Characterization of beta-adrenoceptor mediated smooth muscle relaxation and the detection of mRNA for beta1-, beta2- and beta3-adrenoceptors in rat ileum.
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1. Functional and molecular approaches were used to characterize the beta-AR subtypes mediating relaxation of rat ileal smooth muscle. 2. In functional studies, (-)-isoprenaline relaxation was unchanged by CGP20712A (beta1-AR antagonist) or ICI118551 (beta2-AR antagonist) but shifted by propranolol (pKB=6.69). (+/-)-Cyanopindolol, CGP12177 and ICID7114 did not cause relaxation but antagonized (-)-isoprenaline relaxation. 3. BRL37344 (beta3-AR agonist) caused biphasic relaxation. The high affinity component was shifted with low affinity by propranolol, (+/-)-cyanopindolol, tertatolol and alprenolol. CL316243 (beta3-AR agonist) relaxation was unaffected by CGP20712A or ICI118551 but blocked by SR58894A (beta3-AR antagonist; pA2 = 7.80). Enhanced relaxation after exposure to forskolin and pertussis toxin showed that beta3-AR relaxation can be altered by manipulation of components of the adenylate cyclase signalling pathway. 4. The beta-AR agonist RO363 relaxed the ileum (pEC50=6.18) and was blocked by CGP20712A. Relaxation by the beta2-AR agonist zinterol (pEC50=5.71) was blocked by SR58894A but not by ICI118551. 5. In rat ileum, beta1-, beta2- and beta3-AR mRNA was detected. Comparison of tissues showed that beta3-AR mRNA expression was greatest in WAT>colon=ileum >cerebral cortex>soleus; beta1-AR mRNA was most abundant in cerebral cortex > WAT > ileum = colon > soleus; beta2-AR mRNA was expressed in soleus > WAT > ileum = colon > cerebral cortex. 6. These results show that beta3-ARs are the predominant beta-AR subtype mediating rat ileal relaxation while beta1-ARs may produce a small relaxation. The beta2-AR agonist zinterol produces relaxation through beta3-ARs and there was no evidence for the involvement of beta2-ARs in relaxation despite the detection of beta2-AR mRNA. (+info)
Functional analysis of desensitization of the beta-adrenoceptor signalling pathway in rat cardiac tissues following chronic isoprenaline infusion.
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1. This study examined beta-adrenoceptor signalling in cardiac tissues following infusion of isoprenaline (400 microg kg(-1) h(-1)) or vehicle to rats for 14 days. 2. Isoprenaline infusion caused marked hypertrophy of atria and ventricles and reduced the resting rate of spontaneously beating right atria and the basal force of left atrial contraction. 3. In spontaneously beating right atria, concentration-response curves to isoprenaline and forskolin were shifted 7.9 and 3.2 fold to the right following treatment whereas responses to the cyclic AMP analogue 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole-3', 5'-cyclic monophosphorothioate were unchanged. 4. In electrically driven left atria, concentration-response curves to isoprenaline and forskolin were shifted 4 fold to the right and maximum responses reduced. Responses to dibutyryl cyclic AMP were shifted 3.2 fold to the right but those to Ca2+ were unchanged. 5. Inotropic responses of left and right ventricular papillary muscles to isoprenaline were abolished and markedly reduced respectively by isoprenaline treatment. Responses to forskolin were shifted 5 fold to the right. Responses to dibutyryl cyclic AMP were shifted to the right 3.2 and 2 fold in left and right ventricular papillary muscles. Responses to isobutyl methyl xanthine were shifted to the right 15.8 and 6.3 fold in left and right papillary muscles whereas those to Ca2+ were not significantly altered. 6. This study indicates differences in beta-adrenoceptor desensitization in different regions of the heart following chronic infusion of isoprenaline. Chronotropic responses showed impaired signalling between the receptor and adenylate cyclase whereas inotropic responses exhibited additional desensitization at the level of cyclic AMP dependent protein kinase. (+info)
Abnormal cardiac repolarization and impulse initiation in German shepherd dogs with inherited ventricular arrhythmias and sudden death.
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OBJECTIVE: We tested the hypothesis that delayed afterdepolarization (DAD)-associated rhythms in German shepherd dogs with reduced anteroseptal left ventricular (LV) sympathetic innervation derive from abnormal beta-adrenergic receptor effector coupling. METHODS AND RESULTS: In anteroseptal LV midmyocardium of afflicted dogs, beta-receptor density was greater than that in normal dogs (P < .05), with affinity being equal in both groups. Basal and maximum isoproterenol (ISO) stimulated adenylyl cyclase activity of anteroseptal LV of afflicted dogs was greater than that in normal dogs (P < .05). Isolated anteroseptal M cell preparations of afflicted dogs studied with microelectrodes showed abnormal lengthening, rather than shortening of action potential duration in response to ISO, as well as a 61% incidence of 10(-7) mol/l ISO-induced triggered activity as compared to 12% in normals (P < .05). In contrast, there was no difference between afflicted and control dogs in triggered activity, beta-receptors or adenylyl cyclase activity in a normally innervated region of the ventricles. CONCLUSION: In this model there is an increase in beta-receptor density and beta-adrenergic stimulation of adenylyl cyclase and of triggered activity in anteroseptal myocardium but not in a normally innervated region of the heart. Hence, abnormal beta-adrenergic signal transduction appears associated with the neural abnormality identified in dogs with inherited VT. (+info)
Modulation of the pacemaker current If by beta-adrenoceptor subtypes in ventricular myocytes isolated from hypertensive and normotensive rats.
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OBJECTIVE: Both beta 1- and beta 2-adrenoceptors (beta 1-AR and beta 2-AR) are functionally present in human and rat ventricular myocytes. The two receptor subtypes are differently regulated during the development of myocardial hypertrophy and failure. I(f) is expressed in human and rat ventricular myocytes. In hypertrophied myocytes isolated from old spontaneously hypertensive rats (SHR) the density is much larger than in age-matched normotensive Wistar Kyoto (WKY). Due to the possible relevance of I(f) as an arrhythmogenic mechanism in the rat and human ventricle, we studied and compared the effects of beta 1-AR and beta 2-AR stimulation on I(f) in both hypertrophied and normal left ventricular myocytes of 18-month old SHR and WKY. METHODS: The whole-cell configuration of the patch-clamp technique was employed. Noradrenaline (NA, 1 microM) was used to stimulate beta 1-AR and isoprenaline (ISO, 1 microM) in the presence of the beta 1-AR antagonist CGP 20712A (0.1 microM) to stimulate beta 2-AR. RESULTS: In SHR, NA increased I(f) by causing a 10.8 +/- 0.9 mV (n = 10) positive shift in the voltage of maximal activation (V1/2); this effect was completely reversed by CGP 20712A. beta 2-AR stimulation was effective in seven out of 13 cells tested, where it caused a small positive shift in V1/2 (4.0 +/- 1.7 mV). Cyclopentyladenosine (CPA), a selective A1-receptor agonist, reversed the effect of NA; the antiadrenergic action of CPA was abolished in cells pre-incubated with pertussis toxin (PTX) to block inhibitory G proteins (Gi). In PTX-treated cells the shift in V1/2 caused by both beta 2-AR (9.6 +/- 1.7 mV, n = 6, p < 0.05) and beta 1-AR (17.6 +/- 1.9 mV, n =7, p < 0.05) was significantly greater than in control cells. Both beta-AR subtypes modulated I(f) activation also in WKY: beta 1-AR shifted V1/2 by 16.0 +/- 1.4 mV (n = 15) and beta 2-AR by 4.2 +/- 1.1 mV (n = 7). However, in PTX-treated WKY cells only the beta 2-AR effect was potentiated (shift in V1/2: 11.4 +/- 1.4 mV, n = 9, p < 0.01), while the beta 1-AR response was unchanged (18.9 +/- 4.2 mV, n = 5, n.s.). CONCLUSIONS: I(f) expressed in SHR hypertrophied ventricular myocytes is modulated by catecholamines mainly through the stimulation of the beta 1-AR subtype. The beta 1-AR response is, however, significantly lower than that observed in myocytes from normotensive rats, probably as a consequence of the presence of an increased inhibitory activity of Gi proteins. This post-receptorial control may be seen as a mechanism to limit the arrhythmogenicity of beta-AR stimulation in myocardial hypertrophy and failure. (+info)
No functional beta 3-adrenergic receptors expressed in rat skeletal muscle cells.
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AIM: To determine the functional role of beta 3-adrenoceptors (beta 3-AR) in rat skeletal muscle cells. METHODS: Nonselective beta-AR agonist isoprenaline (isoproterenol, Iso), beta 3-AR agonist CGP12177A which is a beta 1-/beta 2-AR antagonist and selective beta 3-AR antagonist SR59230A on cAMP accumulation was studied in primary cultured rat skeletal muscle cells. RESULTS: Iso stimulated cAMP accumulation in a concentration-dependent manner with EC50 of 1.51 nmol.L-1 and propranolol inhibited cAMP accumulation stimulated by Iso with KB of 3.47 nmol.L-1. CGP12177A had no effect on cAMP accumulation but inhibited cAMP production induced by Iso. SR59230A 10 nmol.L-1 did not inhibit cAMP production induced by Iso. CONCLUSION: The functional beta 3-AR are not present or at least not coupled to adenylyl cyclase activity in skeletal muscle cells. (+info)
In vivo inhibition of elevated myocardial beta-adrenergic receptor kinase activity in hybrid transgenic mice restores normal beta-adrenergic signaling and function.
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BACKGROUND: The clinical syndrome of heart failure (HF) is characterized by an impaired cardiac beta-adrenergic receptor (betaAR) system, which is critical in the regulation of myocardial function. Expression of the betaAR kinase (betaARK1), which phosphorylates and uncouples betaARs, is elevated in human HF; this likely contributes to the abnormal betaAR responsiveness that occurs with beta-agonist administration. We previously showed that transgenic mice with increased myocardial betaARK1 expression had impaired cardiac function in vivo and that inhibiting endogenous betaARK1 activity in the heart led to enhanced myocardial function. METHODS AND RESULTS: We created hybrid transgenic mice with cardiac-specific concomitant overexpression of both betaARK1 and an inhibitor of betaARK1 activity to study the feasibility and functional consequences of the inhibition of elevated betaARK1 activity similar to that present in human HF. Transgenic mice with myocardial overexpression of betaARK1 (3 to 5-fold) have a blunted in vivo contractile response to isoproterenol when compared with non-transgenic control mice. In the hybrid transgenic mice, although myocardial betaARK1 levels remained elevated due to transgene expression, in vitro betaARK1 activity returned to control levels and the percentage of betaARs in the high-affinity state increased to normal wild-type levels. Furthermore, the in vivo left ventricular contractile response to betaAR stimulation was restored to normal in the hybrid double-transgenic mice. CONCLUSIONS: Novel hybrid transgenic mice can be created with concomitant cardiac-specific overexpression of 2 independent transgenes with opposing actions. Elevated myocardial betaARK1 in transgenic mouse hearts (to levels seen in human HF) can be inhibited in vivo by a peptide that can prevent agonist-stimulated desensitization of cardiac betaARs. This may represent a novel strategy to improve myocardial function in the setting of compromised heart function. (+info)
Differentiation-dependent inhibition of proteolysis by norepinephrine in brown adipocytes.
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The objective was to evaluate whether norepinephrine (NE) and other hormonal factors have direct effects on protein degradation in brown fat cells. NE inhibited proteolysis by 35-45% in mouse brown adipocytes differentiated in culture. Insulin also inhibited protein degradation but significantly less than NE, whereas glucagon and leptin had no effect. The inhibitory effect of NE was partially antagonized by propranolol but not by prazosin, and dose-response curves with BRL-37344 (a beta(3)-agonist), isoproterenol (a beta(1)/beta(2)-agonist) and dobutamide (a beta(1)-agonist) were consistent with the involvement of a beta(3)-adrenergic receptor. Furthermore, forskolin mimicked the effects of NE, whereas additions of A-23187 or phorbol esters had no effect, alone or in combination with NE or forskolin. Thus inhibition of proteolysis by NE likely involves a beta(3)-adrenergic receptor-mediated increase in cAMP. In contrast, NE, BRL-37344, and dobutamide had no effect on proteolysis in preadipocytes. Inhibition of proteolysis by NE was due at least in part to inhibition of autophagy. Thus inhibition of proteolysis by NE and insulin in mature brown adipocytes is likely an important process contributing to brown fat growth and atrophy under many physiological or pathological conditions. (+info)
A unique mechanism of desensitization to lipolysis mediated by beta(3)-adrenoceptor in rats with thermal injury.
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Thermal injury causes a hypermetabolic state associated with increased levels of catabolic hormones, but the molecular bases for the metabolic abnormalities are poorly understood. We investigated the lipolytic responses after beta(3)-adrenoceptor (beta(3)-AR) agonists and evaluated the associated changes in beta-AR and its downstream signaling molecules in adipocytes isolated from rats with thermal injury. Maximal lipolytic responses to a specific beta(3)-AR agonist, BRL-37344, were significantly attenuated at post burn days (PBD) 3 and 7. Despite significant reduction of the cell surface beta(3)-AR number and its mRNA at PBD 3 and 7, BRL-37344 and forskolin-stimulated cAMP levels were not decreased. Glycerol production in response to dibutyryl cAMP, a direct stimulant of hormone-sensitive lipase (HSL) via protein kinase A (PKA), was significantly attenuated. Although immunoblot analysis indicated no differences in the expression and activity of PKA or in the expression of HSL, HSL activity showed significant reductions. Finally, beta(3)-AR-induced insulin secretion was indeed attenuated in vivo. These studies indicate that the beta(3)-AR system is desensitized after burns, both in the adipocytes and in beta(3)-AR-induced secretion of insulin. Furthermore, these data suggest a complex and unique mechanism underlying the altered signaling of lipolysis at the level of HSL in animals after burns. (+info)