Beta adrenergic receptor subtypes in the atria: evidence for close coupling of beta-1 and beta-2 adrenergic receptors to adenylate cyclase

The relationship between occupancy of beta adrenergic receptors and stimulation of adenylate cyclase in dog atrial tissue was examined by studying the binding of [125I]iodopindolol and the activation of adenylate cyclase. Computer-assisted nonlinear regression analysis was used to analyze the inhibition of isoproterenol-stimulated adenylate cyclase activity by beta-1- or beta-2-selective antagonists. The Ki values for each subtype of receptor for the selective antagonists resulting from studies of the inhibition of adenylate cyclase activity were similar to those determined in studies of the inhibition of the binding of [125I]iodopindolol. To compare further the occupancy of beta-1 or beta-2 adrenergic receptors with the activation of adenylate cyclase mediated by each class of receptor, computer modeling of the stimulation of adenylate cyclase by the beta-1-selective agonist norepinephrine was carried out. The EC50 values of norepinephrine for each receptor subtype, as measured in studies of norepinephrine-stimulated adenylate cyclase activity, were similar to the Ki values for the inhibition by norepinephrine of the binding of [125I]iodopindolol to each receptor subtype. The data led to the conclusion that beta-1 adrenergic receptors make up about 70% of the total number of beta adrenergic receptors and mediate 70% of the increase in adenylate cyclase activity produced by isoproterenol. These results suggest that the relationship between occupancy of each class of receptor and activation of adenylate cyclase is linear and that, when agonist-stimulated adenylate cyclase activity is used as a functional response, neither spare beta-1 nor spare beta-2 adrenergic receptors exist in the atrium.(ABSTRACT TRUNCATED AT 250 WORDS)     

Selective regulation of beta-1 and beta-2 adrenergic receptors by atypical agonists

The interactions of the atypical agonists pindolol and celiprolol with beta adrenergic receptors were compared with those of the full agonist, isoproterenol. Studies were carried out using intact cells as well as membranes prepared from C6 glioma cells. Computer-assisted analysis of dose-response curves resulting from the inhibition of the binding of [125I]iodopindolol by the beta-1 and beta-2 selective compounds ICI 89,406 and ICI 118,551 revealed that approximately one-third of the beta adrenergic receptors on these cells were beta-1 receptors. Addition of GTP to the binding assay simplified the dose-response curve for inhibition of the binding of [125I]iodopindolol by isoproterenol and diminished the potency of the agonist. GTP had no effect on the binding of pindolol or celiprolol, suggesting that these drugs do not induce the formation of a ternary complex with the receptor and the guanine nucleotide-binding protein for stimulation of adenylate cyclase activity. When added to the growth medium of intact C6 cells, isoproterenol induced a 40-fold increase in cyclic AMP accumulation. Pindolol and celiprolol, however, caused no elevation of enzyme activity. Addition of isoproterenol to the growth medium of intact cells resulted in an 80% decrease in the density of both beta-1 and beta-2 adrenergic receptors within 8 hr. Growing cells in the presence of pindolol or celiprolol induced a 50% decrease in the density of beta-2 receptors, which was inhibited by beta adrenergic antagonists.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effects of clenbuterol on central beta-1 and beta-2 adrenergic receptors of the rat

Repeated administration of the centrally acting beta adrenoceptor agonist, clenbuterol, to rats reduced the ability of isoproterenol to increase the concentration of cyclic AMP (cAMP) in slices of cerebellum. This reduced responsiveness to isoproterenol was accompanied by a marked reduction in the density of beta adrenoceptors as measured by the binding of the beta adrenoceptor antagonist [125I]iodopindolol. In addition, the agonist-binding properties of remaining cerebellar beta adrenoceptors were altered after clenbuterol treatment. The clenbuterol-induced reduction in the density of beta adrenoceptors in the cerebellum is in marked contrast to its inability to do this in cerebral cortex. Comparison of the ability of clenbuterol to that of isoproterenol to increase levels of cAMP in slices of cerebral cortex or cerebellum showed that clenbuterol is a weakly potent agonist in both brain regions. The increase in cAMP induced by isoproterenol in the cortex was significantly reduced in the presence of the selective beta-1 adrenoceptor antagonist, ICI 89,406. In contrast, the clenbuterol-induced increase in cortical cAMP was unchanged by ICI 89,406 but was reduced significantly by the beta-2 adrenoceptor antagonist, ICI 118,551. In cerebellum, both isoproterenol- and clenbuterol-stimulated accumulation of cAMP were antagonized much more potently by ICI 118,551 than by ICI 89,406. Furthermore, clenbuterol antagonized the cAMP response induced by isoproterenol in the presence of ICI 118,551 in a concentration-dependent manner. In terms of measurement of cAMP in brain slices, clenbuterol is weakly potent as an agonist at beta-2 adrenoceptors and has antagonist properties at beta-1 adrenoceptors.     

Stereospecificity of beta adrenergic antagonists: R-enantiomers show increased selectivity for beta-2 receptors in ciliary process

The (+)-stereoisomers of arylethanolamine beta adrenergic agonists and antagonists are usually much less active in biological systems than their corresponding (-)-forms. In the eye, however, prior physiological studies have shown that these (+)-stereoisomers are unexpectedly potent in altering intraocular pressure, results which could be due to a difference in distribution and metabolism or to a difference in receptor interaction. The present experiments evaluated six stereoisomeric pairs of beta adrenergic antagonists for their ability to block rabbit ciliary process and cardiac beta adrenergic receptors activating adenylate cyclase, in vitro, under conditions in which the effects of drug metabolism, distribution and membrane lipid solubility were minimized. In the heart, all six pairs of antagonists demonstrated the expected increased potency of (-)-forms, with isomeric activity ratios of: 33 for metoprolol, 44 for timolol; 48 for bunitrolol; 76 for t-butyl-betaxolol; 100 for t-butyl-didesmethyl-ICI-118,551; and 530 for betaxolol. Under identical assay conditions in the ciliary process, (+)-enantiomers were much more potent relative to (-)-forms, with isomeric activity ratios of: 0.82 for timolol; 3.3 for bunitrolol; 7.4 for t-butyl-didesmethyl-ICI-118,551; 10 for metoprolol; 16 for t-butyl-betaxolol; and 190 for betaxolol. With the exception of metoprolol, all (+)-enantiomers demonstrated a substantially higher absolute affinity for ciliary process receptors (known to be almost exclusively of the beta-2 subtype) than for cardiac receptors.(ABSTRACT TRUNCATED AT 250 WORDS)     

Alpha-1 adrenergic receptors in the nonfailing and failing human heart

We examined alpha-1 adrenergic receptor density in ventricular myocardium from nonfailing and failing human hearts, utilizing the alpha-1 radioligand [125I]IBE2254. The alpha-1 receptor population comprised a relatively small portion of the total adrenergic receptors, 14.6 +/- 1.9%. However, in failing human ventricular myocardium the alpha-1 adrenergic receptor population constituted a much greater portion, 27.3 +/- 2.1% (P less than .01). The reason for the increased proportion of alpha-1 adrenergic receptors was not that the total concentration of alpha-1 receptors was increased, but instead was due to selective down-regulation of the beta-1 adrenergic receptor population. Beta-2 adrenergic receptors behaved similarly to alpha-1 adrenergic receptors in the failing human heart, and were increased in proportion and unchanged in total number. Additionally, the ability of alpha-1 stimulation to increase the incorporation of label from [3H]inositol into inositol phosphates was examined in tissue homogenates. Maximal doses of norepinephrine produced only marginal stimulation of phosphatidylinositol hydrolysis, in contrast to a more substantial response produced by muscarinic stimulation. We conclude that human ventricular myocardium contains alpha-1 adrenergic receptors that 1) are of relatively low density, 2) are unchanged in density by heart failure and 3) mediate relatively low-level stimulation of phosphatidylinositol hydrolysis.     

Behavioral effects of beta adrenergic agonists and antidepressant drugs after down-regulation of beta-2 adrenergic receptors by clenbuterol

Acute treatment with the centrally active beta-2 adrenergic agonist clenbuterol reduced response rate and increased reinforcement rate of rats responding under a differential-reinforcement-of-low-rate (DRL) 72-sec schedule in a dose-dependent manner (ED50 value of about 0.1 mg/kg). With repeated treatment, rapid tolerance developed to this effect of clenbuterol. Redetermination of the dose-response function for clenbuterol, following 2 weeks of repeated daily administration, showed that clenbuterol no longer affected DRL behavior at doses up to 3 mg/kg. Interestingly, tolerance developed to clenbuterol even when it was administered after each daily session. This suggests that behavioral factors did not contribute, in an appreciable manner, to the development of tolerance to clenbuterol, and that neuropharmacological changes were sufficient for tolerance development. Such an interpretation is supported by the finding that the density of beta-2 adrenergic receptors in the cerebral cortices and cerebella of rats receiving the same repeated-treatment regimen was reduced with a time course similar to the loss of behavioral responsiveness. The effects of two additional beta-2 selective agonists, SOM-1122 and zinterol, on DRL behavior also were attenuated after repeated treatment with clenbuterol. By contrast, the effects of the beta-1 selective agonists dobutamine and prenalterol and the antidepressants desipramine, phenelzine and fluoxetine on DRL behavior were unaltered after repeated treatment with clenbuterol. These findings suggest functional independence of the beta adrenergic receptor subtypes and further suggest that, consistent with neuropharmacological data, the behavioral effects of the antidepressants do not depend on functionally responsive beta-2 adrenergic receptors.     

Selective regulation of beta-2 adrenergic receptors by the chronic administration of the lipophilic beta adrenergic receptor agonist clenbuterol: an autoradiographic study

Many antidepressant drugs, when administered chronically to rats, have been shown to produce decreases in the density of beta adrenergic receptors in the central nervous system. The centrally active beta adrenergic receptor agonist clenbuterol is currently being evaluated clinically as an antidepressant. The chronic administration of this drug to rats resulted in a large decrease in the density of beta adrenergic receptors in some areas of the rat brain but not in others. Thus, autoradiographic studies revealed that the total density of beta adrenergic receptors in the molecular layer of the cerebellum, but not in layers 1 to 3 or layer 4 of the cerebral cortex, was decreased. To examine whether this regional selectivity occurred because of differences in plasticity of cerebellum and cortex or because cerebellum contains mainly beta-2 adrenergic receptors and cortex contains mainly beta-1 adrenergic receptors, separate analyses of the subtypes of beta adrenergic receptors were performed in each area. These experiments indicated that the decrease in receptor density was entirely specific for beta-2 adrenergic receptors, whereas the density of beta-1 receptors was unchanged. Thus, even in layers 1 to 3 and layer 4 of the cerebral cortex, beta-2 receptor density was decreased, with no change in beta-1 receptor density. Using the autoradiographic assay for ligand binding, it was shown that clenbuterol has equal affinity for beta-1 and beta-2 adrenergic receptors, indicating that the selective effect of this drug was not due to a selective affinity for beta-2 receptors.(ABSTRACT TRUNCATED AT 250 WORDS)     

Renin-angiotensin system as a potential target for the treatment with heart failure: ACE inhibitors,angiotensin-II receptor blockers and aldosterone antagonists

Recent experimental studies and clinical trials have revealed that the modulation of either systemic or regional(cardiovascular) renin-angiotensin systems(RAS) is one of the potential targets to prevent the progression of heart failure. Either ACE inhibitor, angiotensin-II receptor blocker or aldosterone antagonist differently block RAS. Many clinical trials told us that each of them improves heart failure and might promise the better prognosis. Now, some experimental studies are encouraging us to test the comparative effects or the effects in combination of them. Here we will summarize the current outcomes from the experimental and clinical studies, and discuss the future direction.     

Interaction of beta adrenergic agonists and antagonists with brain beta adrenergic receptors in vivo

There is interest in knowing whether beta adrenergic antagonists or agonists, when administered systemically, can enter the brain to interact with central beta adrenergic receptors. To study this, the reduction in the radioactive content in the brain of rats after administration of (-)-[125I]iodopindolol (IPIN) by systemically administered beta agonists or antagonists was measured. Previous studies show that after the i.v. administration of IPIN the binding in vivo to various areas of the central nervous system has the characteristics expected of binding to beta adrenergic receptors. Of the antagonists tested, pindolol and butylpindolol showed potent interactions with beta receptors in both cortex and cerebellum whereas atenolol and practolol did not interact at doses up to 30 mg/kg. CGP-12177 showed moderate potency in inhibiting IPIN binding in vivo. We have shown previously that propranolol and alprenolol inhibit IPIN binding with high potency in cortex and cerebellum. At high doses, butoxamine, a beta-2 antagonist, reduced the binding of IPIN in the cerebellum but not in the cortex. Of the agonists tested, clenbuterol and prenalterol caused a significant dose-dependent reduction of the binding of IPIN, with clenbuterol being more potent. Isoproterenol, salbutamol, salmefamol and dobutamine had no effect. With the exception of CGP-12177, the affinity of the drugs for central beta adrenergic receptors measured in vitro was correlated significantly with their ability to inhibit IPIN binding in vivo whereas their degree of lipophilicity was not correlated significantly with potency in vivo. The inhibition of IPIN binding in vivo from brain areas can be used to evaluate whether drugs penetrate into brain and interact with central beta adrenergic receptors.     

Effects of extracellular calcium and of the calcium entry blockers flunarizine and nimodipine on contractile responses in human temporal arteries

Contraction induced by 124 mM potassium followed the depolarization of smooth-muscle cells and activation of potential-operated calcium channels in human temporal arteries. The contraction elicited consisted of two phases, one rapid and one slowly developing stable phase; both were affected by the two calcium entry blockers flunarizine and nimodipine but at significantly different concentrations. In calcium-free medium 124 mM potassium resulted in a weak contraction. Addition of calcium caused a concentration-dependent contraction that was attenuated by the calcium entry blockers at concentrations comparable to those inhibiting the second phase. The results suggested that in human temporal arteries flunarizine and nimodipine act as calcium entry blockers; there was good correlation with the therapeutic plasma concentration for nimodipine but not for flunarizine.     

Kinetic analysis of the interactions of agonists and antagonists with beta adrenergic receptors

The affinity of the beta adrenergic receptor for antagonists is frequently higher than that for agonists. It has been assumed that the binding of agonists and antagonists is diffusion limited and that the high affinity of the receptor for typical antagonists is due to slow rates of dissociation. To test this hypothesis, the kinetics of binding of unlabeled agonists and antagonists were determined using the method described by Motulsky and Mahan (Mol. Pharmacol. 25: 1-9, 1984). The time course of the binding of a radioligand in the presence of a competing unlabeled ligand was analyzed in terms of rate constants of association (kon) and dissociation (koff) for binding of the radioligand and the competitor. This approach was validated by showing that the rate constants for binding of [3H]dihydroalprenolol and [3H]CGP-12177 [[3H]-4-(3-tertiarybutylamino-2-hydroxypropoxy)-benzimidazole-2-on ] determined directly were similar to values determined when the binding of [125I]iodopindolol was measured in the presence of [3H]dihydroalprenolol or [3H]CGP-12177. Computer simulations suggested that this method was experimentally limited to competing ligands with rate constants of dissociation below approximately 0.50 min-1. The apparent rate constants for binding of four unlabeled agonists and eight antagonists were determined experimentally at 10 degrees C. Although the values of koff for agonists and antagonists were similar, the values for kon for binding of agonists were consistently lower than the values for binding of antagonists. The relatively slow rate constant for association of agonists may be explained by a two-step mechanism or may involve agonist-induced isomerization of the receptor.     

Lag period for superoxide anion generation and lysosomal enzyme release from human neutrophils: effects of calcium antagonists and anion channel blockers

Responses by human neutrophils to stimulation include superoxide anion generation and lysosomal enzyme release. Both of these responses occur after distinct lag periods. The effects of calcium antagonists and anion channel blockers on these lag periods were examined to discern some activation steps mediating the effects of the chemotactic peptide f-Met-Leu-Phe and the calcium ionophore A23187. The intracellular calcium antagonist TMB-8 and the calmodulin inhibitor trifluoperazine both lengthened the lag periods for superoxide anion generation with either stimulus. This effect was dependent on the concentration of antagonist employed. Similar results were obtained for lysosomal enzyme release in response to A23187, as measured by a rapid centrifugation technique. None of the antagonists altered the lag periods for azurophil granule secretion, as monitored by the 9-aminoacridine technique. These results suggest a role for intracellular free calcium and calmodulin-dependent reactions in activation of the superoxide anion generation system in response to either stimulus, and activation of lysosomal enzyme release system in response to A23187.     

The actions and role of calcium entry blockers in hypertension: cardiac considerations

The calcium antagonists, although initially restricted to the treatment of angina pectoris, are now finding widespread application as safe and effective therapy for hypertension. These drugs reduce systemic vascular resistance and thus address the usual primary physiologic defect in high blood pressure. Reduction of blood pressure with these agents appears to lead to the concomitant reduction of hypertension-induced ventricular hypertrophy and improvement of ventricular function. Unlike other vasodilators, calcium antagonists do not secondarily lead to sodium retention or stimulation of the renin-angiotensin-aldosterone and sympathoadrenal systems. They are effective in the treatment of angina pectoris and may inhibit the onset or progression of atherosclerosis. As calcium antagonists primarily block voltage-operated, as opposed to receptor-operated, sarcolemmal calcium channels, they may be rationally and effectively combined with agents such as converting enzyme inhibitors and adrenergic antagonists. Calcium antagonists have important interactions with drugs relevant to the treatment of cardiac disease. The combination of verapamil and beta blockers or disopyramide and diltiazem with amiodarone should be avoided; caution is advised in the concomitant use of calcium antagonists and digoxin or the major antiarrhythmic agents.     

Vasopressin receptor antagonists: mechanisms of action and potential effects in heart failure

Increased arginine vasopressin (AVP) secretion in heart failure may lead to vasoconstriction, left ventricular remodeling, and water retention-actions that promote afterload, preload, and hyponatremia and thereby cause disease progression. Interfering with AVP-mediated signaling pharmacologically may be beneficial in heart failure. Selective antagonism of the vasopressin 2 (V2) receptor may facilitate a safe diuresis and normalize low serum sodium levels, as demonstrated in preliminary clinical trials. Pure V2 antagonism, however, may stimulate AVP secretion and enhance V1a signaling, while pure V1a receptor antagonism may lead to unwanted V2 stimulation and secondary water retention and volume expansion. Combined V1a and V2 receptor antagonism could potentially prove advantageous as a therapy for heart failure by acting synergistically to facilitate diuresis and improve hemodynamics.     

The clinical effects of vasopressin receptor antagonists in heart failure

The neurohormone arginine vasopressin (AVP) is a promising target in the treatment of heart failure because AVP promotes congestion and hyponatremia, each of which is associated with poor outcomes. Diuretics are standard therapy for heart failure, but they have several limitations, including worsening renal function and hyponatremia. Blocking AVP leads to effective aquaresis, improvements in hemodynamics and renal function parameters, weight loss, and normalization of serum sodium, without changes in blood pressure or heart rate. In placebo-controlled trials in the inpatient and outpatient setting, the AVP receptor antagonist tolvaptan reduced body weight and edema and normalized serum sodium in patients with heart failure.     

Therapeutic role of angiotensin II receptor blockers in the treatment of heart failure

Angiotensin II type 1 receptor blockers (ARBs) are generally as effective as angiotensin-converting enzyme (ACE) inhibitors in patients with hypertension. However, inhibition of angiotensin is not achieved completely through the blocking effects of ACE inhibitors, and the possibility of a non-ACE pathway for generation of angiotensin II has important implications for treating cardiovascular disease. The selective quality of ARBs for the angiotensin II type 1 (AT1) receptor may confer an advantage. In a recently reported trial, the ARB valsartan substantially improved patients' New York Heart Association class, clinical signs and symptoms, and quality of life and provided morbidity and mortality benefits in selected patients. Valsartan was recently approved to treat heart failure in patients who cannot be maintained on an ACE inhibitor. As a class, ARBs are well tolerated and have a good safety profile.     

The role of cardiac beta-1 receptors in the hemodynamic response to a beta-2 agonist

The role of beta 1-receptors in the hemodynamic response to beta 2-stimulation was assessed in seven healthy subjects by infusion of the selective beta 2-agonist terbutaline both with and without selective beta 1-blockade by atenolol (50 mg). Infusion of terbutaline increased heart rate (+28 bpm) and indices of left ventricular (LV) performance associated with a marked decrease in LV end-systolic wall stress. The LV end-diastolic dimension remained unchanged despite the tachycardia, suggesting that venous return had increased. Systolic blood pressure increased, whereas total peripheral resistance and diastolic blood pressure decreased. Atenolol pretreatment caused the hemodynamic changes expected of beta 1-blockade but did not blunt the effects of terbutaline on heart rate, peripheral resistance, or venous return. Increases after terbutaline in LV performance and systolic blood pressure were significantly blunted by atenolol. Stimulation of beta 1-receptors therefore appears to play no role in the chronotropic and only a moderate role in the inotropic response after infusion of a beta 2-agonist. Alternative mechanisms for the cardiac changes with terbutaline include (1) withdrawal of vagal tone, (2) decrease in afterload, and (3) stimulation of cardiac beta 2-receptors.     

Differential inhibition of beta adrenergic receptors in human and rabbit ciliary process and heart

Prior biochemical studies have suggested that beta adrenergic receptors in the ciliary process are mostly of the beta-2 subtype. The present experiments evaluate a number of beta adrenergic antagonists, including several recently developed drugs, for their ability to block rabbit and human ciliary process and heart beta adrenergic receptors activating adenylate cyclase. Three of these agents (alpha-methylpropranolol, IPS 339 and ICI 118,551) demonstrated a high degree of oculoselectivity in both rabbit and human. The other agents (S 37-429, S 32-468, ICI 78,462,H35/25, butoxamine, propranolol, timolol, atenolol and practolol) showed either modest or no oculoselectivity. Structure-activity studies suggested that, among antagonists of the aryloxymethyl type, methylation of the side-chain alpha-carbon or the aromatic ring may enhance oculoselectivity primarily by decreasing potency at cardiac beta adrenergic receptors. Additional physiological studies of cardiac chronotropic response revealed that, compared with nonselective beta blockers, compounds with biochemical oculoselectivity demonstrate decreased physiological effects on cardiac function. This was true when the selective agents were applied either systemically or topically to the eye. On the other hand, the systemic absorption of topical timolol was sufficient to block cardiac chronotropic effects completely. These findings, identifying relatively specific blockers of rabbit and human ciliary process beta adrenergic receptors, have implications for the development of ocular hypotensive agents with fewer systemic side effects on tissues enriched in beta-1 adrenergic receptors.