Beta-adrenergic Receptor Signaling Research Articles

Structure‐Function Relationship and Local Subcellular Signaling using Heart Failure Relevant Molecular Defined Muscle Specific mAKAP Mutants

Beta adrenergic stimulation signals through phosphorylation of downstream proteins such as protein kinase A (PKA). PKA substrate phosphorylation is facilitated through its co localization with its signaling partner by A kinase anchoring proteins (AKAPs). Particularly, mAKAP (muscle selective AKAP) localizes PKA and its target substrates such as phosphodiesterase 4D3 (PDE4D3), ryanodine receptor and protein phosphatase (PP2A). We have recently identified potentially important human mAKAP mutations located within or near key protein binding sites critical to beta adrenergic receptor signaling. Three mutations (P1400S, S2195F and L717V) were cloned for the purpose of comparing whether these substitutions disrupted mAKAP binding to the PKA regulatory subunit RIIα or PDE4D3 binding domain and to understand whether these mutations mediated altered signaling. Interestingly, S2195F, a mutation located in PP2A binding site, showed similar protein expression (p=0.3) to wild type mAKAP, but with a significant increase in binding propensity to RIIα after cell stimulation. This result was corroborated by immunopreciptation (p=0.0003). Consequently, PKA activity showed a significant increase with mAKAP S2195F mutation, as compared to wild type. In conclusion, mAKAP S2195F displayed modified activity and binding propensities and may participate in hypertrophy. This work was supported by NIH (HL085487).

Beta2‐adrenergic receptor signaling in CD4+Foxp3+ regulatory T cells enhances their suppressive function in a PKA‐dependent manner

Beta2-adrenergic receptor (B2AR) signaling is known to impair Th1-cell differentiation and function in a cAMP-dependent way, leading to inhibition of cell proliferation and decreased production of IL-2 and IFN-γ. CD4(+) Foxp3(+) Treg cells play a key role in the regulation of immune responses and are essential for maintenance of self-tolerance. Nevertheless, very little is known about adrenergic receptor expression in Treg cells or the influence of noradrenaline on their function. Here we show that Foxp3(+) Treg cells express functional B2AR. B2AR activation in Treg cells leads to increased intracellular cAMP levels and to protein kinase A (PKA)-dependent CREB phosphorylation. We also found that signaling via B2AR enhances the in vitro suppressive activity of Treg cells. B2AR-mediated increase in Treg-cell suppressive function was associated with decreased IL-2 mRNA levels in responder CD4(+) T cells and improved Treg-cell-induced conversion of CD4(+) Foxp3(-) cells into Foxp3(+) induced Treg cells. Moreover, B2AR signaling increased CTLA-4 expression in Treg cells in a PKA-dependent way. Finally, we found that PKA inhibition totally prevented the B2AR-mediated increase in Treg-cell suppressive function. Our data suggest that sympathetic fibers are able to regulate Treg-cell suppressive activity in a positive manner through B2AR signaling.

BIOMARKER IDENTIFICATION IN BREAST CANCER: BETA-ADRENERGIC RECEPTOR SIGNALING AND PATHWAYS TO THERAPEUTIC RESPONSE

Recent preclinical studies have associated beta-adrenergic receptor (β-AR) signaling with breast cancer pathways such as progression and metastasis. These findings have been supported by clinical and epidemiological studies which examined the effect of beta-blocker therapy on breast cancer metastasis, recurrence and mortality. Results from these studies have provided initial evidence for the inhibition of cell migration in breast cancer by beta-blockers and have introduced the beta-adrenergic receptor pathways as a target for therapy. This paper analyzes gene expression profiles in breast cancer patients, utilising Artificial Neural Networks (ANNs) to identify molecular signatures corresponding to possible disease management pathways and biomarker treatment strategies associated with beta-2-adrenergic receptor (ADRB2) cell signaling. The adrenergic receptor relationship to cancer is investigated in order to validate the results of recent studies that suggest the use of beta-blockers for breast cancer therapy. A panel of genes is identified which has previously been reported to play an important role in cancer and also to be involved in the beta-adrenergic receptor signaling.

Beta2 adrenergic receptor signaling in CD4+ Foxp3+ regulatory T cells enhances their suppressive function in vitro

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124. A comparative examination of the anti-inflammatory effects of SSRI and SNRI antidepressants on microglia

Selective serotonin and serotonin norepinephrine reuptake inhibitors (SSRI; SNRI) are the frontline pharmacological treatment options for major depression. While these drugs have long been assumed to exert their antidepressant effects because of their ability to alter central monoamine levels, they have also been shown to exert anti-inflammatory effects. Recently it has become apparent that these drugs can also exert anti-inflammatory effects on microglia, the principal cells within the CNS that regulate and respond to inflammatory factors. From a pharmacological standpoint, much critical information remains unknown. In particular, the relative efficacy of these drugs in modulating microglial responses is yet to be determined. To address these issues, we evaluated the ability of different SSRIs (fluoxetine, sertraline, paroxetine, fluvoxamine and citalopram) and one SNRI (venlafaxine) to suppress microglial responses to an inflammatory stimulus. Specifically, we examined their ability to alter tumor necrosis factor-alpha (TNF-alpha) and nitric oxide (NO) production after stimulation with lipopolysaccharide. Our results indicated that the SSRIs potently inhibited microglial TNF-alpha and NO production. We then investigated whether these effects might involve either beta-adrenoceptor or cAMP signalling. Using the protein kinase A inhibitor Rp-CAMPs, we found evidence to suggest that cAMP signalling may be partially involved in regulating the anti-inflammatory response. Findings such as these could suggest that antidepressants may owe at least some of their therapeutic effectiveness to their anti-inflammatory properties.

72. A role for beta 2-adrenergic receptor signaling in the exercise-induced re-distribution of Th cells within an asthmatic lung

Moderate aerobic exercise has been documented to modify the immune system, specifically, by mediating a re-distribution of Th cells in vivo . In addition, it is well-established that exercise generates the soluble Beta 2-adrenergic receptor (B2-AR) agonist, epinephrine, in which Th cells can respond by their expression of B2-AR. Studies using a luciferase reporter for OVA-specific Th cells in an OVA-sensitized/challenged allergic asthma mouse model have shown a reduction in OVA-specific Th cell migration into the lungs when exercised, subsequently reducing asthma pathogenesis. In addition, investigations looking at lung-homing chemokine receptor presence on the surface of Th cells (CCR4 and CCR8) by flow cytometry revealed no difference between exercised and sedentary asthmatic mice. However, transmigration experiments demonstrated that exercised Th cell chemokine receptors, CCR4 and CCR8, are less functional than their sedentary counterparts. These data suggest exercised Th cells are less responsive to lung-homing chemokine gradients created by an asthmatic lung. Furthermore, studies using B2-AR −/− Th cells transferred to OVA sensitized/challenged recipient mice, showed a loss in exercise-mediated chemokine receptor desensitization in transmigration assays comparing exercised and sedentary B2-AR −/− Th cells. These studies demonstrate that moderate aerobic exercise training can alter Th cell migration into the asthmatic lung by modifying chemokine receptor function. Additionally, the data suggests a role for B2-AR signaling in mediating this exercise-induced chemokine receptor loss of function.

Optogenetic Therapeutic Cell Implants

Optogenetic Therapeutic Cell Implants

G-Protein Coupled Receptor Kinase 5 Regulates Prostate Tumor Growth

The limited success of cancer therapeutics is largely attributable to the ability of cancer to become resistant to conventional cytotoxic chemotherapy. Thus, further identification of signaling molecules and pathways that influence tumorigenesis is needed to increase the overall therapeutic options. GRKs, originally recognized for their conserved role in GPCR signal control, have now emerged as regulators of additional biological molecules and functions. We used Western blot analysis to determine GRK expression in prostate cancer and RNA interference to establish the role of GRK5 in prostate cancer growth and progression through the cell cycle. GRK5 was expressed highly in the aggressive prostate cancer PC3 cell line and its silencing by RNA interference attenuated in vitro cell proliferation. PC3 cells that stably expressed lentiviral small hairpin RNA and targeted GRK5 evidence reduced xenograft tumor growth in mice. This was reversed by rescuing expression with wild-type but not with kinase inactive K215R GRK5, implying the need of GRK5 kinase activity for tumor growth. To investigate possible cellular mechanism(s) for GRK5 in cell growth regulation we tested whether kinase activity would impact cell cycle progression. Like forced over expression of kinase-inactive K215R GRK5, GRK5 knockdown led to G2/M arrest in the cell cycle. Also, evidence revealed that the loss of GRK5 activity resulted in decreased cyclin D1 expression, Rb protein phosphorylation and E2F target gene expression involved in cell cycle control. Results provide direct evidence that GRK5 has an immediate role in the regulation of prostate tumor growth.

Differential Modulation of Beta-Adrenergic Receptor Signaling by Trace Amine-Associated Receptor 1 Agonists

Trace amine-associated receptors (TAAR) are rhodopsin-like G-protein-coupled receptors (GPCR). TAAR are involved in modulation of neuronal, cardiac and vascular functions and they are potentially linked with neurological disorders like schizophrenia and Parkinson's disease. Subtype TAAR1, the best characterized TAAR so far, is promiscuous for a wide set of ligands and is activated by trace amines tyramine (TYR), phenylethylamine (PEA), octopamine (OA), but also by thyronamines, dopamine, and psycho-active drugs. Unfortunately, effects of trace amines on signaling of the two homologous β-adrenergic receptors 1 (ADRB1) and 2 (ADRB2) have not been clarified yet in detail. We, therefore, tested TAAR1 agonists TYR, PEA and OA regarding their effects on ADRB1/2 signaling by co-stimulation studies. Surprisingly, trace amines TYR and PEA are partial allosteric antagonists at ADRB1/2, whereas OA is a partial orthosteric ADRB2-antagonist and ADRB1-agonist. To specify molecular reasons for TAAR1 ligand promiscuity and for observed differences in signaling effects on particular aminergic receptors we compared TAAR, tyramine (TAR) octopamine (OAR), ADRB1/2 and dopamine receptors at the structural level. We found especially for TAAR1 that the remarkable ligand promiscuity is likely based on high amino acid similarity in the ligand-binding region compared with further aminergic receptors. On the other hand few TAAR specific properties in the ligand-binding site might determine differences in ligand-induced effects compared to ADRB1/2. Taken together, this study points to molecular details of TAAR1-ligand promiscuity and identified specific trace amines as allosteric or orthosteric ligands of particular β-adrenergic receptor subtypes.

A Systems Approach to the Biology of Mood Disorders through Network Analysis of Candidate Genes

Meta analysis of association data of mood disorders has shown evidence for the role of particular genes in genetic risk. Integration of association data from meta analysis with differential expression data in brains of mood disorder patients could heighten the level of support for specific genes. To identify molecular mechanisms that may be disrupted in disease, a systems approach that involves analysis of biological networks created by these selected genes was employed.Interaction networks of hierarchical groupings of selected genes were generated using the Michigan Molecular Interactions (MiMI) software. Large networks were deconvoluted into subclusters of core complexes by using a community clustering program, GLay. Network nodes were functionally annotated in DAVID Bioinformatics Resource to identify enriched pathways and functional clusters. MAPK and beta adrenergic receptor signaling pathways were significantly enriched in the ANK3 and CACNA1C network. The PBRM1 network bolstered the enrichment of chromatin remodeling and transcription regulation functional clusters. Lowering the stringency for inclusion of other genes in network seeds increased network complexity and expanded the recruitment of enriched pathways to include signaling by neurotransmitter and hormone receptors, neurotrophin, ErbB and the cell cycle. We present a strategy to interrogate mechanisms in the cellular system that might be perturbed in disease. Network analysis of meta analysis- generated candidate genes that exhibited differential expression in mood disorder brains identified signaling pathways and functional clusters that may be involved in genetic risk for mood disorders.

Analysis of β‐Adrenergic Receptor Signaling in Gravin (AKAP12) Knockout Mice after Chronic Isoproterenol Stimulation

The purpose of this study was to investigate the effect of knockout of gravin, an A kinase anchoring protein, on the beta‐adrenergic receptor (β‐AR) signaling pathway. We measured heart‐to‐body weight ratio (HW/BW), β‐AR density and affinity and cAMP production in wild‐type (WT) and gravin knockout (KO) mice stimulated with isoproterenol (ISO; 30 mg/kg/day x 14 days). Our results show that the WT ISO group had a significantly higher HW/BW ratio than the WT and gravin KO controls (p=0.02) as well as the gravin KO ISO group (WT control: 4.81 mg/g; WT ISO: 6.05 mg/g; KO control: 5.11 mg/g; KO ISO: 5.54 mg/g; p=0.012)The WT treatment group had significantly higher total β‐AR density (WT control: 103.53 fmol/mg; WT ISO: 209.52 fmol/mg; KO control: 118.62 fmol/mg; KO ISO: 146.25 fmol/mg; p=0.011) but significantly lower affinity both compared to the WT and KO controls as well as the gravin KO treatment group (WT control: 0.30; WT ISO: 0.66; KO control: 0.34; KO ISO: 0.23; p=0.002). However, both the WT and the gravin KO treatment group had significantly lower cAMP production when compared to their respective controls (WT control: 0.68 pmol/mg; WT ISO: 0.32 pmol/mg; KO control: 0.66 pmol/mg; KO ISO: 0.24 pmol/mg; p=0.0001). These data indicate that the absence of gravin attempts to maintain normal conditions of the β‐AR signaling pathway even after chronic β‐AR stimulation. This research was supported by NIH R01HL085487.

Beta-adrenergic receptor signaling by the isomers of isoproterenol and like drugs in retinal endothelial cells and Müller cells

Diabetic retinopathy is the leading cause of blindness to working-age adults. Complications of diabetic retinopathy include pericyte loss, basement membrane thickening of capillaries, microaneurysm formation, and an increase in inflammatory marker levels. We have shown that stimulation of β-adrenergic receptors by β-adrenergic receptor agonists significantly reduces key inflammatory markers in retinal endothelial cells (REC) or Muller cells cultured in high glucose, indicating that restoration of β-adrenergic receptor signaling may be protective to the retina. REC and Muller cells responded to increased glucose concentrations with an increase in inflammatory markers and apoptosis, which is reduced following treatment with β-adrenergic receptor agonists. In our initial studies, we evaluated the ability of optically active R-(−)-isoproterenol (R-1R-1), S-(+)-isoproterenol (S-1S-1S-1), and racemic mixture of (±)-isoproterenol (1), a non-selective β-adrenergic receptor agonist, to reduce the cleavage of caspase 3 and TNFα levels. We observed R-(−)-isoproterenol is more effective than S-(+)-isoproterenol, thus we developed novel optically active analogs of R-(−)-isoproterenol. Of the analogs, we found that compound 12 significantly reduced both caspase 3 and TNFα levels in REC, but did not reduce both markers in Muller cells. Binding data of compound 12 suggest that it is not a β-adrenergic receptor true agonist, but appears to decrease inflammatory levels and apoptosis in REC through an alternate mechanism.

Up‐Regulation and Functional Effect of Cardiac β3‐Adrenoreceptors in Alcoholic Monkeys

Recent studies link altered cardiac beta-adrenergic receptor (AR) signaling to the pathology of alcoholic cardiomyopathy (ACM). However, the alteration and functional effect of beta(3)-AR activation in ACM are unknown. We tested the hypothesis that chronic alcohol intake causes an up-regulation of cardiac beta(3)-AR, which exacerbates myocyte dysfunction and impairs calcium regulation, thereby directly contributing to the progression of ACM. We compared myocyte beta(3)- and beta(1)-AR expression and myocyte contractile ([Ca(2+)](i)), transient ([Ca(2+)](iT)), and Ca(2+) current (I(Ca,L)) responses to beta- and beta(3)-AR stimulation in myocytes obtained from left ventricle (LV) tissue samples obtained from 10 normal control (C) and 16 monkeys with self-administered alcohol for 12 months prior to necropsy: 6 moderate (M) and 10 heavy (H) drinkers with group average alcohol intakes of 1.5 +/- 0.2 and 3.3 +/- 0.2 g/kg/d, respectively. Compared with control myocytes (C), in alcoholic cardiomyocytes, basal cell contraction (dL/dt(max), -39%, H: 69.8 vs. C: 114.6 microm/s), relaxation (dR/dt(max), -37%, 58.2 vs. 92.9 microm/s), [Ca(2+)](iT) (-34%, 0.23 vs. 0.35), and I(Ca,L) (-25%, 4.8 vs. 6.4pA/pF) were all significantly reduced. Compared with controls, in moderate and heavy drinkers, beta(1)-AR protein levels decreased by 23% and 42%, but beta(3)-AR protein increased by 46% and 85%, respectively. These changes were associated with altered myocyte functional responses to beta-AR agonist, isoproterenol (ISO), and beta(3)-AR agonist, BRL-37344 (BRL). Compared with controls, in alcoholic myocytes, ISO (10(-8) M) produced significantly smaller increases in dL/dt(max) (H: 40% vs. C: 71%), dR/dt(max) (37% vs. 52%), [Ca(2+)](iT) (17% vs. 37%), and I(Ca,L) (17% vs. 27%), but BRL (10(-8) M) produced a significantly greater decrease in dL/dt(max) (H: -23% vs. C: -11%), [Ca(2+)](iT) (-30% vs. -11%), and I(Ca,L) (-28% vs. -17%). Chronic alcohol consumption down-regulates cardiac beta(1)- and up-regulates beta(3)-ARs, contributing to the abnormal response to catecholamines in ACM. The up-regulation of cardiac beta(3)-AR signaling enhances inhibition of LV myocyte contraction and relaxation and exacerbates the dysfunctional [Ca(2+)](i) regulation and, thus, may precede the development of ACM.

Application of isoproterenol inhibits diabetic-like changes in the rat retina

Diabetic retinopathy is the leading cause of blindness to working-age adults. We have recently shown that surgical removal or genetic manipulations to eliminate sympathetic neurotransmission produces many of the retinal changes similar to rodent diabetic retinopathy with normal glucose levels. We hypothesized that application of a beta-adrenergic receptor agonist, isoproterenol, could reach the retina to elicit normal cellular signaling and inhibit the functional and morphological markers of early stage diabetic retinopathy in the rat. Rats were made diabetic by injection of 60 mg/kg streptozotocin. Within 3 days of diabetes-induction, rats were placed into 1 of 3 groups (control, diabetes, or diabetic + isoproterenol). Dose and time course studies were done for isoproterenol using a PKA ELISA and CREB analyses. Once the optimal dose and time course were established, electrical activity of the retina was analyzed by electroretinogram each month for the 8-month study. Western blotting was done for insulin receptor signaling and Akt and ELISA analyses for TNFα concentration and cleavage of caspase 3 at 2- and 8-months of diabetes. Diabetes-induced degeneration of neural cells and retinal thickness were assessed at 2 months, while degenerate capillaries were quantitated at 8 months of treatment. Daily application of 50 mM isoproterenol was effective in inhibiting the diabetes-induced loss of a- and b-wave and oscillatory potential amplitudes in the electroretinogram. Isoproterenol blocked the increase in TNFα and apoptosis in the diabetic retina. The numbers of degenerate capillaries were also reduced in the treated + diabetes retina. These data strongly suggest that loss of beta-adrenergic receptor signaling may be a key factors in early stage diabetic retinopathy. Resolution of this loss of adrenergic receptor signaling can inhibit some of the hallmarks of diabetic retinopathy in the retina.

Reduction of Sympathetic Activity via Adrenal-targeted GRK2 Gene Deletion Attenuates Heart Failure Progression and Improves Cardiac Function after Myocardial Infarction

Chronic heart failure (HF) is characterized by sympathetic overactivity and enhanced circulating catecholamines (CAs), which significantly increase HF morbidity and mortality. We recently reported that adrenal G protein-coupled receptor kinase 2 (GRK2) is up-regulated in chronic HF, leading to enhanced CA release via desensitization/down-regulation of the chromaffin cell alpha(2)-adrenergic receptors that normally inhibit CA secretion. We also showed that adrenal GRK2 inhibition decreases circulating CAs and improves cardiac inotropic reserve and function. Herein, we hypothesized that adrenal-targeted GRK2 gene deletion before the onset of HF might be beneficial by reducing sympathetic activation. To specifically delete GRK2 in the chromaffin cells of the adrenal gland, we crossed PNMTCre mice, expressing Cre recombinase under the chromaffin cell-specific phenylethanolamine N-methyltransferase (PNMT) gene promoter, with floxedGRK2 mice. After confirming a significant ( approximately 50%) reduction of adrenal GRK2 mRNA and protein levels, the PNMT-driven GRK2 knock-out (KO) offspring underwent myocardial infarction (MI) to induce HF. At 4 weeks post-MI, plasma levels of both norepinephrine and epinephrine were reduced in PNMT-driven GRK2 KO, compared with control mice, suggesting markedly reduced post-MI sympathetic activation. This translated in PNMT-driven GRK2 KO mice into improved cardiac function and dimensions as well as amelioration of abnormal cardiac beta-adrenergic receptor signaling at 4 weeks post-MI. Thus, adrenal-targeted GRK2 gene KO decreases circulating CAs, leading to improved cardiac function and beta-adrenergic reserve in post-MI HF. GRK2 inhibition in the adrenal gland might represent a novel sympatholytic strategy that can aid in blocking HF progression.

The Role of Lipophilic Bile Acids in the Development of Cirrhotic Cardiomyopathy

Marked hemodynamic changes occur in humans and experimental animals with cirrhotic liver disease. In the heart, basal contractility, responsiveness to beta-adrenoceptor activation, and excitation-contraction coupling (ECC) are negatively affected in models of cirrhosis and portal hypertension with portosystemic shunting (PVS), and comprise what has been called cirrhotic cardiomyopathy. These effects are accompanied by elevated circulating levels of bile acids. We investigated whether elevated bile acids act as a myocardial toxicant by exposing cardiac muscle in vitro to bile acids and compared these results with two models of cirrhotic cardiomyopathy with elevated bile acids: CCl4-induced cirrhosis and PVS. Cholic acid, a lipophilic bile acid, produced a decrease in basal cardiac contractility and responsiveness to beta-adrenoceptor activation, both of which appeared to result from altered ECC. beta-Adrenoceptor density and signaling were unaffected. Acutely, ursodeoxycholic acid, a more hydrophilic bile acid, had no effect. Cirrhosis produced a decrease in basal force, depressed beta-adrenoceptor responsiveness, and altered ECC similar to cholic acid. However, cirrhosis also altered beta-adrenoceptor signaling including decreases in cyclic AMP formation, expression of the stimulatory G protein, GS, and beta-adrenoceptor density. Displacement of lipophilic bile acids by chronic administration of ursodeoxycholic acid to rats during the development of cirrhotic cardiomyopathy produced by PVS produced attenuation of the effect on ECC. These results suggest a possible role for lipophilic bile acids in some, but not all of the myocardial consequences of chronic portal vein stenosis and CCl4-induced cirrhosis.

Noradrenaline activation of neurotrophic pathways protects against neuronal amyloid toxicity

Degeneration of locus coeruleus (LC) noradrenergic forebrain projection neurons is an early feature of Alzheimer's disease. The physiological consequences of this phenomenon are unclear, but observations correlating LC neuron loss with increased Alzheimer's disease pathology in LC projection sites suggest that noradrenaline (NA) is neuroprotective. To investigate this hypothesis, we determined that NA protected both hNT human neuronal cultures and rat primary hippocampal neurons from amyloid-beta (Abeta(1-42) and Abeta(25-35)) toxicity. The noradrenergic co-transmitter galanin was also effective at preventing Abeta-induced cell death. NA inhibited Abeta(25-35)-mediated increases in intracellular reactive oxygen species, mitochondrial membrane depolarization, and caspase activation in hNT neurons. NA exerted its neuroprotective effects in these cells by stimulating canonical beta(1) and beta(2) adrenergic receptor signaling pathways involving the activation of cAMP response element binding protein and the induction of endogenous nerve growth factor (NGF) and brain-derived neurotrophic factor (BDNF). Treatment with functional blocking antibodies for either NGF or BDNF blocked NA's protective actions against Abeta(1-42) and Abeta(25-35) toxicity in primary hippocampal and hNT neurons, respectively. Taken together, these data suggest that the neuroprotective effects of noradrenergic LC afferents result from stimulating neurotrophic NGF and BDNF autocrine or paracrine loops via beta adrenoceptor activation of the cAMP response element binding protein pathway.

Gαq-mediated Activation of GRK2 by Mechanical Stretch in Cardiac Myocytes: THE ROLE OF PROTEIN KINASE C

G protein-coupled receptor kinase-2 (GRK2) is a critical regulator of beta-adrenergic receptor (beta-AR) signaling and cardiac function. We studied the effects of mechanical stretch, a potent stimulus for cardiac myocyte hypertrophy, on GRK2 activity and beta-AR signaling. To eliminate neurohormonal influences, neonatal rat ventricular myocytes were subjected to cyclical equi-biaxial stretch. A hypertrophic response was confirmed by "fetal" gene up-regulation. GRK2 activity in cardiac myocytes was increased 4.2-fold at 48 h of stretch versus unstretched controls. Adenylyl cyclase activity was blunted in sarcolemmal membranes after stretch, demonstrating beta-AR desensitization. The hypertrophic response to mechanical stretch is mediated primarily through the G alpha(q)-coupled angiotensin II AT(1) receptor leading to activation of protein kinase C (PKC). PKC is known to phosphorylate GRK2 at the N-terminal serine 29 residue, leading to kinase activation. Overexpression of a mini-gene that inhibits receptor-G alpha(q) coupling blunted stretch-induced hypertrophy and GRK2 activation. Short hairpin RNA-mediated knockdown of PKC alpha also significantly attenuated stretch-induced GRK2 activation. Overexpression of a GRK2 mutant (S29A) in cardiac myocytes inhibited phosphorylation of GRK2 by PKC, abolished stretch-induced GRK2 activation, and restored adenylyl cyclase activity. Cardiac-specific activation of PKC alpha in transgenic mice led to impaired beta-agonist-stimulated ventricular function, blunted cyclase activity, and increased GRK2 phosphorylation and activity. Phosphorylation of GRK2 by PKC appears to be the primary mechanism of increased GRK2 activity and impaired beta-AR signaling after mechanical stretch. Cross-talk between hypertrophic signaling at the level of PKC and beta-AR signaling regulated by GRK2 may be an important mechanism in the transition from compensatory ventricular hypertrophy to heart failure.

Effects of pain controlling neuropeptides on human fat cell lipolysis

Neuropeptides NPFF and NPSF are involved in pain control, acting through the G-protein coupled receptors (GPR)74 (high affinity for NPFF) and GPR147 (equal affinity for NPFF and NPSF). GPR74 also inhibits catecholamine-induced adipocyte lipolysis and regulates fat mass in humans. The aim of this study was to compare the effects of NPFF and NPSF on noradrenaline-induced lipolysis and to determine the expression of their receptors in human fat cells. Adipose tissue was obtained during surgery. Adipocytes were prepared and kept in primary culture. Lipolysis, protein expression and gene expression were determined. NPFF counteracted noradrenaline-induced lipolysis, which was more marked after 48 h than after 4 h exposure and was solely attributed to inhibition of beta-adrenoceptor signalling. NPSF counteracted noradrenaline-induced lipolysis maximally after 4 h of exposure, which was attributed to a combination of inhibition of beta-adrenoceptor signalling and decreased activation of the protein kinase-A hormone sensitive lipase complex by cyclic AMP. Both neuropeptides were effective in nanomolar concentrations. NPFF and NPSF had no effects on the expression of genes involved in catecholamine signal transduction. Both GPR74 and GPR147 were expressed at the protein level in fat cells from various adipose regions. GPR74 mRNA levels were higher in adipose tissue from obese as compared with non-obese subjects. High gene expression of either receptor correlated with low noradrenaline-induced lipolysis (P<0.05). Pain controlling neuropeptides NPFF and NPSF may be important for the regulation of lipolysis in man probably acting through GPR74 and GPR147. At low concentrations they inhibit catecholamine-induced lipolysis through rapid and long-term post-transcriptional effects at several steps in adrenoceptor signalling in fat cells.

Regulation of T cells in airway disease by beta-agonist

It is widely recognized that Th2 cytokines derived from T cells play a major role in the development of allergic lung inflammation that causes most asthma. Beta-agonists are important rescue and maintenance therapies for asthma, yet our understanding of beta-agonist effects on T cell biology is surprisingly poor. Recent studies using both cell culture and more integrative models are beginning to reveal beta-agonist regulation of T cell signaling and function that may be important in the pathogenesis and treatment of asthma and possibly other inflammatory diseases. Here we provide a comprehensive review of the literature concerning beta-agonist effects on T cells, and discuss the relevance of emerging paradigms of beta-adrenergic receptor signaling to T cell function.