Vol. 293, Issue 2, 522-529, May 2000

Preferential Inhibition by a Novel Na⁺/Ca²⁺ Channel Blocker NS-7 of Severe to Mild Hypoxic Injury in Rat Cerebrocortical Slices: A Possible Involvement of a Highly Voltage-Dependent Blockade of Ca²⁺ Channel

Michiko Oka, Yoshinori Itoh and Yojiro Ukai

Research Laboratories, Nippon Shinyaku Co., Ltd., Nishiohji Hachijo Minami-ku, Kyoto, Japan

The hypoxic injury was induced in rat cerebrocortical slices by the exposure to hypoxia for 45 min in the absence or presence of 3 mM glucose, followed by reoxygenation for 5 h. The injury was more pronounced in the absence of glucose (severe hypoxic injury) than in the presence of glucose (mild hypoxic injury). A novel Na⁺/Ca²⁺ channel blocker, NS-7 [4-(4-fluorophenyl)-2-methyl-6-(5-piperidinopentyloxy) pyrimidine hydrochloride], at 3 to 30 µM inhibited preferentially the severe hypoxic injury, whereas MK-801, -conotoxin GVIA (-CTX), and N^G-nitro-L-arginine methylester suppressed preferentially the mild hypoxic injury. The extracellular cyclic GMP formation, a marker of nitric oxide synthesis, was enhanced during hypoxia, although the extent was greater in the absence of glucose. As observed in the hypoxic injury, NS-7 preferentially inhibited the cyclic GMP formation induced by severe hypoxic insults, whereas MK-801 or -CTX reduced it under mild hypoxic condition. When 30 to 50 mM KCl was applied to normoxic slices, a concentration-dependent increase in the extracellular cyclic GMP formation was observed. NS-7 blocked the cyclic GMP formation induced by 50 mM KCl but not by 30 to 40 mM KCl, whereas -CTX suppressed only the 30 mM KCl-evoked response. In primary neuronal culture, NS-7 reversed KCl-induced increase in intracellular Ca²⁺ in which the inhibition was marked when the KCl concentration was increased. These findings suggest that NS-7, unlike other neuroprotective compounds used in this study, is more effective in severe hypoxic injury. The highly voltage-dependent Ca²⁺ channel blockade may contribute to the mode of neuroprotective action of NS-7.