Capsaicin-evoked release of immunoreactive calcitonin gene-related peptide from the spinal cord is mediated by nitric oxide but not by cyclic GMP

Recent data support a role for nitric oxide (NO) in pain processing at the level of the spinal cord, possibly via regulation of neuropeptide release. The goal of this study was to determine whether capsaicin, which selectively activates primary afferent neurons and evokes neuropeptide release, acts in an NO-dependent manner. Our results indicate that capsaicin (1 μM)-evoked release of immunoreactive calcitonin gene-related peptide (iCGRP) is significantly reduced in the presence of the NO synthase inhibitor, L-NAME (10-400 nM; F_3,45=68.38; P<0.001) and, the selective nNOS inhibitor, 3-bromo-7-nitroindazole (170-680 nM; F_5,48=56.2; P<0.01). D-NAME (200 nM) had no effect on capsaicin-evoked iCGRP release. Hemoglobin (an extracellular scavenger of NO; 3 mg/ml) significantly reduced the effect of capsaicin on the release of iCGRP (F_1,8=9.12; P<0.05). The NOS substrate, L-arginine, effectively reversed the inhibitory effect of 3-bromo-7-nitroindazole on capsaicin-evoked iCGRP release. To determine whether the NO-mediated release was NMDA-driven, we superfused spinal cord slices with competitive and non-competitive NMDA antagonists in the presence and absence of capsaicin. MK-801 (0.1-10 μM; F_4,33=8.49; P<0.0001) and AP-5 (0.01-10 μM; F_4,38=3.34; P<0.05) reduced capsaicin-evoked iCGRP release. CNQX, an AMPA/kainate antagonist (10 nM-10 μM), significantly decreased capsaicin-evoked release of iCGRP (F_6,42=8.76; P<0.01) in a dose-dependent fashion. Additionally, our results demonstrate that while capsaicin-evoked release is significantly reduced in the presence of LY-83583 (10 μM; F_2,18=3.46; P<0.01; a cyclic GMP lowering agent), there is no effect of ODQ (a potent and selective inhibitor of guanylate cyclase). Moreover, the application of a cell permeable analog of cyclic GMP (8-bromo-cGMP; 0.01-1000 μM) is without effect on both basal and evoked iCGRP release. Finally, we observed no colocalization of immunoreactive neuronal NOS (nNOS) with CGRP in the dorsal horn. In summary, these data indicate that capsaicin evokes the release of iCGRP, in part, via the production of NO which enters the extracellular space prior to having an effect. Moreover, iCGRP and nNOS are produced in distinct populations of neurons within the dorsal horn. We conclude that capsaicin-evoked release involves the activation of the NMDA receptor but is also modified by the activation of AMPA or kainate receptors. Finally, these data suggest that while capsaicin-evoked iCGRP release is modified by NO, this release does not require the activation of guanylate cyclase and subsequent production of cyclic GMP. Copyright (C) 2000 Elsevier Science B.V.