Involvement of extracellular and intracellular calcium sources in TRH-induced alpha-MSH secretion from frog melanotrope cells.
The stimulatory effect of thyrotropin-releasing hormone (TRH) on alpha-melanocyte stimulating hormone (MSH) secretion from the frog pars intermedia is mediated through the phospholipase C (PLC) pathway but requires extracellular Ca2+. The aim of the present study was to investigate the respective contribution of extracellular and intracellular Ca2+ in the action of TRH on cytosolic calcium concentration ([Ca2+]i) and alpha-MSH release. In normal conditions, TRH (10(-7) M; 5 s) evoked two types of Ca2+ responses: in 63% of the cells, TRH caused a sustained and biphasic increase in [Ca2+]i while in 37% of the cells, TRH only induced a transient response. In the presence of EGTA or Ni2+, the stimulatory effect of TRH on [Ca2+]i and alpha-MSH secretion was totally suppressed. Nifedipine (10(-6) M) reduced by approximately 50% the amplitude of the two types of Ca2+ responses whereas omega-conotoxin GVIA (10(-7) M) suppressed the plateau-phase of the sustained response indicating that the activation of L-type Ca2+-channels (LCC) is required for initiation of the Ca2+ response while N-type Ca2+-channels (NCC) are involved in the second phase of the response. Paradoxically, neither nifedipine nor omega-conotoxin GVIA had any effect on TRH-induced alpha-MSH secretion. The PLC inhibitor U-73122 (10(-6) M) significantly reduced the transient increase in [Ca2+]i and totally suppressed the sustained phase of the Ca2+ response but had no effect on TRH-induced alpha-MSH secretion. The stimulatory effect of TRH on PLC activity was not effected by nifedipine and omega-conotoxin GVIA but was abolished in Ca2+-free medium. Ryanodine had no effect on the TRH-induced stimulation of [Ca2+]i and alpha-MSH secretion. Concomitant administration of nifedipine/omega-conotoxin GVIA or U-73122/omega-conotoxin GVIA markedly reduced the response to TRH but did not affect TRH-evoked alpha-MSH release. In contrast, concomitant administration of U-73122 and nifedipine significantly reduced the effect of TRH on both [Ca2+]i and alpha-MSH release. Taken together, these data indicate that, in melanotrope cells, activation of TRH receptors induces an initial Ca2+ influx through nifedipine- and omega-conotoxin-insensitive, Ni2+-sensitive Ca2+-channels which subsequently activates LCC and causes Ca2+ mobilization from intracellular pools by enhancing PLC activity. Activation of the PLC causes Ca2+ entry through NCC which is responsible for the plateau-phase of sustained Ca2+ response. Although nifedipine and U-73122, separately used, were devoid of effect on secretory response, Ca2+ entry through LCC and mobilization of intracellular Ca2+ are both involved in TRH-evoked alpha-MSH release because only one source of Ca2+ is sufficient for inducing maximal hormone release. In contrast, the Ca2+ influx through NCC does not contribute to TRH-induced alpha-MSH secretion.
Galas
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L
European Institute for Peptide Research (IFRMP no 23), INSERM U 413, UA CNRS, University of Rouen, Mont-Saint-Aignan, France.
Lamacz
M
M
Garnier
M
M
Roubos
E W
EW
Tonon
M C
MC
Vaudry
H
H
D000818:Animals; D002118:Calcium / Q000378:metabolism* / Q000494:pharmacology; D002121:Calcium Channel Blockers / Q000494:pharmacology; D002478:Cells, Cultured; D004963:Estrenes / Q000494:pharmacology; D015759:Ionomycin / Q000494:pharmacology; D007700:Kinetics; D008297:Male; D008954:Models, Biological; D009543:Nifedipine / Q000494:pharmacology; D010455:Peptides / Q000494:pharmacology; D010726:Phosphodiesterase Inhibitors / Q000494:pharmacology; D010904:Pituitary Gland, Posterior / Q000187:drug effects / Q000378:metabolism / Q000502:physiology*; D011760:Pyrrolidinones / Q000494:pharmacology; D011895:Rana ridibunda; D015398:Signal Transduction; D013973:Thyrotropin-Releasing Hormone / Q000494:pharmacology* / Q000502:physiology; D013997:Time Factors; D010738:Type C Phospholipases / Q000378:metabolism; D000521:alpha-MSH / Q000378:metabolism*; D020866:omega-Conotoxin GVIA
D002121:Calcium Channel Blockers; D004963:Estrenes; D010455:Peptides; D010726:Phosphodiesterase Inhibitors; D011760:Pyrrolidinones; C060229:1-(6-((3-methoxyestra-1,3,5(10)-trien-17-yl)amino)hexyl)-1H-pyrrole-2,5-dione; D015759:Ionomycin; D000521:alpha-MSH; D013973:Thyrotropin-Releasing Hormone; D020866:omega-Conotoxin GVIA; D010738:Type C Phospholipases; D009543:Nifedipine; D002118:Calcium