title: Calcium waves in frog melanotrophs are generated by intracellular inactivation of TTX-sensitive membrane Na+ channel.
abstract: Two models of plasma membrane oscillators may explain the regulation of calcium homeostasis in frog melanotrophs. In the majority (70%) of cells a high frequency and small amplitude fluctuations characterize the spontaneous calcium level. In the 30% of remaining cells a low frequency and high amplitude oscillations were observed. Utilization of EGTA, U73122 and ryanodine suggested that calcium homeostasis in frog melanotrophs is dependent on extra- but not on intracellular calcium pools. EGTA was able to block calcium oscillations and to decrease basal calcium level in non-oscillatory cells. omega-Conotoxin, N-type calcium channels antagonist, stopped calcium oscillations but not modified calcium level in non-oscillatory cells. Nifedipine, antagonist of L-type calcium channels, had no effect either on calcium waves formation or on basal level of calcium in non-oscillatory cells. omega-Conotoxin and nifedipine were able to decrease the spontaneous alpha-MSH release from whole NILs while only omega-conotoxin had inhibitory effect on hormonal output from dispersed melanotrophs. Nickel (Ni2+) provoked dose-dependent effect. At 2 mM concentration Ni2+ blocked either calcium oscillations or alpha-MSH release. In contrast, a 0.5 mM concentration had stimulatory effect on both the phenomenons. Similarly, mibefradil (antagonist of T-type calcium channel), was able to induce an increase in [Ca2+](i) after modification of calcium fluctuations in non-oscillatory cells. Utilization of 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 increase 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+/Ca2+ exchanger) 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 oscillations suggesting implication of intracellular phosphorylation in the generation of calcium waves. Taken together, our results suggest that each type of calcium homeostasis is controlled by different mechanisms. Calcium fluctuations may be ascribed to the high frequency activity of T-type calcium channel, TTX-sensitive and TTX-resistant sodium channels. Calcium oscillations may be generated by the destabilization of the steady-state Na+/Ca2+ gradient 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 responsible for calcium wave. In both types of homeostasis, the calcium and sodium overload is avoided by opening of K+ voltage- and Ca-dependent channels, and by increase in activities of Na+/K+ ATPase and forward mode of Na+/Ca2+ exchanger.
authors.affiliation: Laboratory of Cellular and Molecular Neuroendocrinology, European Institute for Peptide Research (IFRMP no. 23), INSERM U413, Unitée Affiliée au CNRS, University of Rouen, 76821 Mont-Saint-Aignan, France.
authors.forename: L
authors.initials: L
authors.lastname: Galas
authors.forename: M
authors.initials: M
authors.lastname: Garnier
authors.forename: M
authors.initials: M
authors.lastname: Lamacz
mesh_terms: D000818:Animals; D002118:Calcium / Q000378:metabolism / Q000494:pharmacology; D020013:Calcium Signaling / Q000187:drug effects*; D002478:Cells, Cultured; D003600:Cytosol / Q000737:chemistry; D006706:Homeostasis / Q000187:drug effects; D008297:Male; D008856:Microscopy, Fluorescence; D010477:Perfusion / Q000379:methods; D010902:Pituitary Gland / Q000166:cytology*; D015221:Potassium Channels / Q000494:pharmacology; D011898:Ranidae / Q000502:physiology*; D012964:Sodium / Q000494:pharmacology; D015222:Sodium Channels / Q000187:drug effects / Q000494:pharmacology* / Q000502:physiology; D013779:Tetrodotoxin / Q000494:pharmacology
chemical_list: D015221:Potassium Channels; D015222:Sodium Channels; D013779:Tetrodotoxin; D012964:Sodium; D002118:Calcium