Yokoo H, Shiraishi S, Kobayashi H, Yanagita T, Minami S, Yamamoto R, Wada A
Department of Pharmacology, Miyazaki Medical College, Miyazaki 889-1692, Japan.
Br J Pharmacol. 2000 Oct;131(4):779-87. doi: 10.1038/sj.bjp.0703622.
In cultured bovine adrenal chromaffin cells, NS-7 [4-(4-fluorophenyl)-2-methyl-6-(5-piperidinopentyloxy) pyrimidine hydrochloride], a newly-synthesized neuroprotective drug, inhibited veratridine-induced (22)Na(+) influx via voltage-dependent Na(+) channels (IC(50)=11.4 microM). The inhibition by NS-7 occurred in the presence of ouabain, an inhibitor of Na(+),K(+) ATPase, but disappeared at higher concentration of veratridine, and upon the washout of NS-7. NS-7 attenuated veratridine-induced (45)Ca(2+) influx via voltage-dependent Ca(2+) channels (IC(50)=20.0 microM) and catecholamine secretion (IC(50)=25.8 microM). Chronic (>/=12 h) treatment of cells with NS-7 increased cell surface [(3)H]-STX binding by 86% (EC(50)=10.5 microM; t(1/2)=27 h), but did not alter the K(D) value; it was prevented by cycloheximide, an inhibitor of protein synthesis, or brefeldin A, an inhibitor of vesicular transport from the trans-Golgi network, but was not associated with increased levels of Na(+) channel alpha- and beta(1)-subunit mRNAs. In cells subjected to chronic NS-7 treatment, (22)Na(+) influx caused by veratridine (site 2 toxin), alpha-scorpion venom (site 3 toxin) or beta-scorpion venom (site 4 toxin) was suppressed even after the extensive washout of NS-7, and veratridine-induced (22)Na(+) influx remained depressed even at higher concentration of veratridine; however, either alpha- or beta-scorpion venom, or Ptychodiscus brevis toxin-3 (site 5 toxin) enhanced veratridine-induced (22)Na(+) influx as in nontreated cells. These results suggest that in the acute treatment, NS-7 binds to the site 2 and reversibly inhibits Na(+) channels, thereby reducing Ca(2+) channel gating and catecholamine secretion. Chronic treatment with NS-7 up-regulates cell surface Na(+) channels via translational and externalization events, but persistently inhibits Na(+) channel gating without impairing the cooperative interaction between the functional domains of Na(+) channels.
在培养的牛肾上腺嗜铬细胞中，新合成的神经保护药物NS-7 [4-(4-氟苯基)-2-甲基-6-(5-哌啶基戊氧基)嘧啶盐酸盐]可抑制藜芦碱诱导的(22)Na(+)通过电压依赖性Na(+)通道内流(IC(50)=11.4 microM)。NS-7的抑制作用在存在哇巴因（一种Na(+),K(+) ATP酶抑制剂）的情况下发生，但在较高浓度的藜芦碱存在时以及NS-7洗脱后消失。NS-7减弱了藜芦碱诱导的(45)Ca(2+)通过电压依赖性Ca(2+)通道内流(IC(50)=20.0 microM)以及儿茶酚胺分泌(IC(50)=25.8 microM)。用NS-7对细胞进行慢性（≥12小时）处理可使细胞表面[(3)H]-STX结合增加86%（EC(50)=10.5 microM；t(1/2)=27小时），但不改变K(D)值；蛋白质合成抑制剂环己酰亚胺或反式高尔基体网络囊泡运输抑制剂布雷菲德菌素A可阻止这种增加，但这与Na(+)通道α和β(1)亚基mRNA水平升高无关。在接受慢性NS-7处理的细胞中，即使在大量洗脱NS-7后，由藜芦碱（位点2毒素）、α-蝎毒（位点3毒素）或β-蝎毒（位点4毒素）引起的(22)Na(+)内流仍受到抑制，并且即使在较高浓度的藜芦碱存在下，藜芦碱诱导的(22)Na(+)内流仍受到抑制；然而，α-或β-蝎毒或短裸甲藻毒素-3（位点5毒素）与未处理细胞一样增强了藜芦碱诱导的(22)Na(+)内流。这些结果表明，在急性处理中，NS-7与位点2结合并可逆地抑制Na(+)通道，从而减少Ca(2+)通道门控和儿茶酚胺分泌。用NS-7进行慢性处理通过翻译和外化事件上调细胞表面Na(+)通道，但持续抑制Na(+)通道门控而不损害Na(+)通道功能域之间的协同相互作用。
