Identification of α1-adrenergic receptors on sarcolemma from normal subjects and patients with idiopathic dilated cardiomyopathy: Characteristics and linkage to GTP-binding protein

Abstract

Discontinuous density sucrose gradient centrifugation was used to isolate membrane vescicles from the left ventricle of three normal subjects (one prospective organ donor and two traffic victims whose hearts were obtained 1 hour after death) and nine patients undergoing cardiac transplantation as a consequence of idiopathic dilated cardiomyopathy. Sarcolemma-enriched subcellular fractions, detected in the interface between 8.55% and 25% sucrose, were identified by the increased activity of Na+, K+-ATPase and by enrichment in β-adrenergic receptor density. The density of β-adrenergic receptors was lower in vescicles from diseased hearts (610 ± 71 fmol/mg protein) than in vesicles from normal hearts (1,410 ± 226 fmol/mg protein; p < 0.01) α1-Adrenergic receptors were identified in these membrane vescicles by [3H]prazosin binding. Specific binding of [3H]prazosin was about 50% of the total binding at 1 nM, and α1-adrenergic binding sites were saturable at approximately 3 nM. Scatchard analysis revealed 58 ± 5 fmol/mg protein (K(D) = 0.90 ± 0.08 nM) in pathological hearts and 30 ± 5 fmol/mg protein (K(D) = 0.90 ± 0.03 nM) in normal hearts (p < 0.01). The displacement curve of (-)-norepinephrine in membrane vescicles from normal hearts delineated one subpopulation of α1-adrenergic receptors; the addition of 0.1 mM GTP did not cause right shift. In membrane vesicles from diseased heart, the displacement curve of (-)-norepinephrine disclosed two subpopulations of α1-adrenergic receptors. A right shift that occurred after addition of GTP showed that in this case α1-adrenergic receptors were functionally coupled with GTP-binding protein. This study demonstrated the presence of α1-adrenergic receptors in sarcolemma-enriched subcellular fractions from both normal and idiopathic dilated cardiomyopathic hearts. Only α1-adrenergic receptors from pathological hearts were coupled to GTP-binding protein.