Adenosine Modulatesa alpha(2)-Adrenergic Receptors within Specific Subnuclei of the Nucleus Tractus Solitarius in Normotensive and Spontaneously Hypertensive Rats

Adenosine Is known to modulate neuronal activity within the nucleus tractus solitarius (NTS). The modulatory effect of adenosine A, receptors (A(1R)) on alpha(2)-adrenoceptors (Adr(2R)) was evaluated using quantitative radioautography within NTS subnuclei and using neuronal culture of normotensive (WKY) and spontaneously hypertensive rats (SHR). Radioautography was used in a saturation experiment to measure Adr2R binding parameters (B(max), K(d)) In the presence of 3 different concentrations of N(6)-cyclopentyladenosine (CPA), an A(1R) agonist. Neuronal culture confirmed our radioautographic results. [(3)H]RX821002, an Adr(2R) antagonist, was used as a ligand for both approaches. The dorsomedial/dorsolateral subnucleus of WKY showed an increase in B(max) values (21%) Induced by 10 nmol/L of CPA. However, the subpostremal subnucleus showed a decrease in Kd values (24%) induced by 10 nmol/L of CPA. SHR showed the same pattern of changes as WKY within the same subnuclei; however, the modulatory effect of CPA was induced by I nmol/L (increased B(max), 17%; decreased K(d), 26%). Cell culture confirmed these results, because 10(-5) and 10(-7) mol/L of CPA promoted an Increase in [3H]RX821002 binding of WKY (53%) and SHR cells (48%), respectively. DPCPX, an AIR antagonist, was used to block the modulatory effect promoted by CPA with respect to Adr2R binding. In conclusion, our study shows for the first time an interaction between A(1R) that increases the binding of Adr2R within specific subnuclei of the NTS. This may be important In understanding the complex autonomic response induced by adenosine within the NTS. In addition, changes in interactions between receptors might be relevant to understanding the development of hypertension. (Hypertens Res 2008; 31: 2177-2186)

Moxonidine and central alpha(2) adrenergic receptors in sodium intake

Central injections of the alpha(2) adrenergic/imidazoline receptor agonist moxonidine inhibit water and NaCl intake in rats. In the present study, we investigated the possible involvement of central alpha(2) adrenergic receptors on the inhibitory effect of moxonidine in 0.3 M NaCl intake induced by 24 h sodium depletion. Male Holtzman rats with stainless-steel cannulas implanted into the lateral ventricle (LV) were used. Sodium depletion was produced by the treatment with the diuretic furosemide (20 mg/kg of body weight) injected subcutaneously + 24 h of sodium-deficient diet. Intracerebroventricular (icv) injections of moxonidine (20 nmol/l mul) reduced sodium depletion-induced 0.3 M NaCl intake (6.6 +/- 1.9 ml/120 min vs. vehicle: 12.7 +/- 1.7 ml/120 min). Pre-treatment with the alpha(2) adrenoreceptor antagonists RX 821002 (80 nmol/l mul), SK&F 86466 (640 nmol/l mul) and yohimbine (320 nmol/3 mul) injected icv abolished the inhibitory effect of icv moxonidine on sodium depletion-induced 0.3 M NaCl intake (13.3 +/- 1.4, 15.7 +/- 1.7 and 11.8 +/- 2.2 ml/120 min, respectively). The results show that the activation of alpha(2) adrenoreceptors is essential for the inhibitory effect of central moxonidine on sodium depletion-induced NaCl intake. (C) 2003 Elsevier B.V. All rights reserved.

Denervation supersensitivity to phenylephrine in guinea-pig vas deferens in vivo and in vitro - Functional studies on alpha(1)-adrenoceptors

The objective was to estimate alterations in adrenergic receptor sites of guinea pig vas deferens, in vivo and in vitro, induced by chronic denervation. The denervation process induced an increased sensitivity (3-fold at the EC50 level) without alteration in the maximum response to phenylephrine in vitro. The sensitivity alteration was characterized by the decrease in the dissociation constant of phenylephrine for alpha-adrenoceptor [K-A: normal tissue 3.50 (0.75-16.21) x 10(-5) and denervated tissue 0.43 (0.11-1.67) x 10(-5) M, p < 0.05] without changing the dissociation constant of prazosin. A decrease in pD(2)' value for phenylephrine-phenoxybenzamine, probably due to a qualitative rather than a quantitative alteration in the alpha-adrenoceptor, was also shown in vitro [pD(2)': normal tissue (8.2776 +/- 0.0402) and denervated tissue (8.0051 +/- 0.0442), p < 0.05]. No change in sensitivity and maximum response to phenylephrine was observed in vivo after denervation, although an increased resistance of vas deferens to phenoxybenzamine blockade has been evidenced in this condition. (C) 1999 Elsevier B.V. All rights reserved.

Quantitative mRNA analysis of adrenergic receptor subtypes in the intestines of healthy dairy cows and dairy cows with cecal dilatation-dislocation

OBJECTIVE: To investigate the distribution of mRNA coding for 9 adrenoceptor subtypes in the intestines of healthy dairy cows and cows with cecal dilatationdislocation (CDD). SAMPLE POPULATION: Full-thickness specimens of the intestinal wall were obtained from the ileum, cecum, proximal loop of the ascending colon (PLAC), and external loop of the spiral colon (ELSC) of 15 cows with CDD (group 1) and 15 healthy (control) cows (group 2, specimens collected during laparotomy; group 3, specimens collected after slaughter). PROCEDURES: Concentrations of mRNA for 9 adrenoceptor subtypes (alpha(1A), alpha(1B), alpha(1D), alpha(2AD), alpha(2B), alpha(2C), beta(1), beta(2), and beta(3)) were measured by quantitative real-time reverse transcriptase-PCR assay. Results were expressed relative to mRNA expression of a housekeeping gene. RESULTS: Expression of mRNA for alpha(1B)-, alpha(2AD)-, alpha(2B)-, beta(1)-, and beta(2)-adrenoceptors was significantly lower in cows with CDD than in control cows. In the ileum, these receptors all had lower mRNA expression in cows with CDD than in control cows. The same effect was detected in the ELSC for mRNA for alpha(2AD)-, alpha(2B)-, beta(1)-, and beta(2)-adrenoceptors, and in the cecum and PLAC for alpha(2B)- and beta(2)-adrenoceptors. Groups did not differ significantly for alpha(1A)-adrenoceptors. The mRNA expression for alpha(1D)-, alpha(2C)-, and beta(3)-adrenoceptors was extremely low in all groups. CONCLUSIONS AND CLINICAL RELEVANCE: Differences in expression of mRNA coding for adrenoceptors, most pronounced in the ileum and spiral colon, between cows with CDD and control cows support the hypothesis of an implication of adrenergic mechanisms in the pathogenesis of CDD in dairy cows.

Role of selective alpha and beta adrenergic receptor mechanisms in rat jejunal longitudinal muscle contractility

Gut motility is modulated by adrenergic mechanisms. The aim of our study was to examine mechanisms of selective adrenergic receptors in rat jejunum. Spontaneous contractile activity of longitudinal muscle strips from rat jejunum was measured in 5-ml tissue chambers. Dose-responses (six doses, 10(-7) -3 x 10(-5)M) to norepinephrine (NE, nonspecific), phenylephrine (PH, alpha1), clonidine (C, alpha2), prenalterol (PR, beta1), ritodrine (RI, beta2), and ZD7714 (ZD, beta3) were evaluated with and without tetrodotoxin (TTX, nerve blocker). NE(3 x 10(-5)M) inhibited 74 +/- 5% (mean +/- SEM) of spontaneous activity. This was the maximum effect. The same dose of RI(beta2), PH(alpha1), or ZD(beta(3)) resulted in an inhibition of only 56 +/- 5, 43 +/- 4, 33 +/- 6, respectively. The calculated concentration to induce 50% inhibition (EC50) of ZD(beta3) was similar to NE, whereas higher concentrations of PH(alpha1) or RI(beta2) were required. C(alpha2) and PR(beta1) had no effect. TTX changed exclusively the EC50 of RI from 4.4 +/- 0.2 to 2.7 +/- 0.8% (p < 0.04). Contractility was inhibited by NE (nonspecific). PH(alpha1), RI(beta2), and ZD(beta3) mimic the effect of NE. TTX reduced the inhibition by RI. Our results suggest that muscular alpha1, beta2, and beta3 receptor mechanisms mediate adrenergic inhibition of contractility in rat jejunum. beta2 mechanisms seem to involve also neural pathways.

Expression of a β-adrenergic receptor kinase 1 inhibitor prevents the development of myocardial failure in gene-targeted mice

Heart failure is accompanied by severely impaired β-adrenergic receptor (βAR) function, which includes loss of βAR density and functional uncoupling of remaining receptors. An important mechanism for the rapid desensitization of βAR function is agonist-stimulated receptor phosphorylation by the βAR kinase (βARK1), an enzyme known to be elevated in failing human heart tissue. To investigate whether alterations in βAR function contribute to the development of myocardial failure, transgenic mice with cardiac-restricted overexpression of either a peptide inhibitor of βARK1 or the β2AR were mated into a genetic model of murine heart failure (MLP−/−). In vivo cardiac function was assessed by echocardiography and cardiac catheterization. Both MLP−/− and MLP−/−/β2AR mice had enlarged left ventricular (LV) chambers with significantly reduced fractional shortening and mean velocity of circumferential fiber shortening. In contrast, MLP−/−/βARKct mice had normal LV chamber size and function. Basal LV contractility in the MLP−/−/βARKct mice, as measured by LV dP/dtmax, was increased significantly compared with the MLP−/− mice but less than controls. Importantly, heightened βAR desensitization in the MLP−/− mice, measured in vivo (responsiveness to isoproterenol) and in vitro (isoproterenol-stimulated membrane adenylyl cyclase activity), was completely reversed with overexpression of the βARK1 inhibitor. We report here the striking finding that overexpression of this inhibitor prevents the development of cardiomyopathy in this murine model of heart failure. These findings implicate abnormal βAR-G protein coupling in the pathogenesis of the failing heart and point the way toward development of agents to inhibit βARK1 as a novel mode of therapy.

Essential role of β-adrenergic receptor kinase 1 in cardiac development and function

The β-adrenergic receptor kinase 1 (βARK1) is a member of the G protein-coupled receptor kinase (GRK) family that mediates the agonist-dependent phosphorylation and desensitization of G protein-coupled receptors. We have cloned and disrupted the βARK1 gene in mice by homologous recombination. No homozygote βARK1−/− embryos survive beyond gestational day 15.5. Prior to gestational day 15.5, βARK1−/− embryos display pronounced hypoplasia of the ventricular myocardium essentially identical to the “thin myocardium syndrome” observed upon gene inactivation of several transcription factors (RXRα, N-myc, TEF-1, WT-1). Lethality in βARK1−/− embryos is likely due to heart failure as they exhibit a >70% decrease in cardiac ejection fraction determined by direct in utero intravital microscopy. These results along with the virtual absence of endogenous GRK activity in βARK1−/− embryos demonstrate that βARK1 appears to be the predominant GRK in early embryogenesis and that it plays a fundamental role in cardiac development.

Myocardial signaling defects and impaired cardiac function of a human beta 2-adrenergic receptor polymorphism expressed in transgenic mice.

A threonine to isoleucine polymorphism at amino acid 164 in the fourth transmembrane spanning domain of the beta 2-adrenergic receptor (beta 2AR) is known to occur in the human population. The functional consequences of this polymorphism to catecholamine signaling in relevant cells or to end-organ responsiveness, however, are not known. To explore potential differences between the two receptors, site-directed mutagenesis was carried out to mimic the polymorphism. Transgenic FVB/N mice were then created overexpressing wild-type (wt) beta 2AR or the mutant Ile-164 receptor in a targeted manner in the heart using a murine alpha myosin heavy chain promoter. The functional properties of the two receptors were then assessed at the level of in vitro cardiac myocyte signaling and in vivo cardiac responses in intact animals. The expression levels of these receptors in the two lines chosen for study were approximately 1200 fmol/mg protein in cardiac membranes, which represents a approximately 45-fold increase in expression over endogenous beta AR. Myocyte membrane adenylyl cyclase activity in the basal state was significantly lower in the Ile-164 mice (19.5 +/- 2.7 pmol/min/mg) compared with wt beta 2AR mice (35.0 +/- 4.1 pmol/min/mg), as was the maximal isoproterenol-stimulated activity (49.8 +/- 7.8 versus 77.1 +/ 7.3 pmol/min/mg). In intact animals, resting heart rate (441 +/- 21 versus 534 +/- 17 bpm) and dP/dtmax (10,923 +/- 730 versus 15,308 +/- 471 mmHg/sec) were less in the Ile-164 mice as compared with wt beta 2AR mice. Similarly, the physiologic responses to infused isoproterenol were notably less in the mutant expressing mice. Indeed, these values, as well as other contractile parameters, were indistinguishable between Ile-164 mice and nontransgenic littermates. Taken together, these results demonstrate that the Ile-164 polymorphism is substantially dysfunctional in a relevant target tissue, as indicated by depressed receptor coupling to adenylyl cyclase in myocardial membranes and impaired receptor mediated cardiac function in vivo. Under normal homeostatic conditions or in circumstances where sympathetic responses are compromised due to diseased states, such as heart failure, this impairment may have important pathophysiologic consequences.

Contribution of the collagen I alpha 1 and vitamin D receptor genes to the risk of hip fracture in elderly women

Context and Objective: Hip fracture is partially genetically determined. The present study was designed to examine the contributions of vitamin D receptor (VDR) and collagen I alpha 1 (COLIA1) genotypes to the liability to hip fracture in postmenopausal women. Design: The study was designed as a prospective population-based cohort investigation. Subjects: Six hundred seventy-seven postmenopausal women of Caucasian background, aged 70 +/- 7 yr (mean +/- SD), have been followed for up to 14 yr. Sixty-nine women had sustained a hip fracture during the period. Main Outcome: Atraumatic hip fractures were prospectively identified through radiologists' reports. Bone mineral density (BMD) at the hip and lumbar spine was measured by dual-energy x-ray absorptiometry. Genotypes: The TaqI and SpI COLIA1 polymorphisms of the VDR and COLIA1 genes were determined. Using the Single Nucleotide Polymorphism database, VDR TT, Tt, and tt genotypes were coded as TT, TC, and CC, whereas COLIA1 SS, Ss, and ss were coded as GG, GT, and TT. Results: Women with VDR CC genotype (16% prevalence) and COLIA1 TT genotype (5% prevalence) had an increased risk of hip fracture [odds ratio (OR) associated with CC, 2.6; 95% confidence interval (CI), 1.2-5.3; OR associated with TT, 3.8; 95% CI, 1.3-10.8] after adjustment for femoral neck BMD (OR, 3.4 per SD; 95% CI, 2.3-5.0) and age (OR, 1.4 per 5 yr; 95% CI, 1.1-1.7). Approximately 20 and 12% of the liability to hip fracture was attributable to the presence of the CC genotype and TT genotype, respectively. Conclusion: The VDR CC genotype and COLIA1 TT genotype were associated with increased hip fracture risk in Caucasian women, and this association was independent of BMD and age.

Immunochemical approach to the mapping of an assembled epitope of human chorionic gonadotropin: Proximity of CTP-alpha to the receptor binding region of the beta-subunit

A single-step solid-phase RIA (SS-SPRIA) developed in our laboratory using hybridoma culture supernatants has been utilised for the quantitation of epitope-paratope interactions. Using SS-SPRIA as a quantitative tool for the assessment of epitope stability, it was found that several assembled epitopes of human chorionic gonadotropin (hCG) are differentially stable to proteolysis and chemical modification. Based on these observations an approach has been developed for identifying the amino acid residues constituting an epitopic region. This approach has now been used to map an assembled epitope at/near the receptor binding region of the hormone. The mapped site forms a part of the seat belt region and the cystine knot region (C34-C38-C88-C90-H106). The carboxy terminal region of the alpha-subunit forms a part of the epitope indicating its proximity to the receptor binding region. These results are in agreement with the reported receptor binding region identified through other approaches and the X-ray crystal structure of hCG.

Estudo do polimorfismo Ser49Gly do gene do receptor beta adrenérgico 1 (β-adr 1) na população do estado do Rio de Janeiro, Brasil estratificada por cor da pele e ancestralidade genômica

As doenças cardiovasculares possuem a maior taxa de óbitos no mundo, e notavelmente nos últimos anos as pesquisas genéticas sobre as mesmas estão baseadas em estudos de associação, no qual o gene suspeito que esteja em maior frequência entre os pacientes passa a ser considerado um possível fator causal. Os polimorfismos genéticos que ocorrem no receptor beta-adrenérgico podem resultar em mudanças significativas na função do receptor, podendo acarretar fisiopatologias. Neste trabalho, o objetivo foi estimar a diversidade e a frequência do polimorfismo Ser49Gly do gene do receptor beta-adrenérgico 1 a partir de uma amostra de 188 indivíduos da população do Estado do Rio de Janeiro. As frequências também foram analisadas a partir da estratificação da amostra por critério fenotípico em função do padrão de cor da pele em (negros e não negros) ou ancestralidade genética em (afrodescendente e não afrodescendente), definida através da informação dos marcadores de ancestralidade Indels e SNP de cromossomo Y, para avaliar se os padrões de ancestralidade ou cor da pele são fundamentais para a diferenciação e distanciamento genético. Fragmentos de interesse foram amplificados por PCR (reação de cadeia de polimerase) com primers específicos para o marcador Ser49Gly e as reações de genotipagem foram realizadas com enzimas de restrição Eco0109I. Os valores da heterozigosidade variaram entre 0,25-0,50 e 0,20-0,41 nos grupos estratificados por ancestralidade e cor da pele, respectivamente. No que diz respeito à análise do equilíbrio de Hardy-Weinberg, não houve um desvio significativo na distribuição do marcador nas amostras gerais do Estado do Rio de Janeiro, ou mesmo nas amostras estratificadas. A distribuição dos alelos na amostra dos 188 indivíduos da população geral do Rio de Janeiro (AC_RJ) mostrou uma frequência de 80,30% e 19,70% para o alelo selvagem e mutado Ser49Gly, respectivamente. A comparação das análises sobre a distribuição das frequências alélicas para este marcador mostrou a ocorrência de diferenças significativas na distribuição das frequências alélicas entre negros e não negros e afrodescendentes e não afrodescendentes. A diferença significativa observada entre os negros e afrodescendentes, foi em menor grau de distanciamento. A informação obtida em relação à ancestralidade foi crucial para a obtenção dos dados sobre o aumento da variável mutada do polimorfismo Ser49Gly nas populações negras e afrodescendentes do Estado Rio de Janeiro. Tal evidência, em combinação com estudos clínicos podem contribuir para uma análise pormenorizada do padrão de susceptibilidade à doença em questão, em falhas do mecanismo deste receptor.

RESERPINE IMPAIRS SPATIAL WORKING-MEMORY PERFORMANCE IN MONKEYS - REVERSAL BY THE ALPHA-2-ADRENERGIC AGONIST CLONIDINE

Repeated daily treatment with the catecholamine-depleting agent, reserpine, dramatically reduced performance on the delayed response task, a test of spatial working memory that depends upon the integrity of the prefrontal cortex. Delayed response performance fell from an average of 27.2/30 trials correct before reserpine treatment to an average of 20.4/30 trials correct after repeated reserpine administration. Injection of the alpha2-adrenergic agonist, clonidine (0.0001-0.05 mg/kg), to chronic reserpine-treated monkeys significantly restored performance on the delayed response task; performance after an optimal dose averaged 27.8/30 trials correct. Clonidine's beneficial effects on delayed response performance were longlasting; monkeys remained improved for more than 24 h after a single clonidine injection. The finding that clonidine is efficacious in reserpinized animals supports the hypothesis that alpha2-adrenergic agonists improve cognitive function through actions at postsynaptic, alpha2-adrenergic receptors on non-adrenergic cells. In contrast to the delayed response task, reserpine had little effect on performance of a visual discrimination task, a reference memory task which does not depend on the prefrontal cortex. These results emphasize the importance of postsynaptic alpha2-adrenergic mechanisms in the regulation of working memory,

Augmentation of cardiac contractility mediated by the human beta(3)-adrenergic receptor overexpressed in the hearts of transgenic mice.

BACKGROUND: Stimulation of beta(1)- and beta(2)-adrenergic receptors (ARs) in the heart results in positive inotropy. In contrast, it has been reported that the beta(3)AR is also expressed in the human heart and that its stimulation leads to negative inotropic effects. METHODS AND RESULTS: To better understand the role of beta(3)ARs in cardiac function, we generated transgenic mice with cardiac-specific overexpression of 330 fmol/mg protein of the human beta(3)AR (TGbeta(3) mice). Hemodynamic characterization was performed by cardiac catheterization in closed-chest anesthetized mice, by pressure-volume-loop analysis, and by echocardiography in conscious mice. After propranolol blockade of endogenous beta(1)- and beta(2)ARs, isoproterenol resulted in an increase in contractility in the TGbeta(3) mice (30%), with no effect in wild-type mice. Similarly, stimulation with the selective human beta(3)AR agonist L-755,507 significantly increased contractility in the TGbeta(3) mice (160%), with no effect in wild-type mice, as determined by hemodynamic measurements and by end-systolic pressure-volume relations. The underlying mechanism of the positive inotropy incurred with L-755,507 in the TGbeta(3) mice was investigated in terms of beta(3)AR-G-protein coupling and adenylyl cyclase activation. Stimulation of cardiac membranes from TGbeta(3) mice with L-755,507 resulted in a pertussis toxin-insensitive 1.33-fold increase in [(35)S]GTPgammaS loading and a 1.6-fold increase in adenylyl cyclase activity. CONCLUSIONS: Cardiac overexpression of human beta(3)ARs results in positive inotropy only on stimulation with a beta(3)AR agonist. Overexpressed beta(3)ARs couple to G(s) and activate adenylyl cyclase on agonist stimulation.

Enhancement of cardiac function after adenoviral-mediated in vivo intracoronary beta2-adrenergic receptor gene delivery.

Exogenous gene delivery to alter the function of the heart is a potential novel therapeutic strategy for treatment of cardiovascular diseases such as heart failure (HF). Before gene therapy approaches to alter cardiac function can be realized, efficient and reproducible in vivo gene techniques must be established to efficiently transfer transgenes globally to the myocardium. We have been testing the hypothesis that genetic manipulation of the myocardial beta-adrenergic receptor (beta-AR) system, which is impaired in HF, can enhance cardiac function. We have delivered adenoviral transgenes, including the human beta2-AR (Adeno-beta2AR), to the myocardium of rabbits using an intracoronary approach. Catheter-mediated Adeno-beta2AR delivery produced diffuse multichamber myocardial expression, peaking 1 week after gene transfer. A total of 5 x 10(11) viral particles of Adeno-beta2AR reproducibly produced 5- to 10-fold beta-AR overexpression in the heart, which, at 7 and 21 days after delivery, resulted in increased in vivo hemodynamic function compared with control rabbits that received an empty adenovirus. Several physiological parameters, including dP/dtmax as a measure of contractility, were significantly enhanced basally and showed increased responsiveness to the beta-agonist isoproterenol. Our results demonstrate that global myocardial in vivo gene delivery is possible and that genetic manipulation of beta-AR density can result in enhanced cardiac performance. Thus, replacement of lost receptors seen in HF may represent novel inotropic therapy.

Beta-arrestin-mediated beta1-adrenergic receptor transactivation of the EGFR confers cardioprotection.

Deleterious effects on the heart from chronic stimulation of beta-adrenergic receptors (betaARs), members of the 7 transmembrane receptor family, have classically been shown to result from Gs-dependent adenylyl cyclase activation. Here, we identify a new signaling mechanism using both in vitro and in vivo systems whereby beta-arrestins mediate beta1AR signaling to the EGFR. This beta-arrestin-dependent transactivation of the EGFR, which is independent of G protein activation, requires the G protein-coupled receptor kinases 5 and 6. In mice undergoing chronic sympathetic stimulation, this novel signaling pathway is shown to promote activation of cardioprotective pathways that counteract the effects of catecholamine toxicity. These findings suggest that drugs that act as classical antagonists for G protein signaling, but also stimulate signaling via beta-arrestin-mediated cytoprotective pathways, would represent a novel class of agents that could be developed for multiple members of the 7 transmembrane receptor family.