• J D Marsh and T W Smith.
• Mol. Pharmacol. 1985 Jan 1; 27 (1): 10-8.

AbstractTo determine if the two-state, guanine nucleotide-modulated beta-adrenergic receptor model elucidated in erythrocyte membranes accurately describes hormone binding to intact heart cells, and to determine the relationship of agonist binding to physiologic contractile response, we studied beta-adrenergic antagonist and agonist binding to intact cultured heart cells and homogenates of these cells from embryonic chick ventricle and related the binding observations to alterations in amplitude of contraction of intact cells under identical conditions. The levo isomer of the beta-adrenergic antagonist pindolol was radioiodinated, purified, and utilized to characterize the beta-adrenergic receptor in intact, beating heart cells under physiologic conditions. Computer analysis of iodopindolol-binding isotherms revealed a KD = 22 +/- 3 pM with Bmax = 10.3 fmol/mg of protein in intact cells; in homogenates of cells, the KD was 39 +/- 12 pM in the absence of exogenous guanine nucleotides and 19 +/- 7 pM in their presence. Estimation of the dissociation constant for iodopindolol binding to intact cells by kinetic methods yielded KD = 64 pM. Binding was stereospecific, saturable, and identified a beta 1-adrenergic receptor. Computer modeling of agonist competition curves indicated a single receptor state in intact cells with KD = 0.28 microM for isoproterenol. However, in cell homogenates, two receptor states for agonists were identified with the high affinity state of the receptor having a KD = 3 nM. Addition of guanine nucleotides to the cell homogenate reverted the receptor to a single state similar to that in intact cells. Under nonequilibrium binding conditions, a high affinity state for agonist was detected in intact cells with IC50 = 1.1 nM. The EC50 for isoproterenol-enhanced contractility was 6 nM and EC50 for cAMP response was 4 nM. At the isoproterenol concentration causing 50% maximal inotropic response, 67% occupancy of high affinity receptors occurs. Thus, there is a close relationship between high affinity receptor occupancy and augmentation of contractility in intact cells. These findings support the view that agonist interaction with the guanine nucleotide-sensitive, high affinity receptor state initiates the physiologic response of myocardial tissue to beta-adrenergic agonists.

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