It has been reported that tissue acidity corresponds to the severity of hypoxic status, and the tissue acidic environment is a major factor for triggering ischemia related pathological consequences [23, 34, 35]. In terms of pain hypersensitivity, although low tissue pH at the ischemic site has been considered a crucial factor in the development of ischemic pain [8, 14, 36], the contribution of the acidic environment to ischemia-induced thermal hypersensitivity has not been delineated. The present study is the first to demonstrate that under peripheral ischemic conditions an increase in tissue acidity results in the development of thermal hyperalgesia (TH). Here, we injected pH 4.0 saline into the ischemic hind paw to evaluate how an acidic tissue environment affected both thermal hypersensitivity and TRPV1 activity under peripheral ischemic condition. Since hypoxia inducible factor-1α (HIF-1α) is a well-known indicator of hypoxia and carbonic anhydrase II (CA II) has been reported to be a major indicator of pH imbalance [23–29], we examine changes in these two factors in order to evaluate the degree of hypoxic injury and tissue acidity. We observed that repetitive acidic saline injection into the ischemic hind paw increased protein levels of both hypoxia inducible factor-1α (HIF-1a) and carbonic anhydrase II (CA II) in hind paw muscle (Figure 2). These results indicated that the presence of an acidic tissue environment intensified the ischemia-associated insults to the hind paw muscle tissue. Therefore, we postulate that injection of acidic saline into TIIP (AS-TIIP) rats represents a model of the ischemic condition with severe tissue acidosis.
Since the injection of pH 4.0 saline solution itself in sham rats did not affect thermal nociception (Figure 1), these findings indicated that an increased acidic environment in the presence of other pro-algesic substances under ischemic conditions could lead to the development of TH. It has been reported that there is a synergism between low tissue pH conditions and pro-algesic substances in their effect on nociceptor excitation [37, 38]. In the skin-nerve preparation, a strong interaction between acidic pH and inflammatory mediators resulted in an increased prevalence and magnitude of nociceptor excitation . This interaction was also shown in experimental tissue acidosis in human skin; e.g. the injection of additional inflammatory mediators into the acidic skin caused a more significant painful response . In a previous study we demonstrated that the synergism between acidic saline and ATP produced a significant facilitation of nociception (mechanical allodynia and thermal hypersensitivity) in the normal rat hind paw . Accordingly, it is reasonable to hypothesize that ATP may be one of the synergic factors that contribute to acidic pH-mediated pain development under ischemic conditions. Although we do not measure the exact concentration of tissue ATP in TIIP rats in the present study, it is well known that injured tissues possess a high concentration of extracellular ATP especially during early induction phase [39, 40]. In TIIP rats, intraplantar injection of a P2 antagonist effectively alleviated mechanical allodynia at day 3 after surgery . Moreover, in the current study, we injected acidic saline to the ischemic hind paw during this induction phase (D0 to D3), and observed increased thermal hypersensitivity. These results imply that during the early induction phase in which extracellular ATP concentration is elevated, injection of acidic saline could effectively modulate P2Y1 receptors, which could contribute to the development of increased thermal hypersensitivity.
Since previous studies have shown that P2Y1 and TRPV1 receptors are located in the same population of DRG neurons, we have examined the potential role of TRPV1 receptors in this process by examining the effect of injection of the specific TRPV1 antagonist AMG9810, using two different approaches. First, in order to investigate the potential involvement of TRPV1 in maintaining TH, we performed a single intraplantar injection of the selective TRPV1 antagoinst, AMG9810 at day 3. This injection resulted in short-term alleviation of the established TH (Figure 5A). Second, we performed repetitive intraplantar injection to further explore whether each acidic stimulation directly affected TRPV1 or not. AMG9810 were injected once a day (D0-D3 after surgery) 30 min before the acidic saline injection. Interestingly, this pre-blockade of TRPV1 before acidic saline failed prevent the induction of TH (Figure 6B). These results implied that TRPV1 was a final gate for acid induced TH in TIIP rats, but acid stimulation did not directly activate this channel.
In the current study, we discovered that peripheral P2Y1 receptors played a crucial role in acid induced TH, based on the fact that repeated pre-injection of MRS2179, a selective P2Y1 antagonist, effectively prevented the acidic saline-induced TH in AS-TIIP rats (Figure 6D). These results indicate that the acidic environment created by pH 4.0 saline injection in the ischemic hind paw modulates P2Y1 receptors, which in turn contribute to the development of TH. On the other hand, TRPV1 receptors do not appear to play a crucial role in the onset of TH in this model. In addition, we also concluded that ASICs did not contribute to thermal hypersensitivity in AS-TIIP rats (Figure 6A). Previously, we reported that peripheral ASICs were involved in maintaining ischemia-induced mechanical allodynia in TIIP . However, we were unable to investigate their contribution to TH in the present study, since TIIP rats have normal heat sensitivity. In the current study, we observed that low tissue pH in conjunction with ischemia resulted in the development of TH in TIIP rats, and ASICs were not involved in this ischemic thermal hypersensitivity. On the other hand, ASICs did contribute to mechanical allodynia in AS-TIIP rats (data not shown). These results suggest that ASICs are a modality specific contributor to mechanical allodynia in acid-induced nociception, and there are several reports supporting the specific role of ASICs in mechanical sensing and hypersensitivity [20, 41, 42].
Although pH 4.0 is quite an extreme pH not often seen even in pathophysiological conditions, several studies have examined the actual pH level in the plantar tissue, and they demonstrated that the measured tissue pH is considerably higher than solution pH level before injection [21, 41]. For example, Sluka et al. reported that the repeated injection of pH 4.0 saline into gastrocnemius muscle lowered the muscle pH averaged 6.5 with decreases in individual animals to pH 6 . Therefore, we have assumed that a single injection of pH 4.0 saline would not extremely decrease the tissue pH, and further considered that temporary acidic pH tissue condition induced by pH 4.0 saline could mimic the pathophysiological state shown in peripheral ischemic condition. Collectively, the acidic environment is easily buffered by physiological buffering systems [15, 21, 43], it may be difficult to activate TRPV1 receptors directly, since they only respond under relatively low tissue pH (pH < 5–6) [44–46]. Therefore, there is the strong possibility that the interaction of the acidic environment and ATP indirectly activate TRPV1 via P2Y1 receptors which up-regulates intracellular signaling cascades, but this only occurs in an ischemic environment in which tissue acidosis is present.
It is well recognized that activation of TRPV1 receptors contributes to peripheral sensitization, particularly to heat stimuli. Pro-algesic substances such as protons, ATP, bradykinin and prostaglandins sensitize nociceptors either by directly modulating the sensitivity of membrane receptors or by up-regulating intracellular signaling cascades . These signaling cascades include calcium dependent protein kinase (PKC), cyclic AMP-dependent protein kinase (PKA), and calcium-calmodulin dependent protein kinase (CaMKII) dependent phosphorylation . Since PKC dependent modulation of TRPV1 had been reported to be the main pathway stimulated by inflammatory mediators [5, 47, 48], an analysis of phosphorylated TRPV1 that targeted S800 was performed in the present study. Sham (pH 7.0 and 4.0), TIIP and AS-TIIP rats did not show any alterations in TRPV1 expression (Figure 3A), whereas the ratio of pTRPV1/TRPV1 was significantly elevated in AS-TIIP rats (Figure 3B). Although it has been reported that PKC dependent TRPV1 phosphorylation is critical in the TRPV1 response to its agonist in vitro
[7, 49, 50], this is the first evidence that increased phosphorylation of TRPV1 in peripheral tissues directly correlates with behavioral changes in vivo. Since TRPV1 is not only located in peripheral nerve fibers, but also in the keratinocytes distributed in the epidermis of the skin, our initial western blot data contain TRPV1 from both neuronal and non-neuronal origins. Therefore, in attempt to evaluate the contribution of non-neuronal TRPV1 to the up-regulation of the pTRPV1/TRPV1 ratio observed in AS-TIIP rats, we gave resiniferatoxin (RTX) to several groups of rats to destroy TRPV1 containing nerve fibers and subsequently examined the changes in the ratio of TRPV1 and pTRPV1 expression in paw lysates from sham (pH 7.0 and 4.0) and TIIP (pH 7.0 and 4.0) rats. Although TRPV1 was still present in hind paw lysates from RTX treated rats, there were no significant changes in the ratio of pTRPV1/TRPV1 in AS-TIIP rats (Figure 4D). These results indicated that TRPV1 receptors associated with keratinocytes or other non-neuronal sources did not contribute to the increase in pTRPV1/TRPV1 ratios that we observed in AS-TIIP animals. Collectively, these findings suggest that the transitional rate of PKC dependent phosphorylation of TRPV1 in peripheral nerve fibers is an important factor in the development of heat hypersensitivity in chronic ischemic conditions.
P2Y1 receptors are Gq-coupled receptors located in sensory neurons and these receptors have been implicated in sensory transduction [30, 51, 52]. There are several reports suggesting that there is a potential relationship between the P2Y1 and TRPV1 receptors that contributes to increased pain hypersensitivity [7, 30, 53–55]. P2Y1 receptors are located primarily in small diameter sensory neurons, and thus it is perhaps not surprising that over 80% of CGRP neuronal profiles in DRGs contain P2Y1 receptors . Interestingly Gerevich et al. demonstrated that P2X3, TRPV1, and P2Y1 receptors were co-expressed in ~80% of small diameter DRG cells . Furthermore, there was direct evidence supporting a functional relationship between P2Y1 and TRPV1 in DRG neurons; thus, Yousuf et al. demonstrated that ADP-induced activation of P2Y1 receptors facilitated capsaicin-induced currents, and this facilitatory effect was prevented by protein kinase C inhibition . On the other hand, the relationship and interaction between P2Y1 and TRPV1 receptors have not yet been examined in vivo. In the current study, we examined the potential interaction between P2Y1 and TRPV1 receptors and demonstrated that this interaction resulted in the development of a TH response in ischemic rats. As indicated in Figure 7, we found that the creation of a more acidic environment in the ischemic hind paw resulted in the up-regulation of PKC-dependent pTRPV1 expression. Moreover, pre-injection of the P2Y1 receptor antagonist, MRS2179 significantly reduced this increase in pTRPV1 expression. Based on these results, we conclude that activation of P2Y1 receptors in an acidic environment modulates TRPV1 activity (i.e. phosphorylation at the S800 site) and ultimately causes the development of TH under peripheral ischemic conditions. Although we do not delineate the downstream individual molecular steps resulting from activation of P2Y1 receptors in this study, it is feasible to consider that activation of P2Y1 receptors can directly phosphorylate TRPV1 through Gq/G11 related intracellular cascades. When agonists stimulate this type of receptor, phosphatidylinositide-specific phospholipase C (PLC) is activated and subsequently causes hydrolysis of PIP2 in the plasma membrane. Inositol 1,4,5-triphosphate (IP3) and diacylglycerol (DAG) are generated by PIP2 hydrolysis resulting in the release of Ca2+ from intracellular stores and subsequent activation of the protein kinase C (PKC) pathway [56–58].
One of the questions resulting from the current study relates to explaining the mechanism by which acidic tissue conditions such as that associated with ischemia affects P2Y1Rs. One possibility is that the lower pH found in ischemic tissue changes the agonist affinity of the P2Y1 receptor. Many reports have demonstrated that extracellular protonation modulates the affinity of the ATP binding site and enhances the agonist potency of P2 receptors. For example, Li et al. demonstrated that extracellular protons regulated the function of P2X receptors by modulating the affinity of the ATP binding site . Furthermore, extracellular protons have been shown to significantly potentiate the agonist potency of recombinant P2Y4 receptors, indicating the functional potentiation of P2Y receptors by protons . Furthermore, we should also consider the possibility that the concentration of P2Y receptor agonists at the tissue site might be changed by lower tissue pH. In this regard, Dulla et al. reported that ATP hydrolysis to ADP and adenosine (by ecto-nucleotidases) was closely link to the PCO2 level and pH in hippocampal slices, and changes in nucleotides levels ultimately modulated neuronal excitability in the forebrain . In addition, Sowa et al. demonstrated that prostatic acid phosphatase (PAP), which was expressed in nociceptive neurons and functions as an ectonucleotidase, had pH-dependent ectonucleotidase activity. At neutral pH, mPAP primarily dephosphorylated AMP; however, under acidic extracellular conditions, mPAP could dephosphorylate all purine nucleotides (AMP, ADP, ATP) . Collectively, these reports suggest that the acidic condition of the tissue can change nucleotidase activity leading to increased dephosphorylation of ATP, which would result in an increase in ADP concentration and increased activatation of P2Y1 receptors.