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.