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Phosphatidylinositol Depletion in GH₃ Rat Pituitary Cells Inhibits Sustained Responses to Thyrotropin-Releasing Hormone. Reversal with myo-inositol

Division of Endocrinology and Metabolism, Department of Medicine, Cornell University Medical College and The New York Hospital New York, New York 10021

Address requests for reprints to: Marvin C. Gershengorn, M.D., Room A328, Cornell University Medical College, 1300 York Avenue, New York, NY 10021.

Abstract

TRH stimulation of rat pituitary (GH₃) cells causes biphasic changes in cytoplasmic free Ca²⁺ concentration ([Ca²⁺]_i) and PRL secretion. It has been proposed, based primarily on indirect evidence, that the first phase effects are mediated by inositol 1,4,5-trisphosphate, which releases Ca²⁺ from cellular stores, and the sustained effects are mediated by 1,2-diacylglycerol, which activates protein kinase C. To determine more directly if inositol lipid hydrolysis leading to protein kinase C activation is involved in the sustained effects of TRH, GH₃ cells were depleted of phosphatidylinositol (Ptdlns) by prestimulation and incubation in myo-inositol-free, Li⁺-containing medium. Cells depleted of Ptdlns (to 53 ± 3.2% of control) had unchanged Ptdlns 4,5-bisphosphate content, and responded to TRH with a rapid elevation of inositol trisphosphate, and a first phase (or burst) elevation of [Ca²⁺]_i and PRL secretion that was not different from that found in control cells. In contrast, in Ptdlns-depleted cells, the prolonged generation of inositol phosphates, which are produced in equimolar amounts with 1,2-diacylglycerol, caused by TRH was virtually abolished, and the second phase (or sustained) elevation of [Ca²⁺]_i and PRL secretion were inhibited by 50% and 40%, respectively. The inhibition of both sustained effects was reversed by adding 100 mM myo-inositol to the medium, which allowed for synthesis of Ptdlns. Last, in cells in which protein kinase C was down-regulated by pretreatment with a phorbol ester, the sustained effects of TRH were inhibited also. These data provide the first direct evidence in GH₃ cells that changes in Ptdlns content can affect signal transduction by TRH and add support to the hypothesis that the sustained effects of TRH are mediated by inositol lipid hydrolysis leading to activation of protein kinase C.

FOOTNOTES

This work was supported by NIH Grant DK-33468.

Received for publication June 22, 1987. Accepted for publication September 5, 1987.

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