Biphasic changes in intracellular pH induced by thyrotropin-releasing hormone in pituitary cells.
We studied the effects of TRH on intracellular pH (pHi) in individual cells of the GH3 pituitary clonal cell line using the seminaphtorhodafluor pH indicator. We show that, in a majority of cells, TRH action on pHi occurs in two phases: first acidification then alkalinization. Acidification and Ca2+ mobilization are related in time. K+ depolarization (KCl, 50 mM), and Ca2+ ionophores, A23187 (10 microM) or ionomycin (5 microM) lead to acidification. We conclude that a marked increase in [Ca2+]i can induce acidification and that the TRH-induced acidification is due to Ca2+ mobilization. TRH-induced alkalinization is due to Na+/H+ exchanger activation, since it is inhibited by amiloride (200 microM) and Na(+)-free medium. We show that this alkalinization does not occur after a 20-h pretreatment with phorbol myristate acetate (1 microM) which depletes protein kinase C. We also show that blocking Ca2+ entry does not affect the TRH-induced alkalinization, but an increase in [Ca2+]i concomitant with the activation of protein kinase C mimics TRH-induced alkalinization. We conclude that both Ca2+ mobilization and protein kinase C activation are necessary for TRH-induced alkalinization. Studies of secretion in Na(+)-free medium or with amiloride (200 microM) show that pHi does not seem to be involved in PRL short-term release (30 min) but suggest that activation of the Na+/H+ exchanger leading to cytoplasmic alkalinization may have an important role in PRL synthesis.
Laboratoire de Neurophysiologie, Université de Bordeaux 2, France.
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D000584:Amiloride / Q000494:pharmacology; D000818:Animals; D001578:Benzopyrans; D002118:Calcium / Q000378:metabolism; D002352:Carrier Proteins / Q000378:metabolism; D002999:Clone Cells; D003600:Cytosol / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D005456:Fluorescent Dyes; D006863:Hydrogen-Ion Concentration; D007211:Indoles; D007700:Kinetics; D010911:Pituitary Neoplasms; D011493:Protein Kinase C / Q000378:metabolism*; D012964:Sodium / Q000378:metabolism; D017923:Sodium-Hydrogen Exchangers; D013973:Thyrotropin-Releasing Hormone / Q000494:pharmacology*; D013997:Time Factors; D014407:Tumor Cells, Cultured
D001578:Benzopyrans; D002352:Carrier Proteins; D005456:Fluorescent Dyes; D007211:Indoles; C063917:SNARF dye; D017923:Sodium-Hydrogen Exchangers; D013973:Thyrotropin-Releasing Hormone; D000584:Amiloride; D012964:Sodium; D011493:Protein Kinase C; C048960:indo-1; D002118:Calcium