Coupling of beta2-adrenoceptor to Gi proteins and its physiological relevance in murine cardiac myocytes.
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1999-01-08
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Abstract
-Transgenic mouse models have been developed to manipulate beta-adrenergic receptor (betaAR) signal transduction. Although several of these models have altered betaAR subtypes, the specific functional sequelae of betaAR stimulation in murine heart, particularly those of beta2-adrenergic receptor (beta2AR) stimulation, have not been characterized. In the present study, we investigated effects of beta2AR stimulation on contraction, [Ca2+]i transient, and L-type Ca2+ currents (ICa) in single ventricular myocytes isolated from transgenic mice overexpressing human beta2AR (TG4 mice) and wild-type (WT) littermates. Baseline contractility of TG4 heart cells was increased by 3-fold relative to WT controls as a result of the presence of spontaneous beta2AR activation. In contrast, beta2AR stimulation by zinterol or isoproterenol plus a selective beta1-adrenergic receptor (beta1AR) antagonist CGP 20712A failed to enhance the contractility in TG4 myocytes, and more surprisingly, beta2AR stimulation was also ineffective in increasing contractility in WT myocytes. Pertussis toxin (PTX) treatment fully rescued the ICa, [Ca2+]i, and contractile responses to beta2AR agonists in both WT and TG4 cells. The PTX-rescued murine cardiac beta2AR response is mediated by cAMP-dependent mechanisms, because it was totally blocked by the inhibitory cAMP analog Rp-cAMPS. These results suggest that PTX-sensitive G proteins are responsible for the unresponsiveness of mouse heart to agonist-induced beta2AR stimulation. This was further corroborated by an increased incorporation of the photoreactive GTP analog [gamma-32P]GTP azidoanilide into alpha subunits of Gi2 and Gi3 after beta2AR stimulation by zinterol or isoproterenol plus the beta1AR blocker CGP 20712A. This effect to activate Gi proteins was abolished by a selective beta2AR blocker ICI 118,551 or by PTX treatment. Thus, we conclude that (1) beta2ARs in murine cardiac myocytes couple to concurrent Gs and Gi signaling, resulting in null inotropic response, unless the Gi signaling is inhibited; (2) as a special case, the lack of cardiac contractile response to beta2AR agonists in TG4 mice is not due to a saturation of cell contractility or of the cAMP signaling cascade but rather to an activation of beta2AR-coupled Gi proteins; and (3) spontaneous beta2AR activation may differ from agonist-stimulated beta2AR signaling.
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Scholars@Duke
Robert J. Lefkowitz
Dr. Lefkowitz’s memoir, A Funny Thing Happened on the Way to Stockholm, recounts his early career as a cardiologist and his transition to biochemistry, which led to his Nobel Prize win.
Robert J. Lefkowitz, M.D. is Chancellor’s Distinguished Professor of Medicine and Professor of Biochemistry and Chemistry at the Duke University Medical Center. He has been an Investigator of the Howard Hughes Medical Institute since 1976. Dr. Lefkowitz began his research career in the late 1960’s and early 1970’s when there was not a clear consensus that specific receptors for drugs and hormones even existed. His group spent 15 difficult years developing techniques for labeling the receptors with radioactive drugs and then purifying the four different receptors that were known and thought to exist for adrenaline, so called adrenergic receptors. In 1986 Dr. Lefkowitz transformed the understanding of what had by then become known as G protein coupled receptors because of the way the receptor signal for the inside of a cell through G proteins, when he and his colleagues cloned the gene for the beta2-adrenergic receptor. They immediately recognized the similarity to a molecule called rhodopsin which is essentially a light receptor in the retina. This unexpected finding established the beta receptor and rhodopsin as the first member of a new family of proteins. Because each has a peptide structure, which weaves across the cell membrane seven times, these receptors are referred to as seven transmembrane receptors. This super family is now known to be the largest, most diverse and most therapeutically accessible of all the different kinds of cellular receptors. There are almost a thousand members of this receptor family and they regulate virtually all known physiological processes in humans. They include the receptors not only to numerous hormones and neurotransmitters but for the receptors which mediate the senses of sweet and bitter taste and smell amongst many others. Dr. Lefkowitz also discovered the mechanism by which receptor signaling is turned off, a process known as desensitization. Dr. Lefkowitz work was performed at the most fundamental and basic end of the research spectrum and has had remarkable consequences for clinical medicine. Today, more than half of all prescription drug sales are of drugs that target either directly or indirectly the receptors discovered by Dr. Lefkowitz and his trainees. These include amongst many others beta blockers, angiotensin receptor blockers or ARBs and antihistamines. Over the past decade he has discovered novel mechanisms by which the receptors function which may lead to the development of an entirely new class of drugs called “biased agonists”. Several such compounds are already in advanced stages of clinical testing. Dr. Lefkowitz has received numerous honors and awards, including the National Medal of Science, the Shaw Prize, the Albany Prize, and the 2012 Nobel Prize in Chemistry. He was elected to the USA National Academy of Sciences in 1988, the Institute of Medicine in 1994, and the American Academy of Arts and Sciences in 1988.
Walter J. Koch
My research interests are centered on the molecular mechanisms involved in the regulation of signaling through cardiovascular adrenergic receptors (ARs) including the specific in vivo interactions between ARs and myocardial G protein-coupled receptor kinases (GRKs). This includes the role of ARs and GRKs in cardiovascular disease. The β-adrenergic receptor kinase (betaARK1) is a prototypic member of the GRK family that targets and phosphorylates agonist-occupied G protein-coupled receptors which causes functional uncoupling and a dampening of signaling, a process termed desensitization. There is a growing body of evidence demonstrating that the actions of GRKs in the heart are extremely important in modulating myocardial adrenergic signaling and hence cardiac function. Of particular interest to my laboratory, are the biochemical and physiological consequences of altering myocardial AR and GRK signaling. To address this, we are utilizing several in vitro and in vivo model systems. Of special interest, are the endogenous alterations that occur in myocardial GRKs and ARs during cardiovascular diseases such as heart failure (HF). We are also interested in the role of adrenergic signaling in the transplanted heart.
Since heart disease accounts for nearly 40% of all deaths annually in this country, it is of importance to learn more of the molecular pathology present in diseased myocardium. For example, in human HF regardless of the cause, a specific constellation of biochemical defects in cardiac tissue has been noted including loss of specific betaAR density and uncoupling of remaining receptors. Moreover, it has been shown that the levels of betaARK1 rise 3-5 fold in human HF, probably contributing to the dysfunction. Interestingly, it has recently been demonstrated that cardiovascular GRK levels (i.e. betaARK1) and activity were also increased in other disorders including myocardial hypertrophy and hypertension. Thus, a primary objective of my laboratory is to investigate the molecular mechanisms involved in myocardial GRK alterations and how these changes relate to adrenergic signaling. This research includes studying and exploring novel protein binding partners of GRKs in the myocardium using a variety of molecular biochemical assays including GRK-affinity columns.
Over the last several years, my laboratory has been investigating the consequences of altering myocardial ARs and GRKs in several in vivo model systems. Original studies were done in transgenic mice where AR and GRK-based transgenes were targeted specifically to the heart. These studies have been done in close collaboration with Dr. Robert Lefkowitz and Dr. Howard Rockman here at Duke University. We have found that altering AR signaling in the hearts of transgenic mice has profound effects on cardiovascular physiology including the findings that overexpression of beta2ARs or a peptide inhibitor of betaARK1 known as the betaARKct, significantly enhances in vivo cardiac contractility. We now have transgenic animals with myocardial-targeted overexpression of several ARs (alpha and beta) or GRKs and are studying the specific in vivo interactions of these molecules in the heart. Moreover, in collaboration with Drs. Lefkowitz and Rockman, we are generating a host of tissue and temporal specific conditional-knockout mice targeting the myocardial betaAR and GRK systems. Another area of transgenic animals being studied exclusively in my laboratory is altered betaAR and GRK activity specifically targeted to arterial smooth muscle in order to determine the role of AR signaling and desensitization in hypertension and other vascular diseases. These mice have recently been generated in our laboratory and are the target of current investigations.
Over the last year, we have been able to specifically study the role of betaAR desensitization and the role of betaARK1 in HF using genetically engineered mouse models of cardiomyopathy and HF. These studies have lead to the findings that inhibition of betaARK1 through myocardial-targeted betaARKct expression has rescued three separate genetic mouse models of cardiomyopathy, preventing the development of HF. The molecular study of these genetic models through DNA array technology is a logical step in the evolution of these studies and this is a new area of focus in the laboratory. Thus, we are analyzing the hearts of mice with altered betaAR and/or betaARK1 signaling by gene (DNA) chips to determine other genes that have been induced or silenced by our transgenes or gene knockouts. We are excited about using this technology in the laboratory taking advantage of our novel mouse models. Specifically, comparing differential gene expression in a failing mouse heart and comparing the genetic pattern in a heart that has been "rescued" by the betaARKct could lead to the elucidation of specific genes involved in the pathogenesis of HF. Importantly, this may also lead to novel therapeutic approaches to treating this disease as well as other cardiovascular disorders.
Our findings in mice that show that we can genetically enhance the functional contractility of the heart form the basis of another major focus of my research program which is the investigation of enhancing the in vivo function of the compromised heart via acute genetic manipulation using gene therapy. My laboratory heads the Cardiovascular Gene Therapy Program at Duke University Medical Center. The ability to manipulate betaAR density or receptor desensitization in the diseased heart is of great interest since it may provide unique inotropic support and improve existing therapeutic strategies. Gene transfer to the heart in vivo is a powerful approach to study the specific role of GRKs and adrenergic desensitization in both normal and diseased myocardium. Currently, we are employing various in vivo gene delivery techniques using adenoviruses, in order to effectively express specific betaAR and GRK-based transgenes which may produce alterations in myocardial signaling and global cardiac function. We also have established several surgical models of compromised heart function to use in these gene transfer studies including experimental HF in rabbits and pigs, and cardiac transplantation models in rats and rabbits. In addition to studying the feasibility of gene therapy approaches to HF, the Cardiovascular Gene Therapy Program here at Duke has developed a molecular gene therapy strategy to prevention of pathological vascular smooth muscle intimal hyperplasia such as coronary artery restenosis after angioplasty. Our first clinical trial employing adenoviral-mediated gene therapy for restenosis is tentatively planned for the end of 2002.
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