (Petersen et al., 1998; Eckert et al., 1999; Levy & Zochodne, 2000; Yamaguchi-Sase et al., 2003; Rashid et al., 2004). Very recently, it has been reported that B₁ receptors are newly expressed after nerve injury, mainly in myelinated DRG neurons, whereas B₂ receptor expression drastically decreases in DRG (Rashid et al., 2004). Importantly, systemic administration of B₁ receptor antagonist des-Arg⁹-Leu⁸-BK is able to reduce the thermal hyperalgesia and mechanical allodynia produced by sciatic nerve constriction in rats (Levy & Zochodne, 2000; Yamaguchi-Sase et al., 2003). Also, the gene deletion of B₁ receptors practically abolishes the nociceptive hypersensitivity produced by sciatic nerve injury in mice (J.B. Calixto, unpublished results). This effect appears as early as 1 day after lesion and remains significant up to 28 days, suggesting that the B₁ receptor is involved in both development and maintenance of neuropathic pain symptoms. Corroborating these data, intraplantar administration of Lys-des-Arg⁹-BK 7 days after sciatic nerve injury in mice is able to induce both nociception and activation of ERK in DRG neurones (Rashid et al., 2004). Interestingly, oral treatment with the newly developed nonpeptide B₁ receptor antagonist SSR240612 is capable of reducing the thermal hyperalgesia produced by sciatic nerve injury in rats (Gougat et al., 2004).

Painful neuropathy may also be developed in relation to diabetes (Woolf & Mannion, 1999). As discussed earlier, the B₁ receptor seems to be implicated in type I diabetes complications (for review see: Couture et al., 2001; Gabra & Sirois, 2003a, 2003b). In fact, thermal hyperalgesia produced by STZ in mice is blocked by the systemic treatment of the selective B₁ receptor antagonists R715 or R954 (Gabra & Sirois, 2003a, 2003b). In addition, acute administration of des-Arg⁹-BK significantly potentiates diabetes-induced hyperalgesia. Considering these data, it is possible to suggest that the use of selective B₁ receptor antagonists could represent a novel approach for the treatment of chronic pain of inflammatory and neuropathic origin.

Other recent evidence for the involvement of B₁ receptors in painful processes

Other relevant studies have addressed the involvement of B₁ receptors in various models of acute and chronic pain. For instance, intraplantar injection of zymozam produces local increase in the B₁ receptor mRNA expression and mechanical hyperalgesia that has been reversed by the B₁ receptor antagonists des-Arg⁹-Leu⁸-BK and R715 (Bélichard et al., 2000). Moreover, intraplantar injection of the B₁ receptor agonist des-Arg⁹-BK is able to produce hyperalgesia 1 h following administration of IL1-β at the same site, a response that seems to be dependent on p38 MAPK activation (Ganju et al., 2001). A recent report has demonstrated that peripheral B₁ receptor is also involved in the orofacial nociception caused by formalin in rats (Chichorro et al., 2004). B₁ receptors seem likely to be involved in visceral pain production, since the antagonism of the B₁ receptor reduces the late viscero-visceral hyper-reflexia induced by turpentine inflammation. Another recent study has shown that the benzodiazepine-derived nonpeptide B₁ receptor antagonists are able to reduce carrageenan-induced hyperalgesia in rats, apart from their poor bioavailability after systemic treatment (Wood et al., 2003). Moreover, oral treatment with the nonpeptide B₁ receptor antagonist SSR240612 is capable of reducing the nociception produced by formalin and ultra-violet application (Gougat et al., 2004). Based on these data, it is possible to infer that B₁ receptors (expressed constitutively or upregulated) might be related to acute and chronic pain of somatic and visceral origin.

The spinal cord is an important site for B₁ receptor nociceptive action

As previously demonstrated, B₁ receptors have been identified in the spinal cord (Couture & Lindsey, 2000; Wotherspoon & Winter, 2000; Ma & Heavens, 2001; Shughrue et al., 2003). Using an in vitro spinal cord preparation, Pesquero et al. (2000) have shown that B₁ receptor stimulation increases the C fibre component, but not the Aβ fibre-component, of the ventral root potential (VRP) produced by electrical excitation of naïve mouse dorsal root. This indicates that the B₁ receptor functions specifically in nociceptive synaptic pathways and that it may be involved with some forms of central sensitization. However, B₁ receptor activation is not able to produce direct ongoing activation of VRP in rats (Dunn & Rang, 1990). Thus, it is possible to suggest that B₁ receptor activity is not sufficient for the depolarization of unstimulated spinal cord, but that it produces excitation only following C-fibre activation. In fact, intrathecal administration of B₁ receptor agonists induces hyperalgesia after peripheral thermal or mechanical stimulation in mice and rats (Ferreira et al., 2002; Fox et al., 2003). In addition, the B₁ receptor seems to be involved in the late component of the hyperalgesia induced by bradykinin, a potent activator of C fibres (Ferreira et al., 2002; Sot et al., 2002). Moreover, repetitive electrical stimulation of the dorsal root produces an increase in the VRP, a use-dependent facilitation plasticity of the spinal cord neurones named wind-up. Interestingly, in an experiment on B₁ receptor knockout mice conducted by Pesquero et al. (2000), wind-up was significantly reduced (by about 50%) in comparison with the wild-type littermates. These data indicate that the nociceptive impairment observed in knockout B₁ receptor mice might be attributed, at least in part, to a deficit in the pathological plasticity of the spinal neurones. Indeed, it has been shown that spinal B₁ receptor activation greatly contributes to the inflammatory phase of formalin-induced pain and to the chronic inflammatory pain caused by CFA or sciatic nerve injury in mice and rats (McNair et al., 2001; Ferreira et al., 2002; Fox et al., 2003). Moreover, intrathecal administration of B₁ receptor agonist produces thermal hyperalgesia in hyperglycaemic rats (Couture et al., 2001). The data described above strongly supports the notion that the spinal cord represents a relevant site of action for kinins acting at B₁ receptors to produce nociception in both acute and chronic painful processes.

Possible role of B₁ receptors expressed in nonsensory neurons in pain production

Apart from their expression in DRG neurones, B₁ receptors might be expressed and induced in other cells that are involved in pain production, especially in chronic situations. For example, B₁ receptor immunoreactivity has been found in non-neuronal DRG satellite cells after nerve injury in mice (Rashid et al., 2004). Satellite cells (such as fibroblast-like cells and Schwann cells) are closely associated with neurons and may regulate the function of nociceptors (Heblich et al., 2001). However, the role of B₁ receptors in these cells continues to be elusive.

Some painful processes are mediated by sympathetic activity (Woolf & Mannion, 1999). Interestingly, functional B₁ receptors are expressed in sympathetic ganglia, since their activation is able to depolarize superior cervical ganglia neurones in vitro (Seabrook et al., 1995; 1997). Postganglionic sympathetic terminals have been demonstrated to be involved in B₁ receptor agonist-induced hyperalgesia (Khasar et al., 1995). Thus, sympathetic fibres seem to be important to the nociceptive action of B₁ receptor agonists. Glial cells are also involved in pain production, as their activation results in the release of several pronociceptive mediators (including prostaglandins, glutamate and cytokines) (Watkins & Maier, 2003). Furthermore, B₁ receptor upregulation has been reported in rat primary cultured microglia after BK stimulation (Noda et al., 2003). Moreover, the B₁ receptor agonist des-Arg⁹-BK elicits outward membrane current and increases in intracellular calcium in cultured rat astrocytes (Gimpl et al., 1992). Apart from its participation in nociception, the role of glial cell activation by B₁ receptors during painful processes is so far unknown.

Concluding remarks

In the present review, we have described the main advances in the involvement of kinin B₁ receptors in painful and inflammatory processes. The most recent studies are supported by evidence obtained with selective B₁ receptor agonists or antagonists in various distinct models of pain and inflammation. Nevertheless, greater progress achieved in relation to B₁ receptors implies a better understanding of the mechanisms related to their induction. As discussed previously, although pharmacological data suggested early on that B₁ receptors were inducible molecules, only recently has this hypothesis been confirmed by molecular biology studies. In fact, combined functional and molecular studies have permitted a step forward in identifying the signalling pathways implicated in the upregulation of B₁ receptors. These studies have demonstrated that B₁ receptor induction is the result of a strictly regulated cascade of events which involves an interaction among transcriptional factors, protein kinases, cytokines and inflammatory/neuronal cells. Understanding these pathways brings new possibilities for the control of B₁ receptor expression during pathological states. In addition, the efforts to generate mice lacking B₁ receptor gene, together with the recent development of selective peptidic and nonpeptidic B₁ receptor antagonists, has opened up an important option for the development of clinically relevant drugs for the treatment of inflammatory and painful conditions.

With basis on the literature data presented in this review, it is clear that B₁ receptors occupy a relevant place in the inflammatory scenario. In addition, without doubt, B₁ receptors seem to be deeply involved in both acute and chronic nociceptive processes. Given the large number of diseases in which B₁ receptors are expected to be involved, there is much hope that orally active and selective B₁ receptor antagonists could be clinically tested and proved useful in the management of important pathologies, especially in relation to inflammation and pain. In spite of that, the real effectiveness of selective kinin B₁ receptors antagonists for treating inflammatory/nocipetive alterations remains to be investigated by future studies.

Abbreviations

AP-1

activating protein-1

AKT/PKB

protein kinase B

BCG

Mycobacterium bovis bacillus Calmette – Guerin

BK

bradykinin

CFA

complete Freund's adjuvant

COX-2

cyclooxygenase-2

CREB

c-AMP response element-binding

DRG

dorsal root ganglia

ERK

extracellular signal-regulated kinase

IL

interleukin

JNK

c-Jun NH₂-terminal kinase

LPS

lipopolysaccharide

MAPK

mitogen-activated protein kinase

MEK

mitogen-activated protein kinase kinase

MEKK

mitogen-activated protein kinase kinase kinase

iNOS

inducible nitric oxide synthase

NF-κB

nuclear factor-κB

OVA

ovalbumin

PAF

platelet-activating factor

PI3-K

phosphatydilinositol-3 kinase

PLC

phospholipase C

PKC

protein kinase C

STZ

streptozotocin

TLR

Toll-like receptors

TNFα

tumour necrosis factor α

VRP

ventral root potential