Differential addressing of 5-HT1A and 5-HT1B receptors in epithelial cells and neurons.
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The 5-HT1A and 5-HT1B serotonin receptors are expressed in a variety of neurons in the central nervous system. While the 5-HT1A receptor is found on somas and dendrites, the 5-HT1B receptor has been suggested to be localized predominantly on axon terminals. To study the intracellular addressing of these receptors, we have used in vitro systems including Madin-Darby canine kidney (MDCK II) epithelial cells and primary neuronal cultures. Furthermore, we have extended these studies to examine addressing in vivo in transgenic mice. In epithelial cells, 5-HT1A receptors are found on both apical and basolateral membranes while 5-HT1B receptors are found exclusively in intracellular vesicles. In hippocampal neuronal cultures, 5-HT1A receptors are expressed on somatodendritic membranes but are absent from axons. In contrast, 5-HT1B receptors are found on both dendritic and axonal membranes, including growth cones where they accumulate. Using 5-HT1A and 5-HT1B knockout mice and the binary tTA/tetO system, we generated mice expressing these receptors in striatal neurons. These in vivo experiments demonstrate that, in striatal medium spiny neurons, the 5-HT1A receptor is restricted to the somatodendritic level, while 5-HT1B receptors are shipped exclusively toward axon terminals. Therefore, in all systems we have examined, there is a differential sorting of the 5-HT1A and 5-HT1B receptors. Furthermore, we conclude that our in vivo transgenic system is the only model that reconstitutes proper sorting of these receptors. (+info)
In vitro and in vivo characterization of intrinsic sympathomimetic activity in normal and heart failure rats.
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Clinical studies conducted with carvedilol suggest that beta-adrenoceptor antagonism is an effective therapeutic approach to the treatment of heart failure. However, many beta-adrenoceptor antagonists are weak partial agonists and possess significant intrinsic sympathomimetic activity (ISA), which may be problematic in the treatment of heart failure. In the present study, the ISAs of bucindolol, xamoterol, bisoprolol, and carvedilol were evaluated and compared in normal rats [Sprague-Dawley (SD)], in rats with confirmed heart failure [spontaneously hypertensive heart failure (SHHF)], and in isolated neonatal rat cardiomyocytes. At equieffective beta1-adrenolytic doses, the administration of xamoterol and bucindolol produced a prolonged, equieffective, and dose-related increase in heart rate in both pithed SD rats (ED50 = 5 and 40 microgram/kg, respectively) and SHHF rats (ED50 = 6 and 30 microgram/kg, respectively). The maximum effect of both compounds in SHHF rats was approximately 50% of that observed in SD rats. In contrast, carvedilol and bisoprolol had no significant effect on resting heart rate in the pithed SD or SHHF rat. The maximum increase in heart rate elicited by xamoterol and bucindolol was inhibited by treatment with propranolol, carvedilol, and betaxolol (beta1-adrenoceptor antagonist) but not by ICI 118551 (beta2-adrenoceptor antagonist) in neonatal rat. When the beta-adrenoceptor-mediated cAMP response was examined in cardiomyocytes, an identical partial agonist/antagonist response profile was observed for all compounds, demonstrating a strong correlation with the in vivo results. In contrast, GTP-sensitive ligand binding and tissue adenylate cyclase activity were not sensitive methods for detecting beta-adrenoceptor partial agonist activity in the heart. In summary, xamoterol and bucindolol, but not carvedilol and bisoprolol, exhibited direct beta1-adrenoceptor-mediated ISA in normal and heart failure rats. (+info)
Targeted disruption of the beta2 adrenergic receptor gene.
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beta-Adrenergic receptors (beta-ARs) are members of the superfamily of G-protein-coupled receptors that mediate the effects of catecholamines in the sympathetic nervous system. Three distinct beta-AR subtypes have been identified (beta1-AR, beta2-AR, and beta3-AR). In order to define further the role of the different beta-AR subtypes, we have used gene targeting to inactivate selectively the beta2-AR gene in mice. Based on intercrosses of heterozygous knockout (beta2-AR +/-) mice, there is no prenatal lethality associated with this mutation. Adult knockout mice (beta2-AR -/-) appear grossly normal and are fertile. Their resting heart rate and blood pressure are normal, and they have a normal chronotropic response to the beta-AR agonist isoproterenol. The hypotensive response to isoproterenol, however, is significantly blunted compared with wild type mice. Despite this defect in vasodilation, beta2-AR -/- mice can still exercise normally and actually have a greater total exercise capacity than wild type mice. At comparable workloads, beta2-AR -/- mice had a lower respiratory exchange ratio than wild type mice suggesting a difference in energy metabolism. beta2-AR -/- mice become hypertensive during exercise and exhibit a greater hypertensive response to epinephrine compared with wild type mice. In summary, the primary physiologic consequences of the beta2-AR gene disruption are observed only during the stress of exercise and are the result of alterations in both vascular tone and energy metabolism. (+info)
L-type calcium current and contractility in ventricular myocytes from mice overexpressing the cardiac beta 2-adrenoceptor.
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OBJECTIVES: The reported increase in basal activity of hearts from transgenic mice (TG4) overexpressing the human beta 2-adrenoceptor (beta 2-AR) was explained by spontaneously active beta 2-ARs that stimulate the beta-adrenergic cascade in the absence of an agonist. In order to examine altered myocardial function on a cellular level, we have investigated L-type calcium current (ICa,L) and cell shortening in ventricular myocytes from TG4 hearts. Myocytes from littermates (LM) and wild type animals (WT) served as controls. METHODS: Cardiac beta-AR density was measured by [125I]-iodocyanopindolol binding to ventricular membranes. ICa,L was assessed by standard whole-cell voltage clamp technique. Contractility was measured as cell shortening in ventricular myocytes and as force of contraction in electrically stimulated left atria. RESULTS: Overexpression of beta 2-ARs was confirmed by an almost 400-fold increase in beta-AR density. The beta 1:beta 2-AR ratio in WT mice was 71:29. Myocytes from TG4 and LM mice were similar in size as judged by membrane capacitance and two dimensional cell area. ICa,L amplitude was significantly lower in TG4 than in LM myocytes (with 2 mM [Ca2+]o -4.82 +/- 0.48 vs. -6.56 +/- 0.38 pA/pF, respectively). In TG4 myocytes, the ICa,L response to isoproterenol (1 microM) was almost abolished. Cell shortening was not different in physiological [Ca2+]o, but smaller in maximum [Ca2+]o when comparing TG4 to control myocytes. Basal force of contraction in left atria did not differ between TG4 and LM at any age investigated. In TG4 left atria the inotropic response to isoproterenol was also absent, whereas responses to high [Ca2+]o or dibutyryl-cAMP (1 mM) were present but reduced. The rate of spontaneous beating of right atria was elevated in TG4 mice. CONCLUSIONS: Since only spontaneous beating rate but neither basal ICa,L amplitude nor basal contractile activity were elevated, our data fail to reveal evidence for spontaneously active, stimulating beta 2-ARs in left atrium and ventricle. A contractile deficit unrelated to the beta-adrenoceptor pathway is evident in TG4 myocytes and left atria. (+info)
Dihydropyridine and beta adrenergic receptor binding in dogs with tachycardia-induced atrial fibrillation.
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BACKGROUND: We have shown that rapid atrial activation, as occurs during atrial fibrillation (AF), reduces L-type Ca2+ current (ICa) and that this is the principal mechanism of the action potential duration and refractoriness changes that characterize tachycardia-induced atrial remodeling. The present study was designed to determine whether atrial tachycardia alters biochemical indices of the number of L-type Ca2+ channels and/or of the number and binding affinity of beta-adrenergic receptors. METHODS: In canine atrial sarcolemmal preparations, the number and binding affinity of dihydropyridine receptors were determined with the use of 3H-nitrendipine and that of beta-adrenergic receptors with 125I-iodocyanopindolol. Results were obtained with preparations from dogs paced at 400/min for 1 (P1, n = 20), 7 (P7, n = 9), and 42 (P42, n = 9) days, and compared with observations in sham-operated controls (P0, n = 14). RESULTS: Pacing reduced the Bmax of dihydropyridine receptors, from 157 +/- 18 fmol/mg (P0) to 116 +/- 9 fmol/mg (P1, P < 0.05), 100 +/- 14 fmol/mg (P7, P < 0.05) and 94 +/- 9 fmol/mg (P42, P < 0.01). The affinity of dihydropyridine receptors was unchanged, with the Kd averaging 711 +/- 102 pM. 656 +/- 74 pM, 633 +/- 155 pM and 585 +/- 92 pM in P0, P1, P7 and P42 dogs. Neither Bmax nor Kd of beta-adrenergic receptors was altered by rapid pacing. Values of Bmax of dihydropyridine receptors correlated with atrial ICa current density (r2 = 0.95) and ERP (r2 = 0.99). CONCLUSIONS: Rapid atrial activation results in downregulation in the number of dihydropyridine receptors without altering the number or affinity of beta-adrenergic receptors. The reductions in ICa that play an important role in the atrial electrical remodeling by which 'AF begets AF' appear to be due at least in part to a decrease in the number of L-type Ca2+ channels in cardiac cell membranes. (+info)
Heterogeneous cardiac sympathetic innervation in heart failure after myocardial infarction of rats.
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We examined cardiac neuronal function and beta-receptor with a dual-tracer method of [(131)I]meta-iodobenzylguanidine (MIBG) and [(125)I]iodocyanopindolol (ICYP) in rat heart failure after myocardial infarction (MI). In rats with MI, left ventricular (LV) systolic function decreased, and LV dimension and right ventricular (RV) mass increased gradually. MIBG accumulations of the noninfarcted LV (remote region) and RV decreased by 15% at 1 wk compared with sham-operated rats, and these accumulations were restored by 71% and 56%, respectively, at 24 wk compared with age-matched sham rats despite sustained depletion of myocardial norepinephrine contents in these regions. ICYP accumulation of the remote region and of the RV did not decrease at any stages. Myocardial MIBG distribution was heterogeneous at 1 wk when it was lower in the peri-infarcted region than in the remote region, associated with reduced ICYP accumulation in the peri-infarcted region. The heterogeneous distribution of both isotopes disappeared at 12 wk. Thus cardiac sympathetic neuronal alteration was coupled with downregulation of beta-receptors in rat heart failure after MI. The abnormal adrenergic signaling occurred heterogeneously in terms of ventricular distribution and time course after MI. (+info)
Differential effects of dexamethasone and clenbuterol on rat growth and on beta2-adrenoceptors in lung and skeletal muscle.
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Beta-adrenergic agonists increase growth rate, but their efficacy is reduced over time as the number of beta2-adrenoceptors in muscle decreases. Dexamethasone increases beta2-adrenoceptor density in many tissues, but this effect has not been reported in skeletal muscle. In this study, male rats were treated daily for 10 d with either clenbuterol (4 mg/kg of feed), dexamethasone (.2 mg/kg BW, s.c.), or clenbuterol plus dexamethasone. Untreated rats served as controls. Dexamethasone caused a marked suppression of growth rate, which resulted in decreased (P < .001) body weight (-29%), carcass weight (-30%), hind-limb muscles (-22%), omental fat (-22%), and heart weight (-10%). Feed intake was reduced (-26%), but feed conversion efficiency was also impaired (P < .001). Clenbuterol caused a small increase in growth rate (+6%; P < .05), with an increase in leg muscle (+7%; P < .01) and heart mass (+8%; P < .05). Feed efficiency was improved (P < .001) by clenbuterol. Rats given the combined treatment still showed a reduction in growth rate (-81%). Clenbuterol caused only a mild attenuation of the effects of dexamethasone on feed intake, BW, and carcass weight, but reduced the catabolic effect of dexamethasone on hind-limb muscle to only -8%. Clenbuterol caused a slight increase in the affinity beta2-adrenoceptors in lung for binding to the radioligand (-)[125I]iodocyanopindolol. Relative to control values, the density of beta2-adrenoceptors in lung was +31% with dexamethasone treatment, -45% with clenbuterol, and -23% with the combined treatment. Clenbuterol also decreased beta2-adrenoceptors in skeletal muscle (-35%), but so did dexamethasone (-13%), so the effects of the beta-adrenergic agonist were not attenuated through use of the combined treatment (-40%). The results show that the inductive effect of glucocorticoids on beta2-adrenoceptors is tissue-specific and that glucocorticoid treatment is not a useful adjunct to beta-adrenergic agonist treatment in animal production. (+info)
Different intrinsic activities of bucindolol, carvedilol and metoprolol in human failing myocardium.
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1. Clinical studies have shown different effects of beta-blockers on the beta-adrenergic system, tolerability and outcome in patients with heart failure. 2. The study examines beta-adrenoceptor-G-protein coupling and intrinsic activity of bucindolol, carvedilol and metoprolol in human ventricular myocardium. 3. Radioligand binding studies ([(125)I]-Iodocyanopindolol) were performed in membrane preparations of human failing and nonfailing myocardium. Functional experiments were carried out in isolated muscle preparations of human left ventricular myocardium from failing hearts. 4. Bucindolol and carvedilol bound non-selectively to beta(1)- and beta(2)-adrenoceptors and exerted guanine nucleotide modulatable binding. Metoprolol was 35-fold beta(1)-selective and lacked guanine nucleotide modulatable binding. 5. All beta-blockers antagonized isoprenaline-induced enhancement of contractility. 6. In preparations in which the coupling of the stimulatory G-protein to adenylate cyclase was facilitated by forskolin, bucindolol increased force of contraction in three and decreased it in five experiments. Carvedilol increased force in one and decreased it in six experiments. Metoprolol decreased force in all experiments by 89. 4+/-2.2% (P<0.01 metoprolol vs carvedilol and bucindolol). The negative inotropic effect of metoprolol was antagonized by bucindolol. 7. It is concluded that differences in intrinsic activity can be detected in human myocardium and have an impact on cardiac contractility. In human ventricular myocardium, bucindolol displays substantially higher intrinsic activity than metoprolol and carvedilol. Bucindolol can behave as partial agonist or partial inverse agonist depending on the examined tissue. 8. Differences in intrinsic activity may contribute to differences in beta-adrenoceptor regulation and possibly to differences in tolerability and outcomes of patients with heart failure. (+info)