Rapid recovery of cardiac beta-adrenergic receptors after isoproterenol-induced "down"-regulation.

Abstract

Desensitization of beta-receptor-linked adenylate cyclase occurs after prolonged occupancy of the beta-receptors by their agonists. We have followed the development and recovery from "down"-regulation of beta-receptors in enzymatically dissociated cardiac myocytes by using the hydrophilic antagonist [3H]-CGP-12177 to identify surface-bound beta-receptors. After in vitro incubation with (-)-isoproterenol, almost 50% of the beta-receptors are lost within 10 minutes. Isoproterenol-mediated cyclic adenosine monophosphate accumulation by isolated myocytes was also decreased after a 15-minute preincubation with isoproterenol. "Lost" beta-receptors can, however, be recovered when isoproterenol-pretreated, washed cardiac myocytes are incubated at 37 degrees C, 85 +/- 7% of the lost beta-receptors have returned to the cell surface after 20 minutes of incubation. The requirements for such recycling were investigated. Receptor recovery does not depend on de novo protein synthesis, since it is unaffected by prior exposure to cycloheximide. It is, however, dependent on cellular energy, because it is prevented by adenosine triphosphate depletion and involves a lysosomal step since it is inhibited by the lysomotropic agent, chloroquine. In addition, the Golgi apparatus and the microtubules are involved in the beta-receptor recycling to the cell surface, as evidenced by the inhibitory effects of monensin and colchicine, respectively. The mechanism of isoproterenol-induced down-regulation of cardiac beta-receptors involves a rapid, reversible cycling to and from the cytosol and the cell membrane. This intracellular receptor traffic is energy dependent, requires several structures, including lysosomes and microtubules, and may be modified by pathological processes involving the heart.

Demonstration of the suitability of CGP 12177 for in vivo studies of β‐adrenoceptors

“…The data were analysed by the curve fitting routines of the Ligand programme (Munson & Rodbard, 1980 (Limas & Limas, 1984) and cardiac tissue preparations in vitro (Endoh et al, 1989) although higher values (30%) have been observed in myocardial tissue slices (Watson-Wright et al, 1989). Recent studies in anaesthetized rats have given results for uptake of (-)-[3H]-CGP 12177 in lung and heart and for (Kawai et al, 1986), muscle (Watson-Wright & Wilkinson, 1986, fat (Lacasa et al, 1985) and proximal tubules in kidney (Morgunov & Hirsch, 1990).…”

.BETA.-Adrenergic receptor adaptation after an acute exercise in rat myocardium.

“…Acute exercise-induced up-regulation of human lymphocyte f3-AR is also followed by a return to pre-exercise level after supine rest for 25-60 min (BUTLER et al , 1983;BRODDE et al, 1984BRODDE et al, , 1987BRODDE et al, , 1988MAKI et a1.,1988). Similarly, in in vitro study , LIMAS and LIMAS (1984) reported that cell surface fl-AR loss of intact cardiac myocytes, induced by preincubation with isoproterenol, could be almost restored to control level at only 20 min after removal of isoproterenol from incubation medium in rat . Thus redistribution of fl-AR appears to be quick.…”

“…In general, administration of catecholamine(s) causes the rapid sequestration or internalization of /3-AR into light vesicles where the receptors are uncoupled from adenylate cyclase (HARDEN, 1983;LIMAS and LIMAS, 1984;STILES et al, 1984;SIBLEY and LEFKOWITZ, 1985;MAISEL et al, 1987). Thus, an acute exercise-induced up-regulation of /3-AR is an opposite phenomenon to an agonist-induced redistribution of /3-AR.…”

Effects of calcium antagonists on beta-receptors of cultured cardiac myocytes isolated from neonatal rat ventricle.

“…That is, when the cells were preconfluent, cycloheximide could not inhibit the recovery process from down-regulation, but when the cells were confluent, cycloheximide did inhibit the recovery process of 13-receptors. Hence, no definite conclusion regarding the role of protein synthesis in the recovery process of 13-receptors has been established until now. 20,[27][28][29] In our experiments, cycloheximide, an inhibitor of protein synthesis, failed to suppress the action of verapamil, suggesting little involvement of protein synthesis in the drug action. This result is consistent with the results of Hertel and Staehelin29 and Limas and Limas20 who demonstrated the increase in 1-receptors under conditions of no protein synthesis.…”

“…The contribution of microtubules in regulating receptor recycling was also suggested by other investigators. 20,27 One may argue that the following two possibilities should also be considered in the mechanism of action of calcium antagonists. One is the possibility of the block of tonic internalization of [3-receptors similar to [3-blockade.24 The other is that modulation of membrane fluidity through the interaction with membrane lipid30'31 may regulate the increment of assayable [3-receptors.…”