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.     

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.     

Guanine nucleotides regulate beta-adrenergic activation of Na-H exchange independently of receptor coupling to Gs.

We have previously shown that the beta-adrenergic receptor (beta-AR) stimulates activity of the ubiquitous Na-H exchanger (NHE-1) independently of changes in cAMP accumulation and independently of a cholera toxin-sensitive stimulatory GTP-binding protein (Gs). To further investigate the potential role of a GTP-binding protein in coupling the beta-AR to NHE-1, we have used a recently available nonhydrolyzable GTP analog, "caged" guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S), to study time-dependent effects of GTP on NHE-1 in intact cells. By monitoring intracellular pH (pHi) in cells loaded with the fluorescent pH-sensitive dye, 2,7-biscarboxyethyl-5(6)-carboxyfluorescein, we determined NHE-1 activity in primary cultures of canine enteric endocrine cells, which express an endogenous beta-AR, and in mouse L cells stably transfected with either the wild type hamster beta 2-AR or a mutant construct of the hamster beta 2-AR containing a deletion in amino acid residues 222-229. This D(222-229)beta 2-AR is functionally uncoupled from Gs and adenylylcyclase. In all three cell types, NaF and GTP gamma S induced an increase in activity of the exchanger, determined by assessing the rate of pHi recovery from an acute intracellular acid load (dpHi/dt). This increase in pHi recovery was dependent on extracellular Na+ and sensitive to the amiloride analog ethylisopropylamiloride. GTP gamma S, but not NaF, also increased beta-adrenergic stimulation of resting NHE-1 activity. The alkalinization in response to isoproterenol was reversed by propranolol in the absence, but not the presence, of GTP gamma S and was completely blocked by GDP beta S. The ability of guanine nucleotides to regulate beta-adrenergic activation of NHE-1 in cells expressing the mutant D(222-229)beta 2-AR suggests that functional coupling of the beta-AR to NHE-1 may be mediated by a GTP-binding protein other than Gs.     

Phosphoinositide 3-kinase gamma controls autonomic regulation of the mouse heart through Gi-independent downregulation of cAMP level

Cardiac beta-adrenergic and the muscarinic receptors control contractility and heart rate by triggering multiple signaling events involving downstream targets like the phosphoinositide 3-kinase gamma (PI3Kgamma). We thus investigated whether the lack of PI3Kgamma could play a role in the autonomic regulation of the mouse heart. Contractility and ICaL of mutant cardiac preparations appeared increased in basal conditions and after beta-adrenergic stimulation. However, basal and beta-adrenergic stimulated heart rate were normal. Conversely, muscarinic inhibition of heart rate was reduced without alteration of the Gbetagamma-dependent stimulation of IK,ACh current. In addition, muscarinic-mediated anti-adrenergic effect on papillary muscle contractility and ICaL was significantly depressed. Consistently, cAMP level of PI3Kgamma-null ventricles was always higher than wild-type controls. Thus, PI3Kgamma controls the cardiac function by reducing cAMP concentration independently of Gi-mediated signaling.     

β2-肾上腺素受体在新生大鼠心肌成纤维细胞中的分布及其促增殖作用

为了明确β-肾上腺素受体(AR)亚型在新生大鼠心肌成纤维细胞中的分布及其在成纤维细胞增殖反应中的作用,采用放射配体结合实验和[3H]-thymidine掺人法检测了新生大鼠心肌成纤维细胞的β-AR密度和DNA合成速率。结果显示,在培养心肌细胞和心肌成纤维细胞中β-AR密度(Bmtax)和解离常数(Kp)无显著性差异;竞争抑制曲线分析结果提示,心肌成纤维细胞对CGP 20712A和ICI ll8551单位点拟合均显著优于两位点拟合(P＜0．01),表现为对选择性β1-AR拮抗剂CGP 20712A的低亲和性(IC50值：10．1μmol/L)和对选择性β2-AR拮抗剂ICI 118551的高亲和性(IC50值：0.147μmol/L)。异丙肾上腺素(ISO)促心肌成纤维细胞增殖作用可被ICI 118551和心得安(非选择性β-AR拮抗剂)完全抑制,而CGP20812A则无此作用。上述结果提示,在培养心肌成纤维细胞中β-AR亚型占绝对优势,并且ISO引起的心肌成纤维细胞增殖反应是由β2-AR介导的。     

beta1 integrins expression in adult rat ventricular myocytes and its role in the regulation of beta-adrenergic receptor-stimulated apoptosis

We have shown that the stimulation of beta-adrenergic receptors (beta-AR) increases apoptosis in adult rat ventricular myocytes (ARVMs). Integrins, a family of alphabeta-heterodimeric cell surface receptors, are postulated to play a role in ventricular remodeling. Here, we show that norepinephrine (NE) increases beta1 integrins expression in ARVMs via the stimulation of alpha1-AR, not beta-AR. Inhibition of ERK1/2 using PD 98059, an inhibitor of ERK1/2 pathway, inhibited alpha1-AR-stimulated increases in beta1 integrins expression. Activation of beta1 integrins signaling pathway using laminin (LN) inhibited beta-AR-stimulated apoptosis as measured by terminal deoxynucleotidyl transferase-mediated nick end labeling (TUNEL)-staining and flow cytometry. Likewise, ligation of beta1 integrins with anti-beta1 integrin antibodies prevented beta-AR-stimulated apoptosis. Treatment of cells using LN or anti-beta1 integrin antibodies activated ERK1/2 pathway. PD 98059 inhibited activation of ERK1/2 by LN, and prevented the anti-apoptotic effects of LN. Thus (1) stimulation of alpha1-AR regulates beta1 integrins expression via the activation of ERK1/2, (2) beta1 integrins signaling protects ARVMs from beta-AR-stimulated apoptosis, (3) activation of ERK1/2 plays a critical role in the anti-apoptotic effects of beta1-integrin signaling. These data suggest that beta1 integrin signaling protects ARVMs against beta-AR-stimulated apoptosis possibly via the involvement of ERK1/2.     

A novel signaling pathway for beta-adrenergic receptor-mediated activation of phosphoinositide 3-kinase in H9c2 cardiomyocytes

Stimulation of cardiac beta-adrenergic receptors (beta-AR) activates both the G(s)- and G(i)-coupled signaling cascades, including the phosphoinositide 3 kinase (PI3K) pathway, that have important physiological implications. Multiple isoforms of PI3K exist in the heart. The goals of this study were to examine the intracellular signaling pathways linking beta-AR to PI3K and to identify the PI3K isoform mediating this transactivation in a cardiac context. Acute beta-AR stimulation with isoproterenol resulted in increased tyrosine kinase-associated PI3K activity and phosphorylation of Akt and p70S6K in H9c2 cardiomyocytes. Cotreatment with ICI-118,551, but not CGP-20712, abolished the increase in PI3K activity, suggesting a beta(2)-AR-mediated event. PI3K activation was also abrogated by cotreatment with pertussis toxin, 4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolol[3,4-d]pyrimidine (PP2, a selective Src-family tyrosine kinases inhibitor), or AG-1296 [selective platelet-derived growth factor receptor (PDGFR) inhibitor] but not with an inhibitor for protein kinase A, protein kinase C, Ras, adenylyl cyclase, epidermal growth factor receptor, or insulin-like growth factor-1 receptor. beta-AR stimulation induced an increase in tyrosine phosphorylation of PDGFR, which was abolished by inhibition of Src either by PP2 or small interfering RNA. Moreover, H9c2 cardiomyocytes stably transfected with a vector expressing a Gbetagamma sequestrant peptide derived from the COOH-terminus of beta-AR kinase-1 failed to activate PI3K after beta-AR stimulation, suggesting Gbetagamma is required for the transactivation. Furthermore, acute beta-AR stimulation in vivo resulted in increases in PDGFR-associated PI3K and PI3Kalpha isoform activities but not the activities of other isoforms (PI3Kbeta, -delta, -gamma) in adult mouse heart. Taken together, these data provide in vitro and in vivo evidence for a novel mechanism of beta-AR-mediated transactivation of cardiac PI3Kalpha via sequential involvement of Galpha(i)/Gbetagamma, Src, and PDGFR.     

Enhanced in vivo and in vitro contractile responses to beta(2)-adrenergic receptor stimulation in dogs susceptible to lethal arrhythmias.

The response to beta-adrenergic receptor (beta-AR) stimulation was evaluated in both isolated cardiomyocytes (video edge detection) and the intact animal (echocardiography) in dogs either susceptible (S) or resistant (R) to ventricular fibrillation induced by a 2-min coronary occlusion during the last minute of exercise. In the intact animal, velocity of circumferential fiber shortening (Vcf) was evaluated both before (n = 27, S = 12 and R = 15) and after myocardial infarction. Before infarction, increasing doses of isoproterenol provoked similar contractile and heart rate responses in each group of dogs. Either beta(1)-AR (bisoprolol) or beta(2)-AR (ICI-118551) antagonists reduced the isoproterenol response, with a larger reduction noted after the beta(1)-AR blockade. In contrast, after infarction, isoproterenol induced a significantly larger Vcf and heart rate response in the susceptible animals that was eliminated by beta(2)-AR blockade. The single-cell isotonic shortening response to isoproterenol (100 nM) was also larger in cells obtained from susceptible compared with resistant dogs and was reduced to a greater extent by beta(2)-AR blockade in the susceptible dog myocytes (S, -48%, n = 6; R, -15%, n = 9). When considered together, these data suggest that myocardial infarction provoked an enhanced beta(2)-AR response in susceptible, but not resistant, animals.     

Overexpression of beta2-adrenergic receptors in mouse liver alters the expression of gluconeogenic and glycolytic enzymes

In the livers of humans and many other mammalian species, beta2-adrenergic receptors (beta2-ARs) play an important role in the modulation of glucose production by glycogenolysis and gluconeogenesis. In male mice and rats, however, the expression and physiological role of hepatic beta2-ARs are rapidly lost with development under normal physiological conditions. We previously described a line of transgenic mice, F28 (Andre C, Erraji L, Gaston J, Grimber G, Briand P, and Guillet JG. Eur J Biochem 241: 417-424, 1996), which carry the human beta2-AR gene under the control of its own promoter. In these mice, hepatic beta2-AR levels are shown to increase rapidly after birth and, as in humans, be maintained at an elevated level in adulthood. F28 mice display strongly enhanced adenylyl cyclase responses to beta-AR agonists in their livers and, compared with normal mice, have increased basal hepatic adenylyl cyclase activity. In this report we demonstrate that, under normal physiological conditions, this increased beta2-AR activity affects the expression of the gluconeogenic and glycolytic key enzymes phosphoenolpyruvate carboxykinase, glucose-6-phosphatase, and l-pyruvate kinase and considerably decreases hepatic glycogen levels. Furthermore, we show that the effects of beta-adrenergic ligands on liver glycogen observed in humans are reproduced in these mice: liver glycogen levels are strongly decreased by the beta2-AR agonist clenbuterol and increased by the beta-AR antagonist propranolol. These transgenic mice open new perspectives for studying in vivo the hepatic beta2-AR system physiopathology and for testing the effects of beta-AR ligands on liver metabolism.     

Beta2-adrenergic receptors expressed on murine chondrocytes stimulate cellular growth and inhibit the expression of Indian hedgehog and collagen type X

The sympathetic nervous system has been demonstrated to have a role in regulating bone remodeling through beta-adrenergic receptors (beta-AR) expressed on osteoblasts. Studies using beta(2)-adrenergic receptor agonists in vivo have also suggested an effect on endochondral bone development; however, it was not clear if this effect was mediated through osteoblasts or chondrocytes. To more thoroughly examine the role of beta-AR in chondrocytes we characterized the expression and signal transduction systems activated by beta-AR in growth plate chondrocytes prepared from ribs of embryonic E18.5 mice. Using RT-PCR and immunohistochemistry we found that the chondrocytes expressed only beta(2)-AR. The receptors were coupled to stimulation of adenylyl cyclase, phosphorylation of the cyclic AMP response element binding protein (CREB) and extracellular signal-regulated kinase (ERK1/2). Stimulation of ERK1/2 was transient and limited by the concomitant stimulation of the mitogen-activated protein kinase phosphatase (MKP-1). Isoproterenol stimulated the growth of chondrocytes as assessed by increased incorporation of [(3)H]-thymidine into the cells. The cellular expression of two markers of chondrocyte differentiation, Indian hedgehog, expressed in pre-hypertrophic cells and collagen type X, expressed in hypertrophic chondrocytes, were both significantly inhibited after incubation with isoproterenol. Collectively, these findings demonstrate regulation of chondrocytes through beta(2)-AR expressed on the cells that stimulate their growth and inhibit their differentiation, indicating that the sympathetic nervous system may be an important regulator of embryonic cartilage development.     

G(i)-dependent suppression of beta(1)-adrenoceptor effects in ventricular myocytes from NE-treated guinea pigs

It has been suggested that there is a preferential coupling in heart muscle between the inhibitory G protein (G(i)) and the beta(2)-subtype of the beta-adrenergic receptor (beta-AR), since pertussis toxin (which inactivates G(i)) reveals latent beta(2)-ARs in rat and mouse myocytes. We have previously shown that guinea pigs treated with norepinephrine (NE) for 7 days have myocytes that are desensitized to beta-AR-agonist stimulation, and that pertussis toxin restores these responses. The purpose of the present investigation was to determine whether pertussis toxin specifically upregulated beta(2)-ARs in myocytes from NE-treated guinea pigs. The sole beta-AR subtype in control guinea pig myocytes was confirmed as beta(1)-AR by radioligand binding, single-cell autoradiography, and concentration-response curves to isoproterenol in contracting myocytes. In contrast, a minor pool of beta(2)-ARs was observed in rat myocytes by use of the same methods. NE treatment decreased the maximum isoproterenol response (relative to high Ca(2+)) from 0.89 +/- 0.06 to 0.58 +/- 0.08 (n = 7, P < 0.01) and the pD(2) (-log EC(50)) from 8.8 +/- 0.2 to 7.5 +/- 0.2 (n = 7, P < 0.01). Pertussis toxin treatment increased the isoproterenol-to-Ca(2+) ratio to 0.88 +/- 0.04 (n = 6, P < 0.05) and the pD(2) to 8.6 +/- 0.3 (P < 0.01). This was not mediated by increases in either number or function of beta(2)-ARs. G(i) is therefore able to modulate beta(1)-AR responses in guinea pig myocytes.     

A Gs protein couples P2-purinergic stimulation to cardiac Ca channels without cyclic AMP production

P2-purinergic stimulation of the L-type Ca current induced by the external application of 100 microM ATP gamma S was investigated in rat ventricular cardiomyocytes using the whole-cell patch-clamp technique. The purinergic-induced increase in ICa was slow and monophasic and reached a steady state within 3 min. In contrast to beta-adrenergic stimulation, after a brief agonist application the current did not continue to increase on washout; recovery started immediately after agonist removal. The P2-purinergic increase in ICa was significantly less in the presence of GDP beta S, but it occurred much faster and was twice as large when a low dose of GTP gamma S (100 microM) was added to a GTP-containing internal medium. This suggests that the ICa increase was mediated by a G protein. Based on electrophoretic mobility and susceptibility to cholera toxin and anti-G alpha s serum, it is proposed that the G protein involved during purinergic-induced ICa stimulation is an isoform of Gs not coupled to the adenylyl cyclase, since the cyclic AMP level was unaffected. High intracellular GTP gamma S (1 mM) maximally activated ICa so that neither beta-adrenergic nor P2-purinergic agonists further increased ICa. In the absence of GTP and an ATP-regenerating system, GTP gamma S was much more potent in increasing basal ICa and supporting purinergic stimulation. This indicates that a nucleoside diphosphate kinase activity might replenish endogenous GTP; GTP exchange with GTP gamma S on the G protein was promoted by the P2-purinergic stimulation and led to a reversible and reproducible increase in ICa. In the presence of 3 mM internal ATP gamma S, the P2-purinergic stimulation was also reversible and reproducible. Moreover, under these conditions (ATP gamma S or GTP gamma S) the increase in ICa was not maintained during prolonged agonist application. Such an inhibition occurred slowly and irreversibly; it might be related to the threefold increase in cyclic GMP. In conclusion, we propose that extracellular ATP induces both a stimulatory and an inhibitory effect on ICa, probably mediated by subtypes of P2-purinergic receptors. An isoform of the Gs protein is likely to mediate the stimulation.     

RGS2 inhibits β-adrenergic receptor-induced cardiomyocyte hypertrophy

The chronic stimulation of certain G protein-coupled receptors promotes cardiomyocyte hypertrophy and thus plays a pivotal role in the development of human heart failure. The beta-adrenergic receptors (β-AR) are unique among these in that they signal via Gs, whereas others, such as the alpha1-adrenergic (α1-AR) and endothelin-1 (ET-1) receptors, predominantly act through Gq. In this study, we investigated the potential role of regulator of G protein signalling 2 (RGS2) in modulating the hypertrophic effects of the β-AR agonist isoproterenol (ISO) in rat neonatal ventricular cardiomyocytes. We found that ISO-induced hypertrophy in rat neonatal ventricular myocytes was accompanied by the selective upregulation of RGS2 mRNA, with little or no change in RGS1, RGS3, RGS4 or RGS5. The adenylyl cyclase activator forskolin had a similar effect suggesting that it was mediated through cAMP production. To study the role of RGS2 upregulation in β-AR-dependent hypertrophy, cardiomyocytes were infected with adenovirus encoding RGS2 and assayed for cell growth, markers of hypertrophy, and β-AR signalling. ISO-induced increases in cell surface area were virtually eliminated by the overexpression of RGS2, as were increases in α-skeletal actin and atrial natriuretic peptide. RGS2 overexpression also significantly attenuated ISO-induced extracellular signal-regulated kinases 1 and 2 (ERK1/2) and Akt activation, which may account for, or contribute to, its observed antihypertrophic effects. In contrast, RGS2 overexpression significantly activated JNK MAP kinase, while decreasing the potency but not the maximal effect of ISO on cAMP accumulation. In conclusion, the present results suggest that RGS2 negatively regulates hypertrophy induced by β-AR activation and thus may play a protective role in cardiac hypertrophy.     

Endothelial nitric oxide synthase decreases beta-adrenergic responsiveness via inhibition of the L-type Ca2+ current

Signaling via endothelial nitric oxide synthase (NOS3) limits the heart's response to beta-adrenergic (beta-AR) stimulation, which may be protective against arrhythmias. However, mechanistic data are limited. Therefore, we performed simultaneous measurements of action potential (AP, using patch clamp), Ca2+ transients (fluo 4), and myocyte shortening (edge detection). L-type Ca2+ current (ICa) was directly measured by the whole cell ruptured patch-clamp technique. Myocytes were isolated from wild-type (WT) and NOS3 knockout (NOS3-/-) mice. NOS3-/- myocytes exhibited a larger incidence of beta-AR (isoproterenol, 1 microM)-induced early afterdepolarizations (EADs) and spontaneous activity (defined as aftercontractions). We also examined ICa, a major trigger for EADs. NOS3-/- myocytes had a significantly larger beta-AR-stimulated increase in ICa compared with WT myocytes. In addition, NOS3-/- myocytes had a larger response to beta-AR stimulation compared with WT myocytes in Ca2+ transient amplitude, shortening amplitude, and AP duration (APD). We observed similar effects with specific NOS3 inhibition [L-N5-(1-iminoethyl)-ornithine (l-NIO), 10 microM] in WT myocytes as with NOS3 knockout. Specifically, l-NIO further increased isoproterenol-stimulated EADs and aftercontractions. l-NIO also further increased the isoproterenol-stimulated ICa, Ca2+ transient amplitude, shortening amplitude, and APD (all P < 0.05 vs isoproterenol alone). l-NIO had no effect in NOS3-/- myocytes. These results indicate that NOS3 signaling inhibits the beta-AR response by reducing ICa and protects against arrhythmias. This mechanism may play an important role in heart failure, where arrhythmias are increased and NOS3 expression is decreased.     

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

The beta-adrenoceptor (beta-AR) mediated signal transduction pathway in cardiomyocytes is known to involve beta1- and beta2-ARs, stimulatory (Gs) and inhibitory (Gi) guanine nucleotide binding proteins, adenylyl cyclase (AC) and cAMP-dependent protein kinase (PKA). The activation of beta1- and beta2-ARs has been shown to increase heart function by increasing Ca2+ -movements across the sarcolemmal membrane and sarcoplasmic reticulum through the stimulation of Gs-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 beta2-AR has also been shown to affect Gi-proteins, stimulate mitogen activated protein kinase and increase protein synthesis by enhancing gene expression. Beta1- and beta2-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 beta-AR indirectly through the involvement of beta-AR kinase (betaARK), beta-arrestins and Gbeta gamma-protein subunits. Genetic manipulation of different components and regulators of beta-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 PARK in heart dysfunction and therapeutic role of betaARK inhibitors in the treatment of heart failure. Varying degrees of defects in the beta-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 beta1-AR density, an increase in the level of G1-proteins and overexpression of betaARK 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 beta-AR signal transduction pathway in the failing heart. Thus different components and regulators of the beta-AR signal transduction pathway appears to represent important targets for the development of therapeutic interventions for the treatment of heart failure.     

Identification of a Gs activator region of the beta 2-adrenergic receptor that is autoregulated via protein kinase A-dependent phosphorylation.

We have localized a G protein activator region of the human beta 2-adrenergic receptor to region beta III-2 (from Arg259 to Lys273). The synthetic beta III-2, corresponding to the C-terminal end of the third cytoplasmic loop, activates Gs at nanomolar concentrations and weakly activates Gi. beta III-2 activates adenylyl cyclase at nanomolar concentrations in wild-type S49 lymphoma membranes, but not in membranes of unc mutant S49 cells, in which Gs is uncoupled from beta-adrenergic stimulation. Phosphorylation of beta III-2 by cAMP-dependent protein kinase A, which is involved in the desensitization of the beta-adrenergic receptor from Gs, drastically reduces the effect of beta III-2 on Gs while potentiating its action on Gi, resulting in a total loss of adenylyl cyclase-stimulating activity. These findings indicate that this receptor sequence is a multipotential G protein activator whose G protein specificity is regulated by protein kinase A.     

Overexpression of beta 1 and beta 2 adrenergic receptors in rat atrial myocytes. Differential coupling to G protein-gated inward rectifier K(+) channels via G(s) and G(i)/o

G protein-activated inwardly rectifying K(+) (GIRK) channels, expressed in atrial myocytes, various neurons, and endocrine cells, represent the paradigmatic target of beta gamma subunits released from activated heterotrimeric G proteins. These channels contribute to physiological slowing of cardiac frequency and synaptic inhibition. They are activated by beta gamma dimers released upon stimulation of receptors coupled to pertussis toxin-sensitive G proteins (G(i/o)), whereas beta gamma released from G(s) do not converge on the channel subunits. This is in conflict with the finding that dimeric combinations of various beta and gamma subunits can activate GIRK channels with little specificity. In the present study, we have overexpressed the major subtypes of cardiac beta-adrenergic receptors (beta(1)-AR and beta(2)-AR) in atrial myocytes by transient transfection. Whereas in native cells beta-adrenergic stimulation with isoproterenol failed to induce measurable GIRK current, robust currents were recorded from myocytes overexpressing either beta(1)-AR or beta(2)-AR. Whereas the beta(2)-AR-induced current showed the same sensitivity to pertussis toxin as the current evoked by the endogenous G(i/o)-coupled muscarinic M(2) receptor, isoproterenol-activated currents were insensitive to pertussis toxin treatment in beta(1)-AR-overexpressing myocytes. In contrast to a recent publication (Leaney, J. L., Milligan, G., and Tinker, A. (2000) J. Biol. Chem. 275, 921-929), sizable GIRK currents could also be activated by isoproterenol when the signaling pathway was reconstituted by transient transfection in two different standard cell lines (Chinese hamster ovary and HEK293). These results demonstrate that specificity of receptor-G protein signaling can be disrupted by overexpression of receptors. Moreover, the alpha subunit of heterotrimeric G proteins does not confer specificity to G beta gamma-mediated signaling.     

Up-regulation of uncoupling proteins by beta-adrenergic stimulation in L6 myotubes

Catecholamine-induced and beta-adrenergic receptor (beta-AR)-mediated thermogenesis in skeletal muscle is a significant component of whole-body energy expenditure. Skeletal muscle expresses uncoupling protein (UCP) 2 and UCP3, which can dissipate the transmitochondrial electrochemical gradient and thereby may be involved in regulation of energy metabolism. We investigated the effects of beta-AR stimulation on UCP2 and UCP3 expression in L6 myotubes. Stimulation of the cells with epinephrine increased the UCP3 mRNA level transiently at 6 h, and also the UCP2 mRNA level at 6-24 h. The stimulatory effects of epinephrine were also observed in the presence of carbacyclin and 9-cis retinoic acid, and mimicked by isoproterenol and salbutamol (beta2-AR agonists), but abolished by propranolol and ICI-118,551 (beta2-AR antagonists). Pharmacological and mRNA analyses revealed the existence of beta2-AR, but not beta1- and beta3-ARs, in L6 myotubes. These results suggested that catecholamines up-regulate UCP2 and UCP3 expression through direct action on the beta2-AR in skeletal muscle.