title: The role of Gi and the membrane-fluidizing agent benzyl alcohol in modulating the hysteretic activation of human platelet adenylate cyclase by guanylyl 5'-imidodiphosphate.
abstract: The non-hydrolysable GTP analogue guanylyl 5'-imidodiphosphate (p[NH]ppG) elicited a profound increase in the adenylate cyclase activity of human platelets. This occurred after a well-defined lag period of around 6 min, whereupon an enhanced steady-state rate was evident. The duration of the lag period was unchanged over a range of concentrations of p[NH]ppG which gave very different steady-state rates of adenylate cyclase activity. Prior activation of the stimulatory G-protein Gs by cholera-toxin pre-treatment abolished the lag period and elicited a small increase in the steady-state rate. Manipulating function of the inhibitory G-protein Gi also led to profound changes in the lag periods. Thus marked decreases in the lag were seen (approximately 70-81%) when Gi function was ablated through pre-treatment of platelet membranes with pertussis toxin, or by using elevated (25 mM) Mg2+ levels in the assay, or when Mg2+ was replaced by 5 mM Mn2+ in the assay. In contrast with this, potentiation of Gi function led to an increase in the lag period, as seen under conditions of agonist occupancy of inhibitory alpha 2-adrenoceptors (increase approximately 74%) or with the addition of 100 mM NaCl to the assays (increase approximately 44%). The local anaesthetic and membrane-fluidizing agent benzyl alcohol elicited both a profound decrease (around 70% at 80 mM) in the p[NH]ppG-induced lag period and a marked augmentation (around 5-fold) in the steady-state adenylate cyclase activity. When adenylate cyclase assays were done at 35 degrees C instead of 25 degrees C, then the lag period for activation by p[NH]ppG was decreased by around 33% and the steady-state rate increased by around 3-fold. At 35 degrees C, the addition of benzyl alcohol led to the apparent abolition of the lag period for p[NH]ppG activation of adenylate cyclase and amplified the steady-state rate by only around 2.2-fold. It is shown that Gi plays a fundamental role in determining the rate of activation of Gs. The proposal is formulated that such an action may be mediated through the release of beta gamma-subunits. Thus beta gamma-subunit dissociation is proposed as providing the rate-limiting step in Gi activation.
authors: Department of Biochemistry, University of Glasgow, Scotland, U.K.
authors: S
authors: S
authors: Spence
authors: M D
authors: MD
authors: Houslay
mesh_terms: D000246:Adenosine Diphosphate Ribose / Q000378:metabolism; D037361:Adenylate Cyclase Toxin; D000262:Adenylyl Cyclases / Q000097:blood*; D019905:Benzyl Alcohol; D001592:Benzyl Alcohols / Q000494:pharmacology*; D001792:Blood Platelets / Q000187:drug effects / Q000201:enzymology*; D002462:Cell Membrane / Q000187:drug effects / Q000201:enzymology; D004789:Enzyme Activation / Q000187:drug effects; D019204:GTP-Binding Proteins / Q000502:physiology*; D006165:Guanylyl Imidodiphosphate / Q000494:pharmacology*; D006801:Humans; D008274:Magnesium / Q000494:pharmacology; D008560:Membrane Fluidity / Q000187:drug effects*; D037342:Pertussis Toxin; D012965:Sodium Chloride / Q000494:pharmacology; D010566:Virulence Factors, Bordetella / Q000494:pharmacology
chemical_list: D037361:Adenylate Cyclase Toxin; D001592:Benzyl Alcohols; D010566:Virulence Factors, Bordetella; D000246:Adenosine Diphosphate Ribose; D006165:Guanylyl Imidodiphosphate; D012965:Sodium Chloride; D037342:Pertussis Toxin; D019204:GTP-Binding Proteins; D000262:Adenylyl Cyclases; D008274:Magnesium; D019905:Benzyl Alcohol