Evidence for receptor-regulated phosphotransfer reactions involved in activation of the adenylate cyclase inhibitory G protein in human platelet membranes.
The activity of the adenylate cyclase inhibitory guanine-nucleotide-binding regulatory protein (Gi), measured as inhibition of forskolin-stimulated cyclic AMP formation, and its regulation by various nucleotides and the inhibitory alpha 2-adrenoreceptor agonist epinephrine was studied in membranes of human platelets. When adenylate cyclase activity was measured with ATP as substrate and in the absence of a nucleoside-triphosphate-regenerating system, GTP (0.1-10 microM) by itself potently and efficiently inhibited the enzyme. GDP was almost as potent and as effective as GTP. In the additional presence of epinephrine, the potencies of both GTP and GDP were increased about threefold, while maximal inhibition by these nucleotides was only slightly increased by the receptor agonist. In contrast to GTP and GDP, the metabolically stable GDP analog, guanosine 5'-[beta-thio]diphosphate, had only a very small effect, suggesting that GDP but not its stable analog is converted to the active GTP. Addition of UDP (1 mM), used to block the GDP to GTP conversion reaction, completely suppressed the inhibitory effect of GDP, while that caused by GTP was not affected. Most important, the inhibitory receptor agonist epinephrine counteracted the suppressive effect of UDP on GDP's action, suggesting that, while UDP inhibits the formation of GTP from GDP, the activated receptor stimulates this conversion reaction. In the presence of a complete nucleoside-triphosphate-regenerating system, which by itself had no influence on control forskolin-stimulated adenylate cyclase activity, GTP alone, at concentrations up to 10 microM, did not decrease enzyme activity, but required the presence of an inhibitory receptor agonist (epinephrine) to activate the Gi protein. Addition of the regenerating system creatine phosphate plus creatine kinase not only abolished adenylate cyclase inhibition by GTP alone, but also largely reduced both the potency and efficiency of epinephrine to activate the Gi protein in the presence of GTP. Furthermore, the nucleoside-triphosphate-regenerating system also largely delayed the onset of adenylate cyclase inhibition by the GTP analog, guanosine-5'-[beta-thio]triphosphate (10 nM), which was accelerated by epinephrine, and it also decreased the final enzyme inhibition caused by this GTP analog.(ABSTRACT TRUNCATED AT 400 WORDS)
Jakobs
K H
KH
Pharmakologisches Institut der Universität Heidelberg, Federal Republic of Germany.
Wieland
T
T
D000067956:Adenylyl Cyclase Inhibitors; D000262:Adenylyl Cyclases / Q000378:metabolism*; D001792:Blood Platelets / Q000187:drug effects / Q000201:enzymology / Q000378:metabolism*; D002462:Cell Membrane / Q000201:enzymology / Q000378:metabolism; D005576:Colforsin / Q000037:antagonists & inhibitors / Q000494:pharmacology; D004789:Enzyme Activation / Q000187:drug effects; D004837:Epinephrine / Q000494:pharmacology*; D019204:GTP-Binding Proteins / Q000378:metabolism*; D006153:Guanosine Diphosphate / Q000378:metabolism; D006160:Guanosine Triphosphate / Q000096:biosynthesis / Q000494:pharmacology; D006801:Humans; D012965:Sodium Chloride / Q000494:pharmacology
D000067956:Adenylyl Cyclase Inhibitors; D006153:Guanosine Diphosphate; D005576:Colforsin; D012965:Sodium Chloride; D006160:Guanosine Triphosphate; D019204:GTP-Binding Proteins; D000262:Adenylyl Cyclases; D004837:Epinephrine