Beta3-adrenoceptor agonist stimulation of the Na+, K+ -pump in rat skeletal muscle is mediated by beta2- rather than beta3-adrenoceptors.
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ABSTRACT:
SUBMITTER: Murphy KT
PROVIDER: S-EPMC2014662 | biostudies-other | 2006 Nov
REPOSITORIES: biostudies-other
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Project description:Background and purposeThere are two important properties of receptor-ligand interactions: affinity (the ability of the ligand to bind to the receptor) and efficacy (the ability of the receptor-ligand complex to induce a response). Ligands are classified as agonists or antagonists depending on whether or not they have efficacy. In theory, it is possible to develop selective agonists based on selective affinity, selective intrinsic efficacy or both. This study examined the affinity and intrinsic efficacy of 31 beta-adrenoceptor agonists at the three human beta-adrenoceptors to determine whether the current agonists are subtype selective because of affinity or intrinsic efficacy.Experimental approachStable clonal CHO-K1 cell lines, transfected with either the human beta(1), beta(2) or beta(3)-adrenoceptor, were used, and whole-cell [(3)H]-CGP 12177 radioligand binding and [(3)H]-cAMP accumulation were measured.Key resultsSeveral agonists were found to be highly subtype selective because of selective affinity (e.g. salmeterol and formoterol, for the beta(2)-adrenoceptor over the beta(1) or beta(3)), while others (e.g. isoprenaline) had little affinity-selectivity. However, the intrinsic efficacy of salmeterol, formoterol and isoprenaline was similar across all three receptor subtypes. Other ligands (e.g. denopamine for beta(1); clenbuterol, AZ 40140d, salbutamol for beta(2)) were found to have subtype-selective intrinsic efficacy. Several ligands appeared to activate two agonist conformations of the beta(1)- and beta(3)-adrenoceptors.Conclusions and implicationsThere are agonists with subtype selectivity based upon both selective affinity and selective intrinsic efficacy. Therefore, there is scope to develop better selective agonists based upon both selective affinity and selective intrinsic efficacy.
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Project description:A fundamental question regarding receptor-G protein interaction is whether different agonists can lead a receptor to different intracellular signaling pathways. Our previous studies have demonstrated that although most beta(2)-adrenoceptor agonists activate both G(s) and G(i) proteins, fenoterol, a full agonist of beta(2)-adrenoceptor, selectively activates G(s) protein. Fenoterol contains two chiral centers and may exist as four stereoisomers. We have synthesized a series of stereoisomers of fenoterol and its derivatives and characterized their receptor binding and pharmacological properties. We tested the hypothesis that the stereochemistry of an agonist determines selectivity of receptor coupling to different G protein(s). We found that the R,R isomers of fenoterol and methoxyfenoterol exhibited more potent effects to increase cardiomyocyte contraction than their S,R isomers. It is noteworthy that although (R,R)-fenoterol and (R,R)-methoxyfenoterol preferentially activate G(s) signaling, their S,R isomers were able to activate both G(s) and G(i) proteins as evidenced by the robust pertussis toxin sensitivities of their effects on cardiomyocyte contraction and on phosphorylation of extracellular signal-regulated kinase 1/2. The differential G protein selectivities of the fenoterol stereoisomers were further confirmed by photoaffinity labeling studies on G(s),G(i2), and G(i3) proteins. The inefficient G(i) signaling with the R,R isomers is not caused by the inability of the R,R isomers to trigger the protein kinase A (PKA)-mediated phosphorylation of the beta(2)-adrenoceptor, because the R,R isomers also markedly increased phosphorylation of the receptor at serine 262 by PKA. We conclude that in addition to receptor subtype and phosphorylation status, the stereochemistry of a given agonist plays an important role in determining receptor-G protein selectivity and downstream signaling events.
| S-EPMC2654765 | biostudies-literature
Project description:BackgroundSystemic administration of beta-adrenoceptor (beta-AR) agonists has been found to induce skeletal muscle hypertrophy and significant metabolic changes. In the context of energy homeostasis, the importance of beta-AR signaling has been highlighted by the inability of beta(1-3)-AR-deficient mice to regulate energy expenditure and susceptibility to diet induced obesity. However, the molecular pathways and gene expression changes that initiate and maintain these phenotypic modulations are poorly understood. Therefore, the aim of this study was to identify differential changes in gene expression in murine skeletal muscle associated with systemic (acute and chronic) administration of the beta(2)-AR agonist formoterol.ResultsSkeletal muscle gene expression (from murine tibialis anterior) was profiled at both 1 and 4 hours following systemic administration of the beta(2)-AR agonist formoterol, using Illumina 46K mouse BeadArrays. Illumina expression profiling revealed significant expression changes in genes associated with skeletal muscle hypertrophy, myoblast differentiation, metabolism, circadian rhythm, transcription, histones, and oxidative stress. Differentially expressed genes relevant to the regulation of muscle mass and metabolism (in the context of the hypertrophic phenotype) were further validated by quantitative RT-PCR to examine gene expression in response to both acute (1-24 h) and chronic administration (1-28 days) of formoterol at multiple timepoints. In terms of skeletal muscle hypertrophy, attenuation of myostatin signaling (including differential expression of myostatin, activin receptor IIB, phospho-Smad3 etc) was observed following acute and chronic administration of formoterol. Acute (but not chronic) administration of formoterol also significantly induced the expression of genes involved in oxidative metabolism, including hexokinase 2, sorbin and SH3 domain containing 1, and uncoupling protein 3. Interestingly, formoterol administration also appeared to influence some genes associated with the peripheral regulation of circadian rhythm (including nuclear factor interleukin 3 regulated, D site albumin promoter binding protein, and cryptochrome 2).ConclusionThis is the first study to utilize gene expression profiling to examine global gene expression in response to acute beta(2)-AR agonist treatment of skeletal muscle. In summary, systemic administration of a beta(2)-AR agonist had a profound effect on global gene expression in skeletal muscle. In terms of hypertrophy, beta(2)-AR agonist treatment altered the expression of several genes associated with myostatin signaling, a previously unreported effect of beta-AR signaling in skeletal muscle. This study also demonstrates a beta(2)-AR agonist regulation of circadian rhythm genes, indicating crosstalk between beta-AR signaling and circadian cycling in skeletal muscle. Gene expression alterations discovered in this study provides insight into many of the underlying changes in gene expression that mediate beta-AR induced skeletal muscle hypertrophy and altered metabolism.
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Project description:alpha(1)-Adrenoceptors are concentrated in the locus coeruleus (LC) where they appear to regulate various active behaviors but have been difficult to stimulate effectively. The present study examined the behavioral, pharmacological and neural effects of possible stimulation of these receptors with 6-fluoronorepinephrine (6FNE), the only known selective alpha-agonist that has full efficacy at all brain alpha-receptors. Infusion of this compound in the mouse LC was found to produce extreme activation of diverse motivated behaviors of exploration, wheel-running and operant approach responding in different environments consistent with a global behavioral function of the dorsal noradrenergic system. Infusion of selective antagonists of alpha(1)- (terazosin) or alpha(2)- (atipamezole) receptors or of either the partial alpha(1)-agonist, phenylephrine, or full alpha(2)-agonist, dexmedetomidine, indicated that the behavioral effects of 6FNE were due largely due to activation of LC alpha(1)-receptors consistent with the known greater density of alpha(1)- than alpha(2)-adrenoreceptors in the mouse nucleus. Immunohistochemistry of fos in tyrosine hydroxylase-positive LC neurons following IV ventricular infusions indicated that 6FNE markedly depressed whereas terazosin strongly enhanced the apparent functional activity of the nucleus. The changes in fos expression following 6FNE and terazosin were significantly greater than those following dexmedetomidine and atipamezole. It is hypothesized that the alpha(1)-receptors of the mouse LC are strongly activated by 6FNE and serve to potently inhibit its tonic or stress-induced activity which in turn disinhibits prepotent motivated behaviors.
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Project description:Skeletal myopathy is a hallmark of heart failure (HF) and has been associated with a poor prognosis. HF and other chronic degenerative diseases share a common feature of a stressed system: sympathetic hyperactivity. Although beneficial acutely, chronic sympathetic hyperactivity is one of the main triggers of skeletal myopathy in HF. Considering that ?2 -adrenoceptors mediate the activity of sympathetic nervous system in skeletal muscle, we presently evaluated the contribution of ?2 -adrenoceptors for the morphofunctional alterations in skeletal muscle and also for exercise intolerance induced by HF. Male WT and ?2 -adrenoceptor knockout mice on a FVB genetic background (?2 KO) were submitted to myocardial infarction (MI) or SHAM surgery. Ninety days after MI both WT and ?2 KO mice presented to cardiac dysfunction and remodelling accompanied by significantly increased norepinephrine and epinephrine plasma levels, exercise intolerance, changes towards more glycolytic fibres and vascular rarefaction in plantaris muscle. However, ?2 KO MI mice displayed more pronounced exercise intolerance and skeletal myopathy when compared to WT MI mice. Skeletal muscle atrophy of infarcted ?2 KO mice was paralleled by reduced levels of phosphorylated Akt at Ser 473 while increased levels of proteins related with the ubiquitin--proteasome system, and increased 26S proteasome activity. Taken together, our results suggest that lack of ?2 -adrenoceptors worsen and/or anticipate the skeletal myopathy observed in HF.
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Project description:1. The actions of the beta-adrenoceptor agonist isoprenaline on glucose and glycogen metabolism, in the presence of various concentrations of insulin, were investigated in isolated soleus muscle preparations taken from eu-, hyper- and hypothyroid rats. 2. Hyperthyroidism, induced by 3,3',5-tri-iodo-D-thyronine (T3) administration for 5 days, increased the rate of lactate formation and suppressed the rate of glycogen synthesis in soleus muscle in response to isoprenaline, even in the presence of physiological or supraphysiological insulin concentrations. 3. Hypothyroidism, induced by administration of 6-n-propyl-2-thiouracil for 4 weeks, decreased the rate of isoprenaline-stimulated lactate formation at all insulin concentrations, but significantly decreased the responsiveness of lactate formation only at low insulin concentrations. In the presence of 100 or 10,000 mu-units of insulin/ml, the ability of isoprenaline to suppress the rate of glycogen synthesis was markedly impaired (inhibition at 100 mu-units of insulin/ml and 1 micro-M-isoprenaline: eu- 72.6 +/- 2.9%; hypo-41.0 +/- 2.1%; P less than 0.001). 4. Hyperthyroidism had no effect on the number or affinity of beta-adrenoceptors, defined by 125I-pindolol binding, or beta-adrenoceptor- or forskolin-stimulated adenylate cyclase activity in membrane preparations of gastrocnemius muscle, whereas hypothyroidism increased the beta-adrenoceptor density and decreased the beta-adrenoceptor-stimulated adenylate cyclase activity, without affecting the receptor affinity or forskolin-stimulated adenylate cyclase activity. 5. It is concluded that there is a complex interplay between insulin, catecholamines and thyroid hormones to regulate skeletal-muscle glucose metabolism. The changes observed in muscles in hypothyroidism may be explained, at least in part, by changes in beta-adrenoceptor-G-protein-adenylate cyclase coupling affecting the generation of cyclic AMP and the regulation of some of the key enzymes of glycogen metabolism; in contrast, the changes observed in muscles in hyperthyroidism do not appear to result from alterations at the level of the receptor-mediated second-messenger generation.
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Project description:Pumps are critical life-sustaining components for all animals. At the earliest stages of life, the tubular embryonic heart works as a valveless pump capable of generating unidirectional blood flow. Inspired by this elementary pump, we developed an example of a biohybrid valveless pump-bot powered by engineered skeletal muscle. Our pump-bot consists of a soft hydrogel tube connected at both ends to a stiffer polydimethylsiloxane (PDMS) scaffold, creating an impedance mismatch. A contractile muscle ring wraps around the hydrogel tube at an off-center location, squeezing the tube with or without buckling it locally. Cyclic muscle contractions, spontaneous or electrically stimulated, further squeeze the tube, resulting in elastic waves that propagate along the soft tube and get reflected back at the soft/stiff tube boundaries. Asymmetric placement of muscle ring results in a time delay between the wave arrivals, thus establishing a net unidirectional fluid flow irrespective of whether the tube is buckled or not. Flow rates of up to 22.5 μL/min are achieved by the present pump-bot, which are at least three orders of magnitude higher than those from cardiomyocyte-powered valve pumps of similar size. Owning to its simple geometry, robustness, ease of fabrication, and high pumping performance, our pump-bot is particularly well-suited for a wide range of biomedical applications in microfluidics, drug delivery, biomedical devices, cardiovascular pumping system, and more.
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Project description:In brown adipocytes, fundamental cellular processes (cell proliferation, differentiation and apoptosis) are regulated by adrenergic stimulation, notably through beta-adrenergic receptors. The presence of all three beta-receptor subtypes has been demonstrated in brown adipose tissue. Due to the significance of the action of these receptors and indications that the subtypes govern different processes, the adrenergic regulation of the expression of the beta(1)-(,) beta(2)- and beta(3)-adrenoceptor genes was examined in murine brown-fat primary cell cultures. Moderate levels of beta(1)-receptor mRNA, absence of beta(2)-receptor mRNA and high levels of beta(3)-receptor mRNA were observed in mature brown adipocytes (day 6 in culture). Noradrenaline (norepinephrine) addition led to diametrically opposite effects on beta(1)- (markedly enhanced expression) and beta(3)-gene expression (full cessation of expression, as previously shown). beta(2)-Gene expression was induced by noradrenaline, but only transiently (<1 h). The apparent affinities (EC(50)) of noradrenaline were clearly different (7 nM for the beta(1)-gene and</=1 nM for the beta(3)-gene), as were the mediation pathways (solely via beta(3)-receptors and cAMP for the beta(1)-gene and via beta(3)-receptors and cAMP, as well as via alpha(1)-receptors and protein kinase C, for the beta(3)-gene). The half-lives of the corresponding mRNA species were very short but different (17 min for beta(1)-mRNA and 27 min for beta(3)-mRNA), and these degradation rates were not affected by noradrenaline, implying that the mRNA levels were controlled by transcription. Inhibition of protein synthesis also led to diametrically opposite effects on beta(1)- and beta(3)-gene expression, but - notably - these effects were congruent with the noradrenaline effects, implying that a common factor regulating beta(1)-gene expression negatively and beta(3)-gene expression positively could be envisaged. In conclusion, very divergent effects of adrenergic stimulation on the expression of the different beta-receptor genes were found within one cell type, and no unifying concept of adrenergic control of beta-receptor gene expression can be formulated, either concerning different cell types, or concerning the different beta-receptor subtype genes.
| S-EPMC1220999 | biostudies-other
Project description:1 Fluorescence techniques offer a way to circumvent several problems associated with many radioligand binding and functional assays and the need for large numbers of cells. Fluorescent ligands also offer the advantage of allowing real time direct visualisation of ligand - receptors interactions. A fluorescent analogue of CGP 12177 (BODIPY-TMR-CGP) has thus been evaluated as a beta(2)-adrenoceptor ligand in CHO-K1 cells expressing the human beta(2)-adrenoceptor. 2 Studies of (3)H-cAMP accumulation showed that BODIPY-TMR-CGP stimulated an increase in cAMP accumulation and cyclic AMP response element (CRE)-mediated gene transcription with an EC(50) of 21-28 nM. Both of these responses were antagonised by the selective beta(2)-adrenoceptor antagonist ICI 118551. 3 Binding studies with (3)H-CGP 12177, and functional studies of CRE-regulated gene transcription showed that the BODIPY-TMR-CGP interaction with the human beta(2)-adrenoceptor is of very long duration. 4 Visualisation of the binding of BODIPY-TMR-CGP to single living mammalian cells was clearly demonstrated by confocal microscopy and showed that this ligand was able to selectively label cell surface beta(2)-adrenoceptors in living CHO-K1 cells transfected with the human beta(2)-adrenoceptor with an apparent K(D) of 27 nM. Studies with cells expressing a beta(2)-adrenoceptor-green fluorescent protein (GFP) fusion protein provided further strong evidence that BODIPY-TMR-CGP was binding to the beta(2)-adrenoceptor. 5 BODIPY-TMR-CGP is therefore a long-acting fluorescent beta(2)-adrenoceptor agonist that can be used to label beta(2)-adrenoceptors in the plasma membrane of living cells.
| S-EPMC1573863 | biostudies-other
Project description:BackgroundUsing the streptozotocin-induced diabetic rat model, we have recently showed that the expression and function of beta1-adrenoreceptor were decreased in the diabetic rat heart. However, the effect of diabetes on expression of beta-adrenoreceptors in human cardiac tissue remains undefined. Therefore, the focus of the present study was to investigate the effect of diabetes on mRNA encoding beta1- and beta2-ARs in human atrial tissues.MethodsRight atrial appendages from five diabetic (mean age 65 +/- 4.5; 4 female, 1 male) and five nondiabetic patients (mean age 56.2 +/- 2.8; 4 male, 1 female) undergoing coronary artery bypass grafting were collected and assayed using reverse transcriptase-polymerase chain reaction (RT-PCR) for their mRNA content. No patient from these two groups suffered from acute myocardial infarction and/or failure. All diabetic patients received insulin for at least two years and had been diagnosed as diabetics for at least five years.ResultsWhen compared with levels in nondiabetics, steady state levels of mRNA encoding beta1-adrenoreceptor decreased by 69.2 +/- 7.6% in diabetic patients while beta2-adrenoreceptor mRNA decreased by 32.2 +/- 5.5% (p < 0.001).ConclusionsOur findings show a decreased expression of beta1- and beta2-adrenoreceptors in human diabetic atrial appendage.
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