Blockade by NS‐7, a Neuroprotective Compound, of Both L‐Type and P/Q‐Type Ca 2+ Channels Involving Depolarization‐Stimulated Nitric Oxide Synthase Activity in Primary Neuronal Culture

: The effect of4‐(4‐fluorophenyl)‐2‐methyl‐6‐(5‐piperidinopentyloxy)pyrimidine hydrochloride(NS‐7), a neuroprotective compound, on Ca 2+ channels involving theactivation of nitric oxide synthase (NOS) was investigated in primary neuronalculture. The NOS activity was estimated from the cyclic GMP formation. The KCl(25 m M )‐stimulated cyclic GMP formation was totally abolished by acombined treatment with nifedipine and ω‐agatoxin IVA (ω‐Aga),whereas spontaneous cyclic GMP formation was partially but significantlyreduced by nifedipine. In contrast to nifedipine, NS‐7 blocked KCl‐stimulatedcyclic 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 formationstimulated by various concentrations of (±)‐Bay K 8644, whereas NS‐7inhibited the maximal response without affecting the responses induced by lowconcentrations of (±)‐Bay K 8644. The effects of NS‐7 on L‐type andP/Q‐type Ca 2+ channels involving KCl‐stimulated cyclic GMPformation were subsequently examined. NS‐7 suppressed the KCl‐stimulatedcyclic GMP formation measured in the presence of ω‐Aga to almost thesame extent as that determined in the presence of nifedipine. In contrast,NS‐7 had no influence on ionomycin‐induced enhancement of cyclic GMPformation. Finally, NS‐7 reversed KCl‐induced elevation of the intracellularfree Ca 2+ concentration. These findings suggest that NS‐7 inhibitsNOS activation in primary neuronal culture by reducing Ca 2+ entrythrough L‐type and P/Q‐type Ca 2+ channels, in which the inhibitionis largely dependent on Ca 2+ channel activity.