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Trh mobilizes calcium ion from endoplasmic reticulum and mitochondria of gh 3 pituitary cells characterization of cellular calcium ii pools by a method based on digitonin permeabilization


, : Trh mobilizes calcium ion from endoplasmic reticulum and mitochondria of gh 3 pituitary cells characterization of cellular calcium ii pools by a method based on digitonin permeabilization. Proceedings of the National Academy of Sciences of the United States of America 79(20): 6294-6298

Treatment of 45Ca2+-loaded [rat] GH3 pituitary [tumor] cells with various concentrations of digitonin revealed discrete pools (I and II) of cellular 45Ca2+ defined by differing detergent sensitivities. Markers for cytosol and intracellular organelles indicated that the 2 45Ca2+ pools were correlated with the 2 major cellular Ca2+-sequestering organelles, endoplasmic reticulum (I) and mitochondria (II). Studies with various inhibitors were consistent with these assignments. Mitochondrial uncouplers preferentially depleted 45Ca2+ pool II while trifluoperazine selectively depleted 45Ca2+ pool I. Control experiments indicated that translocation of in situ organellar 45Ca2+ during and after permeabilization was negligible. The digitonin-permeabilization method was used to examine the effect of TRH treatment on intracellular Ca2+-pools of GH3 pituitary cells. TRH rapidly depleted both endoplasmic reticulum and mitochondrial exchangeable Ca2+ by 25-30%. The 45Ca2+ loss from both pools was maximal by 1 min after TRH addition and was followed by a recovery phase; mitochondrial 45Ca2+ content returned to control levels by 30 min. Previous treatment of cells with the mitochondrial uncoupler carbonyl cyanide p-trifluoromethoxy-phenylhydrazone blocked TRH-induced 45Ca2+ efflux from mitochondria, while previous treatment with valinomycin, an agent that depleted both 45Ca2+ pools, blocked any additional effect of TRH on these pools, TRH rapidly promotes a net loss of exchangeable Ca2+ from GH3 cells as a result of hormone-induced mobilization of Ca2+ from endoplasmic reticulum and mitochondria.

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