Enhanced G(i) signaling selectively negates beta2-adrenergic receptor (AR)--but not beta1-AR-mediated positive inotropic effect in myocytes from failing rat hearts.
(49/484)
BACKGROUND: Myocardial contractile response to beta1- and beta2-adrenergic receptor (AR) stimulation is severely impaired in chronic heart failure, in which G(i) signaling and the ratio of beta2/beta1 are often increased. Because beta2-AR but not beta1-AR couples to G(s) and G(i) with the G(i) coupling negating the G(s)-mediated contractile response, we determined whether the heart failure-associated augmentation of G(i) signaling contributes differentially to the defects of these beta-AR subtypes and, if so, whether inhibition of G(i) or selective activation of beta2-AR/G(s) by ligands restores beta2-AR contractile response in the failing heart. METHODS AND RESULTS: Cardiomyocytes were isolated from 18- to 24-month-old failing spontaneously hypertensive (SHR) or age-matched Wistar-Kyoto (WKY) rat hearts. In SHR cardiomyocytes, either beta-AR subtype-mediated inotropic effect was markedly diminished, whereas G(i) proteins and the beta2/beta1 ratio were increased. Disruption of G(i) signaling by pertussis toxin (PTX) enabled beta2- but not beta1-AR to induce a full positive inotropic response in SHR myocytes. Furthermore, screening of a panel of beta2-AR ligands revealed that the contractile response mediated by most beta2-AR agonists, including zinterol, salbutamol, and procaterol, was potentiated by PTX, indicating concurrent G(s) and G(i) activation. In contrast, fenoterol, another beta2-AR agonist, induced a full positive inotropic effect in SHR myocytes even in the absence of PTX. CONCLUSIONS: We conclude that enhanced G(i) signaling is selectively involved in the dysfunction of beta2- but not beta1-AR in failing SHR hearts and that disruption of G(i) signaling by PTX or selective activation of beta2-AR/G(s) signaling by fenoterol restores the blunted beta2-AR contractile response in the failing heart. (+info)
Erythrocyte G protein-coupled receptor signaling in malarial infection.
(50/484)
Erythrocytic mechanisms involved in malarial infection are poorly understood. We have found that signaling via the erythrocyte beta2-adrenergic receptor and heterotrimeric guanine nucleotide-binding protein (Galphas) regulated the entry of the human malaria parasite Plasmodium falciparum. Agonists that stimulate cyclic adenosine 3',5'-monophosphate production led to an increase in malarial infection that could be blocked by specific receptor antagonists. Moreover, peptides designed to inhibit Galphas protein function reduced parasitemia in P. falciparum cultures in vitro, and beta-antagonists reduced parasitemia of P. berghei infections in an in vivo mouse model. Thus, signaling via the erythrocyte beta2-adrenergic receptor and Galphas may regulate malarial infection across parasite species. (+info)
Beta 2-adrenergic regulation of ciliary beat frequency in rat bronchiolar epithelium: potentiation by isosmotic cell shrinkage.
(51/484)
Single bronchiolar ciliary cells were isolated from rat lungs. The beta(2)-adrenergic regulation of ciliary beat frequency (CBF) was studied using video-optical microscopy. Terbutaline (a beta(2)-adrenergic agonist) increased CBF in a dose-dependent manner, and it also decreased the volume of the ciliary cells. These terbutaline actions were inhibited by a PKA inhibitor (H-89) and mimicked by forskolin, IBMX and DBcAMP. Ion transport inhibitors were used to isosmotically manipulate the volume of the terbutaline-stimulated bronchiolar ciliary cells. Amiloride (1 microM) and bumetanide (20 microM) potentiated cell shrinkage and the CBF increase, and they shifted the terbutaline dose-response curve to the lower-concentration side. Quinidine (500 microM), in contrast, increased cell volume and suppressed the CBF increase. Moreover, a KCl solution containing amiloride (1 microM) and strophanthidin (100 microM) increased cell volume and suppressed the CBF increase, and then the subsequent removal of either amiloride or strophanthidin decreased cell volume and further increased CBF. NPPB (10 microM) or glybenclamide (200 microM) had no effect on the action of terbutaline. Thus, in terbutaline-stimulated ciliary cells, cell shrinkage enhances the CBF increase; in contrast, cell swelling suppresses it. However, the results of direct manupulation of cell volume by applying osmotic stresses (hyperosmotic shrinkage or hyposmotic swelling) were the opposite of the findings of the isosmotic experiments: hyposmotic cell swelling enhanced the CBF increase, while isosmotic swelling suppressed it. These results suggest that isosmotic and non-isosmotic volume changes in terbutaline-stimulated bronchiolar ciliary cells may trigger different signalling pathways. In conclusion, terbutaline increases CBF and decreases the volume of rat bronchiolar ciliary cells via cAMP accumulation under isosmotic conditions, and the isosmotic cell shrinkage enhances the CBF increase by increasing cAMP sensitivity. (+info)
Beta 2-agonist administration reverses muscle wasting and improves muscle function in aged rats.
(52/484)
The beta2-adrenoceptor agonist (beta2-agonist) fenoterol has potent anabolic effects on rat skeletal muscle. We conducted an extensive dose-response study to determine the most efficacious dose of fenoterol for increasing skeletal muscle mass in adult rats and used this dose in testing the hypothesis that fenoterol may have therapeutic potential for ameliorating age-related muscle wasting and weakness. We used adult (16-month-old) rats that had completed their growth and development, and old (28-month-old) rats that exhibited characteristic muscle wasting and weakness, and treated them daily with either fenoterol (1.4 mg kg(-1), i.p), or saline vehicle, for 4 weeks. Following treatment, functional characteristics of fast-twitch extensor digitorum longus (EDL) and predominantly slow-twitch soleus muscles of the hindlimb were assessed in vitro. Untreated old rats exhibited a loss of skeletal muscle mass and a decrease in force-producing capacity, in both fast and slow muscles, compared with adult rats (P < 0.05). However, there was no age-associated decrease in skeletal muscle beta-adrenoceptor density, nor was the muscle response to chronic beta-agonist stimulation reduced with age. Thus, muscle mass and force-producing capacity of EDL and soleus muscles from old rats treated with fenoterol was equivalent to, or greater than, untreated adult rats. The increase in mass and strength was attributed to a non-selective increase in the cross-sectional area of all muscle fibre types, in both the EDL and soleus. Fenoterol treatment caused a small increase in fatiguability due to a decrease in oxidative metabolism in both EDL and soleus muscles, with some cardiac hypertrophy. Further studies are needed to fully separate the desirable effects on skeletal muscle and the undesirable effects on the heart. Nevertheless, our results demonstrate that fenoterol is a powerful anabolic agent that can restore muscle mass and strength in old rats, and provide preliminary evidence of therapeutic potential for age-related muscle wasting and weakness. (+info)
Agonist and inverse agonist actions of beta-blockers at the human beta 2-adrenoceptor provide evidence for agonist-directed signaling.
(53/484)
Beta-blockers have beneficial effects in heart failure, although the underlying mechanism is unknown. Beta2-adrenoceptors, however, are proportionally higher in the failing human heart. This study shows several clinically used beta-blockers are agonists at the human beta2-adrenoceptor. Although these agonist effects were small at the cAMP level, they were substantial at the level of cAMP response element (CRE)-mediated gene transcription. Some of the effects of "beta-blockers" seen in heart failure may be related to the beta2-agonist actions of these compounds. CRE-gene transcription responses to beta2-agonists, forskolin, and cAMP-analogs were sensitive to p42/44-mitogen-activated protein (MAP) kinase pathway inhibitors. p42/44-MAP kinase activation was also shown directly by western blotting and enzyme-linked immunosorbent assay techniques. N-[2-(4-bromocinnamylamino)ethyl]-5-isoquinoline (H89; a protein kinase A inhibitor) stimulated cAMP accumulation and CRE gene transcription via the beta2-adrenoceptor at concentrations at which protein kinase A was inhibited, providing evidence for an alternative pathway. Propranolol, however, produced paradoxical effects; it reduced basal cAMP accumulation (via beta2-mediated inverse agonism) but stimulated beta2-mediated CRE gene transcription. This cannot be explained by a sequential pathway from Gs-adenylyl cyclase-cAMP to CRE binding protein phosphorylation. Both responses to propranolol were insensitive to pertussis toxin, thus excluding Gi-protein involvement. Propranolol CRE gene transcription responses were attenuated by p42/44-MAP kinase inhibitors and propranolol was also found to directly stimulate the p42/44-MAP kinase pathway. Studies of inositol phosphate accumulation and of protein kinase C or Rho kinase inhibitors on CRE-gene transcription provided no evidence for Gq/11 or G12/13 involvement. These data suggest that propranolol can simultaneously act as an inverse agonist through a Gs-coupled mechanism while stimulating the p42/44-MAP kinase pathway through an alternative G-protein-independent mechanism. (+info)
Real-time analysis of ternary complex on particles: direct evidence for partial agonism at the agonist-receptor-G protein complex assembly step of signal transduction.
(54/484)
We developed a novel and generalized approach to investigate G protein-coupled receptor molecular assemblies. We solubilized a fusion protein consisting of the beta(2)-adrenergic receptor and green fluorescent protein (GFP) for bead-based flow cytometric analysis. beta(2)-Adrenergic receptor GFP bound to dihydroalprenolol-conjugated beads, providing a K(d) for the fusion protein and, in competition with beta(2)-adrenergic receptor ligands, K(d) values for agonists and antagonists. Beads displaying chelated nickel bound purified hexahistidine-tagged G protein heterotrimers and, subsequently, the binary complex of agonist with beta(2)-adrenergic receptor GFP. The dose-response curves of ternary complex formation revealed maximal assembly for ligands previously classified as full agonists and reduced assembly for ligands previously classified as partial agonists. Guanosine 5'-3-O-(thio)triphosphate-induced dissociation rates of the ternary complex were the same for full and partial agonists. Soluble G protein, competing with ternary complexes on beads provided an affinity estimate of agonist-receptor complexes to G protein. When performed simultaneously, the two assemblies discriminated between agonist, antagonist or inactive molecule in a manner appropriate for high throughput, small volume drug discovery. The assemblies can be further generalized to other G protein coupled receptor protein-protein interactions. (+info)
Salmeterol, a beta2-receptor agonist, attenuates lipopolysaccharide-induced lung inflammation in mice.
(55/484)
Lipopolysaccharide is ubiquitously present in the environment. To determine the effect of salmeterol, a long-acting beta(2)-receptor agonist, on lipopolysaccharide-induced lung inflammation, mice received lipopolysaccharide (10 microg) intranasally with or without salmeterol intraperitoneally (5 mg/kg) 30 min earlier and 12 h thereafter. Salmeterol dose- and time-dependently inhibited the lipopolysaccharide-induced influx of neutrophils into bronchoalveolar lavage fluid and lung tissue, and these pulmonary neutrophils displayed a reduced expression of CD11b at their surface. To determine the contribution of the salmeterol effect on neutrophil CD11b in the attenuated neutrophil recruitment, we treated mice intranasally exposed to lipopolysaccharide with salmeterol with or without a blocking anti-CD11b antibody. Anti-CD11b profoundly reduced lipopolysaccharide-induced neutrophil influx in bronchoalveolar lavage fluid, an effect that was modestly enhanced by concurrent salmeterol treatment. These data suggest that salmeterol inhibits lipopolysaccharide-induced neutrophil recruitment to the lungs by a mechanism that possibly in part is mediated by an effect on neutrophil CD11b. (+info)
Differences in endosomal targeting of human (beta)1- and (beta)2-adrenergic receptors following clathrin-mediated endocytosis.
(56/484)
The beta(2)-adrenergic receptor (beta(2)AR) undergoes agonist-mediated endocytosis via clathrin-coated pits by a process dependent on both arrestins and dynamin. Internalization of some G protein-coupled receptors, however, is independent of arrestins and/or dynamin and through other membrane microdomains such as caveolae or lipid rafts. The human beta(1)AR is less susceptible to agonist-mediated internalization than the beta(2)-subtype, and its endocytic route, which is unknown, may be different. We have found that (i) co-expression of arrestin-2 or -3 enhanced the internalization of both subtypes whereas co-expression of dominant-negative mutants of arrestin-2 or dynamin impaired their internalization, as did inhibitors of clathrin-mediated endocytosis. (ii) Agonist stimulation increased the phosphorylation of beta(2)AR but not beta(1)AR. (iii) In response to agonist, each subtype redistributed from the cell surface to a distinct population of cytoplasmic vesicles; those containing beta(1)AR were smaller and closer to the plasma membrane whereas those containing beta(2)AR were larger and more perinuclear. (iv) When subcellular fractions from agonist-treated cells were separated by sucrose density gradient centrifugation, all of the internalized beta(2)AR appeared in the lighter endosomal-containing fractions whereas some of the internalized beta(1)AR remained in the denser plasma membrane-containing fractions. (v) Both subtypes recycled with similar kinetics back to the cell surface upon removal of agonist; however, recycling of beta(2)AR but not beta(1)AR was inhibited by monensin. Based on these results, we propose that the internalization of beta(1)AR is both arrestin- and dynamin-dependent and follows the same clathrin-mediated endocytic pathway as beta(2)AR. But during or after endocytosis, beta(1)AR and beta(2)AR are sorted into different endosomal compartments. (+info)