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.