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

1 In cultured bovine adrenal chromaffin cells, NS-7 [4-(4-fluorophenyl)-2-m ethyl-6-(5-piperidino-pentyloxy) pyrimidine hydrochloride], a newly-synthes ized neuroprotective drug, inhibited veratridine-induced Na-22(+) influx vi a voltage-dependent Na+ channels (IC50=11.4 muM). The inhibition by NS-7 oc curred in the presence of ouabain, an inhibitor of Na+,K+ ATPase, but disap peared at higher concentration of veratridine, and upon the washout of NS-7 . 2 NS-7 attenuated veratridine-induced Ca-45(2+) influx via voltage-dependen t Ca2+ channels (IC50=20.0 muM) and catecholamine secretion (IC50=25.8 muM) . 3 Chronic (greater than or equal to 12 h) treatment of cells with NS-7 incr eased cell surface [H-3]-STX binding by 86% (EC50=10.5 muM; t(1/2)=27 h), b ut did not alter the K-D value; it was prevented by cycloheximide, an inhib itor of protein synthesis, or brefeldin A, an inhibitor of vesicular transp ort from the trans-Golgi network, but was not associated with increased lev els of Na+ channel alpha- and beta (1)-subunit mRNAs. 4 In cells subjected to chronic NS-7 treatment, Na-22(+) influx caused by v eratridine (site 2 toxin), alpha -scorpion venom (site 3 toxin) or beta -sc orpion venom (site 4 toxin) was suppressed even after the extensive washout of NS-7, and veratridine-induced Na-22(+) influx remained depressed even a t higher concentration of veratridine; however, either alpha- or beta -scor pion venom, or Ptychodiscus brevis toxin-3 (site 5 toxin) enhanced veratrid ine-induced Na-22(+) influx as in nontreated cells. 5 These results suggest that in the acute treatment, NS-7 binds to the site 2 and reversibly inhibits Na+ channels, thereby reducing Ca2+ channel gati ng and catecholamine secretion. Chronic treatment with NS-7 up-regulates ce ll surface Na+ channels via translational and externalization events, but p ersistently inhibits Na+ channel gating without impairing the cooperative i nteraction between the functional domains of Na+ channels.