Signal pathways coupled to activation of neuronal nitric oxide synthase in the spinal cord by nociceptin/orphanin FQ

Abstract Nociceptin/orphanin FQ (N/OFQ) was earlier shown to be involved in the maintenance of neuropathic pain by activating neuronal nitric oxide synthase (nNOS). We recently established an ex vivo system to elucidate biochemical and molecular mechanisms for nNOS activation by the use of a combination of isolated intact spinal cord preparations and NADPH-diaphorase histochemistry. Here we examined the N/OFQ signal pathways coupled to nNOS activation in the spinal cord by using this ex vivo system. N/OFQ enhanced nNOS activity in the superficial layer of the spinal cord, as assessed by NADPH-diaphorase histochemistry, in a time- and dose-dependent manner. The maximum effect was observed at 3–10 nM. The N/OFQ-stimulated nNOS activity was inhibited by NMDA receptor antagonists MK-801 and d -AP5, but not by the NR2B-selective antagonist CP-101,606; and the stimulated activity was observed in NR2D −/− mice, but not in NR2A −/− or NR2A −/− /NR2D −/− mice. N/OFQ receptor antagonists attenuated the nNOS activity stimulated by N/OFQ, but not that by NMDA. Furthermore, the potentiation of nNOS by N/OFQ was inhibited by calphostin C and Ro 31–8220, PP2, and KN-62, but not by H-89. These results suggest that N/OFQ stimulated nNOS activity by a biochemical cascade initiated by activation of NMDA receptors containing NR2A.