Inhibition of luteinizing-hormone exocytosis by guanosine 5'-[gamma-thio]triphosphate reveals involvement of a GTP-binding protein distal to second-messenger generation.
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.
van der Merwe
P A
PA
Department of Chemical Pathology, University of Cape Town Medical School, South Africa.
Millar
R P
RP
Wakefield
I K
IK
Davidson
J S
JS
D015056:1-Methyl-3-isobutylxanthine / Q000494:pharmacology; D000255:Adenosine Triphosphate / Q000031:analogs & derivatives / Q000494:pharmacology; D000818:Animals; D002118:Calcium / Q000494:pharmacology; D002463:Cell Membrane Permeability; D002478:Cells, Cultured; D000242:Cyclic AMP / Q000494:pharmacology; D004533:Egtazic Acid / Q000494:pharmacology; D005089:Exocytosis / Q000187:drug effects; D019204:GTP-Binding Proteins / Q000502:physiology*; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000494:pharmacology*; D006165:Guanylyl Imidodiphosphate / Q000494:pharmacology; D007700:Kinetics; D007986:Luteinizing Hormone / Q000378:metabolism*; D010903:Pituitary Gland, Anterior / Q000187:drug effects / Q000378:metabolism*; D015290:Second Messenger Systems; D012756:Sheep; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology
D006165:Guanylyl Imidodiphosphate; C022571:adenosine 5'-O-(3-thiotriphosphate); D016244:Guanosine 5'-O-(3-Thiotriphosphate); D004533:Egtazic Acid; D000255:Adenosine Triphosphate; D007986:Luteinizing Hormone; D000242:Cyclic AMP; D019204:GTP-Binding Proteins; D013755:Tetradecanoylphorbol Acetate; D002118:Calcium; D015056:1-Methyl-3-isobutylxanthine