4- Discussion
The current study illustrates a novel signaling pathway in DRG neurons through which bradykinin can regulate the magnitude of TRPV1 channel responses. It shows that the bradykinin sensitization of capsaicin-evoked calcium responses in rat DRG neurons occurs as a result of formation of PGE2 by COX-1 which mediates sensitization of TRPV1 via the EP4 but not EP3 receptors. Inhibition of DAG-lipase significantly attenuated the bradykinin mediated sensitisation effect on capsaicin responses which confirms the DAG lipase branch of the PLC pathway.
The findings that bradykinin-induced sensitizating effect of capsaicin-evoked calcium responses in the DRG neurons is mediated by the formation of the prostaglandin E2 is consistent with other many in vivo studies demonstrating that bradykinin-evoked hyperalgesia was mediated by prostaglandins28,30-32.
However, this study shows the co-localization of COX-1 with other proteins including EP4, B2 and TRPV1 involved in the proposed signaling pathway providing an anatomical for the proposed signaling pathway. It is particularly noteworthy that the immunocytochemical and morphometric data shows that B2, EP4, TRPV1 and COX-1 are expressed mainly in a subpopulation of putatively defined nociceptors in the rat lumbar DRG, i.e. mall-medium diameter neurons. COX-1 expression in rat DRG neurons was reported some time ago using different techniques such as western blotting33,34,immunocytochemistry35,36,and RT-PCR33,36although it was not demonstrated that it played a role in nociceptor signaling per se. . Likewise, , it has been known for several years that bradykinin, B2 receptor mRNA is present in small DRG neurons as well37,38, and Wang and colleagues showed that B2 expression in occurs mainly in small peripherin positive DRG neurons which are a sub-type of nociceptors39. Several studies have demonstrated EP4 expression and localization in DRG neurons in rats 40-42, and Ma and colleagues reported that the EP4, TRPV1 and the inducible COX-2 protein is expressed in L4-6 DRG and plantar skin in rats. Following the repeated restraint stress, the level of expression of the EP4 and TRPV1 were significantly elevated in both L4-6 DRG and plantar skin, while the levels of COX-2 remained unchanged43.
The finding in this study that COX-1 but not COX-2 is involved in bradykinin-mediated sensitization of capsaicin-evoked calcium responses is consistent with an earlier report studying the potentiating effect of bradykinin on capsaicin elicited calcium uptake in rat DRG22 although the role of specific prostaglandin receptors was not demonstrated in that study Interestingly, several studies showed that the effect of bradykinin on sensory neurons can be abolished by NSAIDs which inhibit COX enzymes (COX-1 and COX-2). For example, bradykinin has a sensitizing effect to heat in C-polymodal nociceptors on rat skin nerve preparations through the cyclooxygenase (COX) pathway44.
In the current study, the bradykinin-mediated sensitization of capsaicin-evoked calcium responses was significantly diminished in presence of EP4 antagonist (GW627368) but not the selective EP3 antagonist (L-798106). Figure 9 shows a summary of the possible signaling pathways that mediate bradykinin-sensitizing effect on TRPV1. These findings confirm the role EP4 receptor in mediating bradykinin effects on TRPV1. In agreement with this finding, deleting EP4 receptors but not the EP1, EP2 or EP3 receptors from mice, reduced inflammation in vivo45. A role of EP4 receptors in nociception has previously demonstrated since the oral administration of the EP4 receptor antagonist, CJ-023,423 significantly diminished thermal hyperalgesia and mechanical allodynia in the carrageenan model of inflammation in rodents46. In addition to the novel pathway suggested here, a previous study has shown that pre-treatment of DRG neurons with PGE2, EP4 and EP2 agonists but not EP3 agonists augments tetrodotoxin-resistant (TTX-R) Na+ currents suggesting that specific prostaglandin moieties can mediate sensitization via different ion channel species..
Importantly the experiments described in this study were conducted in the presence of thapsigargin to inhibit the release of the calcium from intracellular stores in response to the activation by bradykinin. This strategy enabled us to investigate the pathway of prostaglandin formation upon bradykinin application excluding the effects of PLC mediated of calcium release from internal stores via InsP3 receptors in the endoplasmic reticulum which would have interfered with our intracellular measurements of intracellular calcium changes. The finding in this study that blocking DAG-lipase significantly attenuated the bradykinin mediated sensitisation effect on capsaicin responses is consistent with earlier report studying bradykinin activation of PLC in DRG neurons47. The generation of IP3, increase of intracellular calcium, and liberation of DAG activates the PKC pathway. It is suggested, therefore, that bradykinin can modulate TRPV1 through PKC mediated phosphorylation of the channel47,48. In our proposed model, Prostaglandins released in response to BK receptor activation may act on the same cells or it can diffuse freely to other cells or the same cell and bind to prostaglandin receptors on the cell surface. The resulting activation of EP4 receptors will result in TRPV1 phosphorylation via PKA leading to nociceptor sensitization.
The antagonists for EP4 receptors used in previous studies for pharmacological inactivation of EP4 subtype are not selective, and many of these compounds act at multiple prostanoid receptor subtypes. However, the present data indicate that GW627368, a highly potent and selective antagonist of EP4 receptors49, produces antihyperalgesic effects in animal models of inflammatory pain. Thus, specific blockade of the EP4 receptor signaling may represent a novel therapeutic approach for the treatment of inflammatory pain, which retains the therapeutic benefits of NSAIDs and COX inhibitors without the gastrointestinal, cardiovascular, and renal side effects.