(The) role of peripheral P2Y1 receptor-mediated TRPV1 receptor modulation in the development of thermal hyperalgesia

Abstract

BACKGROUND: During the pathological conditions such as ischemia and inflammation, a huge array of endogenous chemicals are released into the damaged tissue that contribute to peripheral sensitization. Since Transient receptor potential vanilloid 1 receptor (TRPV1R) is known for the endpoint target of sensitizing mediators and receptors in periphery, modulation of TRPV1R is the effective way to control the pain initiation at the damage site. P2Y1 receptor (P2Y1R) is a Gq-coupled receptor located in the peripheral nervous system. Preferred agonists for P2Y1R are ADP and ATP which are released from damaged tissues. Recently, putative involvement of P2Y1R in sensory transduction has been documented and the possibility that P2Y1R could modulate the function of TRPV1R was reported in vitro system. However, the underlying mechanisms of P2Y1R and P2Y1R-TRPV1R interaction in pain hypersensitivity are remain to be addressed.

OBJECTIVES: The present study was aimed to 1. Examine whether inflammatory insults would increase the expression of peripheral P2Y1R and blockade of peripheral P2Y1R could prevent the development of inflammatory pain. The modulatory effects of P2Y1R on the expression level of TRPV1R during the inflammation was also investigated. 2. Evaluate whether MAPKs activity in dorsal root ganglion (DRG) would increase in response to the inflammatory insults. This study investigated whether the inhibition of peripheral P2Y1R could affect MAPKs activity, and the possible involvement of MAPKs in the P2Y1R induced up-regulation of TRPV1R expression. 3. Examine whether injection of acidic saline into the hind paw causes the development of TRPV1R mediated thermal hyperalgesia under the ischemic state. In addition, the present study investigated whether functional interactions between TRPV1R and P2Y1R would contribute to the development of this ischemic thermal hyperalgesia.

MATERIALS AND METHODS: All experiments were performed on Sprague-Dawley rats. Inflammation was induced by 2% carrageenan injection to the hind paw, and the ischemic condition was induced by TIIP (thrombus induced ischemic pain) surgery. 20% FeCl2 was applied to the separated femoral artery and the synthesis of thrombus caused the peripheral ischemia in this model. Sensitization to noxious heat stimulation (thermal hyperalgesia) was examined with Hargreaves apparatus, and sensitization to innocuous mechanical stimulation (mechanical allodynia) was examined using von Frey filaments with forces of a 4g. In the present study, MRS2500 and MRS2179 (P2Y1R antagonist), MRS2365 (a P2Y1R agonist), AMG9810 (a TRPV1R antagonist), chelerythrine (a PKC inhibitor), amiloride (an ASICs blocker) and TNP-ATP (a P2Xs antagonist) were intraplantarly injected. SB203580 (a p38 MAPK inhibitor) was intrathecally injected to inhibit p38 MAPK in DRGs. Immunohistochemistry and western blot assay were performed according to each experiment procedure. The computer-assisted image analysis system (Metamorph) was utilized throughout whole experiments.

RESULTS: 1. The expression of P2Y1R and TRPV1R was significantly increased on day 2 following carrageenan injection. Blockade of peripheral P2Y1R by the P2Y1R antagonist, MRS2500 injection significantly reduced the induction of thermal hyperalgesia, but not mechanical allodynia. Simultaneously, MRS2500 injections suppressed up-regulated TRPV1R expression. In addition, repeated injection of P2Y1R agonist, MRS2365 into the naïve rats hind paw dose dependently increase the expression level of TRPV1R in naive rats. 2. Following injection of 2% carrageenan into the hind paw, the phosphorylation rates of both p38 MAPK and ERK but not JNK were increased and peaked at day 2 post-injection. Injection of MRS2500 significantly suppressed the ratio of p38 MAPK phosphorylation in DRGs, while p-ERK signaling was not affected. Furthermore, inhibition of p38 MAPK activation in the DRGs by SB203580 (a p38 MAPK inhibitor) prevented the increase of TRPV1R by inflammation. Furthermore, in naïve rats, repeated stimulation of peripheral P2Y1R dose dependently increased the level of p-p38 MAPK in DRGs. 3. Repeated intraplantar injection of pH 4.0 saline for 3 days following TIIP surgery resulted in the development of thermal hyperalgesia. Moreover, injection of chelerythrine (a PKC inhibitor) and AMG9810 (a TRPV1R antagonist) effectively alleviated the established thermal hyperalgesia. After acidic saline (pH 4.0) injections, there were no changes in the expression of TRPV1R in hind paw skin, whereas a significant increase in TRPV1R phosphorylation was shown in acidic saline injected TIIP animals. Pre-blockade of peripheral P2Y1R significantly prevented the induction of thermal hyperalgesia, and the increase of phosphorylated TRPV1R ratio.

CONCLUSIONS: This study demonstrated that there was a sequential role for P2Y1R, p38 MAPK and TRPV1R in inflammation-induced thermal hyperalgesia. Peripheral P2Y1R activation modulates p38 MAPK signaling and TRPV1R expression, which ultimately leads to the induction of the inflammatory thermal hyperalgesia. I also have addressed that maintenance of an acidic environment in the ischemic sate resulted in the phosphorylation of TRPV1R by P2Y1R, which leads to the development of thermal hyperalgesia mimicking what occurs in chronic ischemic the patients with severe acidosis. Collectively, these data imply that there is a close relationship between P2Y1R and TRPV1R in the development of thermal hypersensitivity, and this connection could be useful therapeutic targets for alleviating thermal hypersensitivity under the conditions of inflammation or ischemia.