Thyrotropin-Releasing Hormone Mobilizes Ca2+from Endoplasmic Reticulum and Mitochondria of GH3Pituitary Cells: Characterization of Cellular Ca2+Pools by a Method Based on Digitonin Permeabilization

Treatment of45Ca2+-loaded GH3pituitary cells with various concentrations of digitonin revealed discrete pools (I and II) of cellular45Ca2+defined by differing detergent sensitivities. Markers for cytosol and intracellular organelles indicated that the two45Ca2+pools were correlated with the two majo...

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Published inProceedings of the National Academy of Sciences - PNAS Vol. 79; no. 20; pp. 6294 - 6298
Main Authors Ronning, Susan A., Heatley, Gregg A., Thomas F. J. Martin
Format Journal Article
LanguageEnglish
Published National Academy of Sciences of the United States of America 15.10.1982
Subjects
Cell biology
Cell membranes
Cells
Chemical suspensions
Cultured cells
Detergents
Endoplasmic reticulum
Hormones
Mitochondria
Organelles
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Summary:Treatment of45Ca2+-loaded GH3pituitary cells with various concentrations of digitonin revealed discrete pools (I and II) of cellular45Ca2+defined by differing detergent sensitivities. Markers for cytosol and intracellular organelles indicated that the two45Ca2+pools were correlated with the two major cellular Ca2+-sequestering organelles, endoplasmic reticulum (I) and mitochondria (II). Studies with various inhibitors were consistent with these assignments. Mitochondrial uncouplers preferentially depleted45Ca2+pool II while trifluoperazine selectively depleted45Ca2+pool I. Control experiments indicated that translocation of in situ organellar45Ca2+during and after permeabilization was negligible. We used the digitonin-permeabilization method to examine the effect of thyrotropin-releasing hormone (TRH) treatment on intracellular Ca2+pools of GH3pituitary cells. TRH was found to rapidly deplete both endoplasmic reticulum and mitochondrial exchangeable Ca2+by 25-30%. The45Ca2+loss from both pools was maximal by 1 min after TRH addition and was followed by a recovery phase; mitochondrial45Ca2+content returned to control levels by 30 min. Previous treatment of cells with the mitochondrial uncoupler carbonyl cyanide p-trifluoromethoxyphenylhydrazone blocked TRH-induced45Ca2+efflux from mitochondria, while previous treatment with valinomycin, an agent that depleted both45Ca2+pools, blocked any additional effect of TRH on these pools. We conclude that TRH rapidly promotes a net loss of exchangeable Ca2+from GH3cells as a result of hormone-induced mobilization of Ca2+from endoplasmic reticulum and mitochondria.
ISSN:0027-8424
1091-6490