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Dual inhibitory and stimulatory actions of guanine nucleotides on luteinizing-hormone (LH) exocytosis were observed in primary sheep gonadotropes permeabilized with staphylococcal alpha-toxin. At resting cytosolic [Ca2+]free (pCa 7), 5'-[gamma-thio]triphosphate (GTP[S]) and guanosine 5'-[beta gamma-imido]triphosphate (p[NH]ppG) stimulated rapid LH exocytosis, which was maximal between 5 and 10 min. GTP[S] and p[NH]ppG had similar potencies (50% of maximum effect at 20-50 microM), but the effect of p[NH]ppG was more prolonged. Experiments carried out in the presence of saturating concentrations of phorbol 12-myristate 13-acetate (PMA), or in PMA-desensitized cells, suggested that stimulation by p[NH]ppG is mediated by a mechanism additional to protein kinase C (PKC) activation. Furthermore, p[NH]ppG stimulated LH exocytosis in the presence of saturating cyclic AMP (cAMP) concentrations, although its effect was less than additive. However, when both PMA and cAMP were present, p[NH]ppG did not stimulate a further increase in the rate of LH exocytosis. In contrast, pretreatment of cells with GTP[S] at low [Ca2+]free markedly inhibited subsequent responses to Ca2+, cAMP, PMA, and cAMP plus PMA. This inhibitory effect required lower GTP[S] concentrations than the stimulatory effect (50% inhibition at 1-10 microM), and was not observed with p[NH]ppG. A similar inhibition was observed with adenosine 5'-[gamma-thio]triphosphate, probably by its conversion into GTP[S]. These results suggest that the stimulatory actions of guanine nucleotides can be accounted for by the combined activation of PKC and generation of cAMP, resulting from activation of conventional signal-transducing GTP-binding proteins. The inhibitory effect of GTP[S] can be clearly distinguished and indicates the involvement of a distinct GTP-binding protein in exocytosis at a site distal to second-messenger generation.

Type

Journal article

Journal

Biochem J

Publication Date

15/04/1991

Volume

275 ( Pt 2)

Pages

399 - 405

Keywords

1-Methyl-3-isobutylxanthine, Adenosine Triphosphate, Animals, Calcium, Cell Membrane Permeability, Cells, Cultured, Cyclic AMP, Egtazic Acid, Exocytosis, GTP-Binding Proteins, Guanosine 5'-O-(3-Thiotriphosphate), Guanylyl Imidodiphosphate, Kinetics, Luteinizing Hormone, Pituitary Gland, Anterior, Second Messenger Systems, Sheep, Tetradecanoylphorbol Acetate