Potentiation of beta-adrenergic signaling by adenoviral-mediated gene transfer in adult rabbit ventricular myocytes.

Our laboratory has been testing the hypothesis that genetic modulation of the beta-adrenergic signaling cascade can enhance cardiac function. We have previously shown that transgenic mice with cardiac overexpression of either the human beta2-adrenergic receptor (beta2AR) or an inhibitor of the beta-adrenergic receptor kinase (betaARK), an enzyme that phosphorylates and uncouples agonist-bound receptors, have increased myocardial inotropy. We now have created recombinant adenoviruses encoding either the beta2AR (Adeno-beta2AR) or a peptide betaARK inhibitor (consisting of the carboxyl terminus of betaARK1, Adeno-betaARKct) and tested their ability to potentiate beta-adrenergic signaling in cultured adult rabbit ventricular myocytes. As assessed by radioligand binding, Adeno-beta2AR infection led to approximately 20-fold overexpression of beta-adrenergic receptors. Protein immunoblots demonstrated the presence of the Adeno-betaARKct transgene. Both transgenes significantly increased isoproterenol-stimulated cAMP as compared to myocytes infected with an adenovirus encoding beta-galactosidase (Adeno-betaGal) but did not affect the sarcolemmal adenylyl cyclase response to Forskolin or NaF. beta-Adrenergic agonist-induced desensitization was significantly inhibited in Adeno-betaARKct-infected myocytes (16+/-2%) as compared to Adeno-betaGal-infected myocytes (37+/-1%, P < 0.001). We conclude that recombinant adenoviral gene transfer of the beta2AR or an inhibitor of betaARK-mediated desensitization can potentiate beta-adrenergic signaling.

Receptor and G betagamma isoform-specific interactions with G protein-coupled receptor kinases.

The G protein-coupled receptor (GPCR) kinases (GRKs) phosphorylate and desensitize agonist-occupied GPCRs. GRK2-mediated receptor phosphorylation is preceded by the agonist-dependent membrane association of this enzyme. Previous in vitro studies with purified proteins have suggested that this translocation may be mediated by the recruitment of GRK2 to the plasma membrane by its interaction with the free betagamma subunits of heterotrimeric G proteins (G betagamma). Here we demonstrate that this mechanism operates in intact cells and that specificity is imparted by the selective interaction of discrete pools of G betagamma with receptors and GRKs. Treatment of Cos-7 cells transiently overexpressing GRK2 with a beta-receptor agonist promotes a 3-fold increase in plasma membrane-associated GRK2. This translocation of GRK2 is inhibited by the carboxyl terminus of GRK2, a known G betagamma sequestrant. Furthermore, in cells overexpressing both GRK2 and G beta1 gamma2, activation of lysophosphatidic acid receptors leads to the rapid and transient formation of a GRK/G betagamma complex. That G betagamma specificity exists at the level of the GPCR and the GRK is indicated by the observation that a GRK2/G betagamma complex is formed after agonist occupancy of the lysophosphatidic acid and beta-adrenergic but not thrombin receptors. In contrast to GRK2, GRK3 forms a G betagamma complex after stimulation of all three GPCRs. This G betagamma binding specificity of the GRKs is also reflected at the level of the purified proteins. Thus the GRK2 carboxyl terminus binds G beta1 and G beta2 but not G beta3, while the GRK3 fusion protein binds all three G beta isoforms. This study provides a direct demonstration of a role for G betagamma in mediating the agonist-stimulated translocation of GRK2 and GRK3 in an intact cellular system and demonstrates isoform specificity in the interaction of these components.

Receptor-specific in vivo desensitization by the G protein-coupled receptor kinase-5 in transgenic mice.

Transgenic mice were generated with cardiac-specific overexpression of the G protein-coupled receptor kinase-5 (GRK5), a serine/threonine kinase most abundantly expressed in the heart compared with other tissues. Animals overexpressing GRK5 showed marked beta-adrenergic receptor desensitization in both the anesthetized and conscious state compared with nontransgenic control mice, while the contractile response to angiotensin II receptor stimulation was unchanged. In contrast, the angiotensin II-induced rise in contractility was significantly attenuated in transgenic mice overexpressing the beta-adrenergic receptor kinase-1, another member of the GRK family. These data suggest that myocardial overexpression of GRK5 results in selective uncoupling of G protein-coupled receptors and demonstrate that receptor specificity of the GRKs may be important in determining the physiological phenotype.

Monoclonal antibodies reveal receptor specificity among G-protein-coupled receptor kinases.

Guanine nucleotide-binding regulatory protein (G protein)-coupled receptor kinases (GRKs) constitute a family of serine/threonine kinases that play a major role in the agonist-induced phosphorylation and desensitization of G-protein-coupled receptors. Herein we describe the generation of monoclonal antibodies (mAbs) that specifically react with GRK2 and GRK3 or with GRK4, GRK5, and GRK6. They are used in several different receptor systems to identify the kinases that are responsible for receptor phosphorylation and desensitization. The ability of these reagents to inhibit GRK- mediated receptor phosphorylation is demonstrated in permeabilized 293 cells that overexpress individual GRKs and the type 1A angiotensin II receptor. We also use this approach to identify the endogenous GRKs that are responsible for the agonist-induced phosphorylation of epitope-tagged beta2- adrenergic receptors (beta2ARs) overexpressed in rabbit ventricular myocytes that are infected with a recombinant adenovirus. In these myocytes, anti-GRK2/3 mAbs inhibit isoproterenol-induced receptor phosphorylation by 77%, while GRK4-6-specific mAbs have no effect. Consistent with the operation of a betaAR kinase-mediated mechanism, GRK2 is identified by immunoblot analysis as well as in a functional assay as the predominant GRK expressed in these cells. Microinjection of GRK2/3-specific mAbs into chicken sensory neurons, which have been shown to express a GRK3-like protein, abolishes desensitization of the alpha2AR-mediated calcium current inhibition. The intracellular inhibition of endogenous GRKs by mAbs represents a novel approach to the study of receptor specificities among GRKs that should be widely applicable to many G-protein-coupled receptors.

G protein beta gamma subunits stimulate phosphorylation of Shc adapter protein.

The mechanism of mitogen-activated protein (MAP) kinase activation by pertussis toxin-sensitive Gi-coupled receptors is known to involve the beta gamma subunits of heterotrimeric G proteins (G beta gamma), p21ras activation, and an as-yet-unidentified tyrosine kinase. To investigate the mechanism of G beta gamma-stimulated p21ras activation, G beta gamma-mediated tyrosine phosphorylation was examined by overexpressing G beta gamma or alpha 2-C10 adrenergic receptors (ARs) that couple to Gi in COS-7 cells. Immunoprecipitation of phosphotyrosine-containing proteins revealed a 2- to 3-fold increase in the phosphorylation of two proteins of approximately 50 kDa (designated as p52) in G beta gamma-transfected cells or in alpha 2-C10 AR-transfected cells stimulated with the agonist UK-14304. The latter response was pertussis toxin sensitive. These proteins (p52) were also specifically immunoprecipitated with anti-Shc antibodies and comigrated with two Shc proteins, 46 and 52 kDa. The G beta gamma- or alpha 2-C10 AR-stimulated p52 (Shc) phosphorylation was inhibited by coexpression of the carboxyl terminus of beta-adrenergic receptor kinase (a G beta gamma-binding pleckstrin homology domain peptide) or by the tyrosine kinase inhibitors genistein and herbimycin A, but not by a dominant negative mutant of p21ras. Worthmannin, a specific inhibitor of phosphatidylinositol 3-kinase (PI3K) inhibited phosphorylation of p52 (Shc), implying involvement of PI3K. These results suggest that G beta gamma-stimulated Shc phosphorylation represents an early step in the pathway leading to p21ras activation, similar to the mechanism utilized by growth factor tyrosine kinase receptors.

Mucosal Allergic Sensitization to Cockroach Allergens Is Dependent on Proteinase Activity and Proteinase-Activated Receptor-2 Activation

We have shown that proteinase-activated receptor-2 (PAR(2)) activation in the airways leads to allergic sensitization to concomitantly inhaled Ags, thus implicating PAR(2) in the pathogenesis of asthma. Many aeroallergens with proteinase activity activate PAR(2). To study the role of PAR(2) in allergic sensitization to aeroallergens, we developed a murine model of mucosal sensitization to cockroach proteins. We hypothesized that PAR(2) activation in the airways by natural allergens with serine proteinase activity plays an important role in allergic sensitization. Cockroach extract (CE) was administered to BALB/c mice intranasally on five consecutive days (sensitization phase) and a week later for four more days (challenge phase). Airway hyperresponsiveness (AHR) and allergic airway inflammation were assessed after the last challenge. To study the role of PAR(2), mice were exposed intranasally to a receptor-blocking anti-PAR(2) Ab before each administration of CE during the sensitization phase. Mucosal exposure to CE induced eosinophilic airway inflammation, AHR, and cockroach-specific IgG1. Heat-inactivated or soybean trypsin inhibitor-treated CE failed to induce these effects, indicating that proteinase activity plays an important role. The use of an anti-PAR(2) blocking Ab during the sensitization phase completely inhibited airway inflammation and also decreased AHR and the production of cockroach-specific IgG1. PAR(2) activation by CE acts as an adjuvant for allergic sensitization even in the absence of functional TLR4. We conclude that CE induces PAR(2)-dependent allergic airway sensitization in a mouse model of allergic airway inflammation. PAR(2) activation may be a general mechanism used by aeroallergens to induce allergic sensitization. The Journal of Immunology, 2011, 186: 3164-3172.

alpha(1A)-adrenoceptors mediate sympathetically evoked pupillary dilation in rats

Evidence suggests that in some species (cats, rabbits, and possibly humans) alpha-adrenoceptors in the iris dilator muscle are "atypical" in that they cannot be readily classified by conventional criteria. This study was undertaken in an attempt to characterize the alpha-adrenoceptor subtype(s) mediating sympathetically elicited mydriasis in rats. Frequency-response pupillary dilator curves were generated by stimulation of the preganglionic cervical sympathetic nerve (1-32 Hz) in pentobarbital-anesthetized rats. Evoked responses were inhibited by systemic administration of nonselective alpha-adrenergic antagonists, phentolamine (0.3-10 mg/kg) and phenoxybenzamine (0.03-1 mg/kg). The selective alpha(1)-adrenergic antagonist, prazosin (0.01-1 mg/kg), also was effective, although alpha(2)-adrenergic antagonism with rauwolscine (0.1-1 mg/kg) was not. alpha(1A)-Adrenoceptor-selective antagonists, 2-([2,6-dimethoxyphenoxyethyl]aminomethyl)-1,4-benzodioxane (WB-4101; 0.1-1 mg/kg) and 5-methylurapidil (0.1-1 mg/kg), as well as the alpha(1D)-adrenoceptor-selective antagonist 8-[2-[4-(2-methoxyphenyl)-1-piperazinyl]ethyl]-8-azaspiro[4.5]decane-7,9-dione (BMY-7378; 1-3 mg/kg), were used to determine the subtype(s) involved. Evoked mydriasis was significantly antagonized by both WB-4101 and 5-methylurapidil but not by BMY-7378. These results suggest that, unlike some other species, adrenoceptors in the rat iris dilator mediating neurogenic mydriasis are "typical" and, in addition, can be characterized as being primarily of the alpha(1A)-adrenoceptor subtype.

Cardiovascular response to beta-adrenergic blockade or activation in 23 inbred mouse strains.

We report the characterisation of 27 cardiovascular-related traits in 23 inbred mouse strains. Mice were phenotyped either in response to chronic administration of a single dose of the beta-adrenergic receptor blocker atenolol or under a low and a high dose of the beta-agonist isoproterenol and compared to baseline condition. The robustness of our data is supported by high trait heritabilities (typically H(2)>0.7) and significant correlations of trait values measured in baseline condition with independent multistrain datasets of the Mouse Phenome Database. We then focused on the drug-, dose-, and strain-specific responses to beta-stimulation and beta-blockade of a selection of traits including heart rate, systolic blood pressure, cardiac weight indices, ECG parameters and body weight. Because of the wealth of data accumulated, we applied integrative analyses such as comprehensive bi-clustering to investigate the structure of the response across the different phenotypes, strains and experimental conditions. Information extracted from these analyses is discussed in terms of novelty and biological implications. For example, we observe that traits related to ventricular weight in most strains respond only to the high dose of isoproterenol, while heart rate and atrial weight are already affected by the low dose. Finally, we observe little concordance between strain similarity based on the phenotypes and genotypic relatedness computed from genomic SNP profiles. This indicates that cardiovascular phenotypes are unlikely to segregate according to global phylogeny, but rather be governed by smaller, local differences in the genetic architecture of the various strains.

Pharmacometrics of neuromuscular blocking agents in anesthetized patients and animals : impact of dose and intravascular mixing phase

Thèse numérisée par la Division de la gestion de documents et des archives de l'Université de Montréal

Functional Analysis of Nuclear beta-Adrenergic Receptors in the Myocardium

Récemment plusieurs récepteurs couplés aux protéines G (RCPGs) ont été caractérisés au niveau des membranes intracellulaires, dont la membrane nucléaire. Notre objectif était de déterminer si les sous-types de récepteurs β-adrénergiques (βAR) et leurs machineries de signalisation étaient fonctionnels et localisés à la membrane nucléaire des cardiomyocytes. Nous avons démontré la présence des β1AR et β3AR, mais pas du β2AR à la membrane nucléaire de myocytes ventriculaires adultes par immunobuvardage, par microscopie confocale, et par des essais fonctionnels. De plus, certains partenaires de signalisation comme les protéines GαS, Gαi, l’adénylate cyclase II, et V/VI y étaient également localisés. Les sous-types de βAR nucléaires étaient fonctionnels puisqu'ils pouvaient lier leurs ligands et activer leurs effecteurs. En utilisant des noyaux isolés, nous avons observé que l'agoniste non-sélectif isoprotérénol (ISO), et que le BRL37344, un ligand sélectif du β3AR, stimulaient l'initiation de la synthèse de l’ARN, contrairement à l'agoniste sélectif du β1AR, le xamotérol. Cette synthèse était abolie par la toxine pertussique (PTX). Cependant, la stimulation des récepteurs nucléaires de type B de l’endothéline (ETB) causaient une réduction de l'initiation de la synthèse d’ARN. Les voies de signalisations impliquées dans la régulation de la synthèse d’ARN par les RCPGs ont ensuite été étudiées en utilisant des noyaux isolés stimulés par des agonistes en présence ou absence de différents inhibiteurs des voies MAP Kinases (proteines kinases activées par mitogènes) et de la voie PI3K/PKB. Les protéines impliquées dans les voies de signalisation de p38, JNK, ERK MAP Kinase et PKB étaient présents dans les noyaux isolés. L'inhibition de PKB par la triciribine, inhibait la synthèse d’ARN. Nous avons ensuite pu mettre en évidence par qPCR que la stimulation par l’ISO entrainait une augmentation du niveau d'ARNr 18S ainsi qu’une diminution de l'expression d’ARNm de NFκB. En contraste, l’ET-1 n’avait aucun effet sur le niveau d’expression de l’ARNr 18S. Nous avons ensuite montré que la stimulation par l’ISO réduisait l’expression de plusieurs gènes impliqués dans l'activation de NFκB, tandis que l’inhibition de ERK1/2 et PKB renversait cet effet. Un microarray global nous a ensuite permis de démontrer que les βARs et les ETRs nucléaires régulaient un grand nombre de gènes distincts. Finalement, les βARs et ETRs nucléaires augmentaient aussi une production de NO de noyaux isolés, ce qui pouvait être inhibée par le LNAME. Ces résultats ont été confirmés dans des cardiomyocytes intacts en utilisant des analogues cagés et perméables d’ISO et de l'ET-1: l'augmentation de NO nucléaire détectée par DAF2-DA, causée par l'ET-1 et l'ISO, pouvait être prévenue par le LNAME. Finalement, l’augmentation de l’initiation de la transcription induite par l'ISO était aussi bloquée par le L-NAME ou par un inbitheur de PKG, le KT5823, suggérant que la voie NO-GC-PKG est impliquée dans la régulation de la transcription par les βAR. En conclusion, les βARs et les ETRs nucléaires utilisent des voies de signalisation différentes et exercent ainsi des effets distincts sur l’expression des gènes cardiaques. Ils représentent donc une avenue intéressante pour le développement de drogues pharmacologiques.

Enhanced /3-adrenergic receptors in the brain and pancreas during pancreatic regeneration in weanling rats

Adrenergic stimulation has an inyortant role in the pancreatic It-cell proliferation and insulin secretion. In the present study. we have investigaled how sympathetic system mgulales the panrrealic n I rnerui nr ht an:ilyiing I'pinephi inn 1111 ), Norepinephrinc (NE) and /1-adrenergic receptor changes in the brain as (%eli is in the I swirls. Fill and NII showed a significant decrease in the brain regions, pancreas and plasma :rt 72Ius iller partial prurcrealectonty. We observed an increase in the circulating insulin levels at 72 hrs. Scatchard analysis using I CHI propranolol showed a significant increase in the number of loth the low affinity and high affinity t-adrenergic receplors in cerebral cortex and hypothalamus of partially pancreatectornised rats during peak DNA synthesis. The affinity of the receptors decrea,ed significantly in the low and high affinity receptors of cerebral cortex and the high affinity hypothalamic receptors. In file brain stein, low affinity receptors were increased significantly during regeneration whereas there was no change in the high affinity receptors. The pancreatic ff-adrenergic receptors were also up regulated at 72 firs after partial panerealectony. In vitro studies showed that /i-adrenergic receptors are positive regulators of islet cell proliferation and insulin secretion. Thus our results suggest that the t-adrenergic receptors are functionally enhanced during pancreatic regeneration, which in turn increases pancreatic ft-cell proliferation an(hilisulin secretion in wean hug rats.

Age-related and sex-related alterations in f3-adrenergic receptors in different regions of rat brain

The binding of (-)[ 3H ]dihydroalprenolol , an antagonist of norepinephrine , to $-adrenergic receptors in different regions of the brain of male and female rats of various ages was measured . The binding to the synaptosomal fraction of corpus striatum , hypothalamus, cerebral cortex, cerebellum and the brainstems shows a significant decrease in the binding in old rats of both sexes . Only in the female corpus striatal region, the binding in the adult and the old is the same . In the case of females, the highest binding is seen in the young. In the male, an increase in binding occurs up to adulthood , after which it declines, suggesting a definite sex-related difference in the Q-adrenergic receptor.

“Adrenergic receptors and monoamines in the brain and pancreatic islets of streptozotoein diabetic rats: Role in insulin secretion as a function of age”

I) To study the changes in the content of brain rrrorroamirres in streptozotocirr-irrduced tliabetes as a lirnction of age and to lirrd the role oliadrenal lrornroncs in diabetic state. 2) To assess the adrenergic receptor function in the brain stem ofstreptozotocin-induced diabetic rats ofdillerent ages. 3) To study the changes in the basal levels of second messenger cAMP in the brain stenr ofstreptozotocin-induced diabetic rats as a function of age. 4) To study the changes occurring in the content ofmorroamines and their metabolites in whole pancreas and isolated pancreatic islets of streptozotocin-diabetic rats as a function ofage and the effect of adrenal hormones. 5) To study the adrenergic receptors and basal levels of cAMP in isolated pancreatic islets in young and old streptozotoein-diabetic rats. 6) The in virro study of CAMP content in pancreatic islets of young and old rats and its ellect on glucose induced insulin secretion. 7) 'lhe in vitro study on the involvement of dopamine and corticosteroids in glucose induced insulin secretion in pancreatic islets as a function of age.

Análisis de polimorfismos genéticos del receptor beta-2 adrenérgico en pacientes pediátricos asmáticos y controles sanos en una muestra de la ciudad de Bogotá

El asma bronquial es una enfermedad inflamatoria crónica, se asocia a hiperrespuesta de la vía aérea, la cual lleva a episodios recurrentes de sibilancias, tos y disnea. La entidad se ha correlacionado con una gran variedad de genes involucrados en su fisiopatología, dentro de los cuales se encuentran genes localizados en el cromosoma 5 (5q23-31), como el del Receptor ß2 Adrenérgico (RB2A). En el presente trabajo se realizó una estimación de las frecuencias de los polimorfismos Arg16Gly, Gln27Glu y Thr164Ile de este receptor, y se estudió la relación existente entre los diferentes polimorfismos y asma, así como su relación con respecto a la severidad de la enfermedad, finalmente se estimó la relación de los haplotipos conformados por estos tres polimorfismos y su asociación con la enfermedad y severidad del fenotipo asmático.

Análisis de polimorfismos del receptor Beta 2 adrenérgico en pacientes colombianos afectados por fibrosis quística

El gen ADRB2 y sus polimorfismos se han asociado al cuadro clínico de los pacientes con Fibrosis Quística. En nuestra población existe asociación de los haplotipos G16E27I164 y R16Q27I164 y la enfermedad, así como la presencia de Pólipos y genotipo E27E y el haplotipo R16E27T164.