Thyrotropin-releasing hormone mobilizes Ca2+ from endoplasmic reticulum and mitochondria of GH3 pituitary cells: characterization of cellular Ca2+ pools by a method based on digitonin permeabilization.

Abstract

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.

Nimodipine block of calcium channels in rat anterior pituitary cells.

“…In summary, nimodipine will have little effect on stimulus-secretion coupling in healthy anterior pituitary cells for three reasons: (1) nimodipine is a very weak inhibitor of Ca entry through the transient Ca channels; (2) high-affinity block of the slowly inactivating Ca channels does not occur to a significant extent under physiological conditions; (3) nimodipine does not inhibit the intracellular release of Ca (as evidenced by the lack of effect on TRH-induced secretion, which involves release of cell Ca in response to an increase of inositol triphosphate (Ronning, Heatley & Martin, 1982;Gershengorn, Geras, Spina, Purello & Rebeechi, 1984)). …”

Requirements for hormone release from permeabilized nerve endings isolated from the rat neurohypophysis.

“…In a study on other tissues, Elias, Goerke, Friend & Brown (1978) showed that digitonin selectively permeabilizes cell membranes and that this is likely to be due to their different cholesterol content compared to intracellular organelles (plasma membrane > endoplasmic reticulum > mitochondria). Beside other uses, digitonin has been utilized to analyse the mechanism of secretion in different types of cells (Ronning, Heatley & Martin, 1982;Dunn & Holz, 1983;Wilson & Kirschner, 1983).…”

The tilapia prolactin cell: A model for stimulus-secretion coupling

“…Rubin 1982). In PRL cells including those of teleost fish, Ca + + influx may be derived from action potentials which involve a Ca + + component and whose frequency can be increased or decreased by stimulators or inhibitors of PRL release (Taraskevich and Douglas 1978;Ronning et al 1982).…”