Pharmacological Characterizations of Adrenergic Receptors in Human Adipocytes
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Pharmacological Characterizations of Adrenergic Receptors in Human Adipocytes Thomas W. Burns, … , J. Adolfo García-Saínz, John N. Fain J Clin Invest. 1981;67(2):467-475. https://doi.org/10.1172/JCI110055. Research Article Three types of adrenergic receptors, beta, alpha-1, and alpha-2, were identified in human adipocytes, isolated from properitoneal adipose tissue, using both the binding of radioactive ligands and the effects of adrenergic agents on receptor-specific biochemical responses. Adrenergic binding studies showed the following results: [3H]dihydroalprenolol 3 binding (beta adrenergic) Bmax 280 fmol/mg protein, KD 0.38 nM; [ H]para-aminoclonidine binding (alpha-2 adrenergic) 3 Bmax 166 fmol/mg protein, KD 0.49 nM; [ H]WB 4101 binding (alpha-1 adrenergic) Bmax 303 fmol/mg protein, KD 0.86 nM. In adipocytes from subcutaneous adipose tissue, [3H]dihydroergocryptine binding indicated the presence of alpha-2 but not alpha-1 receptors. Beta and alpha-2 adrenergic receptors appeared to be positively and negatively coupled to adenylate cyclase, respectively. Cells or cell membranes were incubated with epinephrine (10 μM) alone and in combination with the antagonists yohimbine (alpha-2) and prazosin (alpha-1). Epinephrine alone prompted a modest increase in adenylate cyclase activity, cyclic AMP, and glycerol release, an index of lipolysis. Yohimbine (0.1 μM) greatly enhanced these actions whereas prazosin was without effect. The beta agonist, isoproterenol, stimulated glycerol release, whereas the alpha-2 agonist, clonidine, inhibited lipolysis and cyclic AMP accumulation. To assess further alpha-1 receptors, cells were incubated with [32P]phosphate and epinephrine (10 μM) alone and in combination with prazosin and yohimbine. Epinephrine alone caused a three- to fourfold increase in 32P incorporation into phosphatidylinositol. Prazosin (0.1 μM) blocked […] Find the latest version: https://jci.me/110055/pdf Pharmacological Characterizations of Adrenergic Receptors in Human Adipocytes THOMAS W. BuRNs, PAUL E. LANGLEY, BOYD E. TERRY, DAVID B. BYLUND, BRLAN B. HOFFMAN, MICHAEL D. THARP, ROBERT J. LEFKOWITZ, J. ADOLFO GARCfA-SAINZ, and JOHN N. FAIN, Departments of Medicine, Surgery, and Pharmacology, University of Missouri School of Medicine, Columbia, Missouri 65211; Howard Hughes Medical Institute, Department of Medicine (Cardiology) and Biochemistry, Duke University Medical Center, Durham, North Carolina 27710; Section of Physiological Chemistry, Brown University, Providence, Rhode Island 02912 A B S T R A C T Three types of adrenergic receptors, and epinephrine (10 ,uLM) alone and in combination beta, alpha-i, and alpha-2, were identified in human with prazosin and yohimbine. Epinephrine alone adipocytes, isolated from properitoneal adipose tissue, caused a three- to fourfold increase in 32p incorporation using both the binding of radioactive ligands and the into phosphatidylinositol. Prazosin (0.1 ,uM) blocked effects of adrenergic agents on receptor-specific this action whereas yohimbine (0.1 uM) was without biochemical responses. Adrenergic binding studies effect. Thus, in a homogeneous cell preparation, the showed the following results: [3H]dihydroalprenolol human adipocyte appears to have three different binding (beta adrenergic) Bmax 280 fmol/mg protein, adrenergic receptors, each of which is coupled to a KD 0.38 nM; [3H]para-aminoclonidine binding (alpha-2 distinct biochemical response. adrenergic) Bmax 166 fmol/mg protein, KD 0.49 nM; [3H]WB 4101 binding (alpha-I adrenergic) Bmax 303 INTRODUCTION fmol/mg protein, KD 0.86 nM. In adipocytes from subcutaneous adipose tissue, [3H]dihydroergocryptine Over 30 yr ago, Ahlquist (1) proposed that the binding indicated the presence of alpha-2 but not cellular responses to epinephrine, norepinephrine, and alpha-i receptors. the synthetic catecholamine, isoproterenol, could be Beta and alpha-2 adrenergic receptors appeared to divided into two groups, which he designated alpha be positively and negatively coupled to adenylate and beta, according to their potencies in interacting cyclase, respectively. Cells or cell membranes were with the proposed receptors. With alpha-mediated incubated with epinephrine (10 ,M) alone and in responses, epinephrine was most potent and iso- combination with the antagonists yohimbine (alpha-2) proterenol least potent; with beta-mediated responses, and prazosin (alpha-i). Epinephrine alone prompted a isoproterenol was most active and norepinephrine modest increase in adenylate cyclase activity, cyclic least so. Subsequently, Lands et al. (2) suggested that AMP, and glycerol release, an index of lipolysis. beta receptors could be further divided into two groups, Yohimbine (0.1 ,tM) greatly enhanced these actions beta-i and beta-2. Both beta-i and beta-2 receptors whereas prazosin was without effect. The beta agonist, appear to stimulate adenylate cyclase leading to an isoproterenol, stimulated glycerol release, whereas the increase in cyclic AMP and to an expression of the alpha-2 agonist, clonidine, inhibited lipolysis and tissue-specific function. More recently, alpha receptors cyclic AMP accumulation. To assess further alpha-i have been divided into alpha-i and alpha-2 subtypes receptors, cells were incubated with [32P]phosphate (3-5). Although the initial classification was based on anatomic localization, it appears that a classification Dr. J. A. Garcia-Sainz is an international postdoctoral fellow related to function is more reasonable (3, 4). The sponsored by the National Institutes of Health. Dr. B. B. means by which alpha adrenergic stimulation affects Hoffman is a fellow of the Medical Research Council of cellular function is not completely understood. In Canada. Dr. R. J. Lefkowitz is an investigator of Howard Hughes Medical Institute. several systems, stimulation of alpha-i adrenoceptors Received for publication 25 July 1980 and in revised form increases phosphatidylinositol turnover and calcium 3 October 1980. gating or mobilization whereas activation of alpha-2 J. Clin. Invest. © The American Society for Clinical Investigation, Inc. * 0021-9738/81/02/0467/09 $1.00 467 Volume 67 February 1981 467-475 sites decreases cyclic AMP through adenylate cyclase and then radioactivity retained by the filter paper was inhibition (4, 5). determined for each sample by scintillation spectroscopy. Alpha adrenergic receptor assays with [3H]WB4101 as the The human adipocyte is of interest because it is a radioligand (concentration range, 0.3-4 nM) were per- single cell type that has both alpha and beta receptors. formed as described by U'Prichard et al. (18), and 100 Catecholamines seeril to be physiologically important ,uM norepinephrine was used to determine nonspecific mediators for lipid 'mijbilization in these cells (6-13). binding. Alpha-2 adrenergic receptor binding was deter- Beta increases cy- mined by the procedure of Rouot and Snyder (19) using adrenergic stimulation adenylate [3H]para-aminoclonidine as the radioligand (concentration clase activity, raises cyclic AMP, and enhances the range, 0.1-2.0 nM), and 10 ,uM norepinephrine was used to cyclic AMP-dependent processes such as lipolysis, determine nonspecific binding. The pH of the buffer was 7.4 whereas alpha adrenergic activation, in general, has for the alpha-2 adrenergic receptor binding studies. opposite effects (6-13). These observations support All studies were done using adipocytes isolated from properitoneal adipose tissue at the University of Missouri the hypothesis put forward by Robison et al. (14) that except for the studies with [3H]dihydroergocryptine binding, some cell types have both alpha and beta adreno- which were performed using membranes obtained from ceptors that mediate opposite effects on cyclic AMP adipocytes isolated from subcutaneous adipose tissue at and cell function. The studies reported here were Duke University. The following procedure was used for carried out to characterize further the studies on dihydroergocryptine binding. Adipocytes that adrenergic had been washed three times were homogenized at room receptors of human adipocytes and the metabolic temperature with 10 strokes of a Teflon pestle in buffer functions they subserve by using selective agonists containing 0.25 sucrose, 10 mM Tris HCI, and 1 mM EDTA and antagonists. at pH 7.4. The lysate was spun at 39,000 g for 10 min at 4°C. The pellet was resuspended in 50 mM Tris HCI, 10 mM mgCl2 (pH 7.5), and spun down again at 39,000 g for 10 min. METHODS The resulting pellet was resuspended in 50 mM Tris HCI, 10 mM MgCl2 (pH 7.5) at a protein concentration of 2 mg/ml, Cell preparation. Abdominal adipose tissue samples frozen, and stored under liquid nitrogen until used within were obtained from subjects undergoing elective abdominal 1 wk. Assay conditions for [3Hldihydroergocryptine binding surgery. Informed consent, approved by the institution's and computer modeling techniques were as previously de- Human Experimentation Committee, was obtained from scribed (5). each subject. Because all subjects were infused with 5% Cyclic AMP, glycerol, and adenylate cyclase assays. For dextrose during surgery, they were considered to be in the the determination of cyclic AMP, test substances were in- fed state. Fat cells were obtained by collagenase digestion of cubated in 1 ml of cell suspension for 20 min. At the end of adipose tissue (15). For experiments involving measurement incubation, the contents of each flask were mixed with 1 ml of of cyclic AMP and glycerol release, cells were isolated and 0.6 N perchloric