Rapid kinetics of .alpha.2-adrenergic inhibition of adenylate cyclase. Evidence for a distal rate-limiting step

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Abstract
Activation and inhibition of adenylate cyclase in the presence of GTP, the natural guanine nucleotide regulator, are too fast to study by standard biochemical methods. In order to identify the rate-limiting steps in adenylate cyclase regulation, we measured the kinetics of stimulation and inhibition of the enzyme on a subsecond to second time scale using a novel rapid-mix quench technique. Even using our rapid-mix quench method, activation by PGE1 and forskolin was instantanenous (cAMP accumulation was linear between 0.5 and 30 s). In contrast, we found a lag period of 1.2-10 s for epinephrine-mediated inhibition. The length of the lag depended on the concentration of GTP and monovalent cations present. In the absence of NaCl, the rate constant for the onset of inhibition (kinh) increased only slightly with GTP concentration saturating at a value of 0.16 s-1 (t1/2 4.3 s) at 1 .mu.M GTP. In the presence of 100 mM NaCl, kinh was strongly dependent on GTP concentration, reaching a maximum value of 0.57 s-1 (t1/2 1.2 s) at 100 .mu.M GTP. Thus, activation of both Gi and Gs in intact platelet membranes is much faster (t1/2 < 5 s) than previously reported for reconstituted systems. Also, the strong dependence of the rate of adenylate cyclase inhibition on GTP concentration implies that the rate-limiting step in inhibition is distal to GTP binding. The effect of NaCl to increase the maximal rate of inhibition is specific for sodium since KCl has no effect on kinh. In contrast, both sodium and potassium chloride increased the steady-state EC50 for epinephrine and GTP while neither affected the maximum percentage inhibition. We used these kinetic and steady-state data to these several models of .alpha.2-receptor and Gi-mediated adenylate cyclase inhibition in a quantitative manner. Two distinct models gave similar fits of experimental data in the absence of NaCl. In one, the rate-limiting step is GDP release while in the other, activation of the GTP-liganded G protein is limiting. Only the latter model also explains our prior observation that preincubation of platelet membranes with .alpha.2 agonist in the absence of GTP increases the potency for inhibition [Thomsen et al. (1988) Mol. Pharmacol. 34, 814-822]. Thus, both in the presence and in the absence of sodium chloride, the conformational change of GTP-liganded Gi rather than GDP release is rate limiting. According to this model, sodium chloride increases the rate of three reaction steps: (1) agonist dissociation from receptor; (2) GTP dissociation from Gi; and (3) the rate-limiting conformation change of GTP-liganded G protein. The rapid kinetic studies reported here provide new information regarding regulation of adenylate cyclase by sodium chloride and the natural nucleotide regulator, GTP.

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