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Abstract: Diabetic neuropathy is one of the most common complications of diabetes and causes various problems in daily life. Several investigations have noted that many factors in the spinal cord are involved in the symptoms of painful diabetic neuropathy, and there are very few effective therapeutic regimens. In the present study, we sought to elucidate the role of the RhoA/Rho kinase (ROCK) pathway in thermal hyperalgesia in diabetic mice. The intracellular localization of RhoA and the expression of eNOS were measured by western blotting. Thermal hyperalgesia was assessed by the tail-flick test and mechanical allodynia was assessed by automated von Frey filament test in streptozotocin(STZ)-induced diabetic mice. The spinal cord of STZ-treated diabetic mice showed increased membrane-bound RhoA compared to non-diabetic control. Treatment with the RhoA inhibitor exoenzyme C3, Clostridium botulinum, and the ROCK inhibitor Y27632 attenuated thermal hyperalgesia and mechanical allodynia in diabetic mice. Moreover, daily treatment with simvastatin attenuated all of those changes in diabetic mice. The expression of eNOS and NO metabolite contents in the spinal cord was decreased in diabetic mice, and these changes were normalized by treatment with simvastatin. The present results show that HMG-CoA reductase inhibitors have an inhibitory effect on thermal hyperalgesia in diabetic mice, which is mediated by an increase in NO production through the inhibition of RhoA/ROCK pathways. These results suggest that ROCK inhibitors and HMG-CoA inhibitors may be attractive compounds to relieve the symptoms of painful diabetic neuropathies. [Copyright &y& Elsevier]

Isoprenylation is crucial for the biological activation of small molecular G proteins. Activation of Rho/Rho kinase (ROCK) signaling has been reported to be involved in the initiation and maintenance of hyperalgesia caused by nerve injury and inflammation. The present study was then undertaken to examine whether the protein isoprenylation could affect thermal nociceptive threshold in the mouse spinal cord. Intrathecal administration of mevalonate (0.05-5.0 micromol) dose-dependently decreased the paw-withdrawal latencies for the thermal stimulation, indicating that mevalonate induces thermal hyperalgesia. Intrathecal pretreatment with a geranylgeranyl transferase I inhibitor GGTI-2133 (0.001-1.0 nmol) or a ROCK inhibitor Y27632 (0.001-1.0 nmol) completely blocked the mevalonate-induced thermal hyperalgesia. On the other hand, mevalonate-induced thermal hyperalgesia was only slightly attenuated by a farnesyl transferase inhibitor FTI-277 (0.01-1.0 nmol). Intrathecal injection of mevalonate increased the amount of geranylgeranylated RhoA in the spinal cord, which was completely blocked by intrathecal pretreatment with GGTI-2133. Intrathecal injection of mevalonate also produced RhoA translocation from cytosol to plasma membrane. This mevalonate-induced RhoA translocation was also blocked by intrathecal pretreatment with GGTI-2133, indicating that the RhoA translocation is triggered by RhoA geranylgeranylation. Moreover, inhibition of mevalonate synthesis by HMG-CoA reductase inhibition with simvastatin attenuated the second phase, but not the first phase, of nociceptive response to formalin. Our present results suggest that mevalonate sensitizes the spinal nociceptive transmission, which is mediated by the activation of ROCK following the RhoA geranylgeranylation. [ABSTRACT FROM AUTHOR]

Superficial layers of the dorsal horn receive a dense plexus of nerve fibers immunoreactive to pituitary adenylate cyclase activating polypeptide (PACAP). In vivo experiments were conducted in the mice to evaluate the effects of PACAP-38, herein referred to as PACAP, PACAP receptor antagonist PACAP(6–38) and PACAP-antiserum on nociceptive behaviors induced by radiant heat, intrathecally administered N-methyl-d-aspartate (NMDA) or intraplantarly administered formalin. PACAP (0.05–0.5 μg) dose-dependently decreased the paw-withdrawal latencies induced by thermal stimulation and enhanced the aversive licking and biting behaviors induced by intrathecally injected NMDA. Pretreatment with the PACAP receptor antagonist PACAP(6–38) (0.5–2 μg) or PACAP-antiserum (1:500–2000 dilution) dose-dependently attenuated the second phase, but not the first phase, of nociceptive responses to formalin. Next, the effects of PACAP on NMDA- and kainate-induced currents evoked in single dorsal horn neurons were studied. Whole-cell patch recordings were made from superficial dorsal horn neurons of spinal cord slices from 14- to 20-day-old mice. PACAP at the concentrations of 100 and 200 nM, which caused no significant change of holding currents, increased NMDA-but not kainate-induced currents in superficial dorsal horn neurons. Our results suggest that exogenously applied PACAP sensitizes the dorsal horn neurons to formalin stimulation, and facilitates NMDA receptor-mediated nociceptive response. As a corollary, PACAP, which may be released from primary afferent fibers potentiates nociceptive transmission to the dorsal horn by interacting primarily with NMDA receptors. [Copyright &y& Elsevier]