A gain-of-function polymorphism in a G-protein coupling domain of the human beta1-adrenergic receptor.
The beta1-adrenergic receptor (beta1AR) is a key cell surface signaling protein expressed in the heart and other organs that mediates the actions of catecholamines of the sympathetic nervous system. A polymorphism in the intracellular cytoplasmic tail near the seventh transmembrane-spanning segment of the human beta1AR has been identified in a cohort of normal individuals. At amino acid position 389, Gly or Arg can be found (allele frequencies 0.26 and 0. 74, respectively), the former previously considered as the human wild-type beta1AR. Using site-directed mutagenesis to mimic the two variants, CHW-1102 cells were permanently transfected to express the Gly-389 and Arg-389 receptors. In functional studies with matched expression, the Arg-389 receptors had slightly higher basal levels of adenylyl cyclase activities (10.7 +/- 1.2 versus 6.1 +/- 0.4 pmol/min/mg). However, maximal isoproterenol-stimulated levels were markedly higher for the Arg-389 as compared to the Gly-389 receptor (63.3 +/- 6.1 versus 20.9 +/- 2.0 pmol/min/mg). Agonist-promoted [35S]guanosine 5'-O-(thiotriphosphate) binding was also increased with the Arg-389 receptor consistent with enhanced coupling to Gs and increased adenylyl cyclase activation. In agonist competition studies carried out in the absence of guanosine 5'-(beta, gamma-imido)triphosphate, high affinity binding could not be resolved with the Gly-389 receptor, whereas Arg-389 displayed an accumulation of the agonist high affinity receptor complex (RH = 26%). Taken together, these data indicate that this polymorphic variation of the human beta1AR results in alterations of receptor-Gs interaction with functional signal transduction consequences, consistent with its localization in a putative G-protein binding domain. The genetic variation of beta1AR at this locus may be the basis of interindividual differences in pathophysiologic characteristics or in the response to therapeutic betaAR agonists and antagonists in cardiovascular and other diseases. (+info)
beta2-adrenergic cAMP signaling is uncoupled from phosphorylation of cytoplasmic proteins in canine heart.
BACKGROUND: Recent studies of beta-adrenergic receptor (beta-AR) subtype signaling in in vitro preparations have raised doubts as to whether the cAMP/protein kinase A (PKA) signaling is activated in the same manner in response to beta2-AR versus beta1-AR stimulation. METHODS AND RESULTS: The present study compared, in the intact dog, the magnitude and characteristics of chronotropic, inotropic, and lusitropic effects of cAMP accumulation, PKA activation, and PKA-dependent phosphorylation of key effector proteins in response to beta-AR subtype stimulation. In addition, many of these parameters and L-type Ca2+ current (ICa) were also measured in single canine ventricular myocytes. The results indicate that although the cAMP/PKA-dependent phosphorylation cascade activated by beta1-AR stimulation could explain the resultant modulation of cardiac function, substantial beta2-AR-mediated chronotropic, inotropic, and lusitropic responses occurred in the absence of PKA activation and phosphorylation of nonsarcolemmal proteins, including phospholamban, troponin I, C protein, and glycogen phosphorylase kinase. However, in single canine myocytes, we found that beta2-AR-stimulated increases in both ICa and contraction were abolished by PKA inhibition. Thus, the beta2-AR-directed cAMP/PKA signaling modulates sarcolemmal L-type Ca2+ channels but does not regulate PKA-dependent phosphorylation of cytoplasmic proteins. CONCLUSIONS: These results indicate that the dissociation of beta2-AR signaling from cAMP regulatory systems is only apparent and that beta2-AR-stimulated cAMP/PKA signaling is uncoupled from phosphorylation of nonsarcolemmal regulatory proteins involved in excitation-contraction coupling. (+info)
Cardiovascular and metabolic alterations in mice lacking both beta1- and beta2-adrenergic receptors.
The activation state of beta-adrenergic receptors (beta-ARs) in vivo is an important determinant of hemodynamic status, cardiac performance, and metabolic rate. In order to achieve homeostasis in vivo, the cellular signals generated by beta-AR activation are integrated with signals from a number of other distinct receptors and signaling pathways. We have utilized genetic knockout models to test directly the role of beta1- and/or beta2-AR expression on these homeostatic control mechanisms. Despite total absence of beta1- and beta2-ARs, the predominant cardiovascular beta-adrenergic subtypes, basal heart rate, blood pressure, and metabolic rate do not differ from wild type controls. However, stimulation of beta-AR function by beta-AR agonists or exercise reveals significant impairments in chronotropic range, vascular reactivity, and metabolic rate. Surprisingly, the blunted chronotropic and metabolic response to exercise seen in beta1/beta2-AR double knockouts fails to impact maximal exercise capacity. Integrating the results from single beta1- and beta2-AR knockouts as well as the beta1-/beta2-AR double knock-out suggest that in the mouse, beta-AR stimulation of cardiac inotropy and chronotropy is mediated almost exclusively by the beta1-AR, whereas vascular relaxation and metabolic rate are controlled by all three beta-ARs (beta1-, beta2-, and beta3-AR). Compensatory alterations in cardiac muscarinic receptor density and vascular beta3-AR responsiveness are also observed in beta1-/beta2-AR double knockouts. In addition to its ability to define beta-AR subtype-specific functions, this genetic approach is also useful in identifying adaptive alterations that serve to maintain critical physiological setpoints such as heart rate, blood pressure, and metabolic rate when cellular signaling mechanisms are perturbed. (+info)
Progressive hypertrophy and heart failure in beta1-adrenergic receptor transgenic mice.
Stimulation of cardiac beta1-adrenergic receptors is the main mechanism that increases heart rate and contractility. Consequently, several pharmacological and gene transfer strategies for the prevention of heart failure aim at improving the function of the cardiac beta-adrenergic receptor system, whereas current clinical treatment favors a reduction of cardiac stimulation. To address this controversy, we have generated mice with heart-specific overexpression of beta1-adrenergic receptors. Their cardiac function was investigated in organ bath experiments as well as in vivo by cardiac catheterization and by time-resolved NMR imaging. The transgenic mice had increased cardiac contractility at a young age but also developed marked myocyte hypertrophy (3.5-fold increase in myocyte area). This increase was followed by progressive heart failure with functional and histological deficits typical for humans with heart failure. Contractility was reduced by approximately 50% in 35-week-old mice, and ejection fraction was reduced down to a minimum of approximately 20%. We conclude that overexpression of beta1-adrenergic receptors in the heart may lead to a short-lived improvement of cardiac function, but that increased beta1-adrenergic receptor signalling is ultimately detrimental. (+info)
Potent and selective human beta(3)-adrenergic receptor antagonists.
Although the functional presence of beta(3)-adrenergic receptors (beta(3)-AR) in rodents is well established, its significance in human adipose tissue has been controversial. One of the issues confounding the experimental data has been the lack of potent and selective human beta(3)-AR ligands analogous to the rodent-specific agonist BRL37344. Recently, we described a new class of aryloxypropanolamine beta(3)-AR agonists that potently and selectively activate lipolysis in rhesus isolated adipocytes and stimulate the metabolic rate in rhesus monkeys in vivo. In this article, we describe novel and selective beta(3)-AR antagonists with high affinity for the human receptor. L-748,328 and L-748,337 bind the human cloned beta(3)-AR expressed in Chinese hamster ovary (CHO) cells with an affinity of 3.7 +/- 1.4 and 4.0 +/- 0.4 nM, respectively. They display an affinity of 467 +/- 89 and 390 +/- 154 nM for the human beta(1)-AR. Their selectivity for human beta(3)-AR versus beta(2)-AR is greater than 20-fold (99 +/- 43 nM) and 45-fold (204 +/- 75 nM), respectively. These compounds are competitive antagonists capable of inhibiting the functional activation of agonists in a dose-dependent manner in cells expressing human cloned beta(3)-AR. Moreover, both L-748,328 and L-748,337 inhibit the lipolytic response elicited by the beta(3)-AR agonist L-742,791 in isolated nonhuman primate adipocytes. The aryloxypropanolamine benzenesulfonamide ligands illustrated here and elsewhere demonstrate high-affinity human beta(3)-AR binding. In addition, we describe specific 3'-phenoxy substitutions that transform these compounds from potent agonists into selective antagonists. (+info)
Inducible cyclic AMP early repressor protein in rat pinealocytes: a highly sensitive natural reporter for regulated gene transcription.
Rhythmic activity of arylalkylamine N-acetyltransferase (AANAT) determines melatonin synthesis in rat pineal gland. The transcriptional regulation of AANAT involves the activating and inhibiting transcription factors of the cyclic AMP (cAMP)-signaling pathway, cAMP response element-binding protein and inducible cAMP early repressor (ICER), respectively. Activation of this pathway is centered around norepinephrine, stimulating beta(1)-adrenergic receptors, but various other transmitters can modulate melatonin biosynthesis. To compare the transcriptional impact of norepinephrine with that of other neurotransmitters on melatonin synthesis, we determined ICER protein levels in pinealocytes and, in parallel, hormone secretion. The dose-dependent inductions of ICER protein by norepinephrine, the beta(1)-adrenergic receptor agonist isoproterenol, vasoactive intestinal peptide, pituitary adenylate cyclase-activating polypeptide, and adenosine are correlated to regulatory dynamics in melatonin production. Importantly, ICER protein induction required lower ligand concentrations than the induction of melatonin biosynthesis. Although neuropeptide Y, glutamate, and vasopressin altered norepinephrine-stimulated hormone production without affecting ICER levels, the activation of voltage-gated cation channels increased ICER without affecting hormone synthesis. Sensitivity and versatility of ICER induction in pinealocytes make these neuroendocrine cells a valuable model system in which to study molecular interactions determining a regulated gene expression. (+info)
Inverse agonist activities of beta-adrenoceptor antagonists in rat myocardium.
1. Negative inotropic effects of several beta-adrenoceptor (betaAR) antagonists on electrically-stimulated right atria, left atria, right ventricles and left ventricular papillary muscles from reserpine-treated rats were used as a measure of their inverse agonist activities. 2. Beta1AR antagonists acebutolol, atenolol and metoprolol, beta2AR antagonist ICI-181,551 and nonselective betaAR antagonists alprenolol, nadolol, propranolol and timolol produced negative inotropic effects, which were most marked on the right atria. 3. The nonselective betaAR antagonist pindolol did not exhibit inverse agonist activity but inhibited the negative inotropic activities of ICI-118,551, atenolol and propranolol. 4. The negative inotropic effects of lidocaine, nifedipine and pentobarbitone were similar on all the four myocardial preparations. 5. The positive inotropic efficacy of salbutamol on right and left atria but not on right ventricles and papillary muscles was comparable to that of isoprenaline. The antagonist activity of ICI-118,551 against isoprenaline was greater on right atria than on other cardiac regions. 6. Beta1AR proteins were expressed in all regions of the heart but of beta2AR were primarily localized in the right atrium. 7. It is concluded that beta2AR play a greater role in right atria than in other cardiac regions and almost all betaAR antagonists behave as inverse agonists. (+info)
Characterization of beta-adrenoceptor mediated smooth muscle relaxation and the detection of mRNA for beta1-, beta2- and beta3-adrenoceptors in rat ileum.
1. Functional and molecular approaches were used to characterize the beta-AR subtypes mediating relaxation of rat ileal smooth muscle. 2. In functional studies, (-)-isoprenaline relaxation was unchanged by CGP20712A (beta1-AR antagonist) or ICI118551 (beta2-AR antagonist) but shifted by propranolol (pKB=6.69). (+/-)-Cyanopindolol, CGP12177 and ICID7114 did not cause relaxation but antagonized (-)-isoprenaline relaxation. 3. BRL37344 (beta3-AR agonist) caused biphasic relaxation. The high affinity component was shifted with low affinity by propranolol, (+/-)-cyanopindolol, tertatolol and alprenolol. CL316243 (beta3-AR agonist) relaxation was unaffected by CGP20712A or ICI118551 but blocked by SR58894A (beta3-AR antagonist; pA2 = 7.80). Enhanced relaxation after exposure to forskolin and pertussis toxin showed that beta3-AR relaxation can be altered by manipulation of components of the adenylate cyclase signalling pathway. 4. The beta-AR agonist RO363 relaxed the ileum (pEC50=6.18) and was blocked by CGP20712A. Relaxation by the beta2-AR agonist zinterol (pEC50=5.71) was blocked by SR58894A but not by ICI118551. 5. In rat ileum, beta1-, beta2- and beta3-AR mRNA was detected. Comparison of tissues showed that beta3-AR mRNA expression was greatest in WAT>colon=ileum >cerebral cortex>soleus; beta1-AR mRNA was most abundant in cerebral cortex > WAT > ileum = colon > soleus; beta2-AR mRNA was expressed in soleus > WAT > ileum = colon > cerebral cortex. 6. These results show that beta3-ARs are the predominant beta-AR subtype mediating rat ileal relaxation while beta1-ARs may produce a small relaxation. The beta2-AR agonist zinterol produces relaxation through beta3-ARs and there was no evidence for the involvement of beta2-ARs in relaxation despite the detection of beta2-AR mRNA. (+info)