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JBC, Vol. 252, Issue 3, 1041-1050, Feb, 1977

Cardiac adenosine 3':5'-monophosphate. Free and bound forms in the isolated rat atrium

W. L. Terasaki and G. Brooker

Adenosine 3':5'-monophosphate (cyclic AMP), a mediator of hormone action in a variety of tissues, has been measured in its free and bound forms in intact cardiac tissue. We have used a rapid high dilution technique which involves tissue homogenization, subcellular fractionation, and separation of bound from free cyclic AMP by Millopore filtration. The precision of this method is dependent upon minimization of binding and dissociation of cyclic AMP that occur during the preparation and handling of tissue homogenates. In each experiment, a tracer of cyclic [3H]AMP prebound to isolated cardiac binding protein was freed of unbound cyclic [3H]AMP by Sephadex gel filtration and added to the tissue just prior to homogenization in cold EDTA buffer. This tracer was therefore treated identically to the sample through all subsequent dilution, fractionation, and filtration procedures, and provided an acurate internal monitor for total cyclic AMP dissociation during the course of the free-bound determination. Each tissue sample was then individually corrected for dissociation. Rapid dilution to produce a 1:1000 homogenate was found to lower endogenous cyclic AMP levels sufficiently to make binding (or rebinding) during the procedure negligible (less than 5%). Spontaneously beating rat right atria (controls) contained 5.96 +/- 0.28 pmol of cyclic AMP/mg of protein (n = 19) of which 41 and 14% were bound to soluble and particulate proteins, respectively. The remaining cyclic AMP was free. Pretreatment of the tissue with 1 muM isoproterenol (30 s at 30 degrees) increased both the bound and free forms of cyclic AMP (n = 8). While free cyclic AMP increased 420% with the catecholamine, the bound forms increased 240% (soluble) and 60% (particulate). Similar results were obtained when atria (n = 6) were treated with the phosphodiesterase inhibitor, methylisobutylxanthine (0.5 mM, 10 min at 30 degrees). When both agents were used together, cyclic AMP bound to soluble proteins was elevated 4-fold over control while free cyclic AMP increased 27-fold (n = 7), indicating saturation of the soluble sites. It could be calculated that less than one-third of these sites are occupied in the unstimulated cell. These sites may represent the R subunit of cyclic AMP-dependent protein kinase. The data suggest that half-maximal binding in vivo occurs at an intracellular free cyclic AMP concentration of about 1 muM.
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