Endothelins induce ETB receptor-mediated mechanical hypernociception in rat hindpaw: roles of cAMP and protein kinase C
Introduction
Endothelin-1, endothelin-2 and endothelin-3 are 21-amino acid residue peptides of the endothelin family produced by many cell types, including several implicated in immune defence systems (for review, see Kedzierski and Yanagisawa, 2001). These potent peptides exert widespread biological actions via activation of two specific G protein-coupled receptors, named endothelin ETA and ETB receptors.
Endothelin ETA receptors have higher affinity for endothelin-1 and endothelin-2 than for endothelin-3, and can be blocked by several selective antagonists, including BQ-123 (for review, see Davenport, 2002). In contrast, endothelin ETB receptors do not discriminate between the three isopeptides, but can be selectively activated by agonists such as BQ-3020 or blocked by antagonists such as BQ-788. Both receptor types can couple to multiple intracellular signalling mechanisms, depending on cell type, including phospholipases C, D and A2, adenylyl cyclase and guanylyl cyclase (for extensive review, see Sokolovsky, 1995). Furthermore, activation of protein kinases A and C, as well as intracellular calcium increases also can underlie many effects of endothelins mediated via endothelin ETA and/or ETB receptors (Wu-Wong et al., 1996, Dulin et al., 2001).
Many of these various signalling pathways contribute importantly to inflammatory pain by sensitising nociceptors, a process resulting in a state of hypernociception also known as hyperalgesia or allodynia (for review, see Riedel and Neeck, 2001). There are two groups of hyperalgesic mediators that satisfy the experimental and clinical criteria for agents that directly sensitise nociceptors: eicosanoids and sympathetic amines (Ferreira et al., 1978a, Levine et al., 1986, Nakamura and Ferreira, 1987). Nonetheless, the generation and/or release of these final mediators in mechanical hyperalgesia induced by carrageenin or lipopolysaccharide (LPS) depends on prior recruitment of additional mediators, including cytokines tumour necrosis factor α, interleukin-6, interleukin-1β and cytokine-induced neutrophil chemoattractant-1 (CINC-1) (Cunha et al., 1991, Cunha et al., 1992, Lorenzetti et al., 2002).
Endothelin-1 elicits overt nociceptive behaviour when injected into the human forearm, the knee joint of dogs or rats, as well as the peritoneal cavity and hindpaw foot pad of mice (Ferreira et al., 1989, Dahlof et al., 1990, Raffa and Jacoby, 1991, De-Melo et al., 1998, Piovezan et al., 2000). The peptide also sensitises the human forearm and the rat hindpaw to noxious mechanical stimuli (Ferreira et al., 1989), and the mouse hindpaw to nociception induced by formalin or capsaicin (Piovezan et al., 1997, Piovezan et al., 1998). However, the identity of the receptors implicated in these nociceptive actions of endothelin-1 is still unsettled. Thus, endothelin ETA receptors appear to mediate endothelin-1-induced nociception in the rat hindpaw and knee joint (Davar et al., 1998, De-Melo et al., 1998, Gokin et al., 2001) and both nociception and hyperalgesia to capsaicin and heat in the mouse hindpaw (Piovezan et al., 2000, Menendez et al., 2003). However, both endothelin ETA and ETB receptors mediate endothelin-1-induced hindpaw mechanical hyperalgesia in mice (Baamonde et al., 2004). Abdominal constrictions (i.e. writhes) triggered by endothelins in this species also appear to depend on both endothelin ETA and ETB receptors (Raffa et al., 1996), but writhes induced by i.p. phenylbenzoquinone injection in wild-type mice are suppressed following endothelin ETB (but not ETA) receptor blockade, and are virtually absent in endothelin ETB receptor knockout animals (Griswold et al., 1999). In sharp contrast, other studies have reported that ETB receptors play an antihyperalgesic or antinociceptive role in the hindpaw of mice and rats (Piovezan et al., 2000, Khodorova et al., 2003). On the other hand, the intracellular pathways involved in the nociceptive and hyperalgesic actions of endothelins have not yet been adequately investigated.
In light of these considerations, the present study aimed to further characterise the ability of endothelins to cause mechanical hypernociception in the rat hind paw, by determining which endothelin receptor type(s) is responsible for this effect, as well as the putative contributions of cAMP and of PKA and PKC in the signalling mechanisms involved.
Section snippets
Animals
Male adult Wistar rats (180–200 g) were housed in a temperature-controlled room (22±1 °C), with access to water and food ad libitum, until use. All experiments were conducted in accordance with NIH guidelines on the welfare of experimental animals and with the approval of the Ethics Committee of the Faculty of Medicine of Ribeirão Preto of the University of São Paulo, where the study was undertaken.
Nociceptive test: mechanical hypernociception
Mechanical hypernociception was tested in rats as previously described (Ferreira et al., 1978b).
Mechanical hypernociception induced by intraplantar injection of endothelin-1, endothelin-2, endothelin-3 or BQ-3020
Intraplantar injection of endothelin-1, endothelin-2, endothelin-3 or of the selective ETB receptor agonist BQ-3020 (3, 10 or 30 pmol, in 100 μl) into the hind paws of rats evoked dose- and time-dependent mechanical hypernociception. In all cases, except at doses of 30 pmol of BQ3020, the hypernociceptive responses were already significant 1 h after injection, reached a peak at 3 h and fully returned to control levels within 24 h. The peak of the hypernociceptive response induced by 30 pmol of
Discussion
The results of the current study extend the original finding by Ferreira et al. (1989), by showing that not only endothelin-1, but also endothelin-2 and endothelin-3 are potent inducers of mechanical hypernociception in the rat hindpaw. This study also characterises, to our knowledge for the first time, the endothelin receptor type and some of the putative intracellular signalling mechanisms underlying the nociceptor sensitisation by endothelin peptides.
Intraplantar injection of endothelin-1,
Acknowledgements
The authors gratefully acknowledge the technical assistance of Ieda R. dos Santos Schivo and Sérgio Roberto Rosa. This work was supported by grants from Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP, Brazil), Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq, Brazil) and Programa de Núcleos de Excelência (PRONEX, Brazil).
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