Hormone action at the membrane level. VIII. Adrenergic receptors in rat heart and adipocytes and their modulation by thyroxine.

The regulation of adrenergic receptors in rat heart was measured in rats made hyperthyroid by injection with thyroxine and made hypothyroid by addition of propylthiouracil to the drinking water. Hyperthyroid rats display cardiac hypertrophy and a decrease in epididymal fat pad weight. The maximal beta-receptor level of ventricular membranes, as determined by (-)-[3H]dihydroalprenolol binding, was increased 60% by thyroxine treatment and decreased about 30% by propylthiouracil treatment. The affinity of the beta receptor was unchanged after thyroxine or propylthiouracil treatment. The maximal activity of the isoproterenol-stimulated adenylate cyclase (EC 4.6.1.1) varied with thyroid state in a manner parallel to the increase in beta-adrenergic binding sites. Thyroxine treatment also increases by 2-fold the beta receptors in isolated rat fat cells. Propylthiouracil treatment lowered the level of alpha receptors in heart by 30% as measured by [3H]dihydroergocryptine binding, but increased the affinity about 2.5-fold. The highest level of alpha receptors was seen in control hearts. These studies indicate that thyroxine may control the turnover of beta-adrenergic receptors in heart and fat cells and regulate physiological responses in these tissues via a hormone-hormone interplay system. Thyroxine treatment reduced the activity of the membrane-bound Mg2+-ATPase (EC 3.6.1.3) and 5'-mononucleotidase (EC 3.1.3.5) but appears to increase the activity of the (Na+ + K+)ATPase (EC 3.6.1.4).     

Thyroxine and propylthiouracil effects in vivo on alpha and beta adrenergic receptors in rat heart

Dihydroalprenolol and dihydroergocryptine were used to measure β and α adrenergic receptors respectively in heart ventricles from control, thyroxine (T₄)-treated and propylthiouracil (PTU)-treated rats. Ventricles from T₄-treated rats show an increase in the number of β receptors and a decrease in the number of α receptors. The β to α receptor ratio increases six fold. No change in binding affinity of the β receptor is observed but a decrease occurs in the affinity of the α receptor in ventricles from T₄-treated hearts. Ventricles from PTU-treated hearts show a small decrease in the number of β receptors but a large decrease in the number of α receptors. The binding affinity for both the α and β receptor is increased in the PTU-treated rats. The total number of α plus β receptors is increased in the T₄-treated rats and decreased in the PTU-treated rats.     

Increased number of cardiac adrenergic receptors following local heart irradiation

The effect of local X irradiation on cardiac alpha and beta receptors was studied in Wistar rats. Animals were given local heart irradiation with single doses of 15 or 20 Gy and were examined after a range of latency times of 7 to 400 days. Using the radioactive ligands [3H]CGP-12177 and [3H]prazosin, the maximal binding capacity was determined from saturation experiments. At 7 days after 20 Gy the maximal binding capacity of both alpha and beta receptors was reduced to below the level of untreated control animals. Subsequently it rose continually to a maximum of 160% of the control level for beta receptors and 130% for alpha receptors at 400 days postirradiation. The antagonist affinity as judged from the dissociation constant for [3H]CGP 12177 and [3H]prazosin did not change significantly. A similar effect was observed after 15 Gy. An increase in adrenergic receptors may represent an important pathogenetic link between early morphological and late functional changes in the pathogenesis of radiation-induced heart disease.     

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.     

Changes in adrenergic receptors during the development of heart failure

Moderate and severe stages of congestive heart failure due to the loss of myocardium upon coronary occlusion in rats was associated with an increase in alpha-adrenergic receptors and a decrease in beta-adrenergic receptors in the viable left ventricle. However, at early stages of heart failure the number of beta-adrenergic receptors was decreased without any changes in the number of alpha-adrenergic receptors. The affinities of these receptors to alpha receptor antagonist (³H-prazosin) and beta receptor antagonist (³H-dihydroalprenolol) were not altered in the failing hearts. On the other hand, the pattern of changes in both alpha- and beta-adrenergic receptors in heart membranes treated with oxygen free radical generating system was different from that seen in the failing hearts. In particular, the affinities for these receptors were decreased whereas the number of beta-receptors was increased and the number of alpha-receptors was decreased or unchanged. These results indicate that alterations in the adrenergic receptors in heart failure are not due to the formation of oxygen free radicals.     

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.     

Effects of altered thyroid status on beta-adrenergic actions on skeletal muscle glycogen metabolism

The effects of hypothyroidism on glycogen metabolism in rat skeletal muscle were studied using the perfused rat hindlimb preparation. Three weeks after propylthiouracil treatment, serum thyroxine was undetectable and muscle glycogen and Glc-6-P were decreased. Basal and epinephrine-stimulated phosphorylase a and phosphorylase b kinase activities were also significantly reduced, as were epinephrine-stimulated cAMP accumulation and cAMP-dependent protein kinase activity. Conversely, basal and epinephrine-stimulated glycogen synthase I activities were significantly higher while the Ka of the enzyme for Glc-6-P was lower in hypothyroid animals. Propylthiouracil-treated rats also had increased phosphoprotein phosphatase activities towards phosphorylase and glycogen synthase and decreased activity of phosphatase inhibitor 1. beta-Adrenergic receptor binding and basal and epinephrine-stimulated adenylate cyclase activities were reduced in muscle particulate fractions from hypothyroid rats. Administration of triiodothyronine to rats for 3 days after 3 weeks of propylthiouracil treatment restored the altered metabolic parameters to normal. It is proposed that the decreased beta-adrenergic responsiveness of the enzymes of glycogen metabolism in hypothyroid rat skeletal muscle is due to increased activity of phosphoprotein phosphatases and to reduced beta-adrenergic receptors and adenylate cyclase activity.     

Beta-adrenergic receptor in the brain: comparison of 3H-dihydroalprenolol binding sites and a beta-adrenergic receptor regulating adenylyl cyclase activity in cell free homogenates.

The properties of specific ³H-dihydroalprenolol binding sites resemble the properties of the beta-receptor regulating hormone-sensitive adenylyl cyclase activity in an homogenate of rabbit cerebellum. The rabbit cerebellum has 5 to 6 pmole per gm (wet weight) of high affinity (K_D=1.3 nM) specific binding sites for ³H-dihydroalprenolol. the interaction of several beta-adrenergic agonists and antagonists with the specific binding sites is rapid, reversible, and demonstrates stereospecificity which parallels the properties of the beta receptor. Beta-adrenergic agonists show a similar potency as agonists upon adenylyl cyclase activity and as inhibitors of ³H-dihydroalprenolol binding: i.e. l-isoproterenol > l-epinephrine > l-norepinephrine (suggesting a beta₂ adrenergic receptor). The binding affinities of several beta-adrenergic agonists and antagonists for the specific binding sites approximate the affinities of these compounds for the stimulation of adenylyl cyclase. Thus, the ³H-dihydroalprenolol binding sites have properties similar to the beta-adrenergic receptor regulating adenylyl cyclase activity in a rabbit cerebellar homogenate.     

High affinity thyroxine binding to purified rat liver plasma membranes.

Two sets of high-affinity thyroxine binding sites (K_D 0.39 ± 0.06 nM and 23 ± 5 nM) were detected on purified rat liver plasma membranes. Thyroxine is bound with high stereospecificity regarding iodine substituents and alanine side chain modifications of the molecule. Thyroxine binding is inhibited by -SH blocking agents and proteases. The highest affinity thyroxine binding site is also affected by phospholipase A and is distinct from triiodothyronine binding sites present in the membrane preparations; arguments are given for its plasmalemma origin.     

Biochemical characterization of the BETA-adrenergic receptor of the frog erythrocyte

Summary The beta-adrenergic receptor which is coupled to adenylate cyclase in the frog erythrocycte plasma membrane provides a convenient model system for probing the molecular characteristics of an adenylate cyclase coupled hormone receptor. Direct radioligand binding studies with beta-adrenergic agonists and antagonists such as [³H]hydroxybenzylisoproterenol and [³H]dihydroalprenolol have shed new light on the biochemical properties of the receptor as well as on its mode of interaction with other components of the adenylate cyclase system. Agonist binding to the receptor induces a high affinity state of the receptor which can be selectively reverted to a low agonist affinity state by guanyl nucleotides. This agonist-induced high affinity state of the receptor appears to correspond to a receptor moiety which has larger apparent molecular weight and which is probably a complex of the beta-adrenergic receptor and nucleotide regulatory binding protein. Antagonists do not appear capable of inducing or stabilizing the formation of this high affinity receptor-nucleotide site complex.The beta-adrenergic receptors have been solubilized using the plant glycoside digitonin as the detergent and have been highly purified by biospecific affinity chromatography on an alprenolol-agarose affinity support. These highly purified receptor preparations retain all of the binding characteristics observed in the unpurified soluble receptor preparations.Remarkably, antibodies raised in rabbits against affinity chromatography purified preparations of the receptor, themselves bind beta-adrenergic ligands with typical beta-adrenergic specificity. Such antibodies which possess binding sites similar to those of physiological receptors provide useful model systems for further probing the molecular characteristics of beta-adrenergic binding sites.     

Effect of thyroidectomy on testicular enzymes of the pyruvate/malate cycle involved in lipogenesis

The specific activities of testicular enzymes of the pyruvate/malate cycle involved in lipogenesis after thyroidectomy and thyroxine replacement were studied in prepubertal, pubertal and adult rats. Thyroidectomy induced testicular ATP citrate-lyase, malate dehydrogenase and malic enzyme activities and inhibited isocitrate dehydrogenase (NADP⁺) activity. Thyroxine treatment on thyroidectomized animals reverted all enzyme activities to normal. The result suggests that thyroid hormones have a differential effects on testicular enzymes of the pyruvate/malate cycle involved in lipogenesis.     

Evaluation of the negative cooperativity model for fat cell beta-adrenergic receptors

Cooperative site-to-site interactions among beta-adrenergic receptors of fat cell membranes are probed with the potent beta-adrenergic antagonist (?)-[³H]-dihydroalprenolol according to the kinetic method of De Meyts et al. (De Meyts, P., Roth, J., Neville, Jr., D.M., Gavin, III, J.R. and Lesniak, M.A. (1973) Biochem. Biophys. Res. Commun. 55, 154–161). Dissociation of specific (?)-[³H]dihydroalprenolol binding from fat cell membranes following a 100-fold dilution was rapid at 37°C; only 40% of the initial equilibrium binding remained 30 s after dilution. Dissociation of (?)-[³H]dihydroalprenolol bound under conditions yielding approximately 20% initial occupancy was performed in the absence and in the presence of a large molar excess of beta-adrenergic agonist ((?)-isoproterenol) or beta-adrenergic antagonist ((?)-alprenolol or(?)-propanalol). Neither agonists nor antagonists influenced the rate of (?)-[³H]dihydroalprenolol dissociation from fat cell membranes performed at 4, 22 or 37°C. Although analysis of the steady-state binding of (?)-[³H]-dihydroalprenolol to fat cell membranes yields Hill coefficients, n_H, less than 1.0, the present study indicates that these fat cell beta-adrenergic receptors display no cooperative site-to-site interactions.     

Identification of alpha-2 adrenergic receptors in rabbit adipocytes

Adrenaline, an alpha and beta adrenergic agonist don't modify theophylline induced lipolysis in perirenal and epididymal adipose tissue of the rabbit. Clonidine an alpha 2 adrenergic agonist inhibits theophylline stimulated lipolysis in the two tissue indicating the existence of alpha 2 adrenergic responsiveness. Direct identification of these receptors by radioligand binding studies shows that (H3) yohimbine an alpha 2 adrenergic antagonist binds to rabbit's fat cell membranes with high affinity: KD = 1.7 +/- 0.1 nmoles. The maximal number of binding sites at saturation is low 16 +/- 29 fmoles/mg protein.     

Elevated level of beta-adrenergic receptors in hepatocytes from regenerating rat liver. Time study of [125I]iodocyanopindolol binding following partial hepatectomy and its relationship to catecholamine-sensitive adenylate cyclase

Hepatocytes from regenerating rat liver show an enhanced epinephrine-sensitive adenylate cyclase activity and cAMP response, which may be involved in triggering of the cell proliferation. We have determined adrenergic receptors and adenylate cyclase activity in hepatocytes isolated at various time points after partial hepatectomy. The number of beta-adrenergic receptors, measured by binding of [125I]iodocyanopindolol ([125I]CYP) to a particulate fraction prepared from isolated hepatocytes, increased rapidly after partial hepatectomy as compared with sham-operated or untreated controls. The maximal increase, which was observed at 48 h, was between 5- and 6-fold (from approximately 1 800 to approximately 10 500 sites per cell). Thereafter, the number of beta-adrenergic receptors decreased gradually. Competition experiments indicated beta 2-type receptors. Parallelism was found between the change in the number of beta 2-adrenergic receptors and the isoproterenol-responsive adenylate cyclase activity. The number of alpha 1-adrenergic receptors, determined by binding of [3H]prazosin, was transiently lowered by about 35% at 18-24 h, with no significant change in Kd. Although the results of this study do not exclude the possibility of post-receptor events, they suggest that the increased number of beta 2-adrenergic receptors is a major factor responsible for the enhanced catecholamine-responsive adenylate cyclase activity in regenerating liver.     

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.     

Pharmacokinetic analysis of alpha- and beta-adrenoreceptors in the chick embryo amnion

Following a stimulation with acetylcholine, the beta-adrenergic agonists adrenaline (A), noradrenaline (NA), isoproterenol (Iso) and salbutamol (Sal) induced a concentration-dependent decrease in the tone and (or) rate of amnion contraction with EC50 ISO < NA < A < Sal. Metaprolol, a specific beta 1-antagonist, induced a rightward shift in the dose-response curves of Iso, NA and A, whereas beta-antagonist butoxamine was ineffective. pA2 values for beta-antagonists were propranolol 8.3, metoprolol 7.0, butoxamine 5.6. EC50 values of alpha-adrenergic agonists form a sequence: clonidine < NA < methoxamine < phenylephrine. Specific alpha-antagonists yohimbine and idazoxan were found to antagonise competitively the effects of NA. The data obtained characterize the adrenergic receptors mediating stimulation of amniotic contractile activity as alpha 2-adrenergic receptors. Inhibition of contractile receptors in amnion is mainly mediated by beta 1-adrenergic receptor activation.     

Agonist Interactions with Beta-Adrenergic Receptors Following Chronic Administration of Desipramine or the Atypical Antidepressants,Iprindole and Mianserin

Abstract

Desipramine (DMI), decreased the maximum number of beta-adrenergic receptors by approximately 10, 20, 30, and 20% in groups of rats treated i.p. with 5 mg/kg for 14 days or 10 mg/kg for 7, 14, or 21 days, respectively. In studies of agonist competition for beta-adrenergic receptors labelled with [¹²⁵I]-CYP, chronic DMI administration caused a selective decrease in those receptors normally found in the high affinity conformation in proportion to the dose of DMI administered. No change was observed in either the number of receptors in the agonist low affinity conformation or in the affinity of any drug for the high or low affinity conformations of the receptors. Therefore, chronic DMI caused a selective decrease in the beta-adrenergic receptors linked to adenylate cyclase but did not appear to change other properties of the receptors that would be manifested as a change in their ability to interact with adrenergic agonists. Neither iprindole (15 mg/kg i.p., 14 days) nor mianserin (10 mg/kg i.p., 14 days) decreased the number of receptors, the proportions of agonist high or low affinity receptors, or the affinity of competitor drugs for these receptors, suggesting a different mechanism for the reported loss of adenylate cyclase activity following these drugs than the down-regulation of receptors observed with chronic DMI treatment.     

Phosphorylation of phospholemman (FXYD1) by protein kinases A and C modulates distinct Na,K-ATPase isozymes

Phospholemman (FXYD1), mainly expressed in heart and skeletal muscle, is a member of the FXYD protein family, which has been shown to decrease the apparent K(+) and Na(+) affinity of Na,K-ATPase ( Crambert, G., Fuzesi, M., Garty, H., Karlish, S., and Geering, K. (2002) Proc. Natl. Acad. Sci. U. S. A. 99, 11476-11481 ). In this study, we use the Xenopus oocyte expression system to study the role of phospholemman phosphorylation by protein kinases A and C in the modulation of different Na,K-ATPase isozymes present in the heart. Phosphorylation of phospholemman by protein kinase A has no effect on the maximal transport activity or on the apparent K(+) affinity of Na,K-ATPase alpha1/beta1 and alpha2/beta1 isozymes but increases their apparent Na(+) affinity, dependent on phospholemman phosphorylation at Ser(68). Phosphorylation of phospholemman by protein kinase C affects neither the maximal transport activity of alpha1/beta1 isozymes nor the K(+) affinity of alpha1/beta1 and alpha2/beta1 isozymes. However, protein kinase C phosphorylation of phospholemman increases the maximal Na,K-pump current of alpha2/beta1 isozymes by an increase in their turnover number. Thus, our results indicate that protein kinase A phosphorylation of phospholemman has similar functional effects on Na,K-ATPase alpha1/beta and alpha2/beta isozymes and increases their apparent Na(+) affinity, whereas protein kinase C phosphorylation of phospholemman modulates the transport activity of Na,K-ATPase alpha2/beta but not of alpha1/beta isozymes. The complex and distinct regulation of Na,K-ATPase isozymes by phosphorylation of phospholemman may be important for the efficient control of heart contractility and excitability.     

Interactions of agonists and antagonists with beta-adrenergic receptors on intact L6 muscle cells

A binding assay has been developed to characterize beta-adrenergic receptors on intact L6 muscle cells. The affinity of beta-adrenergic receptors for the radioligand iodohydroxybenzylpindolol (IHYP) was the same in membrane preparations and in intact cells when determined by either equilibrium binding or kinetic analysis. The number of specific IHYP binding sites per cell was approximately the same on intact cells as on membranes. The pharmacological properties of antagonists indicated that the receptors on intact cells were identical to those on membranes. However, the beta-adrenergic receptors on intact cells had a 100-400 fold lower affinity at equilibrium for the agonist isoproterenol than did beta-adrenergic receptors on membranes. This low affinity of the receptor for agonists as measured by inhibition of radioligand binding in intact cells has also been observed in C6 (2) and S49 (3) cells. Our results suggest that beta receptors on intact cells after a 1 minute incubation was similar to the KD value for isoproterenol measured in membranes at equilibrium in the presence of GTP. After 1-2 minutes of exposure to a low concentration of agonist, binding of IHYP was no longer inhibited. These results suggest that agonists rapidly convert the beta receptors on intact cells to a state which has a low affinity for agonists. The affinity of the receptor for antagonists did not change during the incubation.     

Regression of thyroid hormone induced cardiac hypertrophy: Effect on cardiac beta receptors and adenyl cyclase activity

Adrenergic mechanisms may be important in the symptomatic manifestations of hyperthyroidism. The chronic administration of thyroid hormone also results in cardiac hypertrophy and increased numbers of beta-adrenergic receptors in cardiac membranes. The roles of adrenergic mechanisms in the initiation and perpetuation of this hypertrophy has been open to speculation. Rats treated chronically with L-thyroxin were sacrificed after 7 days of treatment and 1–4 days after cessation of treatment. Hearts were removed and weighed and norepinephrine measured. In other groups of identically treated rats, membranes were prepared from the left ventricle for $\text{invitro}$ measurements of beta-adrenergic receptor characteristics and adenyl cyclase activity. Regression of cardiac hypertrophy with a decrease in receptor number to control values was seen as early as 2 days after stopping thyroxine. Cardiac norepinephrine concentrations had also returned to control values at this time. Displacement of bound [H³] dihydroalprenolol by isoproterenol was not changed from control. Basal and isoproterenol stimulated adenyl cyclase activity was not changed by thyroxine administration or its cessation.The rapid reversal of the increased beta-adrenergic receptor number and cardiac hypertrophy raises the possibility that thyroid hormone may play a regulatory role in cardiac function. Although the enhancement of myocardial contractility by thyroid hormone may be mediated through cardiac hypertrophy this effect of thyroid hormone is independent of the catecholamine sensitive adenyl cyclase system.