Treatment of 45Ca24-loaded GH3 pituitary 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 two 45Ca2+ 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 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. We used the digitonin-permeabilization method to examine the effect of thyrotropin-releasing hormone (TRH) treatment on intracellular Ca2+ pools of GH3 pituitary cells. TRH was found to rapidly deplete 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-trifluoromethoxyphenylhydrazone 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. We conclude that 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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GH pituitary cells have been widely utilized for studies of hormone response mechanisms. Studies reported here were motivated by the desirability of isolating characterized GH clones defective in cyclic AMP synthesis or action. Spontaneously occurring GH1 cell variants resistant to the growth-inhibitory effects of cyclic AMP analogs were isolated. Characterization of four variants showed that these were deficient in adenosine kinase and had acquired resistance to the cytotoxic effects of purine nucleoside derivatives formed in the culture medium. A second-stage selection was undertaken with mutagenized adenosine kinase-deficient cells. One 8 Br cAMP-resistant variant was found to have normal cyclic AMP-dependent protein kinase activity but exhibited altered adenylate cyclase activity. Activation of cyclase activity by fluoride, guanyl nucleotides, cholera toxin, and hormone (VIP) was subnormal in the variant. Mn-dependent cyclase activity was also subnormal, suggesting that the 8 Br cAMP-resistant variant may have a deficiency in the catalytic moiety of adenylate cyclase. Surprisingly, adenosine 3':5'-monophosphate and 5'-monophosphate derivatives were found to be equally potent in growth-inhibiting adenosine kinase-deficient cells. Cross-resistance to 8 Br AMP was observed in the 8 Br cAMP-resistant variant. We conclude that cyclic AMP derivatives inhibit growth of GH cells by an unanticipated mechanism that is, nonetheless, related to endogenous cyclic AMP synthesis.

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