Adenylate cyclase in lean and obese (ob/ob) mouse epididymal white adipocytes

The results presented in this study indicate that the defect in catecholamine-stimulated adenylate-cyclase which is characteristic of the ob/ob mouse is associated with a decrease in the sensitivity of the system to guanine nucleotides (guanosine 5'-[beta gamma-imido]triphosphate and guanosine 5'-triphosphate). No difference in the beta-adrenergic receptor activity was found between the lean and obese mice on the basis of their capacity to bind the beta-adrenergic antagonist [3H]dihydroalprenolol. The data suggest that a defect in the activation of the adenylate cyclase by beta-adrenergic agents may reside in the guanyl nucleotide binding site(s).     

Beta-adrenergic receptor alterations in hypertension--physiological and molecular correlates

In hypertensive animals, there is physiological and biochemical evidence that beta-adrenergic responsiveness is diminished. In contrast, in man the physiological evidence of reduced beta-adrenergic responsiveness is not completely convincing and few biochemical studies have been reported. The lymphocyte has been widely used as a model for the human beta-adrenergic receptor complex. In studies comparing young normotensive and mild hypertensive subjects we demonstrated a reduction in beta-adrenergic mediated adenylate cyclase activity in lymphocytes from hypertensive subjects. A parallel reduction in beta-adrenergic receptor affinity for agonists was also seen. These changes are consistent with a functional uncoupling of the receptor from the adenylate cyclase complex. To determine the role of dietary sodium intake on beta-adrenergic receptor properties in hypertension we studied lymphocytes from hypertensive and normotensive subjects fed either a low (10 mequiv.) or high (400 mequiv.) NaCl diet. We demonstrated that a low NaCl diet corrected the defect in lymphocyte beta-adrenergic responsiveness in hypertension. These studies emphasize the utility of biochemical approaches to the study of alterations in beta-adrenergic responsiveness in human hypertension and suggest an important role of dietary sodium in the reduction in beta-adrenergic responsiveness in the hypertensive state.     

Adenosine deaminase normalizes cyclic AMP responses of hypothyroid rat fat cells to forskolin, but not beta-adrenergic agonists

Lipolysis and cyclic AMP accumulation in response to beta-adrenergic agonists or forskolin are severely impaired in fat cells from the hypothyroid rat. Incubating hypothyroid rat fat cells with adenosine deaminase normalizes the cyclic AMP response to forskolin, but not to beta-adrenergic agonists. Increased sensitivity to adenosine action in the hypothyroid state appears to be the basis for the impaired cyclic AMP response to forskolin, but does not appear to be the underlying defect responsible for the impaired response to beta-adrenergic agonists.     

High affinity beta-2-adrenergic receptors in mononuclear leucocytes: similar density in young and old normal subjects

In normal subjects beta-adrenergic responsiveness in the cardiovascular system has been shown to be impaired with increasing age. In order to correlate reduced hormonal responsiveness to an age-related defect at the receptor level, high affinity beta-adrenergic receptors in homogenates of human mononuclear leucocytes have been studied with a (?)-³H-dihydroalprenolol (³H-DHA) binding assay. The binding sites have been characterized by rapid kinetics, saturability, structural and sterospecificity. Binding equilibrium was obtained within 16 minutes at 37° and was reversed by 50% within 10.6 minutes. In 22 healthy subjects a binding capacity of 60 ± 8 fmol/mg protein and an equilibrium dissociation constant (K_D) of 0.6 ± 0.05 nM was found. Beta-adrenergic agonists displaced ³H-DHA binding with a potency order of isoproterenol > adrenaline > noradrenaline. The (?) isomers of beta-adrenergic agonists and antagonists were one to two orders of magnitude more potent as inhibitors of ³H-DHA binding than their corresponding (+) isomers. The binding capacity and affinity of the beta-adrenergic receptors did not differ in old, as compared to young normal subjects. Leucocytes from 14 individuals 18–40 years old had an average density of 53 ± 4 fmol/mg protein, while the average density in leucocytes from 8 individuals aged 53–65 years was 67 ± 8 fmol/mg protein. The K_D was 0.6 ± 0.05 nM in both groups. In conclusion, an age-related decrease of beta-adrenergic receptor-mediated cardiovascular functions does not seem to be reflected in the properties of beta-adrenergic receptors of mononuclear leucocytes.     

Adenylate cyclase activity in brown adipose tissue of the genetically obese (ob/ob) mouse

The activation of brown adipose tissue adenylate cyclase by catecholamines was studied in genetically obese (ob/ob) and lean mice. In obese mice, the maximum activation of the enzyme by several beta-adrenergic agonists was only two-thirds that in lean mice and, as an activator, noradrenaline was only one-eighth as potent. The adenylate cyclase was also less responsive to guanine nucleotides. In these respects, the defect in catecholamine-stimulated adenylate cyclase was similar in both white and brown adipose tissue of the obese mouse. The enzyme in brown adipose tissue differed from that in white adipose tissue in its sensitivity to other beta-adrenergic agonists and in its requirement for Mg2+. It is suggested that this abnormal catecholamine-activated adenylate cyclase in brown adipose tissue may be relate to the thermoregulatory defect of the obese mouse and hence may contribute to the obesity syndrome.     

Reconstitution of catecholamine-sensitive adenylate cyclase activity: interaction of components following cell-cell and membrane-cell fusion

Heterokaryons formed by the fusion of adenylate cyclase-deficient S49 cells and beta-adrenergic receptor-deficient B82 cells in the presence of cycloheximide display a catecholamine-sensitive adenylate cyclase activity. Similar complementation can be observed when receptor-replete membranes from the cyclase-deficient cell are fused with intact B82 cells. Using the cell fusion technique it can be demonstrated that the uncoupled S49 cell variant has a functional beta-adrenergic receptor but that this variant and the adenylate cyclase-deficient variant are not complementary. Hypothetically, both clones may share a common defect in regulatory components of adenylate cyclase or both may lack a specific coupling factor.     

Adipocyte adenylate cyclase and alpha- and beta-adrenergic receptors in one case of multiple symmetric lipomatosis

Adenylate cyclase activity and the number of alpha- and beta-adrenergic receptors (studied by radioligand binding techniques) were compared to adipocyte membranes prepared from lipomatous and normal adjacent adipose tissue of a patient with multiple symmetric lipomatosis. No difference could be detected between the normal and lipomatous adipose biopsies. These data strongly suggest that the impairement of catecholamine-induced lipolysis often reported in lipomas is related to a defect in the enzymatic steps localized beyond rather than before cyclic AMP synthesis.     

Functional desensitisation of beta-adrenergic receptors of avian erythrocytes by catecholamines and adenosine 3',5'-phosphate

Prolonged exposure to beta-adrenergic agonists of pigeon erythrocytes causes a reversible loss (70%) of catecholamine-stimulated adenylate cyclase activity without reduction in the number of beta-adrenergic receptors. In addition a less pronounced decrease in non-stimulated and NaF-stimulated adenylate cyclase activity (15-22%) is observed, appearing at different agonist concentrations and at a different rate. Dibutyryladenosine 3',5'-phosphate and the phosphodiesterase inhibitor methylisobutylxanthine partially mimick the action of the beta-adrenergic agonist, thus pointing to a possible role of adenosine 3',5'-phosphate in establishing desensitization. When adenylate cyclase from desensitized cells is stimulated with 5'-guanylyl-imidodiphosphate in the presence or absence of catecholamines the lag period preceding the attainment of maximal activity is extended. Likewise the rate of reversal by GTP or GTP of persistent activation of adenylate cyclase is slowed down. This is therefore interpreted to mean that the loss in hormonal stimulation on treatment of pigeon red blood cells with beta-adrenergic agonists is due to a delayed exchange of GDP against GTP on the regulatory GTP-binding protein. Furthermore, we conclude that events causing the refractory state in avian erythrocytes should occur at a site distal to the beta-adrenergic receptor.     

External transport of beta-adrenergic binding sites in ischemic myocardium

The properties of beta-adrenergic receptors were studied in normal and in flow restricted regions of the dog heart. Purified cardiac membrane preparations and papillary muscle preparations were isolated from control and ischemic areas and tested a) following chronic beta-receptor blockade with metipranolol or exaprolol, and b) after acute regional myocardial ischemia. A significant reduction in the sensitivity of the heart muscle preparations from compromised heart for isoprenaline resulting in a reduced affinity of beta-adrenergic receptors to exaprolol was observed. Quantitative ligand binding data showed higher numbers of (3H) dihydroalprenolol/(3H) DHA/binding sites in the membrane fraction obtained from compromised compared to control myocardium. The ratio of intra- to extracellular beta-adrenergic receptors decreased from 1.35 to 0.55 in the membrane fractions obtained from the compromised hearts. Pretreatment of experimental animals with metipranolol or propranolol attenuated the observed increase in the total number of beta-adrenergic receptor sites in myocardial membrane fractions from ischemic hearts. These data suggest preferential distribution of beta-adrenergic binding sites from intracellular to membrane fractions in flow restricted regions of the dog heart after coronary occlusion.     

Agonist-dependent recruitment of phosphoinositide 3-kinase to the membrane by beta-adrenergic receptor kinase 1. A role in receptor sequestration

Agonist-dependent desensitization of the beta-adrenergic receptor requires translocation and activation of the beta-adrenergic receptor kinase1 by liberated Gbetagamma subunits. Subsequent internalization of agonist-occupied receptors occurs as a result of the binding of beta-arrestin to the phosphorylated receptor followed by interaction with the AP2 adaptor and clathrin proteins. Receptor internalization is known to require D-3 phosphoinositides that are generated by the action of phosphoinositide 3-kinase. Phosphoinositide 3-kinases form a family of lipid kinases that couple signals via receptor tyrosine kinases and G-protein-coupled receptors. The molecular mechanism by which phosphoinositide 3-kinase acts to promote beta-adrenergic receptor internalization is not well understood. In the present investigation we demonstrate a novel finding that beta-adrenergic receptor kinase 1 and phosphoinositide 3-kinase form a cytosolic complex, which leads to beta-adrenergic receptor kinase 1-mediated translocation of phosphoinositide 3-kinase to the membrane in an agonist-dependent manner. Furthermore, agonist-induced translocation of phosphoinositide 3-kinase results in rapid interaction with the receptor, which is of functional importance, since inhibition of phosphoinositide 3-kinase activity attenuates beta-adrenergic receptor sequestration. Therefore, agonist-dependent recruitment of phosphoinositide 3-kinase to the membrane is an important step in the process of receptor sequestration and links phosphoinositide 3-kinase to G-protein-coupled receptor activation and sequestration.     

Effect of the Tricyclic Antidepressant Desipramine on β-Adrenergic Receptors in Cultured Rat Glioma C6 Cells

Rat glioma C6 cells, cultured in the presence of the tricyclic antidepressant desipramine, lost a significant number of beta-adrenergic receptors in a time- and dose-dependent manner. A similar loss was observed whether binding was determined on intact cells with the hydrophilic beta-adrenergic antagonist (+/-)-[3H]4-(3-tert-butylamino-2-hydroxypropoxyl)benzimidazole-2-o n HCl ([3H]CGP-12177) or on cell lysates with the more hydrophobic antagonists [125I]iodocyanopindolol or [3H]dihydroalprenolol. When stimulated with the agonist isoproterenol, desipramine-treated cells accumulated less cyclic AMP than control cells. The affinity of the beta-adrenergic receptors for either antagonist or agonist was unchanged after desipramine treatment. Desipramine interacted only weakly with the receptors and competed for [125I]iodocyanopindolol binding with a Ki of 30 microM. The presence in the culture medium of alprenolol or propranolol, potent beta-adrenergic antagonists, however, did not prevent the reduction in receptors by desipramine. Desipramine also caused a loss of beta-adrenergic receptors from cells maintained in serum-free medium and the cells themselves did not contain or secrete endogenous catecholamines. Although desipramine is a potent inhibitor of catecholamine uptake, it appears unlikely that the observed loss of beta-adrenergic receptors in rat glioma C6 cells exposed to the drug is due to an increase in extracellular catecholamine levels or to a direct interaction with the receptors.     

Characteristics of beta-adrenergic receptors in isolated cells and in crude membranes of brown adipose tissue

In relation to decreased metabolic sensitivity to catecholamines observed, in vitro, in brown fat of cold-acclimated rats, beta-adrenergic receptors were studied in isolated cells and in a crude membrane preparation from rat interscapular brown adipose tissue. [3H] dihydroalprenolol binding had the same characteristics in both types of preparation; competition studies of [3H] dihydroalprenolol binding led to the characterization of beta 1 subtype adrenergic receptors with a lower affinity of beta-adrenergic agonists for [3H] dihydroalprenolol binding sites in membranes than that found in isolated cells. Cold acclimation produced, in isolated cells only, a decrease of 41% in the [3H] dihydroalprenolol binding sites and a beta-adrenergic agonist affinity increase. It is concluded that beta-adrenergic receptor decrease could be a factor, at the hormone receptor interaction level, in the regulation of the transmission of biological action responsible for the cold-induced decrease in catecholamine responsiveness in brown adipose tissue. For a study of the desensitization process in brown fat, isolated cells seem to offer certain advantages over a crude membrane preparation.     

Effects of elevated prolactin and its normalization on thyroid hormone, cardiac beta-adrenoreceptor number and beta-adrenergic responsiveness

Subcutaneous inoculation of the prolactin secreting MtTW15 adenoma in male Wistar Furth rats for 4 weeks produced a significant increase in serum prolactin and a corresponding decrease in peripheral beta-adrenergic responsiveness. Both the isoproterenol induced drink and heart rate responses used to assess the beta-adrenergic responsiveness were significantly reduced in the hyperprolactinemic rat. Serum T3 and T4 levels were measured as was cardiac beta-adrenergic receptor number to ascertain if an alteration of thyroid hormone and a resultant decrease in beta-adrenergic receptor number was responsible for the attenuated beta-adrenergic responsiveness. Serum T4 was significantly reduced in the hyperprolactinemic group (1.9 +/- 0.3 microgram%) as compared to the control group (6.4 +/- 0.l5 microgram%). However there was no significant difference in serum T3 or in cardiac beta-adrenergic receptor number between the two groups. Removal of the MtTW15 adenoma resulted in a normalization of serum prolactin, T4, and in the responsiveness of the peripheral beta-adrenergic system within 4-6 weeks. These results indicate that the attenuated beta-adrenergic responsiveness associated with hyperprolactinemia is reversible and not dependent on a reduction in beta-adrenergic receptor number.     

Direct inhibition of type 5 adenylyl cyclase prevents myocardial apoptosis without functional deterioration

Adenylyl cyclase, a major target enzyme of beta-adrenergic receptor signals, is potently and directly inhibited by P-site inhibitors, classic inhibitors of this enzyme, when the enzyme catalytic activity is high. Unlike beta-adrenergic receptor antagonists, this is a non- or uncompetitive inhibition with respect to ATP. We have examined whether we can utilize this enzymatic property to regulate the effects of beta-adrenergic receptor stimulation differentially. After screening multiple new and classic compounds, we found that some compounds, including 1R,4R-3-(6-aminopurin-9-yl)-cyclopentanecarboxylic acid hydroxyamide, potently inhibited type 5 adenylyl cyclase, the major cardiac isoform, but not other isoforms. In normal mouse cardiac myocytes, contraction induced by low beta-adrenergic receptor stimulation was poorly inhibited with this compound, but the induction of cardiac myocyte apoptosis by high beta-adrenergic receptor stimulation was effectively prevented by type 5 adenylyl cyclase inhibitors. In contrast, when cardiac myocytes from type 5 adenylyl cyclase knock-out mice were examined, beta-adrenergic stimulation poorly induced apoptosis. Our data suggest that the inhibition of beta-adrenergic signaling at the level of the type 5 adenylyl cyclase isoform by P-site inhibitors may serve as an effective method to prevent cardiac myocyte apoptosis induced by excessive beta-adrenergic stimulation without deleterious effect on cardiac myocyte contraction.     

Reconstitution of beta-adrenergic receptors into phospholipid vesicles: restoration of [125I]iodohydroxybenzylpindolol binding to digitonin-solubilized receptors

beta-adrenergic receptors were solubilized from rat erythrocyte plasma membranes using digitonin. Solubilized receptors were then reconstituted into phospholipid vesicles by the addition of dimyristoylphosphatidylcholine and removal of detergent. Vesicles were separated from residual soluble receptors and detergent by rate-zonal ultracentrifugation. Vesicles were monolamellar, 500-900 A in diameter, and had a lipid content of 6 mumol phospholipid/mg protein. Specific binding of the beta-adrenergic ligand [3H]dihydroalprenolol ([3H]DNA) was 0.9-1.9 pmol/mg protein. Reconstitution of receptors into vesicles restored their ability to bind [125I]iodohydroxybenzylpindolol ([125I]IHYP). This ligand does not bind to detergent-solubilized receptors. [125I]IHYP binding was saturable [Kd = 84 pM] and competed appropriately with (+) and (-) isomers of beta-adrenergic agonists and antagonists. These receptor vesicles therefore appear to be an excellent model system for the study of beta-adrenergic receptor function in a defined lipid milieu.     

The biology of beta-adrenergic receptors: analysis in human epidermoid carcinoma A431 cells.

1. G-protein-linked transmembrane signaling has emerged as a major pathway for information transduction across the cell membrane. 2. In addition to photopigments that propagate the signal from light, cell-surface receptors for hormones, neurotransmitters, and autacoids propagate signals from ligand binding to membrane-bound effector units via G-proteins. 3. Biochemical and molecular features of one prominent member of these receptors, the beta-adrenergic receptor, will be highlighted in the present article. 4. The role of the human epidermoid carcinoma A431 cells as a model for the study of the structure and biology of beta-adrenergic receptors will be emphasized. 5. A model for receptor regulation, gleaned from recent advances in the biochemistry, cell and molecular biology of beta-adrenergic receptors, is discussed.     

Thyroid hormone regulation of beta-adrenergic receptor number.

The effects of exogenous thyroid hormones (thyroxine and triiodothyronine) on beta-adrenergic receptors in the rat myocardium were investigated. The potent beta-adrenergic antagonist, (-)-[3H]dihydroalprenolol, was used to directly estimate the number and affinity of beta-adrenergic receptors in rat heart membranes from control and hyperthyroid rats. Cardiac membranes from hyperthyroid rats contained 196 +/- 7 fmol of (-)-[3H]dihydroalprenolol binding sites/mg of protein which was significantly (p less than 0.005) greater than the number of binding sites (89 +/- 5 fmol/mg of protein) present in control membranes. The equilibrium dissociation constant (KD) for the interaction of receptors with dihydroalprenolol was the same (2 to 15 nM) in membranes from control and hyperthyroid rats. Similarly, there was no significant difference between the control and hyperthyroid membranes in the affinity of the beta-adrenergic receptor binding sites for the beta-adrenergic agonist isoproterenol. The results of this study demonstrate that thyroid hormones can regulate the number of cardiac beta-adrenergic receptors. The increased numbers of receptors may be responsible, at least in part, for the enhanced catecholamine sensitivity of beta-adrenergic-coupled cardiac responses in the hyperthyroid state.     

Development of Phodopus sungorus brown preadipocytes in primary cell culture: effect of an atypical beta-adrenergic agonist, insulin, and triiodothyronine on differentiation, mitochondrial development, and expression of the uncoupling protein UCP

A new cellular model for the study of brown adipocyte development and differentiation in vitro is presented. Preadipocytes isolated from brown adipose tissue (BAT) of the djungarian dwarf hamster Phodopus sungorus are able to proliferate and differentiate in vitro into true brown adipocytes able to express the BAT marker protein the uncoupling protein (UCP). Whereas basal UCP expression is very low, its mRNA levels as well as the UCP detected by immunoblotting are highly increased by beta-adrenergic stimulation. The novel, atypical beta-adrenergic compound D7114 (ICI Pharmaceuticals, Macclesfield, Cheshire, England) was found to increase the number of adipocytes as well as UCP mRNA and UCP content of mitochondria, indicating the involvement of an atypical or beta 3 receptor. Insulin was found to play an important role in brown adipocyte differentiation and mitochondrial development, whereas T3 seemed to be implicated more directly in UCP expression. In a defined, serum-free medium a synergistic stimulatory action of insulin and T3 on UCP expression was found, which seems to involve a pathway different from that of beta-adrenergic UCP stimulation.