High Number of High-Affinity Binding Sites for (-)-[3H]Dihydroalprenolol on Isolated Hamster Brown-Fat Cells. A Study of the beta-Adrenergic Receptors

Abstract

The beta-adrenergic receptors of hamster brown adipocytes have been characterised by binding of the radioactive ligand (-)-[3H]dihydroalprenolol, directly to isolated intact cells in suspension. The brown fat cell contains 57000 specific and saturable binding sites which have a dissociation constant ( K d ) for [3H]dihydroalprenolo1 of 1.4 nM as determined by Scatchard analysis. The kinetically derived Kd, determined from forward and reverse rate constants, is 5 nM. Both of these values are in agreement with the dissociation constant (Kd = 2.2 nM) for alprenolol, determined from competition studies with [3H]dihydroalprenolo1 in these cells. Beta-adrenergic agonists competed for the specific binding sites with a typical b,-adrenergic specificity. The order of potency of agonists agrees well with the ability of these agents to stimulate respiration in isolated brown adipocytes: 50 % stimulation of respiration occurs with apparently less than 10 % occupancy of binding sites.Both the high affinity and high number of specific binding sites of [3H]dihydroalprenolol in brown fat cells presumably reflect the generally accepted dominating role of catecholamines in the regulation of brown fat metabolism and non-shivering thermogenesis.The primary stimulus for induction of heat production by brown adipose tissue is presumably binding of catecholamine molecules to b-adrenergic receptors situated in the plasma membrane [l]. Therefore, the elucidation of the detailed mechanism of such phenomena as cold adaptation and hibernation needs, among other things, delineation of catecholamine/brownadipocyte interaction at the molecular level. The potent beta-adrenergic antagonist (-)-[3H]dihydroalprenolol, has been used here to characterise and quantify the specific binding sites in isolated brown adipocytes. It was found that as many as 57000 receptors are present on a single hamster brown fat cell. Some of these results have been presented elsewhere [2-41. MATERIALS AND METHODS Isolation of Broicn Adipocytes

α1-Adrenergic Stimulation Potentiates the Thermogenic Action of β3-Adrenoreceptor-generated cAMP in Brown Fat Cells

“…This result implies that the ␤ 1 -receptors, earlier characterized on these isolated hamster brown fat cells (21), are not coupled to classical mediation through cAMP, and they appear therefore not to be functional. This may be due to a decoupling occurring during cellular differentiation; at least in mouse and rat brown preadipocytes in culture, the ␤ 1 -receptors are coupled (32)(33)(34) and stimulate cell proliferation (32,35,36) (although the predominant receptor after differentiation also in these cells is the ␤ 3 -receptor (32)).…”

α1- and β-adrenergic receptors in brown adipose tissue of lean (Fa/?) and obese (fa/fa) Zucker rats. Effects of cold-acclimation, sucrose feeding and adrenalectomy

“…;l-Adrenergic receptors There are high concentrations of both al-and yreceptors on brown adipocytes (Svoboda et al, 1979;Mohell et al, 1983a;Raasmaja et al, 1985). Although most of the thermogenic response to noradrenaline is thought to be f-receptor-mediated, up to 20 % of the increase in 02 consumption can be specifically blocked by the a-antagonist prazosin (Mohell et al, 1983b).…”

Membrane responses to norepinephrine in cultured brown fat cells.

“…Norepinephrine activates both a-and /3-adrenergic receptors on brown fat cell membranes (Svoboda et al, 1979;Mohell et al, 1983). We tested whether the inward and outward norepinephrine-induced currents were due to activation of different receptor types by examining the membrane responses to specific adrenergic agonists.…”