beta-adrenergic relaxation of rabbit tracheal smooth muscle: a receptor deficit that improves with corticosteroid administration. (1/142)

beta-Adrenergic agonists are potent relaxing agents of airway smooth muscle; however, they are often incapable of fully reversing agonist-mediated contractions. The present study was designed to quantitate the relationship between beta-adrenoceptor binding, signal transduction, and relaxation in rabbit tracheal smooth muscle (TSM). TSM segments contracted with acetylcholine to 25 to 75% maximal contraction were relaxed with cumulative administration of isoproterenol (ISO). A beta-adrenergic receptor "deficit" was found, such that incomplete relaxation was achieved with full receptor occupancy. Binding studies with [(3)H]dihydroalprenolol demonstrated a beta-adrenoceptor density of 33.1 +/- 8.6 fmol/mg protein in control TSM. Paired studies were performed in TSM from rabbits treated with dexamethasone. Relative to control tissues, dexamethasone-treated TSM displayed twice as much relaxation and cAMP production in response to ISO and twice the beta-adrenoceptor density (82.2 +/- 12.3 fmol/mg protein). Dexamethasone did not affect G(i) function, as assessed by the degree of functional antagonism exerted by acetylcholine on ISO-induced relaxations, or beta-adrenoceptor-G(s) coupling, as reflected in high-affinity beta-agonist binding. Collectively, these results demonstrate that corticosteroid administration exerts parallel potentiating effects on beta-adrenoceptor expression and function in rabbit airway smooth muscle.  (+info)

Rapid electrical stimulation of contraction reduces the density of beta-adrenergic receptors and responsiveness of cultured neonatal rat cardiomyocytes. Possible involvement of microtubule disassembly secondary to mechanical stress. (2/142)

BACKGROUND: Although tachycardia is commonly present in patients with congestive heart failure, its role in the development of congestive heart failure remains unclear. We studied the effect of rapid electrical stimulation of contraction on beta-adrenergic receptor (beta-AR) signal pathway in cultured cardiomyocytes of neonatal rats. METHODS AND RESULTS: Contraction of cardiomyocytes was induced by electrical stimulation at 50 V with twice the threshold pulse width. beta-ARs were identified by [(3)H]CGP-12177 and [(3)H]dihydroalprenolol. Electrical stimulation reduced cell-surface but not total beta-AR density; the effect was dependent on pacing frequency (a reduction of 11%, 28%, and 18% in cells paced at 2.5, 3. 0, and 3.3 Hz, respectively). This reduction was apparent at 3 hours, in contrast to reduced beta-AR density after exposure to isoproterenol (ISP) for 1 hour. The fraction and inhibition constant of beta-AR binding agonist with high affinity were not affected by rapid electrical stimulation. In cardiomyocytes paced at 3.0 Hz for 24 hours, the response to ISP decreased compared with unpaced cells, 142% versus 204% of baseline with 1 micromol/L ISP, whereas the responses to forskolin or acetylcholine were not different. Treatment of cardiomyocytes with 2,3-butanedione monoxime (10 mmol/L) or taxol (10 micromol/L) inhibited the rapid pacing-induced reduction in beta-AR density. CONCLUSIONS: Our results suggest that contractile activity is involved in regulation of cardiac function by modulating the beta-AR system independently of hemodynamic and neurohormonal factors. This may help to elucidate the role of mechanical stress in the development of heart failure.  (+info)

Ligand regulation of green fluorescent protein-tagged forms of the human beta(1)- and beta(2)-adrenoceptors; comparisons with the unmodified receptors. (3/142)

Stable clones of HEK293 cells expressing either FLAG(TM) epitope-tagged, wild type human beta(1)- and beta(2)-adrenoceptors or C-terminally green fluorescent protein (GFP)-tagged forms of these receptors were established. The binding affinity of [(3)H]-dihydroalprenolol and other ligands was little affected by addition of GFP to the C-terminal of either receptor. Isoprenaline induced the internalisation of both beta(1)-adrenoceptor-GFP and beta(2)-adrenoceptor-GFP and following removal of the agonist both constructs were able to recycle to the cell surface. The extent of internalisation of beta(2)-adrenoceptor-GFP produced by isoprenaline was substantially greater than for beta(1)-adrenoceptor-GFP. C-terminal addition of GFP slowed markedly the rate of internalization of both the beta(1)-adrenoceptor and the beta(2)-adrenoceptor in response to isoprenaline. Sustained exposure to isoprenaline (24 h) produced substantially greater levels of downregulation of native beta(2)-adrenoceptor compared to beta(2)-adrenoceptor-GFP although both were equally effectively removed from the plasma membrane. Sustained exposure to isoprenaline resulted in a large fraction of beta(2)-adrenoceptor-GFP becoming trapped in internal vesicles/lysosomes but not degraded. Even after sustained exposure to isoprenaline a significant fraction of beta(1)-adrenoceptor-GFP remained at the cell surface. These results indicate that although GFP tagging of beta-adrenoceptors can provide qualitative visual patterns of agonist-induced receptor trafficking and regulation in HEK293 cells the quantitative details vary markedly from those obtained with the unmodified receptors.  (+info)

Monovalent anions differentially modulate coupling of the beta2-adrenoceptor to G(s)alpha splice variants. (4/142)

The beta2-adrenoceptor (beta2AR) fused to the long splice variant of G(s)alpha (G(s)alphaL), but not the beta2AR fused to the short splice variant of G(s)alpha (G(s)alphaS) shows the hallmarks of high constitutive activity, i.e., strong activation of adenylyl cyclase (AC) by GTP and strong inhibition of AC by inverse agonist. These coupling differences are the result of differences in GDP affinity of G(s)alpha splice variants. The aim of this study was to identify experimental variables that differentially affect beta2AR coupling to G(s)alphaS and G(s)alphaL. NaCl substantially reduced agonist-independent AC activation by GTP and inverse agonist inhibition and enhanced agonist stimulation of AC in Sf9 insect cell membranes expressing the beta2AR-G(s)alphaL fusion protein. Salts reduced inverse agonist inhibition and increased agonist stimulation of AC in the order of efficiency NaI approximately KI > NaBr approximately KBr > NaCl approximately LiCl approximately KCl approximately RbCl approximately CsCl approximately choline chloride, indicating that monovalent anions determine salt effects. Salts inhibited guanosine 5'-O-(3-thiotriphosphate)-mediated AC activation by G(salphaL) without beta2AR in the order of efficiency NaI > NaBr > NaCl. NaCl enhanced the affinity of G(s)alphaL for GDP. Salts had much smaller effects on beta2AR ligand regulation of AC in membranes expressing beta2AR-G(s)alphaS than in membranes expressing beta2AR-G(s)alphaL. These data are explained by a model in which anions increase the GDP affinity of G(s)alphaL more efficiently than the GDP affinity of G(s)alphaS, and, thereby, decrease the efficiency of the agonist-free beta2AR and increase the efficiency of the agonist-occupied beta2AR at promoting GDP dissociation from G(s)alphaL. Thus, monovalent anions differentially regulate beta2AR-coupling to G(s)alphaS and G(s)alphaL.  (+info)

A possible role of decreased relaxation mediated by beta-adrenoceptors in bladder outlet obstruction by benign prostatic hyperplasia. (5/142)

1. To explore mechanisms of urinary obstruction in benign prostatic hyperplasia (BPH), the features of contraction and relaxation in human hyperplastic and non-hyperplastic (control) prostatic tissues were investigated for beta- and alpha 1-adrenoceptors by radioligand binding and in vitro isometric tension experiments. 2. Hyperplastic and control prostatic tissues had a similar number (per mg protein) of prazosin binding sites with similar affinities. Noradrenaline (NA) induced dose-dependent contraction in both tissues. Contraction induced by either exogenous NA or transmural stimulation was inhibited by prazosin in both tissues, indicating that the same contractile mechanisms mediated by alpha 1-adrenoceptors exist in hyperplastic and control tissues. 3. The number of dihydroalprenolol (DHA) binding sites (per mg protein) was less in hyperplastic tissues than in controls, whereas the affinity to the ligand was identical in both tissues. Isoprenaline caused a marked relaxation of the tonic contraction induced by KCl in control tissues, but not in hyperplastic tissues. Propranolol enhanced exogenous NA- or transmural stimulation-induced contraction more in control tissues than in hyperplastic tissues. Both tissues, however, similarly responded to forskolin by relaxation. 4. These results indicate that decreased beta-adrenoceptor-mediated relaxation in hyperplastic prostatic tissues, which is attributable at least in part to the decreased number of beta-adrenoceptors, may play a role in the urinary obstruction of BPH in addition to mechanical compression of the urethra by the enlarged prostate.  (+info)

Co-expression of the beta2-adrenoceptor and dopamine D1-receptor with Gsalpha proteins in Sf9 insect cells: limitations in comparison with fusion proteins. (6/142)

The G-protein G(salpha) exists in three isoforms, the G(salpha) splice variants G(salphashort) (G(salphaS)) and G(salphalong) (G(salphaL)), and the G-protein G(alphaolf) that is not only involved in olfactory signaling but also in extrapyramidal motor regulation. Studies with beta(2)-adrenoceptor (beta(2)AR)-G(salpha) fusion proteins showed that G(salpha) proteins activate adenylyl cyclase (AC) in the order of efficacy G(salphaS)>G(salphaL) approximately G(alphaolf) and that G(salpha) proteins confer the hallmarks of constitutive activity to the beta(2)AR in the order of efficacy G(salphaL)>G(alphaolf)>G(salphaS). However, it is unclear whether such differences between G(salpha) proteins also exist in the nonfused state. In the present study, we co-expressed the beta(2)AR and dopamine D(1)-receptor (D(1)R) with G(salpha) proteins at different ratios in Sf9 insect cells. In agreement with the fusion protein studies, nonfused G(alphaolf) was less efficient than nonfused G(salphaS) and G(salphaL) at activating AC, but otherwise, we did not observe differences between the three G(salpha) isoforms. Thus, it is much easier to dissect differences between G(salpha) isoforms using beta(2)AR-G(salpha) fusion proteins than nonfused G(salpha) isoforms.  (+info)

Enhancement of membrane insertion and function in a type IIIb membrane protein following introduction of a cleavable signal peptide. (7/142)

The human beta 2 adrenergic receptor is a type IIIb membrane protein. It has a putative seven-transmembrane topology but lacks an amino-terminal cleavable signal sequence. The mechanism by which the amino terminus of the beta 2 receptor is translocated across the endoplasmic reticulum membrane is unknown. Furthermore, it is not known if translocation as a type IIIb protein is essential for the proper folding. Our studies indicate that conversion of beta 2 receptor from a type IIIb to a type IIIa membrane protein by introducing an NH2-terminal cleavable signal sequence enhances translocation of the receptor into the endoplasmic reticulum membrane, thereby facilitating expression of functional receptor.  (+info)

Beta-agonist-induced inhibitory-guanine-nucleotide-binding regulatory protein coupling to adenylate cyclase in mollusc Anodonta cygnea foot muscle sarcolemma. (8/142)

In the sarcolemma fraction of foot muscles of a fresh-water bivalve mollusc, Anodonta cygnea, a direct inhibitory, rather than stimulatory, effect of the beta-adrenergic agonist isoproterenol, at micromolar concentration, on cAMP level and adenylate cyclase activity, was revealed. It was blocked by beta- but not alpha-adrenergic antagonists. A single class of [3H]dihydroalprenolol-binding sites with binding properties of beta-adrenergic receptor was detected in mollusc sarcolemma. Potentiation of the inhibitory effect of isoproterenol on mollusc adenylate cyclase activity by GTP or guanosine 5'-[beta,gamma-imido]triphosphate at micromolar concentrations, and its elimination in the presence of guanosine 5'-[beta-thio]diphosphate, were shown. The pertussis-toxin-induced ADP-ribosylation of sarcolemma 40-kDa protein [immunochemically related in the C-terminal part to pertussis-toxin-sensitive guanine-nucleotide-binding regulatory protein (G-protein) alpha subunits of vertebrates], as well as the treatment of mollusc sarcolemma with antisera responsive to the C-terminus of vertebrate inhibitory G-protein (G(i)) alpha subunit led to elimination of the inhibitory effect of isoproterenol on adenylate cyclase activity. The results obtained suggest that beta-agonist-induced inhibition of adenylate cyclase in A. cygnea foot muscle may be realized via the beta-adrenoreceptor/G(i) signalling pathway.  (+info)