Identification and regulation of alpha- and beta-adrenergic receptors.

Direct radioligand binding methods for studying the alpha- and beta-adrenergic receptors have been developed over the past several years. These techniques use radioactively labeled adrenergic antagonists and agonists to identify the receptors in appropriate membrane fractions from catecholamine-sensitive tissues. In the case of the beta-adrenergic receptors, confident receptor identification has been aided by the close correlation of binding data with data on adenylate cyclase activation. Such direct binding studies are providing new insights about the molecular characteristics and regulatory properties of the receptors.     

Labetalol: potent antihypertensive agent that blocks both alpha- and beta-adrenergic receptors.

Labetalol was administered as the sole antihypertensive agent to 20 ambulatory patients with mild to moderate hypertension. The mean systolic and diastolic blood pressures (+/- standard error of the mean) with the patients sitting fell significantly (P < 0.001), from 145.5 +/- 3.2 and 103.7 +/- 1.6 mm Hg respectively at the start of labetalol therapy (after a period free of antihypertensive medication) to 125.7 +/- 2.0 and 87.2 +/- 1.1 mm Hg by the end of the trial. The diastolic blood pressure was well controlled (90 mm Hg or less) with labetalol therapy in 90% of the patients. The medication was well tolerated, and no orthostatic fall in the diastolic blood pressure was observed. Pharmacologically labetalol most closely resembles a combination of a nonselective beta-adrenergic blocker like propranolol and a postsynaptic alpha-adrenergic blocker like prazosin.     

Epinephrine-mediated stimulation of glucose uptake and lactate release by the perfused rat heart. Evidence for alpha- and beta-adrenergic mechanisms

Epinephrine treatment of the perfused rat heart led to an increase in the rate of glucose uptake and lactate release as well as increases in the rate of beating and the activity ratio of phosphofructokinase. The dose of epinephrine required for half maximal increases in the rate of beating, and glucose uptake and the activity ratio of phosphofructokinase was approx.10^?7M. Glucose uptake, lactate release and the activity ratio of phosphofructokinase were increased by the α-agonists methoxamine and phenylephrine, and the β agonist, isoproterenol. Propranolol and phenoxybenzamine each partially blocked the stimulatory effects of epinephrine on glucose uptake and lactate production. Phenoxybenzamine blocked the stimulatory effects of methoxamine but had no effect on those produced by isoproterenol which were blocked by propranolol. It is concluded that dual α and β adrenergic control of glycolysis occurs in cardiac muscle. It is proposed that the previously reported α-adrenergic control of phosphofructokinase plays a key role in the control of heart muscle glycolysis.     

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.     

Circadian rhythms in rat brain alpha- and beta-adrenergic receptors are modified by chronic imipramine

Circadian rhythms of α- and ß-adrenergic receptor number, with different wave forms, as well as differences in timing of maximal binding, are present in rat brain. Chronic treatment with the tricyclic antidepressant drug imipramine modifies these rhythms: peak binding of both receptors occurs 4–12 hours later than in controls, the 24-hour mean is decreased by 15–30%, and the amplitude is increased by 20–30%. Delaying of the phase position of neurotransmitter receptor rhythms by a tricyclic antidepressant may be relevant to its clinical mode of action, since depressive patients appear to have abnormally phase-advanced circadian rhythms.     

Effect of age and hypoxia on beta-adrenergic receptors in rat heart.

Previous reports suggest that hypoxia downregulates cardiac beta-adrenergic receptors from young rats. Because aging alters response to stress, we hypothesized an age-related alteration in the response to hypoxia. Male Fischer-344 rats, aged 3 and 20 mo, were divided into control and hypoxic groups. The hypoxic rats were exposed to hypobaric hypoxia (0.5 atm) for 3 wk. After hypoxic exposure, body weight decreased, hematocrit increased, right ventricular weight increased, and left ventricular weight decreased in all animals. beta-Adrenergic receptor density declined after hypoxic exposure in the young but not in the older animals, a change that was confined to the left ventricle. beta-Adrenergic receptor density in the right ventricle was significantly lower in the older animals than in the young animals. Plasma catecholamines (norepinephrine, epinephrine) drawn after the animals were killed (stress levels) decreased in young rats and increased in old rats after the exposure to hypoxia. Hypoxia is a useful physiological stress that elucidates age-related changes in cardiac beta-adrenergic receptor and catecholamine regulation that have not previously been described.     

Adrenergic and cholinergic mediated glucose oxidation by rat parotid gland acinar cells during aging

A₂-phospholipase from Crotalus Adamanteus venom has been assayed using the fluorescent probe 2-all trans-parinoyllecithin. The hydrolysis of the lecithin in an “albumin-rich” incubation medium is paralleled with a fluorescence hyperpolarization. The mechanism of the hyperpolarization is discussed. The application of the method to assay phospholipases, allows detection of an activity under 1 nanomole/min and continuous monitoring of the hydrolysis.     

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.     

Developmental regulation in the expression of rat heart glucose transporters.

Antibodies against human erythrocyte glucose transporters (GLUT-1) were used to determine if the transporters of embryonic and adult rat hearts have similar reactivity. On the basis of immunoblotting, these antibodies react more strongly with embryonic transporters than with adult ones. To determine if this phenomenon may be correlated with changes in the expression of transporter types during development, RNA isolated from either the embryonic or the adult rat heart was amplified by polymerase chain reaction (PCR) to identify the transporter species. Both GLUT-1 and GLUT-4 fragments were obtained among the PCR products. They were used for Northern blot analysis. The results indicate that the embryonic heart is rich in GLUT-1 mRNA; whereas the adult heart contains predominantly GLUT-4 mRNA. Thus, it appears that the major type of glucose transporter in rat heart switches from GLUT-1 to GLUT-4 during development.     

Differential alpha- and beta-adrenergic responsiveness of beating rat heart myocytes after stationary and non-stationary cultivation

Spontaneously beating myocytes from the heart ventricles of 1 to 4-day old rats were maintained for 8 days in stationary or in rocker culture under otherwise identical conditions and exposed to phenylephrine and isoproterenol for comparison of their positive chronotropic responses to alpha and to beta-adrenoceptor stimulation, respectively. The heart myocytes in rocked culture were more sensitive to phenylephrine and less sensitive to isoproterenol than were the myocytes in stationary culture and they also displayed a greater maximal response to the alpha-agonist. Addition of 1 mM L(+)-lactate to the rocker cultures abolished alpha-adrenergic responsiveness in one third of the cases while persistently increasing beta-adrenergic sensitivity.     

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).     

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 displayed cardiac hypertrophy and a decrease in epididymal gat 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).     

The effects of ovarian hormones on beta-adrenergic and muscarinic receptors in rat heart

The in vitro and in vivo effects of estrogen and progesterone on muscarinic and beta-adrenergic receptors of cardiac tissue were studied in ovariectomized (OVX) rats. The binding assay for muscarinic receptors was performed under a nonequilibrium condition; whereas the binding assay for beta-adrenergic receptors, under an equilibrium condition. Estrogenic compounds and progesterone were found to have no effect on the binding of the radioligand, [3H]-dihydroalprenolol, to beta-adrenergic receptors in vitro. However, progestins but not estrogenic compounds inhibited the binding of the radioligand, [3H]-quinuclidinyl benzilate, to muscarinic receptors in vitro, with progesterone as the most potent inhibitor (IC50 = 37 microM, apparent Ki = 13 microM). Progesterone was found to decrease the apparent affinity of muscarinic receptors for [3H](-)QNB in vitro. Daily treatment of OVX rats with estradiol benzoate (4 micrograms) or progesterone (2.5 mg) for 4 days had no effect on the muscarinic or beta-adrenergic receptors with respect to the binding affinity and receptor density. However, administrations of these hormones together for 4 days caused an increase in the receptor density of muscarinic receptors without a significant effect on their apparent binding affinity; also these hormones induced a decrease in the binding affinity and an increase in the receptor density of beta-adrenergic receptors. The results of this study demonstrate that progestins are capable of interacting with the cardiac muscarinic receptors in vitro, and indicate that estrogen and progesterone have a synergistic effect to increase the receptor densities of muscarinic and beta-adrenergic receptors as well as to cause a decrease in the binding affinity of beta-adrenergic receptors in vivo.     

Adrenergic modulation of the type 1 IP3 receptors in the rat heart

Inositol 1,4,5-trisphosphate (IP3) receptors are calcium-releasing channels localized on the sarcoplasmic reticulum. IP3 receptors mediate the calcium mobilizing effect of a wide range of hormones, cytokines, and neurotransmitters and play an important role in variety of cell functions. The aim of this work was to study, how partial depletion of catecholamines affects the gene expression and protein levels of the type 1 IP3 receptors in rat heart. The type 1 IP3 receptor mRNA levels were studied in the left cardiac atrium and ventricle of rats treated with 6-hydroxydopamine (6-OHDA) in control and stressed conditions. The 6-OHDA produces anatomical and functional denervation resulting in decreased levels of noradrenaline and adrenaline. We also used corticoliberin (CRH) knockout mice, where secretion of adrenaline is significantly suppressed. Administration of 6-OHDA significantly decreases mRNA levels of the type 1 IP3 receptor in both, the left atrium and the left ventricle, while the gene expression of the sarcoplasmic reticular Ca2+-ATPase (SERCA 2) was unaffected. CRH knockout mice possess markedly lower levels of the type 1 IP3 receptor mRNA compared to wild-type mice in both, control and stressed conditions. These data point to the adrenergic modulation of the type 1 IP3 receptors in the rat hearts.     

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.     

Sex differences in expression of calcium-handling proteins and beta-adrenergic receptors in rat heart ventricle

Human studies reveal sex differences in myocardial function as well as in the incidence and manifestation of heart disease. Myocellular Ca(2+) cycling regulates normal contractile function; whereas cardiac dysfunction in heart failure has been associated with alterations in Ca(2+)-handling proteins. Beta-adrenergic receptor (beta-AR) signaling regulates activity of several Ca(2+)-handling proteins and alterations in beta-AR signaling are associated with heart disease. This study examines sex differences in expression of beta(1)-AR, beta(2)-AR, and Ca(2+)-handling proteins including: L-type calcium channel (Ca(v)1.2) , ryanodine calcium-release channels (RyR), sarcoplasmic reticular Ca(2+) ATPase (SERCA2), phospholamban (PLB) and Na(+)-Ca(2+) exchange protein (NCX) in healthy hearts from male and female Sprague-Dawley rats. Protein levels were examined using Western blot analysis. Abundance of mRNA was determined by real time RT-PCR normalized to abundance of GAPDH mRNA. Contraction parameters were measured in right ventricular papillary muscle in the presence and absence of isoproterenol. Results demonstrate that female ventricle has significantly higher levels of Ca(v)1.2, RyR, and NCX protein compared to males. Messenger RNA abundance for RyR, and NCX protein was significantly higher in females whereas Ca(v)1.2 mRNA was higher in males. No differences were detected in beta-ARs, SERCA2 or PLB. Female right papillary muscle had a faster maximal rate of force development and decline (+/- dF/dt). There were no sex differences in response to isoproterenol. Results show significant sex differences in expression of key ventricular Ca(2+)-handling proteins that are associated with small functional differences in +/- dF/dt. Further studies will determine whether differences in the abundance of these key proteins play a role in sex disparities in the incidence and manifestation of heart disease.