Thyroid-hormone modulation of the number of beta-adrenergic receptors in rat fat-cell membranes.

Adipocytes from thyroidectomized rats contain 3 times less [3H]dihydroalprenolol-binding sites (beta-adrenergic receptors) than adipocytes from euthyroid animals. This alteration is not solely due to cell-size differences, but also to a thyroidectomy-induced defect in beta-adrenergic receptor density per adipocyte surface area, a defect that is furthermore corrected by tri-iodothyronine treatment.     

A characterization of beta-adrenergic receptors on cellular and perigranular membranes of rat peritoneal mast cells

Beta-adrenergic receptors were characterized by measuring the specific binding of 3H-dihydroalprenolol (DHA) on intact isolated rat peritoneal mast cells (RPMC) and on perigranular membranes derived from purified RPMC granules. The specific binding of 3H-DHA reaches an equilibrium within 30 min at 5 degrees C and is linear with cell number. Scatchard analysis reveals two populations of binding sites on intact cells: with KD = 10.6 +/- 2.6 and 129 +/- 4.7 nM and Bmax of 186 +/- 38 and 1200 +/- 415 fmol/10(6) cells, respectively. Each cell contains 120 X 10(3) high-affinity binding sites and 720 X 10(3) low-affinity binding sites. There appears to be neither alpha-adrenergic nor muscarinic cholinergic receptors on the RPMC. Specific binding of 3H-DHA also occurred to isolated granules with perigranular membranes. The binding was saturable with a single population of binding sites with an affinity (KD) of 7.0 +/- 0.45 nM. Maximum binding (Bmax) was calculated at 56.6 +/- 1.9 fmol/10(9) granules. Subfractionation of granule components demonstrated that the specific binding sites appear to be localized exclusively on the perigranular membrane.     

Characterization of pancreastatin receptors and signaling in adipocyte membranes.

Pancreastatin (PST), a chromogranin A derived peptide with an array of effects in different tissues, has a role as a counterregulatory hormone of insulin action in hepatocytes and adipocytes, regulating glucose, lipid and protein metabolism. We have previously characterized PST receptors and signaling in rat hepatocytes, in which PST functions as a calcium-mobilizing hormone. In the present work we have studied PST receptors as well as the signal transduction pathways generated upon PST binding in adipocyte membranes. First, we have characterized PST receptors using radiolabeled PST as a ligand. Analysis of binding data indicated the existence of one class of binding sites, with a B(max) of 5 fmol/mg of protein and a K(d) of 1 nM. In addition, we have studied the G protein system that couples the PST receptor by gamma-(35)S-GTP binding studies. We have found that two G protein systems are involved, pertussis toxin-sensitive and -insensitive respectively. Specific anti-G protein alpha subtype sera were used to block the effect of pancreastatin receptor activation. Galpha(q/11) and to a lesser extent Galpha(i1,2) are activated by PST in rat adipocyte membranes. On the other hand, adenylate cyclase activity was not affected by PST. Finally, we have studied the specific phospholipase C isoform that is activated in response to PST. We have found that PST receptor is coupled to PLC-beta(3) via Galpha(q/11) activation in adipocyte membranes.     

Differential response of adenylate cyclase to beta-adrenergic agonists in white adipocyte membranes from lean and obese (ob/ob) mice

The quality of the beta-adrenergic response, as measured by the activation of adenylate cyclase, was found to differ in adipocyte membranes from lean and obese mice. In the tissue from lean mice, the response was similar to that in rat adipose tissue and could, by analogy, be classified as beta 1-receptor response. In the tissue preparations from the obese mice, the rank order of potency of the three classical agonists (isoproterenol, epinephrine, and norepinephrine) was more typical of a beta 2-receptor response.     

Effect of hydrocortisone on beta-adrenergic receptors in lung membranes.

It has been observed that glucocorticoids potentiate beta-adrenergic stimulation of cardiovascular and airway tissues. In order to investigate the mechanism of this potentiating action, we examined the effect of glucocorticoids on the number and affinity of beta-adrenergic receptors in animal lung tissues, by a direct binding technique using [125]I-Iodohydroxybenzylpindolol ([125]I-HYP), a potent beta-adrenergic receptor antagonist. Specific binding of [125]I-HYP to rat lung membranes was saturable with 386 fmol of [125]I-HYP/mg protein at saturation. The apparent equilibrium dissociation constant of [125]I-HYP for beta-receptors was 221 nM. Chronic administration of hydrocortisone increased the density of beta-adrenergic receptors by 70% from 386 fmol to 657 fmol/mg with some decrease in the affinity of [125]I-HYP for beta-adrenergic receptors. By contrast, adrenalectomy produced a 29% fall in the number of beta-adrenergic receptors without altering the affinity of [125]I-HYP for beta-receptors, and this change was reversed by exogenous adminstration of hydrocortisone. The present study suggests that glucocorticoids may participate in regulating the density of beta-adrenergic receptors, and may potentiate beta-adrenergic receptors stimulation, at least in part by increasing beta-receptor density in tissue membranes.     

Effects of androgen on alpha- and beta-adrenergic receptors in membranes from the rat seminal vesicle.

The effects of castration and androgen-replacement on adrenergic receptors in membranes from the rat seminal vesicle were studied. Membranes from seminal vesicles showed saturable and high-affinity binding sites for the beta-adrenergic receptor antagonist, [3H]dihydroalprenolol ([3H]DHA), and the alpha 1-adrenergic receptor antagonist, [3H]prazosin. Castration markedly reduced beta-adrenergic receptors with decreasing the effect of GTP modulating the receptor-ligand affinity, suggesting defects in both the receptor per se and the guanine-nucleotides-regulating mechanism after castration. In contrast, castration increased alpha 1-adrenergic receptors and androgen-replacement reversed this change. The effects of GTP decreasing the alpha 1-receptor binding affinity to the radioligand were observed to a similar extent in the castrated and control membranes. These results demonstrate an inverse regulation by androgen on beta- and alpha 1-adrenergic receptors in membranes of the rat seminal vesicle.     

Down-regulation of rat liver beta-adrenergic receptors by cysteine

Down-regulation of hepatic beta-adrenergic receptors was indicated by a 56% decrease in the specific activity of 125I-iodocyanopindolol bound to rat liver membrane preparations from rats fed diets containing 15% of casein supplemented with cysteine, instead of methionine or unsupplemented. Down-regulation of hepatic beta-adrenergic receptors by cysteine appears to be mediated through an effect of cysteine on the tissue concentration of S-adenosyl methionine (SAM). The liver tissue concentration of SAM in rats fed cysteine-supplemented diets decreased 53% compared to those fed diets supplemented with methionine. The decrease in liver SAM in rats fed the diet supplemented with cysteine appears to reflect a non-competitive inhibition of methionine adenosyl-transferase by cysteine. Lineweaver-Burk plots demonstrated a dose-related Vmax response to cysteine but did not change the apparent Km at any concentration tested.     

Age-related parallel decline in beta-adrenergic receptors, adenylate cyclase and phosphodiesterase activity in rat erythrocyte membranes.

The activities of three components of the cyclic AMP system were compared in erythrocyte ghost membranes prepared from the blood of rats at various ages from 1.5 to 15 months. The apparent number of β-adrenergic receptor sites, adenylate cyclase activity and cyclic AMP phosphodiesterase activity all declined about 50% in the membranes from the older animals (>5 months) as compared to the 1.5 month ones. The soluble erythrocyte phosphodiesterase also declined with age, but the decline did not parallel that of the membrane-associated activity. In contrast, there was no age-related change in the number of β-adrenergic receptors in membranes from the brains of the same animals. In erythrocyte ghosts, both the ratio of isoproterenol-stimulated adenylate cyclase activity to basal activity and the ratio of sodium fluoride-stimulated activity to basal were constant with age. Neither the dissociation constant for the β-adrenergic receptor nor the Michaelis constant for the phosphodiesterase changed as a function of age. Together with other data in the literature, these results suggest a close functional association of the components of the cyclic AMP system in the mature erythrocyte membrane, and support a physiological role for the cyclic AMP mediated β-adrenergic effects in the red blood cell.     

"Antibodies raised against beta-adrenergic receptors stimulate adenylate cyclase"

Antibodies raised in mice against β-adrenergic receptors purified from turkey erythrocyte membranes, specifically bind to cells which possess a β-adrenergic receptor and immunoprecipitate radiolabelled purified receptor. These antibodies stimulate the adenylate cyclase activity of the turkey erythrocytes, although they do not compete with the catecholamine hormones for binding to the β-adrenergic receptor. Thus the receptor-antibody interaction, although occuring at another site than the receptor-hormone interaction, may still trigger the enzymatic activity.     

Stoichiometry for the binding of insulin to insulin receptors in adipocyte membranes

Insulin receptor molecules in rat adipocyte plasma membranes were shown to be monovalent with respect to their capacity to bind insulin. The 1:1 stoichiometry for insulin binding was determined by a "double-probe labeling" procedure, wherein 125I-insulin (probe 1) was affinity cross-linked to its receptor in the presence of an excess saturating concentration of an unlabeled biotinylated insulin derivative (probe 2). If the receptor were competent to bind more than one insulin molecule, any receptor molecule that was cross-linked to probe 1 also should have been cross-linked to probe 2 in the double probe labeling procedure. The monovalent character of the insulin receptor was indicated by the failure of the probe 1-linked receptor to be cross-linked to probe 2. This was indicated by the failure of succinylavidin to increase the molecular weight of the probe 1-linked receptor. Control experiments indicated that succinylavidin increased the molecular weight of receptor that had been cross-linked to probe 2. The 1:1 stoichiometry for insulin binding demonstrated here indicates that if insulin receptors contain more than one insulin binding subunit, the binding of insulin to its receptor must be a highly negatively cooperative process.     

Hypoxia-induced downregulation of beta-adrenergic receptors in rat heart.

To test the desensitization hypothesis of cardiac beta-adrenergic receptors (beta-AR) in chronic hypoxia, the effect of 1, 3, 7, 15, and 21 days of exposure to hypobaric hypoxia (380 Torr) was evaluated in Wistar rats. Exposure to hypoxia for 1-15 days did not induce any change in right and left ventricular beta-AR density (Bmax) determined with [125I]iodocyanopindolol or in antagonist affinity. After 21 days, Bmax decreased by 24% in the left ventricle. In contrast, no change in beta-AR was shown in the right hypertrophied ventricle. Agonist affinity in the left ventricle was not altered, as shown by the analysis of displacement curves of isoproterenol (normoxia 185 +/- 26 nM, hypoxia 170 +/- 11 nM). Moreover, there was no significant decrease in adenylate cyclase activity (pmol.mg-1.min-1) in the left ventricle. In the right ventricle, a 21-day exposure to hypoxia led to a decrease in basal and maximal activity when stimulated by isoproterenol. A decrease in tissue norepinephrine content was observed after 7 days of hypoxia. In conclusion, these data support the beta-AR downregulation hypothesis as one of the mechanisms of myocardial adaptation to high altitude occurring after 2-3 wk of exposure to hypoxia. The regulation pathways of beta-AR may differ between left nonhypertrophied and right hypertrophied ventricles. No evidence of profound abnormality of signal transduction was shown.     

Production of insulinomimetic antibodies against rat adipocyte membranes by hybridoma cells

SJL mice were injected intraperitoneally with adipocyte plasma membranes or with intrinsic membrane proteins obtained by extraction of plasma membranes with dimethylmaleic anhydride. Three days after the boost injection, the spleens were removed and fused with NS-1, a thioguanine-resistant myeloma cell line derived from P₃X63 Ag⁸ (Balb/c). Following selection for hybrids with hypoxanthine, aminopterin, and thymidine, medium of the hybrid cells was tested for its ability to bind to the plasma membrane of the adipocyte and to stimulate the oxidation of D-(1-¹⁴C) glucose to ¹⁴CO₂. Approximately 40% of the wells containing hybridomas derived from splenocytes of SJL mice immunized with plasma membranes produced immunoglobulin that bound to adipocyte plasma membranes. About 30% of these mimicked the ability of insulin to stimulate the oxidation of D-(1-¹⁴C) glucose to ¹⁴CO₂ in adipocytes. Media from 51% of the wells containing hybridomas derived from splenocytes of SJL mice immunized with intrinsic membrane proteins produced immunoglobulin that bound to the plasma membrane and 48% of those stimulated glucose oxidation. The bioactivity of the hybrid cell media could be blocked by adsorption with intrinsic membrane proteins or by the removal of immunoglobulins using formalin-fixed Staphylococcus aureus. The hybrids generated in this study can be divided into three categories: (1) hybrids that secrete antibodies that can bind to plasma membranes and mimic insulin action of glucose transport; (2) hybrids that secrete antibodies that bind to plasma membranes but do not stimulate the oxidation of D-(1-¹⁴C) glucose to ¹⁴CO₂; and (3) hybrids that produce no antimembrane antibodies. The data suggest that interaction of immunoglobulins with specific membrane proteins is essential in mimicking the action of insulin on glucose transport and oxidation in the rat adipocyte.     

An acute exercise-induced translocation of beta-adrenergic receptors in rat myocardium

The effect of acute exercise (treadmill running) on rat myocardium beta-adrenergic receptors (beta-AR) was studied. beta-AR was identified in purified sarcolemmal membrane fractions and light vesicle fractions. In control hearts, the number of beta-AR was 21.25 +/- 2.25 and 20.89 +/- 2.89 fmol/mg protein (mean +/- SE) in sarcolemmal membranes and light vesicles, respectively. Immediately after a single bout of dynamic exercise, about 35% of beta-AR was transferred from light vesicles to sarcolemmal membranes (p less than 0.05); concomitantly, isoproterenol-stimulated adenylate cyclase activity also significantly increased in sarcolemmal membranes (p less than 0.05). These results suggest that acute exercise provokes the translocation of beta-AR from a presumably intracellular site (light vesicles) to functional membrane fractions (sarcolemmal membranes) in rat myocardium.