Modification of β-adrenoceptor signal transduction pathway by genetic manipulation and heart failure

The -adrenoceptor (-AR) mediated signal transduction pathway in cardiomyocytes is known to involve ₁- and ₂-ARs, stimulatory (G_s) and inhibitory (G_i) guanine nucleotide binding proteins, adenylyl cyclase (AC) and cAMP-dependent protein kinase (PKA). The activation of ₁- and ₂-ARs has been shown to increase heart function by increasing Ca²⁺-movements across the sarcolemmal membrane and sarcoplasmic reticulum through the stimulation of G_s-proteins, activation of AC and PKA enzymes and phosphorylation of the target sites. The activation of PKA has also been reported to increase phosphorylation of some myofibrillar proteins (for promoting cardiac relaxation) and nuclear proteins (for cardiac hypertrophy). The activation of ₂-AR has also been shown to affect G_i-proteins, stimulate mitogen activated protein kinase and increase protein synthesis by enhancing gene expression. ₁- and ₂-ARs as well as AC are considered to be regulated by PKA- and protein kinase C (PKC)-mediated phosphorylations directly; both PKA and PKC also regulate -AR indirectly through the involvement of -AR kinase (ARK), -arrestins and G-protein subunits. Genetic manipulation of different components and regulators of -AR signal transduction pathway by employing transgenic and knockout mouse models has provided insight into their functional and regulatory characteristics in cardiomyocytes. The genetic studies have also helped in understanding the pathophysiological role of ARK in heart dysfunction and therapeutic role of ARK inhibitors in the treatment of heart failure. Varying degrees of defects in the -AR signal transduction system have been identified in different types of heart failure to explain the attenuated response of the failing heart to sympathetic stimulation or catecholamine infusion. A decrease in ₁-AR density, an increase in the level of G_i-proteins and overexpression of ARK are usually associated with heart failure; however, these attenuations have been shown to be dependent upon the type and stage of heart failure as well as region of the heart. Both local and circulating renin-angiotensin systems, sympathetic nervous system and endothelial cell function appears to regulate the status of -AR signal transduction pathway in the failing heart. Thus different components and regulators of the -AR signal transduction pathway appears to represent important targets for the development of therapeutic interventions for the treatment of heart failure.     

Differential changes in beta-adrenoceptor signal transduction in left and right ventricles of infarcted rats

Although different experimental and clinical studies have revealed varying degrees of defects in beta-adrenoceptors (beta-ARs) during the development of heart failure, the mechanisms for differences in beta-AR signal transduction between the left (LV) and right ventricle (RV) are not understood. Because biochemical alterations in the myocardium depend on the stage of heart disease, this study was undertaken to assess the status of beta-ARs in the LV and RV at different stages of heart failure. Myocardial infarction was induced in rats by occluding the left coronary artery for 8 and 24 weeks. The beta-AR signal transduction was monitored by measuring beta1-AR density, the isoproterenol-induced positive inotropic effect, the increase in [Ca2+]i in cardiomyocytes, and the activation of adenylyl cyclase. The beta-AR signal transduction parameters in the 8- and 24-week failing LV were depressed, whereas the RV showed upregulation at 8 weeks and downregulation at 24 weeks of these mechanisms. These results suggest that beta-AR-mediated signal transduction in the LV and RV are differentially regulated and are dependent upon the stage of development of congestive heart failure due to myocardial infarction.     

Changes in beta-adrenoceptors in heart failure due to myocardial infarction are attenuated by blockade of renin-angiotensin system

Earlier studies have revealed an improvement of cardiac function in animals with congestive heart failure (CHF) due to myocardial infarction (MI) by treatment with angiotensin converting enzyme (ACE) inhibitors. Since heart failure is also associated with attenuated responses to catecholamines, we examined the effects of imidapril, an ACE inhibitor, on the beta-adrenoceptor (beta-AR) signal transduction in the failing heart. Heart failure in rats was induced by occluding the coronary artery, and 3 weeks later the animals were treated with g/(kg x day) (orally) imidapril for 4 weeks. The animals were assessed for their left ventricular function and inotropic responses to isoproterenol. Cardiomyocytes and crude membranes were isolated from the non-ischemic viable left ventricle and examined for the intracellular concentration of Ca2+ [Ca2+]i and beta-ARs as well as adenylyl cyclase (AC) activity, respectively. Animals with heart failure exhibited depressions in ventricular function and positive inotropic response to isoproterenol as well as isoproterenol-induced increase in [Ca2+]i in cardiomyocytes; these changes were attenuated by imidapril treatment. Both beta1-AR receptor density and isoproterenol-stimulated AC activity were decreased in the failing heart and these alterations were prevented by imidapril treatment. Alterations in cardiac function, positive inotropic effect of isoproterenol, beta1-AR density and isoproterenol-stimulated AC activity in the failing heart were also attenuated by treatment with another ACE inhibitor, enalapril and an angiotensin II receptor antagonist, losartan. The results indicate that imidapril not only attenuates cardiac dysfunction but also prevents changes in beta-AR signal transduction in CHF due to MI. These beneficial effects are similar to those of enalapril or losartan and thus appear to be due to blockade of the renin-angiotensin system.     

Upregulation of beta-adrenergic receptors in heart failure due to volume overload

To examine the mechanisms of changes in beta-adrenergic signal transduction in heart failing due to volume overload, we studied the status of beta-adrenoceptors (beta-ARs), G protein-coupled receptor kinase (GRK), and beta-arrestin in heart failure due to aortocaval shunt (AVS). Heart failure in rats was induced by creating AVS for 16 wk, and beta-AR binding, GRK activity, as well as their protein content, and mRNA levels were determined in both left and right ventricles. The density and protein content for beta1-ARs, unlike those for beta2-ARs, were increased in the failing hearts. Furthermore, protein contents for GRK isoforms and beta-arrestin-1 were decreased in membranous fractions and increased in cytosolic fractions from the failing hearts. On the other hand, steady-state mRNA levels for beta1-ARs and GRK2, as well as protein content for Gbetagamma-subunits, did not change in the failing heart. Basal cardiac function was depressed; however, both in vivo and ex vivo positive inotropic responses of the failing hearts to isoproterenol were augmented. Treatment of AVS animals with imidapril (1 mg.kg(-1).day(-1)) or losartan (20 mg.kg(-1).day(-1)) retarded the progression of heart failure; partially prevented changes in beta1-ARs, GRKs, and beta-arrestin-1 in the failing myocardium; and attenuated the increase in positive inotropic effect of isoproterenol. These results indicate that upregulation of beta1-ARs is associated with subcellular redistribution of GRKs and beta-arrestin-1 in the failing heart due to volume overload. Furthermore, attenuation of alterations in beta-adrenergic system by imidapril or losartan may be due to blockade of the renin-angiotensin system in the AVS model of heart failure.     

Differential targeting of beta -adrenergic receptor subtypes and adenylyl cyclase to cardiomyocyte caveolae. A mechanism to functionally regulate the cAMP signaling pathway

Differential modes for beta(1)- and beta(2)-adrenergic receptor (AR) regulation of adenylyl cyclase in cardiomyocytes is most consistent with spatial regulation in microdomains of the plasma membrane. This study examines whether caveolae represent specialized subdomains that concentrate and organize these moieties in cardiomyocytes. Caveolae from quiescent rat ventricular cardiomyocytes are highly enriched in beta(2)-ARs, Galpha(i), protein kinase A RIIalpha subunits, caveolin-3, and flotillins (caveolin functional homologues); beta(1)-ARs, m(2)-muscarinic cholinergic receptors, Galpha(s), and cardiac types V/VI adenylyl cyclase distribute between caveolae and other cell fractions, whereas protein kinase A RIalpha subunits, G protein-coupled receptor kinase-2, and clathrin are largely excluded from caveolae. Cell surface beta(2)-ARs localize to caveolae in cardiomyocytes and cardiac fibroblasts (with markedly different beta(2)-AR expression levels), indicating that the fidelity of beta(2)-AR targeting to caveolae is maintained over a physiologic range of beta(2)-AR expression. In cardiomyocytes, agonist stimulation leads to a marked decline in the abundance of beta(2)-ARs (but not beta(1)-ARs) in caveolae. Other studies show co-immunoprecipitation of cardiomyocytes adenylyl cyclase V/VI and caveolin-3, suggesting their in vivo association. However, caveolin is not required for adenylyl cyclase targeting to low density membranes, since adenylyl cyclase targets to low buoyant density membrane fractions of HEK cells that lack prototypical caveolins. Nevertheless, cholesterol depletion with cyclodextrin augments agonist-stimulated cAMP accumulation, indicating that caveolae function as negative regulators of cAMP accumulation. The inhibitory interaction between caveolae and the cAMP signaling pathway as well as domain-specific differences in the stoichiometry of individual elements in the beta-AR signaling cascade represent important modifiers of cAMP-dependent signaling in the heart.     

The cytosolic phospholipase A2 pathway, a safeguard of beta2-adrenergic cardiac effects in rat

We have recently demonstrated that in human heart, beta2-adrenergic receptors (beta2-ARs) are biochemically coupled not only to the classical adenylyl cyclase (AC) pathway but also to the cytosolic phospholipase A2 (cPLA2) pathway (Pavoine, C., Behforouz, N., Gauthier, C., Le Gouvello, S., Roudot-Thoraval, F., Martin, C. R., Pawlak, A., Feral, C., Defer, N., Houel, R., Magne, S., Amadou, A., Loisance, D., Duvaldestin, P., and Pecker, F. (2003) Mol. Pharmacol. 64, 1117-1125). In this study, using Fura-2-loaded cardiomyocytes isolated from adult rats, we showed that stimulation of beta2-ARs triggered an increase in the amplitude of electrically stimulated [Ca2+]i transients and contractions. This effect was abolished with the PKA inhibitor, H89, but greatly enhanced upon addition of the selective cPLA2 inhibitor, AACOCF3. The beta2-AR/cPLA2 inhibitory pathway involved G(i) and MSK1. Potentiation of beta2-AR/AC/PKA-induced Ca2+ responses by AACOCF3 did not rely on the enhancement of AC activity but was associated with eNOS phosphorylation (Ser1177) and L-NAME-sensitive NO production. This was correlated with PKA-dependent phosphorylation of PLB (Ser16). The constraint exerted by the beta2-AR/cPLA2 pathway on the beta2-AR/AC/PKA-induced Ca2+ responses required integrity of caveolar structures and was impaired by Filipin III treatment. Immunoblot analyses demonstrated zinterol-induced translocation of cPLA and its cosedimentation with MSK1, eNOS, PLB, and sarcoplasmic reticulum Ca2+ pump (SERCA) 2a in a low density caveolin-3-enriched membrane fraction. This inferred the gathering of beta2-AR signaling effectors around caveolae/sarcoplasmic reticulum (SR) functional platforms. Taken together, these data highlight cPLA as a cardiac beta2-AR signaling pathway that limits beta2-AR/AC/PKA-induced Ca2+ responses in adult rat cardiomyocytes through the impairment of eNOS activation and PLB phosphorylation.     

The cardiac beta2-adrenergic signalling a new role for the cPLA2

The cardiac actions of catecholamines have long been attributed to the predominant beta(1)-AR subtype that couples to the classical Gs/AC/cAMP pathway. Recent research clearly indicates that cardiac beta(2)-ARs play a functional role in healthy heart and assume increasing importance in failing and aged heart. beta(2)-ARs are primarily coupled to an atypical compartmentalized cAMP pathway, regulated by phosphorylation and/or oligomerization of beta(2)-ARs, and under the control of additional beta(2)-AR/Gi-coupled lipidic pathways, the impact of which seems to vary depending on the animal species, the developmental and pathophysiological state. This review focuses, more especially, on one of the last identified beta(2)-AR/Gi pathway, namely the cPLA(2).     

Coupling of beta-adrenergic receptors to cardiac L-type Ca2+ channels: preferential coupling of the beta1 versus beta2 receptor subtype and evidence for PKA-independent activation of the channel.

Beta1- and beta2-adrenergic receptors (beta-ARs) co-exist in mammalian heart, and it is generally accepted that both activate adenylyl cyclase (AC), resulting in increased levels of cAMP and subsequent activation of L-type Ca2+ channels (CaCh). To investigate the contribution of each beta-AR subtype in AC and CaCh coupling, we stably expressed cardiac CaCh alpha1 and beta2 subunits along with either beta1-AR or beta2-AR in CHW fibroblasts. Co-expression of either beta-AR with CaCh subunits conferred responsiveness of AC and CaCh to isoproterenol (ISO), which was not observed in non-transfected cells. ISO-promoted cAMP formation occurred at a lower EC50 through the beta2-AR than through the beta1-AR (0.13 +/- 0.01 vs. 0.6 +/- 0.14 nM). In contrast, activation of CaCh was more efficacious via the beta1-AR than the beta2-AR (EC50 for CaCh activation = 238 +/- 33 vs. 1057 +/- 113 nM). Pre-treatment with pertussis toxin (PTX) had no effect upon the responsiveness of either cAMP formation or CaCh activation through either receptor. We conclude (1) that beta1-ARs exhibit preferential coupling to CaCh activation, versus that observed for the beta2-AR; (2) that this preferential coupling cannot be explained solely by cAMP-dependent processes; and (3) that the relative attenuation of beta2-AR-promoted CaCh activation is not due to receptor coupling to PTX-sensitive G proteins. Thus, it is likely that other subtype-specific, cAMP-independent coupling of the beta-AR to CaCh is present.     

Changes in β-adrenoceptors in heart failure due to myocardial infarction are attenuated by blockade of renin–angiotensin system

Earlier studies have revealed an improvement of cardiac function in animals with congestive heart failure (CHF) due to myocardial infarction (MI) by treatment with angiotensin converting enzyme (ACE) inhibitors. Since heart failure is also associated with attenuated responses to catecholamines, we examined the effects of imidapril, an ACE inhibitor, on the -adrenoceptor (-AR) signal transduction in the failing heart. Heart failure in rats was induced by occluding the coronary artery, and 3 weeks later the animals were treated with 1 mg/(kg·day) (orally) imidapril for 4 weeks. The animals were assessed for their left ventricular function and inotropic responses to isoproterenol. Cardiomyocytes and crude membranes were isolated from the non-ischemic viable left ventricle and examined for the intracellular concentration of Ca²⁺ [Ca²⁺]_i and -ARs as well as adenylyl cyclase (AC) activity, respectively. Animals with heart failure exhibited depressions in ventricular function and positive inotropic response to isoproterenol as well as isoproterenol-induced increase in [Ca²⁺]_i in cardiomyocytes; these changes were attenuated by imidapril treatment. Both ₁-AR receptor density and isoproterenol-stimulated AC activity were decreased in the failing heart and these alterations were prevented by imidapril treatment. Alterations in cardiac function, positive inotropic effect of isoproterenol, ₁-AR density and isoproterenol-stimulated AC activity in the failing heart were also attenuated by treatment with another ACE inhibitor, enalapril and an angiotensin II receptor antagonist, losartan. The results indicate that imidapril not only attenuates cardiac dysfunction but also prevents changes in -AR signal transduction in CHF due to MI. These beneficial effects are similar to those of enalapril or losartan and thus appear to be due to blockade of the renin–angiotensin system. (Mol Cell Biochem 263: 11–20, 2004)     

Inhibition of spontaneous beta 2-adrenergic activation rescues beta 1-adrenergic contractile response in cardiomyocytes overexpressing beta 2-adrenoceptor

Cardiac-specific overexpression of the human beta(2)-adrenergic receptor (AR) in transgenic mice (TG4) enhances basal cardiac function due to ligand-independent spontaneous beta(2)-AR activation. However, agonist-mediated stimulation of either beta(1)-AR or beta(2)-AR fails to further enhance contractility in TG4 ventricular myocytes. Although the lack of beta(2)-AR response has been ascribed to an efficient coupling of the receptor to pertussis toxin-sensitive G(i) proteins in addition to G(s), the contractile response to beta(1)-AR stimulation by norepinephrine and an alpha(1)-adrenergic antagonist prazosin is not restored by pertussis toxin treatment despite a G(i) protein elevation of 1.7-fold in TG4 hearts. Since beta-adrenergic receptor kinase, betaARK1, activity remains unaltered, the unresponsiveness of beta(1)-AR is not caused by betaARK1-mediated receptor desensitization. In contrast, pre-incubation of cells with anti-adrenergic reagents such as muscarinic receptor agonist, carbachol (10(-5)m), or a beta(2)-AR inverse agonist, ICI 118,551 (5 x 10(-7)m), to abolish spontaneous beta(2)-AR signaling, both reduce the base-line cAMP and contractility and, surprisingly, restore the beta(1)-AR contractile response. The "rescued" contractile response is completely reversed by a beta(1)-AR antagonist, CGP 20712A. Furthermore, these results from the transgenic animals are corroborated by in vitro acute gene manipulation in cultured wild type adult mouse ventricular myocytes. Adenovirus-directed overexpression of the human beta(2)-AR results in elevated base-line cAMP and contraction associated with a marked attenuation of beta(1)-AR response; carbachol pretreatment fully revives the diminished beta(1)-AR contractile response. Thus, we conclude that constitutive beta(2)-AR activation induces a heterologous desensitization of beta(1)-ARs independent of betaARK1 and G(i) proteins; suppression of the constitutive beta(2)-AR signaling by either a beta(2)-AR inverse agonist or stimulation of the muscarinic receptor rescues the beta(1)-ARs from desensitization, permitting agonist-induced contractile response.     

Overexpression of beta2-adrenergic receptors cAMP-dependent protein kinase phosphorylates and modulates slow delayed rectifier potassium channels expressed in murine heart: evidence for receptor/channel co-localization

The cardiac slow delayed rectifier potassium channel (IKs), comprised of (KCNQ1) and beta (KCNE1) subunits, is regulated by sympathetic nervous stimulation, with activation of beta-adrenergic receptors PKA phosphorylating IKs channels. We examined the effects of 2-adrenergic receptors (beta2-AR) on IKs in cardiac ventricular myocytes from transgenic mice expressing fusion proteins of IKs subunits and hbeta2-ARs. KCNQ1 and beta2-ARs were localized to the same subcellular regions, sharing intimate localization within nanometers of each other. In IKs/B2-AR myocytes, IKs density was increased, and activation shifted in the hyperpolarizing direction; IKs was not further modulated by exposure to isoproterenol, and KCNQ1 was found to be PKA-phosphorylated. Conversely, beta2-AR overexpression did not affect L-type calcium channel current (ICaL) under basal conditions with ICaL remaining responsive to cAMP. These data indicate intimate association of KCNQ1 and beta2-ARs and that beta2-AR signaling can modulate the function of IKs channels under conditions of increased beta2-AR expression, even in the absence of exogenous beta-AR agonist.     

Genetic and phenotypic targeting of beta-adrenergic signaling in heart failure

Heart failure is a leading cause of hospitalization worldwide. No major significant improvements in prognosis have been achieved for heart failure over the last several decades despite advances in disease management. Heart failure itself represents a final common endpoint for several disease entities, including hypertension and coronary artery disease. On a molecular level, certain biochemical features remain common to failing myocardium. Among these are alterations in the beta-adrenergic receptor (beta-AR) signaling cascade. Recent advances in transgenic and gene therapy techniques have presented novel therapeutic strategies for management of heart failure via genetic manipulation of beta-AR signaling including the targeted inhibition of the beta-AR kinase (betaARK1 or GRK2). In this review, we will discuss the beta-AR signaling changes that accompany heart failure as well as corresponding therapeutic strategies. We will then review the evidence from transgenic mouse work supporting the use of beta-AR manipulation in the failing heart and more recent in vivo applications of gene therapy directed at reversing or preventing heart failure.     

Phosphoinositide 3-kinasegamma (PI3Kgamma) controls L-type calcium current (ICa,L) through its positive modulation of type-3 phosphodiesterase (PDE3)

The modulation of L-type calcium current (ICa,L) is mainly due to mediators acting through activation of G protein-coupled receptors (GPCR) and different protein kinases; among them, phosphoinositide 3-kinasegamma (PI3Kgamma) has been recently discovered to play an important role in the regulation of cardiac contractility and beta-adrenergic signal transduction. Recent reports have demonstrated that, in the heart, different subtypes of beta-adrenergic receptors are coupled to both Gi and/or Gs proteins. While beta1-adrenergic receptors (beta1-AR) couple only to Gs and evoke a strong ICa,L, beta2-adrenergic receptors (beta2-AR) can activate both Gs and Gi proteins and trigger only a limited ICa,L. Here we demonstrate that (i) PI3Kgamma-/- ventricular myocytes are characterized by an higher basal ICa,L density, even if the responsiveness of adenylyl cyclase to Forskolin is comparable to that observed in PI3Kgamma+/+ cardiomyocytes; (ii) both in basal conditions and after beta-AR stimulation, the activity of phosphodiesterase (PDE) type 3 depends on PI3Kgamma; (iii) in PI3Kgamma-/- cardiac myocytes, specific stimulation of beta2-AR is followed by a increase in ICa,L stronger than in wild-type controls. Taken together, our results suggest that the higher values of ICa,L observed both in basal conditions and after beta-AR stimulation in PI3Kgamma-/- ventricular myocytes are mainly due to a positive modulation of PDE3 activity exerted by PI3Kgamma. As observed in PI3Kgamma-/- neonatal cardiomyocytes, cells lacking PI3Kgamma are more sensitive to stimulation of beta2-adrenergic receptors.     

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.     

Expression and functional analysis of beta-adrenoceptor subtypes in rabbit submandibular gland

beta-Adrenoceptors (beta-ARs) mediate important physiological functions in salivary glands. Here we investigated the expression and function of beta-AR subtypes in rabbit submandibular gland (SMG). Both beta(1)- and beta(2)-ARs, but not beta(3)-AR, were strongly expressed in rabbit SMG. beta(1)-AR proteins were widely expressed in acinar and ductal cells whereas beta(2)-AR proteins were mainly detected in ductal cells. A [(3)H]-dihydroalprenolol binding assay revealed that beta-AR B(max) was 186+/-11.9 fmol/mg protein and K(d) was 2.71+/-0.23 nM. A competitive binding assay with CGP 20712A, a beta(1)-AR antagonist, indicated that the proportion of beta(1)-AR and beta(2)-AR was 71.9% and 28.1%, respectively. Gland perfusion with the beta-AR agonist isoproterenol induced a significant increase in saliva secretion which was abolished by pretreatment with the non-selective beta-AR antagonist propranolol. Pretreatment with beta(1)- or beta(2)-AR selective antagonists, CGP 20712A or ICI 118551, diminished isoproterenol-induced increase in saliva secretion by 71.2% and 28.8%, respectively. The expression of alpha-amylase mRNA was significantly stimulated by isoproterenol, which was eliminated by propranolol and CGP 20712A. Perfusion with isoproterenol decreased alpha-amylase protein storage in SMG and increased alpha-amylase activity in saliva. These alterations became less significant after pretreatment with propranolol and CGP 20712A. Our results suggest that both beta(1)- and beta(2)-ARs are expressed in rabbit SMG. beta(1)-AR is the predominant subtype and may play an important role in regulating saliva and alpha-amylase secretion.     

Assembly of an A kinase-anchoring protein-beta(2)-adrenergic receptor complex facilitates receptor phosphorylation and signaling

Phosphorylation of G-protein-coupled receptors by second-messenger-stimulated kinases is central to the process of receptor desensitization [1-3]. Phosphorylation of the beta(2)-adrenergic receptor (beta(2)-AR) by protein kinase A (PKA), in addition to uncoupling adenylate cyclase activation, is obligatory for receptor-mediated activation of mitogen-activated protein kinase (MAP kinase) cascades [4] [5]. Although mechanisms for linking G-protein-coupled receptor kinases to the activated receptor are well established, analogous mechanisms for targeting second messenger kinases to the beta(2)-AR at the plasma membrane have not been elucidated. Here we show that the A-kinase-anchoring protein, AKAP79/150, co-precipitates with the beta(2)-AR in cell and tissue extracts, nucleating a signaling complex that includes PKA, protein kinase C (PKC) and protein phosphatase PP2B. The anchoring protein directly and constitutively interacts with the beta(2)-AR and promotes receptor phosphorylation following agonist stimulation. Functional studies show that PKA anchoring is required to enhance beta(2)-AR phosphorylation and to facilitate downstream activation of the MAP kinase pathway. This defines a role for AKAP79/150 in the recruitment of second-messenger-regulated signaling enzymes to a G-protein-coupled receptor.     

Are developing beta-adrenoceptors able to desensitize? Acute and chronic effects of beta-agonists in neonatal heart and liver

During fetal and neonatal development, beta-adrenergic receptors (beta-ARs) appear to be resistant to desensitization by beta-agonist drugs. To determine the mechanisms underlying the regulatory differences between adults and neonates, we administered isoproterenol, a mixed beta(1)/beta(2)-AR agonist, and terbutaline, a beta(2)-selective agonist. Effects were examined in the ensuing 4 h after a single injection, or after the last of four daily injections. We prepared cell membranes from heart (predominantly beta(1)-ARs) and liver (predominantly beta(2)-ARs) and assessed signal transduction in the adenylyl cyclase (AC) pathway. In the first few hours after a single administration of isoproterenol to adult rats, cardiac beta-ARs showed activation of G proteins (elevated AC response to forskolin) and desensitization of beta-AR-mediated responses; after the fourth injection, heterologous desensitization emerged, characterized by a loss of signaling mediated either through beta-ARs or glucagon receptors. Terbutaline evoked an increase in the forskolin response but no desensitization of receptor-mediated responses. When we gave the same treatments to neonatal rats, we observed cardiac G protein activation, but there was neither homologous nor heterologous desensitization of beta-ARs or glucagon receptors. In the adult liver, isoproterenol and terbutaline both failed to evoke desensitization, regardless of whether the drugs were given once or for 4 days. In neonates, however, acute or chronic treatment elicited homologous desensitization of beta-AR-mediated AC signaling, while sensitizing the response to glucagon. These results show that neonatal beta-ARs are inherently capable of desensitization in some, but not all, cell types; cellular responses can be maintained through heterologous sensitization of signaling proteins downstream from the receptor. Differences from adult patterns of response are highly tissue selective and are likely to depend on ontogenetic differences in subtypes of beta-ARs and AC.     

Role of Beta-adrenergic Receptors and Sirtuin Signaling in the Heart During Aging,Heart Failure,and Adaptation to Stress

In the heart, catecholamine effects occur by activation of beta-adrenergic receptors (β-ARs), mainly the beta 1 (β₁-AR) and beta 2 (β₂-AR) subtypes, both of which couple to the Gs protein that activates the adenylyl cyclase signaling pathway. The β₂-ARs can also couple to the Gi protein that counterbalances the effect of the Gs protein on cyclic adenosine monophosphate production and activates the phosphatidylinositol 3-kinase (PI3K)–Akt signaling pathway. In several cardiovascular disorders, including heart failure, as well as in aging and in animal models of environmental stress, a reduction in the β₁/β₂-AR ratio and activation of the β₂-AR-Gi-PI3K–Akt signaling pathway have been observed. Recent studies have shown that sirtuins modulate certain organic processes, including the cellular stress response, through activation of the PI3K–Akt signaling pathway and of downstream molecules such as p53, Akt, HIF1-α, and nuclear factor-kappa B. In the heart, SIRT1, SIRT3, and β₂-ARs are crucial to the regulation of the cardiomyocyte energy metabolism, oxidative stress, reactive oxygen species production, and autophagy. SIRT1 and the β₂-AR-Gi complex also control signaling pathways of cell survival and death. Here, we review the role played by β₂-ARs and sirtuins during aging, heart failure, and adaptation to stress, focusing on the putative interplay between the two. That relationship, if proven, merits further investigation in the context of cardiac function and dysfunction.     

Vascular beta-adrenoceptor function in hypertension and in ageing

Functional beta-adrenoceptors (beta-AR) have been identified and characterized in blood vessels under in vivo conditions as well as in vascular smooth muscle cells (SMC) grown in culture. Agonist occupancy of beta-AR activates adenylyl cyclase (AC) via the stimulatory guanine nucleotide-binding protein (Gs) and leads to elevations in intracellular adenosine 3'5'-cyclic monophosphate levels (cAMP). Increased cAMP activates the cAMP-dependent protein kinase (PKA), with subsequent phosphorylation of various target proteins. This beta-AR pathway interacts with several other intracellular signalling pathways via cross-talk, so that activation by beta-AR agonists may also modulate other second messengers and protein kinases. SMC beta-AR play an important role in SMC function. In intact blood vessels they mediate SMC relaxation by various intracellular mechanisms, ultimately causing a decrease in intracellular Ca2+ levels. In cultured SMC, activation of the beta-AR pathway results in inhibition of cellular proliferation, the development of SMC polyploidy, and SMC apoptosis. Blood vessels from hypertensive animals are characterized by an increase in SMC cell mass, a greater incidence of SMC polyploidy in the aorta, and an impairment in the beta-agonist-mediated SMC relaxation. Some of these changes may result from an attenuation of beta-AR function due to agonist-induced receptor desensitization caused by the uncoupling of receptors from the Gs-AC system. The phosphorylated beta-AR may in turn trigger new signals and activate different intracellular pathways. However, the details of these mechanisms are still unresolved. Since functional beta-AR play such a prominent and multi-faceted role in SMC function, it is important to understand how these diverse physiological effects are mediated by this receptor system, and how they contribute to the development of hypertension. With ageing, a decrease in beta-AR-Gs-AC coupling is observed, and this is implicated in the reduced responsiveness of SMC. The similarities in SMC beta-AR functional changes in hypertension and in ageing suggest that the underlying mechanisms are also analogous.