A novel Na+/Ca2+ channel blocker, NS-7, suppresses hypoxic injury in rat cerebrocortical slices

Abstract

The substance 4-(4-fluorophenyl)-2-methyl-6-(5-piperidinopentyloxy) pyrimidine hydrochloride (NS-7) has been developed recently as a cerebroprotective compound with Na+ and Ca2+ channel blocking action. In the present study, the effect of NS-7 in an in vitro model of hypoxic injury was examined and the possible involvement of Na+ and Ca2+ channels in the hypoxic injury subsequently determined. When slices of rat cerebral cortex were exposed to hypoxia/glucose deprivation followed by reoxygenation and restoration of the glucose supply, marked leakage of lactate dehydrogenase (LDH) occurred 3-6 h after reoxygenation. This hypoxia/reoxygenation-induced injury was blocked almost completely by the removal of extracellular Ca2+ or by chelating intracellular Ca2+ with 1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid tetra(acetoxymethyl)ester (BAPTA/AM). In addition, combined treatment with the N-type Ca2+ channel blocker omega-conotoxin GVIA and the P/Q-type Ca2+ channel blocker omega-agatoxin IVA significantly reduced LDH leakage, although neither of these Ca2+ channel blockers alone, nor nimodipine, an L-type Ca2+ channel blocker, was effective. On the other hand, several Na+ channel blockers, including tetrodotoxin, local anaesthetics and antiepileptics, significantly reduced the hypoxic injury. NS-7 (3-30 microM) concentration-dependently inhibited LDH leakage caused by hypoxia/reoxygenation, but had no influence on the reduction of tissue ATP content and energy charge during hypoxia and glucose deprivation. It is suggested that blockade of Na+ and Ca2+ channels is implicated in the cerebroprotective action of NS-7.

Short‐ and long‐term differential effects of neuroprotective drug NS‐7 on voltage‐dependent sodium channels in adrenal chromaffin cells

“…NS‐7 [4‐(4‐fluorophenyl)‐2‐methyl‐6‐(5‐piperidinopentyloxy) pyrimidine hydrochloride] is a newly‐synthesized neuroprotective agent. Previous in vivo and in vitro studies in cerebral cortex have shown that NS‐7 attenuated hypoxia‐induced degradation of cytoskeletal protein fodrin ( Takagaki et al ., 1997 ) and cell injury ( Tatsumi et al ., 1998b ) at 10–30 μ M , and the cellular mechanisms of NS‐7 for neuroprotection are postulated to be attributed to the blockade of voltage‐dependent Na + and Ca 2+ channels. In brain, NS‐7 bound with a K i value of 1 μ M ( Shimidzu et al ., 1997 ) to the toxin binding site 2 of the Na + channel α‐subunit, a major subunit forming the ion‐pore and the toxin binding sites 1–5 ( Catterall, 1992 ); NS‐7 at 10–30 μ M diminished the overflow of glutamate ( Shimidzu et al ., 1997 ) and dopamine ( Itoh et al ., 1998 ) caused by veratridine, a toxin that interacts with the site 2 in the transmembrane segment 6 of domain I (IS6) of the Na + channel α‐subunit ( Trainer et al ., 1996 ) and activates Na + channels ( Catterall, 1992 ).…”

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

“…In addition, intracerebroventricular injection of ω‐Aga reduces the size of cerebral infarction caused by a transient middle cerebral artery occlusion in rats (Asakura et al, 1997). More recently, we have found that the tissue injury induced in rat cerebrocortical slices by a transient exposure to hypoxia and glucose deprivation is significantly inhibited by a combined treatment with ω‐Aga and ω‐CTX (Tatsumi et al, 1998 b ). NS‐7 (3‐30 μ M ) has a cerebroprotective action in this model of hypoxic injury (Tatsumi et al, 1998 b ).…”

“…More recently, we have found that the tissue injury induced in rat cerebrocortical slices by a transient exposure to hypoxia and glucose deprivation is significantly inhibited by a combined treatment with ω‐Aga and ω‐CTX (Tatsumi et al, 1998 b ). NS‐7 (3‐30 μ M ) has a cerebroprotective action in this model of hypoxic injury (Tatsumi et al, 1998 b ). Therefore, the blockade of the P/Q‐type Ca 2+ channel observed in the present study may contribute at least in part to the cerebroprotective action of NS‐7.…”