Epac1 participates in β1-adrenoreceptor autoantibody-mediated decreased autophagic flux in cardiomyocytes

Decreased autophagic flux in cardiomyocytes is an important mechanism by which the β₁-adrenoreceptor (β₁-AR) autoantibody (β₁-AA) induces heart failure. A previous study found that β₁-AA imparts its biological effects via the β₁-AR/Gs/AC/cAMP/PKA canonical signaling pathway, but PKA inhibition does not completely reverse β₁-AA-induced reduction in autophagy in myocardial tissues, suggesting that other signaling molecules participate in this process. This study confirmed that Epac1 upregulation is indeed involved β₁-AA-induced decreased cardiomyocyte autophagy through CE3F4 pretreatment, Epac1 siRNA transfection, western blot and immunofluorescence methods. On this basis, we constructed β₁-AR and β₂-AR knockout mice, and used receptor knockout mice, β₁-AR selective blocker (atenolol), and the β₂-AR/Gi-biased agonist ICI 118551 to show that β₁-AA upregulated Epac1 expression through β₁-AR and β₂-AR to inhibit autophagy, and biased activation of β₂-AR/Gi signaling downregulated myocardial Epac1 expression to reverse β₁-AA-induced myocardial autophagy inhibition. This study aimed to test the hypothesis that Epac1 acts as another effector downstream of cAMP on β₁-AA-induced reduction in cardiomyocyte autophagy, and β₁-AA upregulates myocardial Epac1 expression through β₁-AR and β₂-AR, and biased activation of the β₂-AR/Gi signaling pathway can reverse β₁-AA-induced myocardial autophagy inhibition. This study provides new ideas and therapeutic targets for the prevention and treatment of cardiovascular diseases related to dysregulated autophagy.     

Stress-induced effects, which inhibit host defenses, alter leukocyte trafficking

Acute cold restraint stress (ACRS) has been reported to suppress host defenses against Listeria monocytogenes, and this suppression was mediated by beta1-adrenoceptors (β1-ARs). Although ACRS appears to inhibit mainly early innate immune defenses, interference with leukocyte chemotaxis and the involvement of β1-AR (or β2-AR) signaling had not been assessed. Thus, the link between sympathetic nerve stimulation, release of neurotransmitters, and changes in blood leukocyte profiles, including oxidative changes, following ACRS was evaluated. The numbers of leukocyte subsets in the blood were differentially affected by β1-ARs and β2-ARs following ACRS; CD3⁺ (CD4 and CD8) T-cells were shown to be decreased following ACRS, and the T cell lymphopenia was mediated mainly through a β2-AR mechanism, while the decrease in CD19⁺ B-cells was influenced through both β1- and β2-ARs, as assessed by pharmacological and genetic manipulations. In contrast to the ACRS-induced loss of circulating lymphocytes, the number of circulating neutrophils was increased (i.e., neutrophilia), and this neutrophilia was mediated through β1-ARs. The increase in circulating neutrophils was not due to an increase in serum chemokines promoting neutrophil emigration from the bone marrow; rather it was due to neutrophil release from the bone marrow through activation of a β1-AR pathway. There was no loss of glutathione in any of the leukocyte subsets suggesting that there was minimal oxidative stress; however, there was early production of nitric oxide and generation of some protein radicals. Premature egress of neutrophils from bone marrow is suggested to be due to norepinephrine induction of nitric oxide, which affects the early release of neutrophils from bone marrow and lessens host defenses.     

The role of β-adrenergic receptor signaling in the proliferation of hemangioma-derived endothelial cells

Background

Infantile hemangioma (IH) is a benign vascular neoplasm that arises from the abnormal proliferation of endothelial cells and enhanced angiogenesis. Recently, propranolol has been found to be effective in the management of IH, suggesting that β-adrenergic receptors (β-ARs) may play an important role in the pathogenesis of IH.

Results

In the present study, we investigated the β-adrenergic signaling that is associated with hemangioma-derived endothelial cell (HemEC) proliferation. The results showed that both β₁- and β₂-ARs were expressed in HemECs. Stimulation of the β-ARs by isoprenaline induced cell proliferation and elevation of second messenger cAMP levels. The proliferation-promoting action of isoprenaline was abolished by a β₁-selective antagonist and was more effectively abolished by a β₂-selective antagonist; the mechanism for the action of the antagonists was a G₀/G₁ phase cell cycle arrest which was associated with decreased cyclin D1, CDK-4, CDK-6 and phospho-Rb expression. Pre-treatment of the cells with VEGFR-2 or ERK inhibitors also prevented the isoprenaline-mediated proliferation of cells. In agreement with the involvement of β-ARs and VEGFR-2 in the HemEC response, β-AR antagonists and the VEGFR-2 inhibitor significantly attenuated isoprenaline-induced ERK phosphorylation. Moreover, treating the cells with isoprenaline markedly increased VEGF-A expression and VEGFR-2 activity in a β₂-AR-dependent manner.

Conclusions

We have demonstrated that the activation of the β-ARs in the ERK pathway may be important mechanisms in promoting HemEC growth. Furthermore, stimulation of the β-AR may transactivate VEGFR-2 signaling and further increase HemEC proliferation.     

Role of G(i/o)-Src kinase-PI3K/Akt pathway and caveolin-1 in β₂-adrenoceptor coupling to endothelial NO synthase in mouse pulmonary artery

Activation of the β₂-adrenoceptor (β₂-AR) elicits an endothelial nitric oxide synthase (eNOS)-dependent relaxation in mouse pulmonary artery, which, contrary to the muscarinic receptor-dependent relaxation, is preserved in hypoxic pulmonary arterial hypertension. We therefore characterized the signaling pathways underlying the β₂-AR-mediated eNOS activation, with special focus on G_i/o proteins, protein kinases and caveolae. Functional studies (for evaluation of vasorelaxant response), Western blotting (for assessment of eNOS and caveolin-1 phosphorylation) and transmission electron microscopy (for visualization of caveolae) were conducted in pulmonary arteries from wild-type or caveolin-1 knockout mice. In wild-type isolated arteries, relaxation to the selective β₂-AR agonist procaterol was reduced by inhibitors of G_i/o proteins (pertussis toxin, PTX), phosphatidylinositol 3-kinase (PI3K; wortmannin or LY 294002), Akt (Akt inhibitor X) and Src-kinase (PP2) and by cholesterol depletion (using methyl-β-cyclodextrin). Procaterol induced eNOS phosphorylation at Ser¹¹⁷⁷, which was prevented by PTX, PP2 or Akt inhibitor. Procaterol also promoted caveolin-1 phosphorylation at Tyr¹⁴, which was decreased by PTX or PP2. Caveolin-1 gene deletion resulted in endothelial caveolae disruption in mouse pulmonary artery and in potentiation of procaterol-induced relaxation. Unlike procaterol, acetylcholine-induced relaxation was unaffected by PTX, methyl-β-cyclodextrin or caveolin-1 gene deletion. To conclude, the mouse pulmonary endothelial β₂-AR is coupled to a G_i/o-Src kinase-PI3K/Akt pathway to promote eNOS phosphorylation at Ser¹¹⁷⁷ leading to a NO-dependent vasorelaxation. Caveolin-1 exerts a negative control on this response that is abrogated by its phosphorylation at Tyr¹⁴, through a G_i/o-Src kinase pathway. Since pulmonary β₂-AR- and muscarinic receptor-mediated relaxations differentiate in their respective signaling pathways leading to eNOS activation and sensitivities during hypoxia-induced pulmonary arterial hypertension, mechanisms underlying eNOS activation might be key determinants of pulmonary endothelial dysfunction.     

β2-Adrenergic receptor (β2-AR) agonist formoterol suppresses differentiation of L6 myogenic cells by blocking PI3K–AKT pathway

β₂-Adrenergic receptor (β₂-AR) is implicated in muscle metabolic activities such as glycogen metabolism, glucose uptake, lipolysis and muscle growth. However, the functional role of β₂-AR in the differentiation of skeletal muscle is largely unknown. Here, we examined the functional role of β₂-AR in L6 myoblast differentiation using the long-term-acting β₂-AR-specific agonist formoterol. We observed that formoterol treatment strongly suppressed L6 myoblast differentiation and the expression of myosin heavy chain (MHC) in a dose- and time-dependent manner. Showing that both long-acting agonist (formoterol) and short-acting agonist (terbutaline) inhibited the induction of MHC protein, whereas β₂-AR antagonist (ICI-118,551) upregulated MHC expression, we clearly demonstrated that β₂-AR is involved in L6 myoblast differentiation. Furthermore, our pharmacological inhibition study revealed that the PI3K–AKT pathway is the main signaling pathway for myotube formation. Formoterol inhibited the activation of PI3K–AKT signaling, but not that of ERK signaling. Moreover, formoterol selectively inhibited AKT activation by IGF-I, but not by insulin. Collectively, our findings reveal a previously undocumented role of β₂-AR activation in modulating the differentiation of L6 myoblasts.     

α1A-Adrenergic receptor differentially regulates STAT3 phosphorylation through PKCϵ and PKCδ in myocytes

Previous studies demonstrated α₁-adrenergic receptors (ARs) increase STAT3 activation in transfected and non-cardiac primary cell lines. However, the mechanism used by α₁-ARs resulting in STAT3 activation is unknown. While other G-protein-coupled receptors (GPCRs) can couple to STAT3, these mechanisms demonstrate coupling through SRC, TYK, Rac, or complex formation with Gq and used only transfected cell lines. Using normal and transgenic mice containing constitutively active mutations (CAM) of the α_1A-AR subtype, neonatal mouse myocytes and whole hearts were analyzed for the mechanism to couple to STAT3 activation. α₁-ARs stimulated time-dependent increases in p-SRC, p-JAK2, and p-STAT3 in normal neonatal myocytes. Using various kinase inhibitors and siRNA, we determined that the α_1A-AR coupled to STAT3 through distinct and unique pathways in neonatal myocytes. We found that PKC? inhibition decreased p-ERK and p-Ser STAT3 levels without affecting p-Tyr STAT3. In contrast, we found that PKCδ inhibition affected p-SRC and p-JAK2 resulting in decreased p-Tyr and p-Ser STAT3 levels. We suggest a novel α_1A-AR mediated PKC?/ERK pathway that regulates the phosphorylation status of STAT3 at Ser-727 while PKCδ couples to SRC/JAK2 to affect Tyr-705 phosphorylation. Furthermore, this pathway has not been previously described in a GPCR system that couples to STAT3. Given cell survival and protective cardiac effects induced by PKC, STAT3 and ERK signaling, our results could explain the neuroprotective and cardiac protective pathways that are enhanced with α_1A-AR agonism.     

Cardiac pressure overload hypertrophy is differentially regulated by β-adrenergic receptor subtypes

In isolated myocytes, hypertrophy induced by norepinephrine is mediated via α(1)-adrenergic receptors (ARs) and not β-ARs. However, mice with deletions of both major cardiac α(1)-ARs still develop hypertrophy in response to pressure overload. Our purpose was to better define the role of β-AR subtypes in regulating cardiac hypertrophy in vivo, important given the widespread clinical use of β-AR antagonists and the likelihood that patients treated with these agents could develop conditions of further afterload stress. Mice with deletions of β(1), β(2), or both β(1)- and β(2)-ARs were subjected to transverse aortic constriction (TAC). After 3 wk, β(1)(-/-) showed a 21% increase in heart to body weight vs. sham controls, similar to wild type, whereas β(2)(-/-) developed exaggerated (49% increase) hypertrophy. Only when both β-ARs were ablated (β(1)β(2)(-/-)) was hypertrophy totally abolished. Cardiac function was preserved in all genotypes. Several known inhibitors of cardiac hypertrophy (FK506 binding protein 5, thioredoxin interacting protein, and S100A9) were upregulated in β(1)β(2)(-/-) compared with the other genotypes, whereas transforming growth factor-β(2), a positive mediator of hypertrophy was upregulated in all genotypes except the β(1)β(2)(-/-). In contrast to recent reports suggesting that angiogenesis plays a critical role in regulating cardiac hypertrophy-induced heart failure, we found no evidence that angiogenesis or its regulators (VEGF, Hif1α, and p53) play a role in compensated cardiac hypertrophy. Pressure overload hypertrophy in vivo is dependent on a coordination of signaling through both β(1)- and β(2)-ARs, mediated through several key cardiac remodeling pathways. Angiogenesis is not a prerequisite for compensated cardiac hypertrophy.     

Syntrophin isoforms play specific functional roles in the α1D-adrenergic receptor/DAPC signalosome

α_1D-Adrenergic receptors, key regulators of cardiovascular system function, are organized as a multi-protein complex in the plasma membrane. Using a Type-I PDZ-binding motif in their distal C-terminal domain, α_1D-ARs associate with syntrophins and dystrophin-associated protein complex (DAPC) members utrophin, dystrobrevin and α-catulin. Three of the five syntrophin isoforms (α, β₁ and β₂) interact with α_1D-ARs and our previous studies suggest multiple isoforms are required for proper α_1D-AR function in vivo. This study determined the contribution of each specific syntrophin isoform to α_1D-AR function. Radioligand binding experiments reveal α-syntrophin enhances α_1D-AR binding site density, while phosphoinositol and ERK1/2 signaling assays indicate β₂-syntrophin augments full and partial agonist efficacy for coupling to downstream signaling mechanisms. The results of this study provide clear evidence that the cytosolic components within the α_1D-AR/DAPC signalosome significantly alter the pharmacological properties of α₁-AR ligands in vitro.     

β3-Adrenergic Regulation of L-Type Ca2+ Current and Force of Contraction in Human Ventricle

β₃-Adrenergic receptor (β₃-AR) is expressed in human atrial and ventricular tissues. Recently, we have demonstrated that it was involved in the activation of L-type Ca²⁺ current (I _Ca,L) in human atrial myocytes and the force of contraction of human atrial trabeculae. In the present study, we examined the effect of β₃-AR agonist CGP12177 which also is a β₁-AR/β₂-AR antagonist on I _Ca,L in human ventricular myocytes (HVMs) and the force of contraction of human ventricular trabeculae. CGP12177 stimulated I _Ca,L in HVMs with high potency but much lower efficacy than isoprenaline. The β₃-AR antagonist L-748,337 inhibited the effect of CGP12177. CGP12177 and L748,337 competed selectively on β₃-ARs because L748,337 had no effect on isoprenaline-induced stimulation of I _Ca,L, while CGP12177 completely blocked the effect of isoprenaline. The activation of β₃-ARs by CGP12177 does not involve the activation of G_i proteins because CGP12177 had no effect on forskolin-induced stimulation of I _Ca,L. CGP12177 had no effect on the force of contraction of human ventricular trabeculae. L-NMMA, an inhibitor of NO synthase, and IBMX, a nonselective inhibitor of phosphodiesterases, did not potentiate the effect of CGP12177 either on contraction of human ventricular trabeculae or on I _Ca,L in HVMs. We conclude that in human ventricles β₃-AR activation has no inotropic effect, while it slightly increases I _Ca,L. In contrast to human atrium, the activation of β₃-ARs in human ventricle is not accompanied by increased activity of phosphodiesterases.     

Adrenergic Receptors and Fat Cells: Differential Recruitment by Physiological Amines and Homologous Regulation

The control of fat cell lipolysis by the catecholamines involves at least four different adrenoceptor subtypes; three β (β1-, β2-, and β3-ARs) and one α2-adrenoceptor(α2-AR). The physiological importance of the β- and α2A-ARs varies according to the species, the sex, the age, the anatomical location of fat deposits and the degree of obesity in humans and animals. The physiological amines operate through differential recruitment of these sites on the basis of their relative affinities. This point has been assessed by in vitro studies and has partly been confirmed in in vivo experiments using selected a/β-AR antagonists and in situ microdialysis. The affinity of the β3-AR for catecholamines is less than that of the classical β1- and β2-ARs in the various species investigated. Conversely, it is the α2-AR which exhibit the highest affinity for the physiological amines in all fat cells. The relative order of affinity of the various fat cell ARs for the physiological amines defined in binding studies and in vitro ass ays is α2 > β1 > β2 > β3 for norepinephrine and α2 >β2 > β1> β3 for epinephrine. When considering differential β-AR recruitment by catecholamines, it is the β1-AR which is always activated at the lowest norepinephrine levels, whatever the species, while the activation of the β3-AR requires higher norepinephrine levels. In addition to the differential recruitment, differential regulation by hormones could also occur for each fat cell AR subtype. The α2-and β3-ARs are less prone to desensitization and down-regulation by comparison with the β1- and β2-AR.     

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).     

β2-Adrenoceptor transfection enhances contractile reserve of isolated rat ventricular myocytes exposed to chronic isoprenaline stimulation by improving β1-adrenoceptor responsiveness

Context: Heart failure (HF) is a progressive deterioration in heart function associated with overactivity of the sympathetic nervous system. Elevated sympathetic nervous system activity down regulates the β-adrenergic signal system, suppressing β-adrenoceptors (β-ARs)-mediated contractile support in the failing heart.

Objective: We investigated the effects of β₂-AR gene transfer on shortening amplitude of isolated ventricular myocytes under chronic exposure to isoprenaline (ISO), and further determine the contributions of β₁-AR and β₂-AR to the contraction.

Materials and methods: Cardiomyocytes were isolated from adult rat hearts and then transfected with β₂-AR gene using an adenovirus vector. Four hours after the infection, cardiomyocytes were treated with ISO for another 24 hours to imitate high levels of circulating catecholamines in HF. Western blotting was performed to measure myocardial protein expression of β₂-AR. Video-based edge-detection system was used to evaluate basal and ISO-stimulated shortening amplitudes of cardiomyocytes.

Results: β₂-AR gene transfer increased β₂-AR protein content. Chronic ISO stimulation produced a negative inotropic response, whereas acute ISO stimulation showed a positive inotropic response. β₂-AR gene transfer had no significant effects on shortening amplitude of cardiomyocytes under normal conditions, but enhanced the blunted contraction of cardiomyocytes under pathological conditions induced by chronic ISO stimulation, and the effect was inhibited by β₁-AR antagonist, CGP 20712A, instead of β₂-AR antagonist, ICI 118,551.

Discussion and conclusions: We conclude that β₂-AR gene transfer in isolated ventricular myocytes under chronic ISO stimulation improves cellular contraction, and the beneficial effects might be mediated by improving β₁-adrenoceptor responsiveness.     

Rab1 GTPase promotes expression of β-adrenergic receptors in rat pulmonary microvascular endothelial cells

It is known that Rab1 regulates the expression and function of beta-adrenoceptors (β-ARs) in many cells. However, the effect of these changes in rat pulmonary microvascular endothelial cells (RPMVECs) is not known. In the present study, we investigated the role of Rab1, a Ras-like GTPase that coordinates protein transport from the endoplasmic reticulum (ER) to the Golgi body and regulates the cell-surface targeting and function of endogenous β-ARs in RPMVECs in the presence of lipopolysaccharide (LPS).We found that lentivirus-driven expression of wild-type Rab1 (Rab1WT) in RPMVECs strongly enhanced the amount of β-ARs on the cell surface, whereas the dominant-negative mutant Rab1N124I significantly attenuated β-ARs expression on the cell surface. In addition, LPS stimulation significantly reduced β-ARs expression on the cell surface in RPMVECs; however, this effect was reversed by over-expression of wild-type Rab1WT. Fluorescent microscopy analysis demonstrated that expression of Rab1N124I and Rab1 small interfering RNA (siRNA) significantly induced the accumulation of green fluorescent protein (GFP)-tagged β₂-AR in the ER. Consistent with their effects on β-ARs export, Rab1WT and Rab1N124I differentially modified the β-AR-mediated activation of extracellular signal-regulated kinase1/2 (ERK1/2). Importantly, over-expression of Rab1WT markedly reduced LPS-induced hyper-permeability of RPMVECs by increasing the expression of β₂-AR on the cell surface. These data reveal that β-ARs function in RPMVECs could be modulated by manipulating β-ARs traffic from the ER to the Golgi body. We propose the ER-to-Golgi transport as a regulatory site for control of permeability of RPMVECs.     

β-Adrenergic inhibition of contractility in L6 skeletal muscle cells

The β-adrenoceptors (β-ARs) control many cellular processes. Here, we show that β-ARs inhibit calcium depletion-induced cell contractility and subsequent cell detachment of L6 skeletal muscle cells. The mechanism underlying the cell detachment inhibition was studied by using a quantitative cell detachment assay. We demonstrate that cell detachment induced by depletion of extracellular calcium is due to myosin- and ROCK-dependent contractility. The β-AR inhibition of L6 skeletal muscle cell detachment was shown to be mediated by the β(2)-AR and increased cAMP but was surprisingly not dependent on the classical downstream effectors PKA or Epac, nor was it dependent on PKG, PI3K or PKC. However, inhibition of potassium channels blocks the β(2)-AR mediated effects. Furthermore, activation of potassium channels fully mimicked the results of β(2)-AR activation. In conclusion, we present a novel finding that β(2)-AR signaling inhibits contractility and thus cell detachment in L6 skeletal muscle cells by a cAMP and potassium channel dependent mechanism.     

Exogenous NAD Blocks Cardiac Hypertrophic Response via Activation of the SIRT3-LKB1-AMP-activated Kinase Pathway

Since the discovery of NAD-dependent deacetylases, sirtuins, it has been recognized that maintaining intracellular levels of NAD is crucial for the management of stress response of cells. Here we show that agonist-induced cardiac hypertrophy is associated with loss of intracellular levels of NAD, but not exercise-induced physiologic hypertrophy. Exogenous addition of NAD was capable of maintaining intracellular levels of NAD and blocking the agonist-induced cardiac hypertrophic response in vitro as well as in vivo. NAD treatment blocked the activation of pro-hypertrophic Akt1 signaling, and augmented the activity of anti-hypertrophic LKB1-AMPK signaling in the heart, which prevented subsequent induction of mTOR-mediated protein synthesis. By using gene knock-out and transgenic mouse models of SIRT3 and SIRT1, we showed that the anti-hypertrophic effects of exogenous NAD are mediated through activation of SIRT3, but not SIRT1. SIRT3 deacetylates and activates LKB1, thus augmenting the activity of the LKB1-AMPK pathway. These results reveal a novel role of NAD as an inhibitor of cardiac hypertrophic signaling, and suggest that prevention of NAD depletion may be critical in the treatment of cardiac hypertrophy and heart failure.     

β1-Adrenergic receptor vs adenylyl cyclase 6 expression in cardiac myocytes: Differences in transgene localization and intracellular signaling

Adenylyl cyclase type 6 (AC6) and the β₁ adrenergic receptor (β₁AR) are pivotal proteins in transmembrane βAR-signaling in cardiac myocytes. Increased expression of AC6 has beneficial effects on the heart, but increased β₁AR expression has marked deleterious effects. Why do these two elements of the βAR pathway have such different effects? Using adenovirus-mediated gene transfer of the two transgenes in neonatal rat cardiac myocytes, we assessed cellular distribution and performed selected biochemical assays. β₁AR was found predominantly in the plasma membrane. In contrast, AC6 was found in the plasma membrane but also was associated with the nuclear envelope, sarcoplasmic reticulum, mitochondria, and cytoplasm. Increased β₁AR, but not AC6, increased follistatin expression, p38 phosphorylation, phosphatidylserine translocation to the PM, and apoptosis. In contrast, increased AC6, but not β₁AR, inhibited PHLPP2 activity, activated PI3K and Akt, and increased p70S6 kinase phosphorylation and Bcl-2 expression; apoptosis was unchanged. The distribution of AC6 to multiple cellular compartments appears to enable interactions with other proteins (e.g., PHLPP2) and activates cardioprotective signaling (PI3K/Akt). In contrast, β₁AR, confined to the plasma membrane, increased phosphatidylserine translocation and apoptosis. These data provide a potential underlying mechanism for the beneficial vs deleterious effects of these two related βAR-signaling elements.     

α₂-Adrenoceptors activate noradrenaline-mediated glycogen turnover in chick astrocytes

In the brain, glycogen is primarily stored in astrocytes where it is regulated by several hormones/neurotransmitters, including noradrenaline that controls glycogen breakdown (in the short term) and synthesis. Here, we have examined the adrenoceptor (AR) subtype that mediates the glycogenic effect of noradrenaline in chick primary astrocytes by the measurement of glycogen turnover (total (14) C incorporation of glucose into glycogen) following noradrenergic activation. Noradrenaline and insulin increased glycogen turnover in a concentration-dependent manner. The effect of noradrenaline was mimicked by stimulation of α(2) -ARs (and to a lesser degree by β(3) -ARs), but not by stimulation of α(1) -, β(1) -, or β(2) -ARs, and occurred only in astrocytes and not neurons. In chick astrocytes, studies using RT-PCR and radioligand binding showed that α(2A) - and α(2C) -AR mRNA and protein were present. α(2) -AR- or insulin-mediated glycogen turnover was inhibited by phosphatidylinositol-3 kinase inhibitors, and both insulin and clonidine caused phosphorylation of Akt and glycogen synthase kinase-3 in chick astrocytes. α(2) -AR but not insulin-mediated glycogen turnover was inhibited by pertussis toxin pre-treatment indicating involvement of Gi/o proteins. These results show that the increase in glycogen turnover caused by noradrenaline is because of activation of α(2) -ARs that increase glycogen turnover in astrocytes utilizing a Gi/o-PI3K pathway.     

β2- and β3-, but not β1-adrenergic receptors are involved in osteogenesis of mouse mesenchymal stem cells via cAMP/PKA signaling

The osteogenic capacity of mesenchymal stem cells (MSCs) and the importance of β-adrenergic signals in bone formation and resorption have been well investigated. However, little is known about the development of β-adrenergic receptor (β-AR) systems and the role of β-adrenergic signals in osteogenic differentiation of MSCs, which is critically important in bone physiology and pharmacology. In this study, we demonstrated that both the mRNA and protein levels of β2- and β3-AR are up-regulated following osteogenesis of mouse MSCs. We also established that β-AR agonists negatively while antagonists positively affect MSC osteogenesis. Both β2- and β3-AR are involved in MSC osteogenesis, with β2-AR being dominant. The effect of β-ARs on MSC osteogenesis is partly mediated via the cAMP/PKA signaling. These findings suggest that MSC is also a target for β-adrenergic regulation and β-adrenergic signaling plays a role in MSC osteogenesis.