Functionality of beta-adrenergic receptors in patients with cirrhosis treated chronically with non-selective beta-blockers.

BACKGROUND: In patients with cirrhosis, beta-adrenoceptors expressed on peripheral blood mononuclear cells have a reduced response to catecholamine stimulation. This study aimed to determine if chronic treatment with beta-blockers influences these changes. METHODS: Blood samples were collected from patients with cirrhosis treated in outpatient clinics. Differences in cyclic AMP production before and after stimulation of mononuclear cells with epinephrine and/or N-Formylmethionine-leucyl-phenylalanine (fMLP) was used as a marker of beta-adrenoceptors activity in patients treated (N = 19) versus not treated (N = 55) with beta-blockers. In addition, we studied the gene expression of different types of adrenoceptors and possible associations with the activity of beta-adrenoceptors, the serum concentrations of catecholamines and cytokines, and the presence of bacterial antigens such as DNA or gram-negative bacterial endotoxin in patients' blood. RESULTS: The increase in intracellular cAMP concentrations after stimulation of adrenergic receptors with epinephrine was significantly higher in samples from patients treated with beta-blockers. Older patients showed lower responses to epinephrine stimulus, while the response increased linearly with the duration of the beta-blocker treatment. mRNA expression levels of adrenoceptors ß1, ß2, ß3 and α1-A, B and D showed no significant differences according to treatment with beta-blockers. Neither serum cytokines nor catecholamines levels were significantly associated with the intracellular production of cAMP after adrenergic stimulation. CONCLUSION: Chronic treatment with beta-blockers in patients with cirrhosis enables beta-adrenoceptors to respond to catecholamine stimulation irrespective of the degree of systemic adrenergic or immune activations of the patient at the time of sampling.

Sex-Related Effects of Prenatal Stress on Region-Specific Expression of Monoamine Oxidase A and β Adrenergic Receptors in Rat Hearts / Efeitos Sexo-Específicos de Estresse Pré-Natal na Expressão Região-Específica de Monoamina Oxidase A e Receptores Adrenérgicos Β no Coração de Ratos

Abstract Background: Prenatal stress may increase risk of developing cardiovascular disorders in adulthood. The cardiotoxic effects of catecholamines are mediated via prolonged adrenergic receptor stimulation and increased oxidative stress upon their degradation by monoamine oxidase A (MAO-A). Objectives: We investigated long-term effects of prenatal stress on β (1, 2, 3) adrenergic receptors and MAO-A gene expression in the hearts of adult rat offspring. Methods: Pregnant rats were exposed to unpredictable mild stress during the third week of gestation. RNA was isolated from left ventricular apex and base of adult offspring. Quantitative PCR was used to measure gene expression in collected ventricular tissue samples. The level of significance was set to p < 0.05. Results: β3 adrenergic receptor mRNA was undetectable in rat left ventricle. β1 adrenergic receptor was the predominantly expressed subtype at the apical and basal left ventricular myocardium in the control females. Male offspring from unstressed mothers displayed higher apical cardiac β1 than β2 adrenergic receptor mRNA levels. However, β1 and β2 adrenergic receptor mRNAs were similarly expressed at the ventricular basal myocardium in males. Unlike males, prenatally stressed females exhibited decreased β1 adrenergic receptor mRNA expression at the apical myocardium. Prenatal stress did not affect cardiac MAO-A gene expression. Conclusions: Collectively, our results show that prenatal stress may have exerted region- and sex-specific β1 and β2 adrenergic receptor expression patterns within the left ventricle.

Resumo Fundamento: Estresse pré-natal pode aumentar os riscos de desenvolver doenças cardiovasculares na idade adulta. Os efeitos cardiotóxicos de catecolaminas são mediados pela estimulação prolongada dos receptores adrenérgicos e pelo aumento do estresse oxidativo após sua degradação pela monoamina oxidase A (MAO-A). Objetivos: Investigamos os efeitos a longo prazo de estresse pré-natal nos receptores β (1, 2, 3) adrenérgicos e na expressão do gene MAO-A nos corações da prole adulta de ratos. Método: Ratas prenhes foram expostas a estresse crônico moderado imprevisível durante a terceira semana de gestação. O RNA foi isolado do ápice e da base do ventrículo esquerdo da prole adulta. Utilizou-se PCR quantitativa em tempo real para medir a expressão gênica nas amostras de tecido ventricular coletadas. O nível de significância foi estabelecido em p < 0,05. Resultados: Foi indetectável o mRNA do receptor adrenérgico β3 no ventrículo esquerdo dos ratos. O receptor adrenérgico β1 foi o subtipo mais expresso no miocárdio ventricular esquerdo apical e basal nas fêmeas controle. A prole masculina das mães não estressadas apresentou níveis cardíacos apicais de mRNA do receptor adrenérgico β1 mais altos do que os de β2. Porém, mRNAs dos receptores adrenérgicos β1 e β2 foram expressos de forma semelhante no miocárdio basal ventricular na prole masculina em geral. Ao contrário da prole masculina, a prole feminina exposta ao estresse pré-natal exibiu uma expressão diminuída do mRNA do receptor adrenérgico β1 no miocárdio apical. O estresse pré-natal não afetou a expressão gênica de MAO-A cardíaca. Conclusões: Coletivamente, nossos resultados mostram que estresse pré-natal pode ter exercido padrões de expressão região- e sexo-específica dos receptores adrenérgicos β1 e β2 no ventrículo esquerdo.

Sex-Related Effects of Prenatal Stress on Region-Specific Expression of Monoamine Oxidase A and ß Adrenergic Receptors in Rat Hearts.

BACKGROUND: Prenatal stress may increase risk of developing cardiovascular disorders in adulthood. The cardiotoxic effects of catecholamines are mediated via prolonged adrenergic receptor stimulation and increased oxidative stress upon their degradation by monoamine oxidase A (MAO-A). OBJECTIVES: We investigated long-term effects of prenatal stress on ß (1, 2, 3) adrenergic receptors and MAO-A gene expression in the hearts of adult rat offspring. METHODS: Pregnant rats were exposed to unpredictable mild stress during the third week of gestation. RNA was isolated from left ventricular apex and base of adult offspring. Quantitative PCR was used to measure gene expression in collected ventricular tissue samples. The level of significance was set to p < 0.05. RESULTS: ß3 adrenergic receptor mRNA was undetectable in rat left ventricle. ß1 adrenergic receptor was the predominantly expressed subtype at the apical and basal left ventricular myocardium in the control females. Male offspring from unstressed mothers displayed higher apical cardiac ß1 than ß2 adrenergic receptor mRNA levels. However, ß1 and ß2 adrenergic receptor mRNAs were similarly expressed at the ventricular basal myocardium in males. Unlike males, prenatally stressed females exhibited decreased ß1 adrenergic receptor mRNA expression at the apical myocardium. Prenatal stress did not affect cardiac MAO-A gene expression. CONCLUSIONS: Collectively, our results show that prenatal stress may have exerted region- and sex-specific ß1 and ß2 adrenergic receptor expression patterns within the left ventricle.

Relative quantification of beta-adrenergic receptor in peripheral blood cells using flow cytometry.

Beta-adrenergic receptors (ß-ARs) play a critical role in many diseases. Quantification of ß-AR density may have clinical implications in terms of assessing disease severity and identifying patients who could potentially benefit from beta-blocker therapy. Classical methods for ß-AR quantification are based on labor-intensive and time-consuming radioligand binding assays. Here, we report optimization of a flow cytometry-based method utilizing a biotinylated ß-AR ligand alprenolol as a probe and use of this method to quantify relative receptor expression in healthy controls (HC). Quantum™ MESF beads were used for quantification in absolute fluorescence units. The probe was chemically modified by adding a spacer moiety between biotin and alprenolol to stabilize receptor binding, thus preventing binding decay. Testing of three different standard cell fixation and permeabilization methods (formaldehyde fixation and saponin, Tween-20, or Triton-X 100 permeabilization) showed that the formaldehyde/Triton-X 100 method yielded the best results. ß-AR expression was significantly higher in granulocytes compared to mononuclear cells. These data show that flow cytometric quantification of relative ß-AR expression in circulating leukocytes is a suitable technology for large-scale clinical application. © 2018 International Society for Advancement of Cytometry.

Cardiac voltage gated calcium channels and their regulation by ß-adrenergic signaling.

Voltage-gated calcium channels (VGCCs) are the predominant source of calcium influx in the heart leading to calcium-induced calcium release and ultimately excitation-contraction coupling. In the heart, VGCCs are modulated by the ß-adrenergic signaling. Signaling through ß-adrenergic receptors (ßARs) and modulation of VGCCs by ß-adrenergic signaling in the heart are critical signaling and changes to these have been significantly implicated in heart failure. However, data related to calcium channel dysfunction in heart failure is divergent and contradictory ranging from reduced function to no change in the calcium current. Many recent studies have highlighted the importance of functional and spatial microdomains in the heart and that may be the key to answer several puzzling questions. In this review, we have briefly discussed the types of VGCCs found in heart tissues, their structure, and significance in the normal and pathological condition of the heart. More importantly, we have reviewed the modulation of VGCCs by ßARs in normal and pathological conditions incorporating functional and structural aspects. There are different types of ßARs, each having their own significance in the functioning of the heart. Finally, we emphasize the importance of location of proteins as it relates to their function and modulation by co-signaling molecules. Its implication on the studies of heart failure is speculated.

Increased Atrial ß-Adrenergic Receptors and GRK-2 Gene Expression Can Play a Fundamental Role in Heart Failure After Repair of Congenital Heart Disease with Cardiopulmonary Bypass.

Surgeries to correct congenital heart diseases are increasing in Brazil and worldwide. However, even with the advances in surgical techniques and perfusion, some cases, especially the more complex ones, can develop heart failure and death. A retrospective study of patients who underwent surgery for correction of congenital heart diseases with cardiopulmonary bypass (CPB) in a university tertiary-care hospital that died, showed infarction in different stages of evolution and scattered microcalcifications in the myocardium, even without coronary obstruction. CPB is a process routinely used during cardiac surgery for congenital heart disease. However, CPB has been related to increased endogenous catecholamines that can lead to major injuries in cardiomyocytes. The mechanisms involved are not completely understood. The aim of this study was to evaluate the alterations induced in the ß-adrenergic receptors and GRK-2 present in atrial cardiomyocytes of infants with congenital heart disease undergoing surgical repair with CPB and correlate the alterations with functional and biochemical markers of ischemia/myocardial injury. The study consisted of right atrial biopsies of infants undergoing surgical correction in HC-FMRPUSP. Thirty-three cases were selected. Atrial biopsies were obtained at the beginning of CPB (group G1) and at the end of CPB (group G2). Real-time PCR, Western blotting, and immunofluorescence analysis were conducted to evaluate the expression of ß1, ß2-adrenergic receptors, and GRK-2 in atrial myocardium. Cardiac function was evaluated by echocardiography and biochemical analysis (N-terminal pro-brain natriuretic peptide (NT-ProBNP), lactate, and cardiac troponin I). We observed an increase in serum lactate, NT-proBNP, and troponin I at the end of CPB indicating tissue hypoxia/ischemia. Even without major clinical consequences in cardiac function, these alterations were followed by a significant increase in gene expression of ß1 and ß2 receptors and GRK-2, suggesting that this is one of the mechanisms responsible for the exacerbated response of cardiomyocytes to circulating catecholamines. These alterations could explain the irreversible myocardial damage and lipid peroxidation of membranes classically attributed to catecholamine excess, observed in some infants who develop heart failure and postoperative death. Although other factors may be involved, this study confirms that CPB acts as a potent inducer of increased gene expression of ß- adrenergic receptors and GRK-2, making the myocardium of these infants more susceptible to the effects of circulating endogenous catecholamines, which may contribute to the development of irreversible myocardial damage and death.

[Criteria for selecting patients with resistant hypertension for a renal sympathetic denervation]. / Kriterii otbora bol'nykh s rezistentnoi arterial'noi gipertoniei dlya simpaticheskoi denervatsiya pochek.

AIM: to define and develop a procedure that can select patients with resistant hypertension (RH) for a renal sympathetic denervation (RSD) procedure, by being orienting to the specific initial values of ß-adrenoreactivity (ß-AR) and systolic blood pressure (SBP). SUBJECTS AND METHODS: The analysis included 23 RH patients receiving the maximally tolerable doses of 4 antihypertensive drugs. The investigations involving BP control and a Russian spectrophotometric procedure for determining ß-AR in terms of the change in the osmoresistance of erythrocyte membranes (EM) were performed at baseline and 4 and 24 weeks. RSD was carried out using endovascular radiofrequency ablation of the renal arteries. The therapy was not changed during the observation. RESULTS: If SBP was >170 mm Hg and ß-AR of EM >40 conditional units (CUs) at baseline, ΔSBP was 17.68±3.24 mm Hg and the efficiency of RSD was 100%. When SBP was <170 mm Hg and ß-AR of EM <40 CUs at baseline, ΔSBP was 0.97±4.21 mm Hg (p>0.05) and the efficiency of RSD was low. CONCLUSION: The overall estimate of baseline SBP and ß-AR of EM in patients with RH could determine the expediency of the RSD procedure in order to lower BP. The proposed procedure can optimize the selection of patients and enhance the efficiency of RSD in the treatment of RH.

[Possibility of evaluating the effectiveness of renal artery sympathetic denervation in resistant hypertension early after radiofrequency ablation]. / Vozmozhnost' otsenki effektivnosti simpaticheskoi denervatsii pochechnykh arterii pri rezistentnoi arterial'noi gipertonii v rannie sroki posle provedeniya radiochastotnoi ablyatsii.

AIM: to investigate time course of changes in the adrenoreactivity (AR) of erythrocyte membranes (EM) after radiofrequency ablation of the synaptic nerves of the renal arteries (RA) in patients with resistant hypertension (RH) and to assess whether this indicator can be used for the early evaluation of the efficiency of an invasive intervention into the RA. SUBJECTS AND METHODS: 24 patients with RH, who received full-dose antihypertensive therapy with at least three drugs, including a diuretic, were examined. Renal sympathetic denervation (RSD) was carried out by endovascular radiofrequency ablation (RFA) of the RA. 24-hour blood pressure (BP) monitoring and determination of the ß-adrenoreactivity (ß-AR) of EM were performed, by taking into account the change in erythrocyte osmoresistence at baseline and 1 and 24 weeks after RFA. The therapy was not changed during the observation. RESULTS: The patients included in the study were divided into 2 groups. One week following RSD, 15 patients of Group 1 were noted to have a decrease in the ß-AR of EM by 10 conditional units or more; average daily systolic BP (SBP) and diastolic BP (DBP) reduced by 8.3 and 2.8 mm Hg, respectively. In 9 patients of Group 2, the ß-AR of EM was unchanged in this observation period or increased compared with baseline. In this group, the decrease in average daily SBP and DBP was noted to be less pronounced than that in Group 1 (by 1.4 and 1.5 mm Hg, respectively). At 24 weeks after RSD, Group 1 was seen to have an effective daily decrease in average daily SBP and DBP by 25.6 and 14.3 mm Hg, respectively (p=0.01 and 0.05). The average value of the ß-AR of EM significantly declined compared with baseline. In Group 2, no statistically significant changes were reported; SPB and DBP lowered by 7.0 and 3.0 mm Hg, respectively. There was a significant decrease in the ß-AR of EM compared with that at week 1. CONCLUSION: The decline in the ß-AR of EM within the first week after RFA is suggestive of the decreased activity of the sympathoadrenal system and may be used as an early efficiency index of RSD after the procedure.

Expression of ß-adrenoceptors in human lower esophageal sphincter.

BACKGROUND/AIMS: Beta-adrenoceptor is considered to be an important modulator of smooth muscle function. It is widely present in the mammalian gastrointestinal tract and nervous system. The aim of this study was to explore the expression of 1-adrenoceptors (ß1-AR, ß2-AR, ß3-AR) in sling fibers and clasp fibers from human lower esophageal sphincter (LES). METHODOLOGY: Sling and clasp fibers from the LES were obtained from patients undergoing esophagogastrectomy; circular muscle strips from the esophagus and stomach were used as controls. Reverse transcription-polymerase chain reaction and western blotting were used to determine the expression of the three subtypes of ß-adrenoceptors. RESULTS: Messenger RNA and protein for three subtypes of ß-adrenoceptors were all identified in the sling and clasp fibers of the LES. Expression was highest for ß3-AR, then ß1-AR and ß2-AR in decreasing levels. CONCLUSIONS: ß1-AR, ß2-AR, ß3-AR can be detected in human lower esophageal sphincter and contribute to LES function.

Heart rate changes mediate the embryotoxic effect of antiarrhythmic drugs in the chick embryo.

A significant increase in cardiovascular medication use during pregnancy occurred in recent years. Only limited evidence on safety profiles is available, and little is known about the mechanisms of adverse effect on the fetus. We hypothesized that drug-induced bradycardia is the leading mechanism of developmental toxicity. Embryotoxicity was tested in ovo after administration of various doses of metoprolol, carvedilol, or ivabradine. Embryonic day (ED) 4 and 8 chick embryos were studied by video microscopy and ultrasound biomicroscopy ex ovo after intraamniotic injection of the drug for a period of 30 min. Stroke volume was calculated by the Simpson method and prolate ellipsoid formula. Significant dose-dependent mortality was achieved in embryos injected with carvedilol and ivabradine. In ED4 embryos, metoprolol, carvedilol, and ivabradine reduced the heart rate by 33%, 27%, and 55%, respectively, compared with controls (6%). In ED8 embryos this effect was more pronounced with a heart rate reduction by 71%, 54%, and 53%, respectively (controls, 36%). Cardiac output decreased in all tested groups but only proved significant in the metoprolol group in ED8 embryos. The number of ß-adrenergic receptors showed a downward tendency during embryonic development. A negative chronotropic effect of metoprolol, carvedilol, and ivabradine was increasingly pronounced with embryonic maturity despite a downward trend in the number of ß-adrenergic receptors. This effect was associated with reduced cardiac output in chick embryos, probably leading to premature death. Although standard doses of these drugs appear relatively safe, high doses have a potentially adverse effect on the fetus through reduced heart rate.

Zilpaterol hydrochloride alters abundance of ß-adrenergic receptors in bovine muscle cells but has little effect on de novo fatty acid biosynthesis in bovine subcutaneous adipose tissue explants.

We predicted that zilpaterol hydrochloride (ZH), a ß-adrenergic receptor (AR) agonist, would depress mRNA and protein abundance of ß-AR in bovine satellite cells. We also predicted that ZH would decrease total lipid synthesis in bovine adipose tissue. Bovine satellite cells isolated from the semimembranosus muscle were plated on tissue culture plates coated with reduced growth factor matrigel or collagen. Real-time quantitative PCR was used to measure specific gene expression after 48 h of ZH exposure in proliferating satellite cells and fused myoblasts. There was no effect of ZH dose on [(3)H]thymidine incorporation into DNA in proliferating myoblasts. Zilpaterol hydrochloride at 1 µM decreased (P < 0.05) ß1-AR mRNA, and 0.01 and 1 µM ZH decreased (P < 0.05) ß2-AR and ß3-AR mRNA in myoblasts. The expression of IGF-I mRNA tended to increase (P = 0.07) with 1 µM ZH. There was no effect (P > 0.10) of ZH on the ß-AR or IGF-I gene expression in fused myotube cultures at 192 h or on fusion percentage. The ß2-AR antagonist ICI-118, 551 at 0.1 µM attenuated (P < 0.05) the effect of 0.1 µM ZH to reduce expression of ß1- and ß2-AR mRNA. The combination of 0.01 µM ZH and 0.1 µM ICI-118, 551 caused an increase (P < 0.05) in ß1-AR gene expression. There was no effect (P > 0.10) of ICI-118, 551 or ZH on ß3-AR or IGF-I. Western blot analysis revealed that the protein content of ß2-AR in ZH-treated myotube cultures decreased (P < 0.05) relative to control. Total lipid synthesis from acetate was increased by ZH in bovine subcutaneous adipose tissue explants in the absence of theophylline but was decreased by ZH when theophylline was included in the incubation medium. These data indicate that ZH alters mRNA and protein concentrations of ß-AR in satellite cell cultures, which in turn could affect responsiveness of cells to prolonged ZH exposure in vivo. Similar to other ß-adrenergic agonists, ZH had only modest effects on lipid metabolism in adipose tissue explants.

Probing cellular protein complexes using single-molecule pull-down.

Proteins perform most cellular functions in macromolecular complexes. The same protein often participates in different complexes to exhibit diverse functionality. Current ensemble approaches of identifying cellular protein interactions cannot reveal physiological permutations of these interactions. Here we describe a single-molecule pull-down (SiMPull) assay that combines the principles of a conventional pull-down assay with single-molecule fluorescence microscopy and enables direct visualization of individual cellular protein complexes. SiMPull can reveal how many proteins and of which kinds are present in the in vivo complex, as we show using protein kinase A. We then demonstrate a wide applicability to various signalling proteins found in the cytosol, membrane and cellular organelles, and to endogenous protein complexes from animal tissue extracts. The pulled-down proteins are functional and are used, without further processing, for single-molecule biochemical studies. SiMPull should provide a rapid, sensitive and robust platform for analysing protein assemblies in biological pathways.

Identification and characterization of ß-adrenergic receptors in isolated primary equine tracheal epithelial cells.

Responses and functions of airway epithelial cells are stimulated by ß2-agonists via the ß2-adrenergic receptors (ß2-ARs)-G(s)-protein-cAMP-system, thus, affecting airway inflammation such as in asthma and equine recurrent airway obstruction (RAO). Though horses can be used as large animal model for human asthma, evaluation of the expression and functions of the ß-AR system in primary equine airway epithelial cells has not been yet carried out. Thus, for the first time, we determined the ß-AR density and subtype distribution by [¹²5I]-iodocyanopindolol (ICYP) binding, examined ß-AR function by cAMP assay as well as their expression by western blot analysis and immunocytochemical staining in primary equine tracheal epithelial cells (ETEC). Cells were collected from 19 horses and cultured subsequently. The specific ICYP binding was saturable and of high affinity: in freshly isolated cells the receptor density (B(max)) and ICYP affinity (K(D)) for ß-ARs were 12727 ± 883 binding sites/cell and 31.78 ± 6.57 pM, respectively, and in cultured ETEC 3730 ± 212 binding sites/cell and 15.26 ± 3.37 pM, respectively. The ß-AR subtype assessed by ß1-selective (CGP 20712A) and ß2-selective (ICI 118.551) adrenergic receptor antagonists demonstrated that the ß2-AR subtype predominated (>95%) in both cell populations (p < 0.001). The ß-AR agonists increased cAMP formation with a rank order of potency: isoproterenol > epinephrine > norepinephrine. ICI 118.551 (100 nM) significantly blocked (p < 0.05) isoproterenol-induced cAMP accumulation but not CGP 20712A (300 nM). Western blot analyses and immunocytochemical staining further indicated the expression of the ß(2)-AR subtype in both cell preparations. Our data indicate that in acutely dissociated and primary cultured ETEC the ß(2)-AR-AC system is expressed, but varies considerably between the two preparations.

Diffraction cartography: applying microbeams to macromolecular crystallography sample evaluation and data collection.

Crystals of biological macromolecules often exhibit considerable inter-crystal and intra-crystal variation in diffraction quality. This requires the evaluation of many samples prior to data collection, a practice that is already widespread in macromolecular crystallography. As structural biologists move towards tackling ever more ambitious projects, new automated methods of sample evaluation will become crucial to the success of many projects, as will the availability of synchrotron-based facilities optimized for high-throughput evaluation of the diffraction characteristics of samples. Here, two examples of the types of advanced sample evaluation that will be required are presented: searching within a sample-containing loop for microcrystals using an X-ray beam of 5 microm diameter and selecting the most ordered regions of relatively large crystals using X-ray beams of 5-50 microm in diameter. A graphical user interface developed to assist with these screening methods is also presented. For the case in which the diffraction quality of a relatively large crystal is probed using a microbeam, the usefulness and implications of mapping diffraction-quality heterogeneity (diffraction cartography) are discussed. The implementation of these techniques in the context of planned upgrades to the ESRF's structural biology beamlines is also presented.

Involvement of the beta-adrenergic system in the cardiac chronic form of experimental Trypanosoma cruzi infection.

Changes in the cardiac beta-adrenergic system in early stages of Trypanosoma cruzi infection have been described. Here, we studied an early (135 days post-infection-p.i.) and a late stage (365 days p.i.) of the cardiac chronic form of the experimental infection (Tulahuen or SGO-Z12 strains), determining plasma epinephrine and norepinephrine levels, beta-receptor density, affinity and function, cardiac cAMP concentration and phosphodiesterase activity, cardiac contractility, and the presence of beta-receptor autoantibodies. Tulahuen-infected mice presented lower epinephrine and norepinephrine levels; lower beta-receptor affinity and density; a diminished norepinephrine response and higher cAMP levels in the early stage, and a basal contractility similar to non-infected controls in the early and augmented in the late stage. The Tulahuen strain induced autoantibodies with weak beta-receptor interaction. SGO-Z12-infected mice presented lower norepinephrine levels and epinephrine levels that diminished with the evolution of the infection; lower beta-receptor affinity and an increased density; unchanged epinephrine and norepinephrine response in the early and a diminished response in the late stage; higher cAMP levels and unchanged basal contractility. The SGO-Z12 isolate induced beta-receptor autoantibodies with strong interaction with the beta-receptors. None of the antibodies, however, acted a as beta-receptor agonist. The present results demonstrate that this system is seriously compromised in the cardiac chronic stage of T. cruzi infection.

Lack of specificity of antibodies directed against human beta-adrenergic receptors.

The present study was designed to investigate if antibodies against beta-adrenergic receptors (betaARs) can be used to determine expression of betaAR in human myocardium. Western blotting was performed to investigate the specificity of antibodies directed against beta(1)AR and beta(2)AR in human left ventricular tissue. A comparison was made between cardiac tissue from patients with idiopathic dilated cardiomyopathy and ischemic heart disease and nonfailing donors. The antibodies directed against beta(1)AR and beta(2)AR recognized several protein bands at different molecular weights. Moreover, both antibodies also recognized multiple proteins in Chinese hamster ovary cells expressing beta(1)AR, beta(2)AR, and even beta(3)AR. betaAR antibodies are not specific and are not suited to study expression of betaAR in human myocardium.

Tissue-type plasminogen activator release in healthy subjects and hypertensive patients: relationship with beta-adrenergic receptors and the nitric oxide pathway.

The relationship between adrenergic stimuli and NO in modulating tissue-type plasminogen activator (t-PA) release from endothelial cells was investigated in normotensive subjects and essential hypertensive patients. Sympathetic activation, a well-known stimulus for endogenous fibrinolysis, is also involved in the determination of cardiovascular risk in essential hypertension. However, the existence of cross-talk between adrenergic stimuli and NO availability in modulating t-PA release is not well established yet. We assessed the release of t-PA in the forearm microcirculation of 58 normotensive subjects (mean age: 47+/-9 years) and 44 essential hypertensive patients (mean age: 48+/-11 years) under specific intra-arterial adrenergic stimuli. Intrabrachial infusion of epinephrine (0.1 to 0.3 microg/100 mL per minute) induced greater t-PA release in normotensive subjects as compared with essential hypertensive patients (P<0.05). However, inhibition of NO synthase with N(G)-monomethyl-L-arginine (100 microg/100 mL per minute) infusion blunted epinephrine-induced t-PA release in normotensive subjects (P<0.05) but not in essential hypertensive patients. In normotensive subjects, t-PA release by epinephrine was not affected by phentolamine (8 microg/100 mL per minute) coinfusion and was abolished in the presence of propanolol (10 microg/100 mL per minute). Intrabrachial isoproterenol (0.03 microg/100 mL per minute) induced a significant increase in t-PA release (P<0.01), an effect blunted by N(G)-monomethyl-L-arginine (P<0.05). In essential hypertensive patients, the response to isoproterenol was impaired as compared with normotensive subjects and was unaffected by N(G)-monomethyl-L-arginine coinfusion. In conclusion, the results of the present study demonstrate that adrenergic-induced t-PA release is mediated by beta-adrenoreceptors via a mechanism involving the NO pathway. Our results show an impaired adrenergic-stimulated t-PA release among essential hypertensive patients, probably mediated via a reduced NO availability. This impaired fibrinolytic activity might contribute to the increased cardiovascular risk associated with hypertension.

[Cardiac receptor imaging].

Molecular imaging has been focused in the field of cardiovascular medicine. With use of suitable radiopharmaceuticals, neuronal function in the cardiovascular system has been assessed in vivo. Of particular, positron emission tomography(PET) plays an important role for in vivo quantification of various neurotransmitter and receptor functions. We have recently developed 11C-labeled CGP12177, hydrophilic beta-adrenoreceptor antagonist, to measure myocardial beta-receptor density(Bmax) in vivo. The basic study showed high uptake in the lung and myocardium which was significantly suppressed by propranolol pretreatment in the rat model, suggesting specific binding of this ligand in the beta-receptors. Bmax was significantly reduced in patients with congestive heart failure. In addition, Bmax was inversely correlated with washout rate of 123I-MIBG from the myocardium in these patients. These new imaging technique has a potential role for assessing severity of heart failure and providing appropriate treatment strategy.

A noninflammatory interleukin-1beta fragment stimulates fetal lung fluid absorption in guinea pigs.

We have previously demonstrated that full-length interleukin (IL)-1beta can induce and stimulate lung fluid absorption in near-term guinea pig fetuses via stimulation of fetal cortisol synthesis and release. To develop a potentially clinically useful drug, we tested the hypothesis that maternal administration of a noninflammatory IL-1beta-fragment (IL-1beta(Fr)) induced cortisol synthesis and stimulated lung fluid absorption in preterm fetuses. IL-1beta(Fr) was administered s.c. daily to timed-pregnant guinea pigs for 3 days with and without simultaneous cortisol synthesis inhibition by metyrapone. Fetuses were obtained by abdominal hysterotomy at 61 and 68 days gestation and instilled with isosmolar 5% albumin into the lungs, and lung fluid absorption was measured over 1 h by mass balance. Lung fluid absorption was induced at 61 days and stimulated at 68 days gestation by IL-1beta(Fr), which both were attenuated by cortisol synthesis inhibition. Moreover, induction of labor by oxytocin stimulated lung fluid absorption at 61 days but had no stimulatory effect at 68 days gestation when given with the IL-1beta(Fr). Plasma adrenocorticotropin and cortisol concentrations were increased by IL-1beta(Fr) at 61 days gestation and remained high but unstimulated by IL-1beta(Fr) at 68 days gestation, and metyrapone always reduced cortisol concentrations. Prenatal lung fluid absorption, when present as well as IL-1beta(Fr)-induced, was always propranolol- and amiloride-sensitive, suggesting that beta-adrenoceptor stimulation and the epithelial Na(+) channel (ENaC) were critical for the induced/stimulated lung fluid absorption. ENaC expression was increased by IL-1beta(Fr) and attenuated by cortisol synthesis inhibition. Thus, our results suggest a potential clinical use of IL-1beta(Fr) therapeutically to induce lung fluid absorption in fetuses at risk of preterm delivery.

Chronic direct stimulation of adenylyl cyclase induces cardiac desensitization to catecholamine and beta-adrenergic receptor downregulation in rabbits.

Chronic stimulation of beta-adrenergic receptors (betaARs) induces betaAR downregulation. However, it is not known whether continuous activation of adenylyl cyclase without direct stimulation of betaARs leads to receptor downregulation. This study investigated the effects of chronic stimulation of adenylyl cyclase with colforsin, on hemodynamic variables, and on myocardial betaAR density. In all, 55 rabbits received intravenous colforsin (1.6 microg/kg/min, n = 20), isoproterenol (ISO; 0.4 microg/kg/min, n = 16), or saline (n = 19) for two weeks. After chronic drug administration, responses of systolic (Delta% peak LV +dP/dt) and diastolic function (Delta% peak LV -dP/dt), and heart rate (Delta% heart rate), to acute administration of ISO (0.05 to 0.2 microg/kg/min) or colforsin (5 to 20 nmol/kg/min) were decreased compared to those before chronic administration. betaAR density in the colforsin group (69.8 +/- 13.8 fmol/ml protein) was less than that in the saline group (79.8 +/- 15.0 fmol/ml protein, P < 0.05), but was greater than that in the ISO group (56.3 +/- 8.4 fmol/ml protein, P < 0.05). Thus, chronic direct stimulation of adenylyl cyclase elicited systolic and diastolic functional desensitization to betaAR stimulation or adenylyl cyclase stimulation, and myocardial betaAR downregulation.