Calcium waves in frog melanotrophs are generated by intracellular inactivation of TTX-sensitive membrane Na+ channel

Two models of plasma membrane oscillators may explain the regulation of cal cium homeostasis in frog melanotrophs. In the majority (70%) of cells a hig h frequency and small amplitude fluctuations characterize the spontaneous c alcium level. In the 30% of remaining cells a low frequency and high amplit ude oscillations were observed. Utilization of EGTA, U73122 and ryanodine s uggested that calcium homeostasis in frog melanotrophs is dependent on extr a- but not on intracellular calcium pools. EGTA was able to block calcium o scillations and to decrease basal calcium level in non-oscillatory cells. o mega -Conotoxin, N-type calcium channels antagonist, stopped calcium oscill ations but not modified calcium level in non-oscillatory cells. Nifedipine, antagonist of L-type calcium channels, had no effect either on calcium wav es formation or on basal level of calcium in non-oscillatory cells. omega - Conotoxin and nifedipine were able to decrease the spontaneous alpha -MSH r elease from whole NILs while only omega -conotoxin had inhibitory effect on hormonal output from dispersed melanotrophs. Nickel (Ni2+) provoked dose-d ependent effect. At 2 mM concentration Ni2+ blocked either calcium oscillat ions or alpha -MSH release. In contrast, a 0.5 mM concentration had stimula tory effect on both the phenomenons. Similarly, mibefradil (antagonist of T -type calcium channel), was able to induce an increase in [Ca2+](i) after m odification of calcium fluctuations in non-oscillatory cells. Utilization o f veratridine and TTX, agonist and antagonist of Na channels, respectively, indicated that mobilization of extracellular sodium, by TTX-sensitive and TTX-resistant Na channels, stimulates a hormonal output resulting from incr ease of [Ca2+](i). In the presence of TTX, veratridine was able to generate a calcium oscillations, which were also observed after inactivation of TTX -sensitive channel. Bepridil (antagonist of Na-Na exchange of the Na+/Ca2exchanger) and Na-free medium had powerful effect on increase of [Ca2+](i). The same observations obtained after administration of ouabain, antagonist of Na+/K+ dependent ATPase, confirmed dependence of calcium homeostasis on sodium distribution. Furthermore, dibutyryl-cAMP induced calcium oscillati ons suggesting implication of intracellular phosphorylation in the generati on of calcium waves. Taken together, our results suggest that each type of calcium homeostasis is controlled by different mechanisms. Calcium fluctuat ions may be ascribed to the high frequency activity of T-type calcium chann el, TTX-sensitive and TTX-resistant sodium channels. Calcium oscillations m ay be generated by the destabilization of the steady-state Na+/Ca2+ gradien t provoked by intracellular inactivation of TTX-sensitive Na channel. This ionic unbalance would increase Ca-Ca exchange of Na+/Ca2+ exchanger, which by local depolarization promotes opening of N-type calcium channel responsi ble for calcium wave. In both types of homeostasis, the calcium and sodium overload is avoided by opening of K+ voltage- and Ca-dependent channels, an d by increase in activities of Na+/K+ ATPase and forward mode of Na+/Ca2+ e xchanger. (C) 2000 Elsevier Science Ireland Ltd. All rights reserved.