Characterization of high-density lipoprotein binding to rat adipocytes and adipocyte plasma membranes

The interaction of high-density lipoproteins (HDL) with adipocytes is important in the regulation of cellular cholesterol flux. To study the mechanisms of HDL binding and cellular processing, we incubated adipocytes isolated from epididymal and perirenal adipose tissue of male Wistar rats (300 g) with HDL1 (1.07-1.10 g/mL) and HDL2 (1.10-1.14 g/mL) fractions separated from rat plasma by gradient ultracentrifugation. Freshly isolated adipocytes were incubated with 125I-labeled HDL for 2 h at 37 degrees C to determine cell-associated uptake and degradation. Adipocytes from both fat regions showed significant cell-associated HDL1 and HDL2 uptake and very high medium degradation (2- to 6-fold higher than uptake). To assess 125I-labeled HDL binding independent of cellular metabolism, we purified adipocyte plasma membranes from isolated adipocytes and used them in binding assays. Binding of HDL1 and HDL2 in the membrane system was 85-95% specific, sensitive to high NaCl concentrations, and abolished by pronase treatment. In contrast to HDL2 binding, the maximum HDL1 binding to perirenal plasma membranes was significantly higher than its binding to epididymal membranes (7.2 +/- 1.3 vs. 4.4 +/- 0.2 micrograms/mg, n = 6, p less than 0.05). This increment in HDL1 binding to perirenal membranes represented an EDTA- sensitive, calcium-dependent component. These results indicate that HDL binding to adipocyte plasma membranes depends on both adipose tissue region and HDL subtype. The membrane binding characteristics, taken together with the cellular uptake results, suggest that adipocytes bind and metabolize HDL and that this interaction may involve a protein receptor.     

Localization of estrogen receptors in uterine cells. An appraisal of translocation.

The nuclear localization of estrogen receptors has been examined under conditions which minimize redistribution and localization artifacts. A procedure is presented which rapidly lyses suspensions of cells from immature rat uteri by using 0.04% Triton X-100 in isotonic buffer. The ‘nuclei’ which are obtained after lysis have a median diameter of 1μm and are devoid of nuclear membranes. There is close agreement between the number of cells before lysis and the number of nuclear particles after lysis. Triton X-100 gave no interference with quantitative binding of estradiol to receptor and no alteration in the sedimentation behavior of receptor on sucrose gradients containing high or low salt. Using this procedure to monitor the dynamics of estrogen receptor distribution within uterine cells after exposure to estradiol, translocation of estrogen receptor to the nucleus was observed to occur at a rate slightly slower than the rate at which estradiol was specifically bound to free cells or receptors. The difference in these rates is compatible with a model in which estradiol must first bind to the receptor before the binding complex moves to the nucleus. The rate of nuclear translocation was temperature-dependent and was observed to occur at 0 °C, provided that enough time was allowed for steroid entry, receptor charging and transit to the nucleus. Two distinct phases were observed to characterize nuclear receptor localization. In the first phase after hormone exposure, estrogen receptor progressively accumulated in the nucleus; afterwards, estrogen receptor was progressively lost from the nucleus but could not be detected in other subcellular compartments in a form still binding hormone. Since high cell viability was maintained during these manipulations, loss of nuclear receptor was not due to cell damage during in vitro incubation. These studies suggest that this decline in nuclear receptor level after hormone interaction, which is known to occur in vivo, may be a normal event during estrogen interaction with target cells.     

Resistin mRNA levels are downregulated by estrogen in vivo and in vitro

Resistin, a hormone secreted by adipocytes, is suggested to be an important link between obesity and diabetes. The aim of this study was to evaluate the regulatory effect of estrogen on adipocyte resistin gene expression in ovariectomized (OVX) rats and in isolated rat adipocytes in vitro. Subcutaneous injection of estradiol benzoate reduced resistin mRNA levels in adipocytes isolated from the inguinal, parametrial, perirenal, retroperitoneal, or periovarian fat deposits of OVX rats, while an in vitro study showed that estradiol treatment decreased resistin mRNA levels in cultured rat periovarian fat adipocytes. Results of Western blotting analysis also showed that estrogen decreased adipose resistin contents in vivo and in vitro. These data suggest that estrogen is a pivotal negative regulator of resistin gene expression.     

Enucleation of human endometrial cells: nucleo-cytoplasmic distribution of DNA polymerase alpha and estrogen receptor

Nucleo-cytoplasmic distribution of estrogen receptors and DNA polymerase alpha activity in human endometrial adenocarcinoma cells (HEC-50 line) was evaluated after separation of nuclei following either homogenization or enucleation with cytochalasin B. About 30% of the estrogen receptor was found in the nuclear fraction after homogenization whereas 86% was found in the karyoplasts after enucleation. The total amounts of estrogen receptor per cell after homogenization and enucleation were not significantly different (14,000-17,000 binding sites/cell). Receptor measurements were carried out using the hydroxylapatite method after labeling with [3H]estradiol (5 nM [3H]E2 +/- 500 nM E2) at 30 degrees C for 3 h. About 20% of the DNA polymerase alpha activity was found in the nuclear fraction after homogenization, whereas 96% was found in the karyoplasts after enucleation. The average total activity (0.84 Units/10(6) cells) in homogenized cells was about 1/8 of the activity in karyoplasts. These results indicate that estrogen receptor and DNA polymerase alpha activity reside in the nucleus in intact HEC-50 cells. DNA polymerase alpha is translocated to the cytoplasmic fraction and inactivated after homogenization.     

Estrogen receptors in ovary and uterus of immature hamster and rat: effects of estrogens

Cytosolic and nuclear estrogen receptors in the ovary and uterus of immature rats and hamsters were determined to evaluate why exogenous estrogens were ineffective in stimulating follicular maturation in the hamster compared to the rat. Animals were injected sc with oil or single injection of 1 mg estradiol cyclopentylpropionate (ECP) on Day 23 or a daily injection of 2 mg diethylstilbestrol (DES) on Days 23-25 and killed on Day 26. Total binding sites for estrogen in ovarian cytosol of control hamsters were half the number in the rat ovary (28 fmole/mg protein) and about 50% of the receptors were occupied in the hamster. The apparent affinity of the estrogen-cytosol receptor complex was also lower in the hamster (Kd; 1.41 nM) than in the rat (Kd; 0.52 nM). After ECP treatment, there was a tendency for translocation in all 4 tissues examined even though some differences were not statistically significant. However, after DES treatment both cytosol and nuclear estrogen receptors decreased in both species. This discrepancy may be due to the difference in the time course of the nuclear translocation, the difference in metabolism and difference in the binding potencies of ECP and DES. The lack of ovarian responsiveness to estrogen in the hamster thus appears to be due to the reduced number of cytosol receptor sites which have a low affinity for estrogen and are already partially occupied.     

Androgen-induced nuclear accumulation of the estrogen receptor.

The effect of androgens on the nuclear uptake of both tritiated estradiol (3H-E2) and the estrogen receptor was studied in immature rat uteri. It was demonstrated that in vitro preincubation of immature rat uteri with various androgens (1 muM to 50 muM) followed by incubation with 3H-E2 (20 nM) resulted in a greatly decreased specific nuclear uptake of 3H-E2. Non-androgenic steroids had no effect. It was also confirmed that 5alpha-dihydrotestosterone (DHT) causes the accumulation of the estrogen receptor in the nuclei of uterine tissue. In vitro incubations of rat uteri with DHT (1muM and 50muM) were found to cause maximal nuclear estrogen receptor accumulation to the same degree as caused by estradiol, i.e. the nuclear uptake of approximately 100% of the estrogen receptor. Antiandrogens, which block the binding of androgens to the testosterone receptor in various tissues, did not inhibit the DHT - induced decrease in the 3H-E2 uptake by the uterine nuclei or the DHT - caused accumulation of the estrogen receptor in nuclei. These results seem to indicate that the uterine testosterone receptor has no role in the androgen - induced nuclear uptake of the estrogen receptor. However, the non-steroidal antiestrogens inhibited the DHT - induced nuclear accumulation of the estrogen receptor. This would seem to indicate that the estrogen - and androgen - induced nuclear accumulation of the estrogen receptor share a common mechanism.     

Effects of serum proteins on estrogen action in the perfused rat liver

To determine the effects of serum proteins on the biologic activity of estrogens, we perfused isolated livers from ovariectomized female rats with oxygenated Krebs-Henseleit-bicarbonate buffer (KHBB), with and without 4% human serum albumin (4% HSA), with and without added estrogens, or with charcoal-stripped human serum (CSHS) with and without added estradiol. At the end of the perfusions, the cytosolic and nuclear estrogen receptors were measured by an exchange assay. When added to KHBB, estradiol 10(-9) or 10(-8) M or estrone 10(-8) M did not cause any significant increase in the percent of receptors measured in the nucleus. When the livers were perfused with KHBB containing 4% HSA and estradiol 10(-9) to 10(-7) M or estrone 10(-8) M, there was an increase in nuclear receptors. Perfusion with estradiol 10(-8) M in CSHS resulted in significantly less receptor in the nucleus than after estradiol in KHBB plus 4% HSA. We conclude that the presence of 4% HSA in the perfusion medium increases the biologic activity of estradiol and estrone on the isolated rat liver, and this increase is inhibited in the presence of sex hormone-binding globulin. The exact mechanism by which HSA increases the biologic activity is uncertain, but may be due in part to better diffusion of estrogen through the liver.     

Translocatable estrogen receptors in rat splenocytes

Estradiol has previously been shown to suppress the response of the cellular immune system of the rat while enhancing the production of IgM antibodies. Analysis of the cytosol from rat splenocytes showed saturation of specific binding sites at concentrations of between 80 and 160 nM [3H]-estradiol with an approximate Kd of 12 nM. Competitive binding studies showed a dose-dependent decrease in the binding of [3H]-estradiol to the receptor in the presence of increasing concentrations of unlabeled estradiol. Dexamethasone, progesterone and R1881 (synthetic androgen) had no effect on the binding of [3H]-estradiol. The in vivo administration of estradiol resulted in increased nuclear binding of [3H]-estradiol as compared to vehicle treated controls. These results indicate that rat splenocytes possess specific, translocatable estrogen receptors which may be responsible for the observed modulation of the immune system.     

Receptor binding and biological potency of despentapeptide insulin

The receptor binding and biological potency of despentapeptide insulin (DPI) was assessed in human adipocytes, rat adipocytes and rat hepatocytes. DPI displayed a lower affinity for binding to both human adipocytes (half-maximum displacement at 0.89 +/- 0.04 and 0.20 +/- 0.02 nmol/l for DPI and insulin respectively; P less than 0.001) and rat adipocytes (half-maximum displacement at 7.12 +/- 1.06 and 1.14 +/- 0.18 nmol/l respectively, P less than 0.05). However, although DPI was less potent than unmodified insulin in stimulating glucose uptake in rat adipocytes (half-maximal stimulation at 2.0 +/- 0.67 and 0.47 +/- 0.18 nmol/l respectively; P less than 0.05), DPI was equipotent with insulin in human adipocytes (half-maximal stimulation at 0.034 +/- 0.001 and 0.027 +/- 0.001 nmol/l respectively; P greater than 0.2). In rat hepatocytes, DPI was twofold less potent in binding displacement activity (half-maximum displacement at 3.8 +/- 0.9 and 1.7 +/- 0.3 nmol/l respectively; P less than 0.01) but appeared to be equivalent in stimulating amino butyric acid uptake (half-maximum stimulation at 0.98 +/- 0.12 and 0.95 +/- 0.26 nmol/l respectively). The difference in affinity of DPI binding to rat liver membranes was less marked (1.3 fold decreased compared with insulin: 5.3 +/- 0.7 and 4.2 +/- 0.6 nmol/l respectively; P less than 0.001). Thus, the decreased receptor affinity of DPI was reflected in decreased biological potency in rat adipocytes, but not in human adipocytes nor rat hepatocytes. These data suggest differences in the binding-action linking in the cells of different tissues and different species.     

Hormone independent activation of rat uterine estrogen receptor by exposure of isolated uteri to anaerobic conditions

Incubation of isolated rat uteri under anaerobic conditions, which consisted of either an atmosphere of carbon monoxide or nitrogen, caused an increase in nuclear estrogen binding which was not dependent on added estrogen. The incubation of uteri in the absence of added estrogen under aerobic conditions (atmosphere of oxygen or oxygen-carbon dioxide [95-5%]) did not increase uterine nuclear estrogen binding levels. High salt (0.5-M KCl) extracts of the nuclear estrogen binding moiety induced by anaerobiosis were shown to possess a sedimentation coefficient on sucrose-glycerol gradients of 4.8S, a binding specificity restricted to estrogens and an apparent affinity constant of 1.35 nM. These data confirm that the nuclear binding moiety induced by anaerobiosis possesses the characteristics of an estrogen receptor. The enhanced nuclear estrogen receptor retention induced under anaerobic conditions could be accounted for by a significant increase in nuclear receptor extracted by high salt (0.5 M KCl) and by ethanol (salt resistant fraction). Furthermore, sequential extraction of nuclear estrogen receptor from uteri exposed to aerobic conditions in the presence of added estradiol paralleled the results obtained with anaerobiosis. Total receptor retained under anaerobiosis represented 25% of that observed under aerobic conditions in the presence of estrogen. These results indicate that anaerobic conditions can cause an activation of uterine estrogen receptor. This activation process represents a pathway for receptor activation which does not require steroid.     

Characterization of estrogen receptor in estrogen-dependent transplantable rat pituitary tumor MtT/F84

Properties of nuclear and cytosolic estrogen receptors (ERs) were examined in a new transplantable rat pituitary tumor designated as MtT/F84, of which growth is stimulated by estrogen. The optimal incubation conditions of both nuclear and cytosolic exchange were found to be at 37 degrees C for 15 min and at 25 degrees C for 2 hr, respectively. Molybdate increased a specific binding of estradiol (E2) as determined by [3H]E2-binding assay. Sucrose density gradient analyses of crude cytosol revealed specific peaks of radioactivity in both 4-5S and 8-10S areas. However, only a single 5S peak was present in 0.4M KCl-extractable nuclear ER. Molybdate also enhanced the stability of cytosolic 8-10S receptor in density gradient sedimentation behavior. Scatchard plot analysis for nuclear ER yielded a single class of binding sites with a dissociation constant (Kd) of 0.317 nM and the maximum number of binding sites (NBSmax) of 25.4 fmol/mg protein. Saturation analysis of [3H]estrogen binding to cytosolic ER also yielded a straight line with a Kd of 0.146 nM and NBSmax of 58.5 fmol/mg protein. The effect of E2 administration on the intracellular distribution of ER was also examined. A marked disappearance in the ER binding in cytosol with a concomitant increase in binding in nuclear fraction was found after the administration of the unlabeled E2 in vivo, whereas the total number of ER did not change. Thus, it is concluded that properties of ER in the MtT/F84 were very similar to those in other target organs such as uterus and pituitary gland.     

Estrogen receptors in the rat uterus. Retention of hormone-receptor complexes.

The retention pattern and biochemical characteristics of estrogen receptors in the nuclei of uterine cells were studied as a function of time after the in vivo injection of estradiol (E2) to immature female rats. One hour after the injection of 0.1 mug of tritiated E2, approximately 0.20 pmol per uterus of receptor bound hormone is retained in uterine nuclei. This dose of E2 produces a maximal uterotrophic response. Six hours after E2 administration, uterine nuclei retain 0.04-0.08 pmol of hormone per uterus. Hormone receptor complexes extracted from uterine nuclei 1, 3, and 6 h after in vivo injection of hormone have similar structural and binding characteristics. Receptors extracted at all three times sediment at 5S in high salt gradients and have a dissociation binding constant of approximately 3 nM for E2. The wash-out curves of receptors as a function of salt concentration are identical for uterine nuclei from animals treated for 1 or 6 h with estradiol, suggesting that the nature of the nuclear binding of receptors is not altered during this time interval. Experiments utilizing the injection of unlabeled estradiol, followed by an in vitro exchange procedure with tritiated estradiol, indicated that the total nuclear estrogen receptor sites, i.e., filled and vacant, decreased similarly.     

Comparison of formation, activation, and nuclear translocation of receptor . estradiol (R . E2) complex at 0 degrees C and 37 degrees C in intact uterine cells.

The present study was undertaken to establish whether molecular events leading to binding, transformation-activation, and nuclear translocation of cytoplasmic uterine estrogen receptor described for cell-free systems also occur in intact uterine cells. Cell suspensions were incubated at 0 degrees C or 37 degrees C with estradiol (E2) and specific binding to intracellular receptors was measured. The data demonstrate that saturation of specific estrogen binding sites occurs within 60 min at 37 degrees C and within 22 h at 0 degrees C, with a total of approximately 24,000 to 30,000 receptor sites per cell. At equilibrium, the total number and subcellular distribution of receptor . estradiol (R . E2) complexes formed in cells incubated at 0 degrees C or 37 degrees C were identical. Scatchard analysis of the equilibrium binding data yielded the same association constants for cytoplasmic and nuclear R . E2 formed in intact cells incubated at either temperature. Sucrose density gradient analysis of nuclear and cytoplasmic R . E2 formed in intact cells at 0 degrees C or 37 degrees C showed that at both temperatures, the nuclear R . E2 had a 5 S sedimentation coefficient; at both temperatures, a 5 S cytosol R . E2 was detected; only in the 0 degrees C incubation, an additional 4 S cytosol R . E2 was found. These results suggest that the molecular interactions regulating the dynamics of estrogen binding in the intact cell are similar at both physiological and low temperatures.     

Characterization of a [3H]methyltrienolone (R1881) binding protein in rat liver cytosol

The binding of radiolabelled methyltrienolone 17 beta-hydroxy-17 alpha-methyl-estra-4,9,11-trien-3-one (R1881) to adult male rat liver cytosol has been characterized in the presence of Na-molybdate to stabilize steroid-hormone receptors, and triamcinolone acetonide to block progestin receptors. Using sucrose density gradient analysis, male liver cytosol contains a [3H] R1881 macromolecular complex which sediments in the 8-9S region. 8S binding of R1881 to male rat serum, female liver cytosol or cytosol from a tfm rat cannot be demonstrated. Further metabolism of [3H] R1881 following 20h incubation with male rat liver cytosol was excluded: In the 8S region 97% of [3H] R1881 was recovered by thin layer chromatography. Characteristics of this [3H] R1881-8S binding protein include high affinity (Kd = 2.3 +/- 41 nM) and low binding capacity (18.8 +/- 3.3 fmol/mg cytosol protein), precipitability in 0-33% ammonium sulfate, and translocation to isolated nuclei following in vivo R1881 treatment. Whereas, the cytosol R1881-receptor is competed for by dihydrotestosterone, testosterone, and estradiol, [3H] estradiol binding in the 8S region is not competitive with androgens but does compete with diethylstilbestrol. The nuclear androgen binding site has a Kd = 2.8 nM for [3H] R1881, and is androgen specific (testosterone greater than 5 alpha-dihydrotestosterone greater than estradiol greater than progesterone greater than cyproterone acetate greater than diethylstilbestrol greater than dexamethasone greater than triamcinolone). Since a number of liver proteins including the drug and steroid metabolizing enzymes are, in part, influenced by the sex-hormone milieu, the presence of a specific androgen receptor in male rat liver may provide valuable insight into the regulation of these proteins.     

The characteristic binding of catechol estrogens to estrogen receptors in 7,12-dimethylbenz(a)anthracene-induced rat mammary tumors

The binding of catechol estrogens (2-hydroxyestrone, 4-hydroxyestrone, 2-hydroxyestradiol, and 4-hydroxyestradiol) to estrogen receptors in 7,12-dimethylbenz(a)anthracene (DMBA)-induced rat mammary tumor cytosols was investigated. Cytosol estrogen receptors exhibited high affinities (Ka = 1.12-1.88 X 10(8) M-1) for all catechol estrogens as well as estradiol. The receptor level of catechol estrogens (46.1-97.5 fmol/mg protein) was 1.6-3.0 times higher than that of estradiol; especially the binding of 4-hydroxyestrone to estrogen receptors was the highest of all catechol estrogens and estradiol. In judging the receptor level of more than 20 fmol/mg protein to be positive, the binding of catechol estrogens to estrogen receptors was approximately correlated with that of estradiol. The positive receptor level of catechol estrogens was found in a half of tumor cytosols which showed the negative receptor level of estradiol. These results suggested that characteristic estrogen receptors indicating high affinities for catechol estrogens might be present in rat mammary tumor cytosols.     

Estrogen receptors and androgen receptors in the mammalian liver

An estrogen receptor and an androgen receptor are present in the mammalian liver. In the liver of the rat, the estrogen receptor concentration increases markedly at puberty and this change correlates with enhanced estrogen stimulation of plasma renin substrate synthesis. High doses of estrogen are required for nuclear binding in liver when compared to doses for the uterus. The high dose requirement appears to be predominantly due to extensive metabolism in the hepatocyte of the estrogen to inactive derivatives. Furthermore, estradiol is much weaker than ethinyl estradiol for promoting nuclear binding in the liver. This is due to extremely rapid and extensive metabolism of estradiol. In human liver the concentration of estrogen receptor is low. An androgen receptor is present in high concentration in rabbit liver and is located predominantly in the nucleus after androgen administration. High concentrations of a putative androgen receptor are also present in human liver cytosol. Preliminary studies indicate that synthetic progestins can attach to the human liver androgen receptor. To date, a progesterone receptor has not been found in the mammalian liver. Thus, it appears that extensive steroid metabolism in liver preferentially diminishes sex steroid interaction with liver receptors and that androgen receptors may mediate progestin effects in liver. These observations provide a scientific basis for improved safety of oral contraceptives. Lowering the estrogen and progestin doses in oral contraceptives will decrease the major side-effects, which are liver mediated, and still maintain the desired effects at the hypothalamic-pituitary axis and uterus. Furthermore, it is likely that by selecting which estrogen, progestin or androgen is administered as well as by utilizing a parenteral route of administration that sex steroid effects on the liver could be minimized.     

Non-stoichiometric nuclear-cytoplasmic redistribution of estrogen receptor in adult rat uterus, following estradiol injection

In immature and ovariectomized rats acutely injected with estradiol (E2), accumulation of estradiol receptor complexes (E2R) from the uterine cytosol to the nucleus has been shown to be quantitative by numerous investigators. In the present study, translocation of E2R from the cytosol to the nuclear fraction in adult and ovariectomized estrogen prestimulated rats was analyzed. Twenty micrograms of E2, dissolved in saline containing 10% ethanol and 1 g% bovine serum albumin (B.S.A.) were injected intraperitoneally to the animals and 2 h later E2R in the cytosol and crude nuclear fractions were assayed by exchange techniques. Unlike a 91% recovery of the depleted cytosol E2R in the nuclear fraction of ovariectomized rats, only 39.2 and 27.5% were recovered in the adult and ovariectomized estrogen prestimulated rat uterus respectively. Moreover, depending on the temperature and duration of nuclear suspension incubation, from 18 up to 80% of the recovered nuclear E2R were solubilized in the incubation medium and nuclear post-incubation washes and could be measured by hydroxylapatite treatment (HAP). Saturation assays showed a plateau from 12 nM E2 3H onwards up to 80 nM. The Kd values computed for the receptors in the nucleus and HAP in all the three groups were of the order of 2 X 10(-9) M. In conclusion, after E2 administration to adult or ovariectomized estrogen prestimulated rats, a stoichiometric recovery of the depleted cytosol E2R in the nuclear fraction was not observed, even when leakage of nuclear receptor into the medium in course of exchange was taken into account. Chronic estrogenization appeared to modify the dynamics of uterine receptor.     

An assay for beta-adrenergic receptors in isolated human fat cells

The beta-adrenergic receptors have been characterized in isolated human adipocytes using a potent beta-adrenergic antagonist (-)-[3H]dihydroalprenolol. Binding of (-)-[3H]dihydroalprenolol to isolated fat cells was stereospecific and saturable, the maximum number of binding sites calculated being 7.8 +/- 2.2 pmol of bound ligand/10(7) cells, corresponding to 450,000 binding sites/cell. The dissociation constant was estimated to be 2.7 +/- 1.1 nM. The results with competition-inhibition experiments using beta-adrenergic agonists and antagonists indicated that the binding sites in isolated adipocytes were predominantly of the beta1-subtype; about 80% of the receptors were of this type. With the present method, specific beta-adrenergic receptor number and affinity in isolated human adipocytes could be determined in about 1 g of human adipose tissue.     

Direct assessment of beta-adrenergic receptors in intact rat adipocytes by binding of [3H]CGP 12177. Evidence for agonist high-affinity binding complex and for beta 1 and beta 2 receptor subtypes

The characteristics of the binding of the hydrophilic beta-adrenergic antagonist [3H]CGP 12177 to intact rat adipocytes were studied at 37 degrees C and 6 degrees C. At both temperatures and at 90% saturation, the non-specific binding was less than 30% of the total binding. At 37 degrees C, specific [3H]CGP 12177 binding was rapid, reversible of high affinity (1.8 +/- 0.4 nM) and saturable. The number of specific binding sites per adipocyte increased with the fat cell size (about 35 000 and 115 000 sites per cell in adipocytes with diameters of 60 microns and 88 microns, respectively) but remained constant when expressed per unit fat cell surface area. Displacement of [3H]CGP 12177 bound to adipocytes by unselective and selective beta-antagonists was stereospecific, had the same characteristics as those found in adipocyte membranes and showed a heterogeneous specificity for beta 1 and beta 2 adrenergic subtypes. In contrast, beta agonist competition curves, which modeled to two affinity-states of binding, showed high-affinity-state Kd values for agonists 10-25-times higher than those found in membranes under the same experimental conditions. At 6 degrees C, although the number and affinity of the specific binding sites for [3H]CGP 12177 were the same as those found at 37 degrees C, the Kd value for (-)-isoproterenol binding to the high affinity state of these sites (3.0 +/- 0.5 nM) was 25-times lower than at 37 degrees C and similar to the value found in membrane preparations (1.5-4 nM). These results show that the [3H]CGP 12177 specific binding sites detected on intact adipocytes represent the physiological beta-adrenergic receptors. Moreover, this study extends to the adipocyte the validity of the model recently proposed for other cell lines, according to which in intact cells, but not in membranes, agonist-binding promotes a rapid and temperature-dependent conformational change of the beta-adrenergic receptors, leading to a progressive loss of capacity of agonists to form a high-affinity complex.