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 Ca2+ 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 KCl (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 KCl‐stimulated cyclic GMP formation without affecting spontaneous cyclic GMP formation. Subsequently, the effects of nifedipine and NS‐7 on L‐type Ca2+ 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 Ca2+ channels involving KCl‐stimulated cyclic GMP formation were subsequently examined. NS‐7 suppressed the KCl‐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 KCl‐induced elevation of the intracellular free Ca2+ concentration. These findings suggest that NS‐7 inhibits NOS activation in primary neuronal culture by reducing Ca2+ entry through L‐type and P/Q‐type Ca2+ channels, in which the inhibition is largely dependent on Ca2+ channel activity.