Rapid reciprocal changes in adrenergic receptors in intact isolated hepatocytes during primary cell culture
Schwarz, K.R.; Lanier, S.M.; Carter, E.A.; Homcy, C.J.; Graham, R.M.
Molecular Pharmacology 27(2): 200-209
1985
ISSN/ISBN: 0026-895X PMID: 3969069 Document Number: 246735
In hepatocytes freshly isolated from adult female rat livers, catecholamine-stimulated glycogenolysis was mediated predominantly by .alpha.1-adrenergic receptors, and to only a minimal extent by .beta.2 receptors. Primary cell culture of these hepatocytes resulted in a switch in the adrenergic control of glycogenolysis from an .alpha.1 to a predominant .beta.2 type of response. To investigate whether this switch was due to an alteration in the plasma membrane receptor composition, .alpha.1 and .beta.2-adrenergic receptors were characterized in both freshly isolated and cultured hepatocytes, using radioligand-binding techniques. Binding of the selective .alpha.1-adrenergic antagonist = 93,000 .+-. 5500 sites/cell) and pindolol (Kd = 66 .+-. 6 pM; Bmax = 2000 .+-. 700 sites/cell). In intact hepatocytes and in membranes prepared from these hepatocytes, competitive inhibition curves revealed the coexistence of 2 different sites with high and low affinities for agonists at both .alpha.1- and .beta.2-adrenergic receptors. When isolated hepatocytes were kept in monolayer cell culture for up to 72 h, the switch in adrenergic control of glycogenolysis (phosphorylase a activation) from an .alpha. to a .beta. pathway was confirmed and was associated with a progressive decrease in the number of .alpha.1 receptors and an increase in .beta.2-adrenergic receptor density, without marked change in the affinity of agonists or antagonists. To investigate the mechanism(s) of this reciprocal change, a number of perturbations were examined including alterations in the composition of the culture medium and the influence of various hormones and inhibitors of cellular function. De novo protein synthesis is implicated in both receptor alterations as the inhibitors cycloheximide and actinomycin D prevented the increase in .beta.- and attenuated the decrease in .alpha.-adrenergic sites. The other perturbations were without effect. These studies provide evidence for a coupling of the functional alteration in glycogenolysis to changes at the receptor level per se. The mechanism underlying the reciprocal changes in hepatocyte adrenergic receptors during culture remains undefined. As similar reciprocal changes also occur in a variety of pathophysiologic states in vivo, the cultured hepatocytes is a useful model for the study of this phenomenon in vitro.