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Fluctuation of cyclic AMP-binding site occupancy as a consequence of hormone-dependent cyclic AMP formation in rat renal cortex
The initial event in cyclic AMP action is thought to be binding to the regulatory subunit and subsequent activation of protein kinases. As a measure of hormone action distal to cyclic AMP generation, cyclic AMP binding to postmicrosomal supernatant fraction from rat renal cortex was studied. Renal cortical slices were incubated under a variety of conditions in Krebs-Ringer bicarbonate buffer and cyclic AMP binding by 100,000 x g supernatant fraction was determined by trapping [3H]-cAMP - protein complexes on nitrose cellulose filters. Binding characteristics were evaluated by the method of Scatchard. After correcting for nonspecific binding, results were consistent with binding by a single class of sites with an apparent binding constant of 39 nH. There were 1.56 + .20 x 1012 sites per milligram protein and treatment with PTH or PGE1 in doses insufficient to cause significant elevation of cyclic AMP content, decreased these unoccupied binding sites. Affinity for cyclic AMP was the same regardless of the degree of binding site depletion or mode of cyclic AMP elevation. Maximal unoccupied binding site depletion was not increased by additional agonist, despite further increases in cyclic AMP formation. Fluctuations in physiologic concentrations of hormone may therefore regulate cellular activity by inducing only modest changes in intracellular cyclic AMP. Time course studies showed that unoccupied binding site number follows changes in cyclic AMP concentration with a nadir at 10 minutes and restoration to basal levels by 45 minutes. At maximally effective concentrations of PTH and PGe1 cyclic AMP formation was additive, but unoccupied binding site depletion was not greater than that seen with either agonist alone. Persistence of 35% of unoccupied binding sites despite maximal stimulation of cyclic AMP by hormone suggests a physiological role in the binding equilibrium. Depletion of cyclic AMP binding sites appears to fluctuate as a direct consequence of elevations in ambient cyclic AMP concentration and does not appear to be independently regulated.