Effects Of Dynorphin A_1-17 On The Activities, Immunoreactivities And MRNA Expression Of CNOS And INOS In Rat Spinal Cord And Their Correlations With NMDA Receptor, Calcium And C-fos

Dynorphin A_1-17(Dyn), an endogenous κ opioid ligand, produces a potent analgesia upon intrathecal injection (i.t.) but induces transient or permanent paralysis of hindlimbs and tail at higher dosages. The dual effect of Dyn has obvious clinical implications. After spinal cord injury(SCI) and peripheral neuropathy with allodynea, both content and gene expression of endogenous Dyn were markedly increased in the spinal cord, implying mat Dyn is implicated in SCI and spinal pain modulation. However, the underlying mechanisms for Dyn dualism in spinal neurotoxicity and antinociception remain ellusive.Over the years, nitric oxide(NO) has absorbed universal attention as a new neuronal messenger of broad physiological and pathophysiological functions, especially in central nervous system injury and pain modulation. In order to elucidate the differential roles of cNOS and iNOS in Dyn-induced SCI and analgesia, the temporal profile of their expressions and their interactions with NMDA receptor and Ca²⁺ as well as c-fos oncogene were studied to provide new supportive evidence in the basic research of SCI and NO and their clinical implications. The temporal profile of NOS expression was studied by NADPH-diaphorase(NADPH-d) histochemical staining; the immunoreactivity(IR) of bcNOS, iNOS and c-Fos proteins and the expression of their mRNAs were studied by immunocytochemistry and in situ hybridization. The activities of cNOS and iNOS and the functional activity of NMDA receptor in the ventral and dorsal spinal cord were measured with ³H-L-Arginine conversion and ³H-MK801 binding respectively. In the primary spinal cord neuronal culture, the effects of different concentrations of Dyn on the concentrations of intracellular free calcium([Ca²⁺]i) in single spinal neurons and its receptor mechanism were studied by microspectrofluorometry. We have also investigated the pharmacological effects of selective cNOS and iNOS inhibitor, NOS substrate and NO donor on Dyn spinal neurotoxicity and antinociception via intrathecal injection.1. Dose-dependent effects of Dyn spinal neurotoxicity and antinociceptionDyn 1.25 20nmol i.t. produced dose-dependent paralysis of hindlimbs and tail as well as inhibition of tail flick(TF) and foot flinch(FF) reflexes. The paralysis of hindlimb usually came together with the paralysis of tail, and the inhibition of FF was often associated with inhibition of TF. Tail movement often recovered earlier than TF; but the hindlimb motor function and FF always recovered simultaneously. Dyn 1.25nmol had no analgesic effect and 2.5 5.0nmol induced potent analgesia. Dyn 10nmol induced only transient paralysis with no obvious pathological changes(by Nissl stain), but Dyn 20nmol produced permanent paraplegia with irreversible spinal cord damage, characterized by central and progressive necrosis. Dyn 15nmol caused an intermediate neuronal dysfunction and pathological abnormalities in between Dyn 10 and 20nmol2. Effects of Dyn on NOS activities, immunoreactivities and mRNA expression in dorsal(horn of) spinal cordDyn 10 and 20nmol apparently reduced the NADPH-d reaction, bcNOS-IR and bcNOS...