Inhibition of luteinizing-hormone exocytosis by guanosine 5'-[gamma-thio]triphosphate reveals involvement of a GTP-binding protein distal to second-messenger generation.

Journal article

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.

Authors: Van Der Merwe, P; Millar, R; Wakefield, I; Davidson, J

• Journal: Biochemical journal
• Volume: 275 ( Pt 2)
• Pages: 399-405
• Publication date: 1991-04-01
• EISSN: 1470-8728
• ISSN: 0264-6021
• Language: English
• Keywords: Adenosine Triphosphate; Kinetics; Tetradecanoylphorbol Acetate; Second Messenger Systems; Luteinizing Hormone; Calcium; Exocytosis; Animals; Cells, Cultured; Egtazic Acid; Cyclic AMP; Guanosine 5'-O-(3-Thiotriphosphate); Sheep; GTP-Binding Proteins; Guanylyl Imidodiphosphate; Pituitary Gland, Anterior; Cell Membrane Permeability; 1-Methyl-3-isobutylxanthine