Inhibition of Depolarization-Induced Nitric Oxide Synthase Activation by NS-7, a Phenylpyrimidine Derivative, in Primary Neuronal Culture

Abstract: Neuronal nitric oxide synthase (NOS) is considered to be involved in the pathogenesis of ischemic brain damage. In the present study, the effect of a novel neuroprotective phenylpyrimidine derivative, 4-(4-fluorophenyl)-2-methyl-6-(5-piperidinopentyloxy)pyrimidine hydrochloride (NS-7), on depolarization-stimulated NOS activity was examined in cultured neurons of mouse cerebral cortex. Various depolarizing stimuli such as veratridine, KCl, and N -methyl- d -aspartate increased the NOS activity determined by cyclic GMP formation. NS-7 concentration-dependently inhibited both the veratridine- and KCl-induced NOS activation with IC₅₀ values of 9.3 and 9.6 µ M , respectively. The reversal of KCl-evoked NOS activity by NS-7 was also observed under blockade of both ionotropic glutamate receptors and the Na⁺ channel with MK-801, 6-cyano-7-nitroquinoxaline-2,3-dione, and tetrodotoxin. In contrast, NS-7, even at 100 µ M , did not affect N -methyl- d -aspartate-stimulated NOS activity, nor did it have any influence on NOS activity determined in the soluble fraction of rat hippocampus. Because NS-7 has already been shown to block both Na⁺ and Ca²⁺ channels, the present findings suggest that this compound inhibits depolarization-induced NOS activation by reducing Ca²⁺ influx through blockade of Na⁺ and Ca²⁺ channels in primary neuronal culture.