β‐Adrenergic Receptors in Brown‐Adipose Tissue. Characterization and Alteratios during Acclimation of Rats to Cold

Abstract

The capacity of brown adipose tissue to respond calorigenically to catecholamines increases markedly during cold‐acclimation of adult rats. To investigate this phenomenon, we have used the potent radioactive ligand (‐)‐[3H]dihydroalprenolol to directly estimate the number, the density and the affinity of β‐adrenergic receptors in brown adipose tissue membranes from control, cold‐exposed and cold‐acclimated rats. Binding of (−)‐[3H]dihydroalprenolol to unfractionated membranes was rapid, stable, saturable and reversible. It displayed the affinity, specificity and stereoselectivity expected of binding to adenylate cyclase‐coupled β‐adrenergic receptors. β‐Adrenergic agonists competed for binding sites with an order of potency typical of the β1 subtype of adrenergic receptors: (−)‐Isoproterenol > (−)‐norepinephrine ≥ (−)‐epinephrine. Binding exhibited a remarkable stereoselectivity, the (−)‐isomers of β‐adrenergic agonists and antagonists being 34 to 280 times more potent than the (+)‐isomers in competing for (−)‐[3H]dihydroalprenolol binding sites. Total interscapular brown adipose tissue of the adult, warm‐acclimated rat contained 1.12 ± 0.08 pmol of (−)‐[3H]dihydroalprenolol binding sites. During cold‐acclimation, growth of the tissue was accompanied by a 4–5‐fold increase in the total number of recptor sites. However, this increase did not keep pace with the increse in brown adipose tissue cellularity (as estimated by total tissue DNA content), resulting in a 40–50% reduction in receptor density. The decrease in receptor density was associated with cold‐exposure rather than with cold‐acclimation. The affinity of (−)‐[3H]dihydroalprenolol receptor sites was not significantly altered by cold‐acclimation. The results of this study are interpreted as indicating that catecholamines released via activation of the sympathetic nervous system regulate both the density and the number of their own receptors in brown adipose tissue of cold‐exposed animals. The development of the hyperadrenergic response of this thermogenic tissue during cold‐acclimation may result from a marked organ hyperplasia associated with an increased number of β‐adrenergic receptor sites and cannot be explained by alterations in receptor density or affinity.

Characterization of β1‐ and β3‐adrenoceptors in intact brown adipocytes of the rat

“…Histological cellular frequency studies have demonstrated that typical brown adipocytes represent about 40% of the total cell population in BAT (Geloen et al, 1988 (Bukowiecki et al, 1978;Dax & Partilla, 1982;Senault et al, 1984;Raasmaja, 1990;Langin et al, 1991;Galitzky et al, 1993b). However, these two radioligands were inadequate for quantifying P-adrenoceptors in intact cells due to their high lipophilicity.…”

“…One hundred nanomolar is approximately the noradrenaline concentration that is required to stimulate thermogenesis maximally in isolated brown adipocytes (Bukowiecki et al, 1980;1981). On the other hand, it is known that PI-adrenoceptors may be desensitized (internalized or down-regulated) by chronic cold exposure (Bukowiecki et al, 1978) or prolonged exposure to P-agonists (Granneman, 1992). In contrast, P3-adrenoceptors are particularly resistant to catecholamine-induced desensitization (Granneman, 1992;Carpene et al, 1993).…”

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

“…Further, as seen in Table 3 However, in a recent article, a rather high Kd (95 nM) for alprenolol has been determined for the /3-receptor in brown fat homogenates [20]. This value was determined from titrations with alprenolol up to concentrations of 240 nM.…”

“…However, these studies were mostly confined to avian and amphibian erythrocytes and to isolated mammalian plasma membranes [lo, 15,17,19,20]. Relatively few studies have been devoted to the investigation of intact mammalian cells [22,23].…”

“…This indicates that binding of [3H]dihydroalprenolol occurs to the physiological P-adrenergic receptor. In a recent study on brown fat homogenates [20], a concentration of 13 nM dihydroalprenolol was used for competition studies for the reported dihydroalprenolol binding site with a Kd of 95 nM. From these values and the E G O values for the competitors obtained experimentally, the authors calculated very high apparent K i values for the respective competing agents.…”