Blockade by NS-7, a neuroprotective compound, of both L-type and P/Q-type Ca2+ channels involving depolarization-stimulated nitric oxide synthase activity in primary neuronal culture.

The effect of 4-(4-fluorophenyl)-2-methyl-6-(5-piperidinopentyloxy)pyrimidine hydrochloride (NS-7), a neuroprotective compound, on Ca 2+ channels involving the activation of nitric oxide synthase (NOS) was investigated in primary neuronal culture. The NOS activity was estimated from the cyclic GMP formation. The KCI (25 mM)-stimulated cyclic GMP formation was totally abolished by a combined treatment with nifedipine and ω-agatoxin IVA (ω-Aga), whereas spontaneous cyclic GMP formation was partially but significantly reduced by nifedipine. In contrast to nifedipine, NS-7 blocked KCI-stimulated cyclic GMP formation without affecting spontaneous cyclic GMP formation. Subsequently, the effects of nifedipine and NS-7 on L-type Ca 2+ channels were compared. Nifedipine blocked equally the cyclic GMP formation stimulated by various concentrations of (±)-Bay K 8644, whereas NS-7 inhibited the maximal response without affecting the responses induced by low concentrations of (±)-Bay K 8644. The effects of NS-7 on L-type and P/Q-type Ca 2+ channels involving KCI-stimulated cyclic GMP formation were subsequently examined. NS-7 suppressed the KCI-stimulated cyclic GMP formation measured in the presence of ω-Aga to almost the same extent as that determined in the presence of nifedipine. In contrast, NS-7 had no influence on ionomycin-induced enhancement of cyclic GMP formation. Finally, NS-7 reversed KCI-induced elevation of the intracellular free Ca 2+ concentration. These findings suggest that NS-7 inhibits NOS activation in primary neuronal culture by reducing Ca 2+ entry through L-type and P/Q-type Ca 2+ channels, in which the inhibition is largely dependent on Ca 2+ channel activity.