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  • Title: Mechanisms underlying the stimulation of PTH release by GppNHp in permeabilized bovine parathyroid cells.
    Author: Leboff MS, Oetting M, Brown EM.
    Journal: J Bone Miner Res; 1990 Jul; 5(7):683-9. PubMed ID: 2168660.
    Abstract:
    We examined changes in cAMP and inositol phosphate metabolism to assess the contribution of the guanine nucleotide regulatory (G) protein(s) regulating adenylate cyclase and phospholipase C in mediating the stimulatory effects of GppNHp on PTH release from permeabilized bovine parathyroid cells. To examine the role of Gs, the G protein stimulating adenylate cyclase, and cAMP on PTH release, permeabilized cells were incubated with either GppNHp or isoproterenol, and the effects of these agents on PTH release and cellular cAMP content were determined by RIA. To study the effects of GppNHp on inositol phosphate accumulation, permeabilized cells prelabeled with [3H]inositol were exposed to GppNHp, and inositol phosphates were measured using ion-exchange chromatography. These studies revealed that isoproterenol produced a dose-dependent increment in cAMP content in permeabilized cells with no significant effect on PTH release. Conversely, GppNHp rapidly and markedly elevated PTH release with a smaller and delayed rise in cAMP content. GppNHp- also promoted a dose-dependent increase in inositol monophosphate (IP), inositol bisphosphate (IP2), and inositol trisphosphate (IP3) accumulation, suggesting activation of phosphoinositide hydrolysis. Addition of dioctanoylglycerol, however, a synthetic diacylglycerol (DG) that activates protein kinase C, produced a much smaller increment in PTH release than GppNHp. Moreover, reducing the free calcium concentration to less than 10(-9) M by adding 10 mM EGTA to the permeabilization medium dissociated the effects of GppNHp and DG on secretion, increasing GppNHp-stimulated PTH release while reducing PTH secretion evoked by DG.(ABSTRACT TRUNCATED AT 250 WORDS)
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