ß3-Adrenoreceptors as ROS Balancer in Hematopoietic Stem Cell Transplantation.

In the last decades, the therapeutic potential of hematopoietic stem cell transplantation (HSCT) has acquired a primary role in the management of a broad spectrum of diseases including cancer, hematologic conditions, immune system dysregulations, and inborn errors of metabolism. The different types of HSCT, autologous and allogeneic, include risks of severe complications including acute and chronic graft-versus-host disease (GvHD) complications, hepatic veno-occlusive disease, lung injury, and infections. Despite being a dangerous procedure, it improved patient survival. Hence, its use was extended to treat autoimmune diseases, metabolic disorders, malignant infantile disorders, and hereditary skeletal dysplasia. HSCT is performed to restore or treat various congenital conditions in which immunologic functions are compromised, for instance, by chemo- and radiotherapy, and involves the administration of hematopoietic stem cells (HSCs) in patients with depleted or dysfunctional bone marrow (BM). Since HSCs biology is tightly regulated by oxidative stress (OS), the control of reactive oxygen species (ROS) levels is important to maintain their self-renewal capacity. In quiescent HSCs, low ROS levels are essential for stemness maintenance; however, physiological ROS levels promote HSC proliferation and differentiation. High ROS levels are mainly involved in short-term repopulation, whereas low ROS levels are associated with long-term repopulating ability. In this review, we aim summarize the current state of knowledge about the role of ß3-adrenoreceptors (ß3-ARs) in regulating HSCs redox homeostasis. ß3-ARs play a major role in regulating stromal cell differentiation, and the antagonist SR59230A promotes differentiation of different progenitor cells in hematopoietic tumors, suggesting that ß3-ARs agonism and antagonism could be exploited for clinical benefit.

ß3 -Adrenoceptor as a potential immuno-suppressor agent in melanoma.

BACKGROUND AND PURPOSE: Stress-related catecholamines have a role in cancer and ß-adrenoceptors; specifically, ß2 -adrenoceptors have been identified as new targets in treating melanoma. Recently, ß3 -adrenoceptors have shown a pleiotropic effect on melanoma micro-environment leading to cancer progression. However, the mechanisms by which ß3 -adrenoceptors promote this progression remain poorly understood. Catecholamines affect the immune system by modulating several factors that can alter immune cell sub-population homeostasis. Understanding the mechanisms of cancer immune-tolerance is one of the most intriguing challenges in modern research. This study investigates the potential role of ß3 -adrenoceptors in immune-tolerance regulation. EXPERIMENTAL APPROACH: A mouse model of melanoma in which syngeneic B16-F10 cells were injected in C57BL-6 mice was used to evaluate the effect of ß-adrenoceptor blockade on the number and activity of immune cell sub-populations (Treg, NK, CD8, MDSC, macrophages, and neutrophils). Pharmacological and molecular approaches with ß-blockers (propranolol and SR59230A) and specific ß-adrenoceptor siRNAs targeting ß2 - or ß3 -adrenoceptors were used. KEY RESULTS: Only ß3 -, but not ß2 -adrenoceptors, were up-regulated under hypoxia in peripheral blood mononuclear cells and selectively expressed in immune cell sub-populations including Treg, MDSC, and NK. SR59230A and ß3 -adrenoceptor siRNAs increased NK and CD8 number and cytotoxicity, while they attenuated Treg and MDSC sub-populations in the tumour mass, blood, and spleen. SR59230A and ß3 -adrenoceptor siRNAs increased the ratio of M1/M2 macrophages and N1 granulocytes. CONCLUSIONS AND IMPLICATIONS: Our data suggest that ß3 -adrenoceptors are involved in immune-tolerance, which opens the way for new strategic therapies to overcome melanoma growth. LINKED ARTICLES: This article is part of a themed section on Adrenoceptors-New Roles for Old Players. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v176.14/issuetoc.

Effects of long-term active immunization with the second extracellular loop of human ß1- or ß3-adrenoceptors in thoracic aorta and mesenteric arteries in Lewis rats.

OBJECTIVE: To evaluate whether active immunization producing ß1- or ß3-antibodies (ß1-ABs and ß3-ABs) detected in sera of patients with dilated cardiomyopathies has deleterious effects on vascular reactivity in Lewis rat thoracic aorta (TA) and small mesenteric arteries (SMA). DESIGN AND METHOD: Lewis rats were immunized for 6months with peptidic sequences corresponding to the second extracellular loop of ß1- and ß3-adrenoceptors (ARs). During the immunization, systolic blood pressure (SBP) was monitored using the tail cuff method. The vascular reactivity of immunized rats was assessed by ex vivo studies on SMA and TA using various ß-AR agonists, phenylephrine and KCl. RESULTS: The immunizations producing functional ß1-ABs and ß3-ABs did not affect the SBP. However, in TA from ß1-AR-immunized rats, the relaxations mediated by dobutamine and salbutamol were significantly impaired in comparison with adjuvant rats whereas nebivolol-induced relaxation was not modified. Moreover, phenylephrine and KCl-mediated contractions were enhanced in these rats. In contrast, immunization with ß3-AR peptide led to the increase of relaxations induced by dobutamine in TA but did not change those induced by salbutamol and nebivolol. Surprisingly, in SMA from both rats immunized with ß1- or ß3-peptides, relaxations induced by the various ß-agonists were not changed whereas phenylephrine and KCl-mediated contractions were impaired. CONCLUSIONS: Our study shows that ß1- and ß3-ABs can affect vascular reactivity. ß1-ABs would have a pathogenic action whereas ß3-ABs would have a beneficial effect on aorta reactivity.

Cardiac effects of long-term active immunization with the second extracellular loop of human ß1- and/or ß3-adrenoceptors in Lewis rats.

ß1- and ß3-adrenoceptor (AR) auto-antibodies were detected in patients with dilated cardiomyopathy. Many studies have shown that ß1-AR auto-antibodies with partial agonist-like effect play an important role in the pathogenesis of this disease. Moreover, a recent study carried out in our laboratory has shown that ß3-AR antibodies (ß3-ABs), produced in rats, were able to reduce cardiomyocyte contractility via ß3-AR activation. The aims of this study were (1) to investigate, in isolated cardiomyocytes from rabbit, the role of Gi proteins in the ß3-ABs-induced cardiac negative inotropy, (2) to determine whether ß3-ABs may exhibit ß3-AR antagonistic property which is characteristic of partial agonists, and (3) to determine whether long-term active immunization producing both ß1-ABs and/or ß3-ABs leads to the development of cardiac dysfunction in Lewis rats. Lewis rats were immunized for 6 months with peptidic sequences corresponding to the second extracellular loop of human ß3-AR and/or ß1-AR. Agonistic effect of ß3-ABs was evaluated on electrically field-stimulated isolated cardiomyocytes from adult rabbit by measuring the cell shortening. Echocardiography and ex vivo isolated perfused heart studies were conducted on immunized rats. Finally, ß-AR expression was quantified by immunofluorescence and RT-qPCR. SR58611A (10 nM), a preferential ß3-AR agonist, and purified ß3-ABs (25 µg/ml) induced a decrease in cell shortening (-39.71±4.9% (n=10) and -17.06±3.9% (n=10) respectively). This effect was significantly inhibited when the cardiomyocytes were preincubated with pertussis toxin (0.3 µg/ml), a Gi protein inhibitor (p<0.05). In addition, SR58611A-mediated negative inotropic effect was decreased when cardiomyocytes were preincubated with ß3-ABs (p<0.0001). Echocardiography revealed a decrease in the fractional shortening and ejection fraction in rats immunized against ß1-AR and both ß1- and ß3-AR. However, the study on isolated heart showed a decrease of the isoproterenol-induced lusitropic and inotropic effects in the 3 groups of immunized rats. These systolic and diastolic dysfunctions are correlated with a decrease in the expression of ß1-ARs and an increase of ß3-ARs in rats immunized against the ß1-AR and an increase of both ß3-AR and ß1-AR in rats immunized against the ß3-AR. For the first time, these results showed that ß3-ABs had a ß3-AR partial agonist-like activity which might play a role in the pathogenesis of cardiac dysfunction.

The expression of ß3-adrenoceptor and muscarinic type 3 receptor immuno-reactivity in the major pelvic ganglion of the rat.

Bladder afferent outflow, linked to sensation, plays a critical role in bladder pathology: abnormal outflow results in altered sensation, leading to increased voiding frequency, urge and often incontinence. ß3-adrenoceptor agonists have been suggested to be beneficial in treating these symptoms. However, the absence of a significant sympathetic innervation of the detrusor and only a modest relaxation of bladder muscle by ß3 agonists has questioned the therapeutic site of action of ß3 agonists in the bladder. The present study was done to explore the possibility that ß3-adrenoceptors might be located in the pelvic plexus. Using the rat, where the pelvic plexus is located primarily within a single ganglion, the major pelvic ganglion (MPG), immuno-histochemical approaches were used to identify structures expressing ß3-adrenoceptor immuno-reactivity (ß3AR-IR). The only structures found to express ß3AR-IR were small-diameter tyrosine hydroxylase and vesicular mono-amine transporter immuno-reactive (TH-IR and vmat-IR) neurones. These neurones, found in clusters or singly on the periphery of the ganglion, or dispersed in smaller clumps throughout the MPG, are similar to the small intensely fluorescent (SIF) cells described previously. Not all small cells expressed ß3AR-IR. A population of the small cells were also immuno-reactive to the type 3 muscarinic receptor (M3R-IR) and the P2X3 purinergic receptor (P2X3-IR). Clumps of small cells were associated with calcitonin gene-related peptide immuno-reactive (CGRP-IR) nerve fibres (putative sensory fibres) and a small number were contacted by putative cholinergic nerves expressing immuno-reactivity to vesicular acetylcholine transporter (vacht-IR). These observations are consistent with the idea that small cells are interneurons and one of the components making up complex neural circuits within the MPG. The precise physiological role of these neural elements in the MPG is unknown. However, as one therapeutic action of ß3-adrenoceptor agonists is to modulate sensation, it is possible that these neural circuits may be involved in the regulation of afferent outflow and sensation.

Rat ß3-adrenoceptor protein expression: antibody validation and distribution in rat gastrointestinal and urogenital tissues.

ß3-Adrenoceptors play important roles in the regulation of urogenital and probably gastrointestinal function. However, despite recent progress, their detection at the protein level has remained difficult due to a lack of sufficiently validated selective antibodies. Therefore, we have explored the selectivity of two antibodies for the detection of rodent ß3-adrenoceptors in immunoblots and immunohistochemistry. Of two reportedly promising candidates, antibody AB15688 did not exhibit subtype selectivity in immunoblots. In contrast, the antibody Sc1473 exhibited at least some selectivity in immunoblots and more promising results in immunocytochemical and immunohistochemical stains in cells transfected with cloned ß-adrenoceptor subtypes and in rat and mouse tissues. In a systematic screening of rat gastrointestinal and urogenital tissues, Sc1473 produced selective staining in the epithelial cell lining of the stomach and the urothelium of ureter and bladder. We conclude that the two tested antibodies are inappropriate or at least insufficient for immunoblotting applications, but Sc1473 appears to be useful for immunohistochemical detection of ß3-adrenoceptor protein in rodent tissues. The ß3-adrenoceptor protein exhibits a distinct expression pattern in the rat gastrointestinal and urogenital tract, which is at least partly in line with previously reported functional data.

[Cardiac protective effect of the autoantibody against ß3-adrenoceptor in rats with experimental heart failure].

OBJECTIVE: To explore the effect of the autoantibody against the ß3-adrenoceptor on rats with experimental heart failure. METHOD: The peptide corresponding to the sequence of ß3 adrenoceptor was synthesized to actively immunize the rats, ELISA was used to detect the serum level of autoantibody against the ß3-adrenoceptor (ß3AA). Total IgGs were extracted from the serum containing ß3AA in immunized rats. Aortic banding surgery was used to establish the heart failure model in male Wistar rats and rats were divided into the sham group (n = 8), heart failure group(n = 8),ß3AA-immunized heart failure group (HF+ß3AA, n = 8) and corresponding negative IgG-immunized heart failure group (HF+ IgG, n = 8).In 6 weeks and 8 weeks after aortic banding surgery, the serum levels of NT-pro brain natriuretic peptide (NT-proBNP) were assayed with ELISA assay and cardiac function was assessed by echocardiography. RESULTS: ß3AA was used to immunize rat with heart failure, the serum level of ß3AA was stable at 50 days post immunization. At 8 weeks after aortic banding surgery, heart failure group showed significantly increased LVEDD [(6.92 ± 0.22) mm vs.(5.62 ± 0.19) mm, P < 0.001], LVESD [(4.63 ± 0.23) mm vs.(3.50 ± 0.20) mm, P < 0.01] and IVS [(2.44 ± 0.06) mm vs.(2.28 ± 0.05) mm, P < 0.05], and decreased LVEF[(62.07 ± 3.99)% vs.(79.63 ± 3.02)%, P < 0.01] and LVFS [(31.46 ± 3.22)% vs.(43.65 ± 2.68) %, P < 0.05] compared with the sham group.HF+ß3AA IgG group showed decreased LVEDD [(6.07 ± 0.30) mm vs.(6.92 ± 0.24) mm, P < 0.05] and LVESD [(3.92 ± 0.22) mm vs.(4.68 ± 0.23) mm, P < 0.05], and higher LVEF [(70.29 ± 1.78)% vs.(61.95 ± 3.03)%, P < 0.05] and LVFS [(38.08 ± 2.32)% vs.(30.50 ± 1.82)%, P < 0.05] compared to the HF+ IgG group.In addition, compared with the HF+ IgG group, HF+ß3AA IgG group showed decreased serum levels of NT-proBNP [(196.43 ± 6.56) pg/ml vs.(242.13 ± 7.86) pg/ml, P < 0.01]. CONCLUSION: Our results demonstrate that ß3AA can improve cardiac function and reduce the serum levels of NT-proBNP in rat with heart failure.

[Cardiovascular effects of beta1 and beta3-adrenergic receptor autoantibodies in Lewis rat]. / Effets cardiovasculaires des auto-anticorps anti-récepteurs béta1 et béta3-adrénergiques chez le rat Lewis.

PURPOSE: To analyze vascular reactivity changes in response to immunization protocols with antigens corresponding to the second extracellular loop of -ß3 and -ß1 and 3 adrenergic receptors (AR). METHODS: Lewis rats were immunized for 3months with peptidic sequences corresponding to the second extracellular loop of ß3-AR or ß1 and 3-AR. Specific ß3-AR antibodies were characterized by Elisa and purified using "Proteus Protein G" kit. Their functionality were tested in rabbit isolated ventricular cardiomyocytes. Aortic and mesenteric artery rings isolated from control or immunized rats were mounted in organ baths and precontracted with phenylephrine. Then, relaxant curves were established. RESULTS: SR58611A (10nM), a preferential ß3-AR agonist and purified ß3-AR antibodies (25µg/mL) induced a decrease of cell shortening (-39.56±4.4% [n=11] and -18.45±3.9% [n=10] respectively) in isolated cardiomyocytes. This decrease was significantly inhibited when the cardiomyocytes were pre-incubated with the L-748337 (1µM), a selective ß3-AR antagonist (P<0.05). In contrast with what was observed in rats immunized against the ß1-AR, vasorelaxations induced by acetylcholine and SR58611A in both aorta and mesenteric arteries were unaltered in rats immunized against the ß3-AR and ß1 and 3-AR. CONCLUSION: These results show, for the first time, that ß3-AR antibodies induced a ß3-AR agonist-like activity. They would not have a vascular pathogenic action but would offset the endothelial dysfunction caused by ß1-AR antibodies.

Regulation of cardiac ß3-adrenergic receptors in hyperglycemia.

Beta-adrenoceptors (ß-AR), members of the G protein-coupled receptors play important roles in the regulation of heart function. A positive inotropic action of catecholamines is mediated through their interaction with ß-AR, located on the sarcolemma, while they can also mediate some deleterious effects, such as cardiac arrhythmias or myocardial apoptosis. The well-known ß-AR-associated signaling in heart is composed of a coupled mechanism among both ß1- and ß2-AR and stimulatory G protein (G(s)). This coupled mechanism further leads to the activation of adenylyl cyclase and thereby increases in intracellular cAMP level. However, recent studies have emphasized the contribution of constitutive ß3-AR coupling to G(i) proteins, thereby initiating additional signal transduction pathways, particularly under physiopathological conditions. Diabetic cardiomyopathy, as a distinct entity is recognized due to its diminished responsiveness to ß1-AR agonist stimulation in the heart from diabetic rats with no important changes in the responses mediated with ß2-AR. Furthermore, an upregulation of ß3-AR has been shown in diabetic rat heart with a strong negative inotropic effect on left ventricular function. Experimental data provide evidences that the mechanisms for the negative inotropic effect with ß3-AR activation appear to involve a pertussis toxin (PTX)-sensitive G protein and the activation of a nitric oxide synthase pathway. On the other hand, ß-blockers demonstrate marked beneficial effects in heart dysfunction with scavenging free radicals and/or acting as an antioxidant with both sex- and dose-dependent manner. However, further investigations are needed to clarify the roles of both altered expression and/or responsiveness of ß-AR and the benefits with ß-blocker treatment in diabetes. This review discusses the role of ß-AR activation, particularly ß3-AR in cardiac pathological remodeling under hyperglycemia.

Autoantibodies against the ß3-adrenoceptor protect from cardiac dysfunction in a rat model of pressure overload.

ß3-Adrenoceptors (ß3-ARs) mediate a negative inotropic effect in human ventricular cardiomyocytes, which is opposite to that of ß1- and ß2-ARs. It has been previously demonstrated that autoantibodies against the ß1/ß2-AR exist in the sera of some patients with heart failure (HF) and these autoantibodies display agonist-like effects. Our aim in this study was to observe whether autoantibodies against the ß3-AR (ß3-AR Abs) exist in the sera of patients with HF and to assess the effects of ß3-AR Abs on rat model of pressure overload cardiomyopthy. In the present study, the level of ß3-AR Abs in the sera of HF patients was screened by ELISA. ß3-AR Abs from HF patients were administrated to male adult rats with abdominal aortic banding (AAB), and the cardiac function was measured by echocardiographic examination and hemodynamic studies. The biological effects of this autoantibody on cardiomyocytes were evaluated using a motion-edge detection system, intracellular calcium transient assay, and patch clamp techniques. Compared to healthy subjects, the frequency of occurrence and titer of ß3-AR Abs in the sera of HF patients were greatly increased, and ß3-AR Abs could prevent LV dilation and improve the cardiac function of rats with AAB. ß3-AR Abs exhibited negative chronotropic and inotropic effects and were accompanied by a decreased intracellular Ca(2+) transient and membrane L-type Ca(2+) current in cardiomyocytes. Our results demonstrated the existence of ß3-AR Abs in the sera of patients with HF and found that this autoantibody could alleviate the cardiac dysfunction induced by pressure-overload in AAB rats.

Relationship between the autoantibody and expression of ß3-adrenoceptor in lung and heart.

BACKGROUND: Evidences suggest that ß3 -adrenoceptor (ß3-AR) plays an important role in heart failure (HF), although no data is reported indicating how these effects may change with the increasing age. Pulmonary congestion and edema are the major life-threatening complications associated with HF. The purpose of this study is to explore the relationship between the anti-ß3-AR autoantibody and the expression of ß3-AR in the lungs and heart for both aged patients and rats with HF. METHODS: Synthetic ß3-AR peptides served as the target antigens in ELISA were used to screen the anti-ß3-AR autoantibody in aged patients and rats. Two aged rat models were constructed based on aortic banding and sham-operation. The expression of ß3-AR mRNA and protein in the lung and heart was measured in intervention and non-intervention groups by Western blot analysis at the baseline, 5(th), 7(th), 9(th) and 11(th) week, respectively. RESULTS: The frequency and titer of anti-ß3-AR autoantibody in aged patients and rats with HF were higher than those in the control group (p<0.05). The expression of ß3-AR mRNA and protein in pulmonary tissues decreased continually from the 7(th) week (p<0.05), followed by HF observed during the 9(th) week. The expression of ß3-AR in myocardial tissues continued to increase after the 9(th) week (p<0.05), and the expression of both ß3-AR mRNA and protein in the BRL group [HF group with BRL37344 (4-[-[2-hydroxy-(3-chlorophenyl)ethyl-amino] phenoxyacetic acid) (a ß3-AR agonist) injection] was positively correlated with BRL37344 when compared with non-BRL group (HF group without BRL37344 injection) (p<0.05). CONCLUSION: Anti-ß3-AR autoantibody was detected in aged patients and rats with HF. The expression of ß3-AR mRNA and protein in pulmonary tissues decreased continually, and began earlier than in the heart, but its expression in myocardial tissues increased continually and could be further promoted by ß3-AR agonist.

Regulation of endothelial nitric-oxide synthase (NOS) S-glutathionylation by neuronal NOS: evidence of a functional interaction between myocardial constitutive NOS isoforms.

Myocardial constitutive No production depends on the activity of both endothelial and neuronal NOS (eNOS and nNOS, respectively). Stimulation of myocardial ß(3)-adrenergic receptor (ß(3)-AR) produces a negative inotropic effect that is dependent on eNOS. We evaluated whether nNOS also plays a role in ß(3)-AR signaling and found that the ß(3)-AR-mediated reduction in cell shortening and [Ca(2+)](i) transient amplitude was abolished both in eNOS(-/-) and nNOS(-/-) left ventricular (LV) myocytes and in wild type LV myocytes after nNOS inhibition with S-methyl-L-thiocitrulline. LV superoxide (O(2)(·-)) production was increased in nNOS(-/-) mice and reduced by L-N(ω)-nitroarginine methyl ester (L-NAME), indicating uncoupling of eNOS activity. eNOS S-glutathionylation and Ser-1177 phosphorylation were significantly increased in nNOS(-/-) myocytes, whereas myocardial tetrahydrobiopterin, eNOS Thr-495 phosphorylation, and arginase activity did not differ between genotypes. Although inhibitors of xanthine oxidoreductase (XOR) or NOX2 NADPH oxidase caused a similar reduction in myocardial O(2)(·-), only XOR inhibition reduced eNOS S-glutathionylation and Ser-1177 phosphorylation and restored both eNOS coupled activity and the negative inotropic and [Ca(2+)](i) transient response to ß(3)-AR stimulation in nNOS(-/-) mice. In summary, our data show that increased O(2)(·-) production by XOR selectively uncouples eNOS activity and abolishes the negative inotropic effect of ß(3)-AR stimulation in nNOS(-/-) myocytes. These findings provide unequivocal evidence of a functional interaction between the myocardial constitutive NOS isoforms and indicate that aspects of the myocardial phenotype of nNOS(-/-) mice result from disruption of eNOS signaling.

Specificity evaluation of antibodies against human ß3-adrenoceptors.

ß(3)-Adrenoceptors are a promising drug target for the treatment of urinary bladder dysfunction, but knowledge about their expression at the protein level and their functional role is limited, partly due to a lack of well validated tools. As many antibodies against G-protein-coupled receptors, including those against ß(3)- and other ß-adrenoceptor subtypes, lack selectivity for their target, we have evaluated the specificity of five antibodies raised against the full-length protein of the human ß(3)-adrenoceptor (H155-B01), its N-terminus (LSA4198 and TA303277) and its C-terminus (AB5122, Sc1472) in immunoblotting and immunocytochemistry. Our primary test system were Chinese hamster ovary cells stably transfected to express each of the three human ß-adrenoceptor subtypes at near physiological levels (100-200 fmol/mg protein). None of the five antibodies exhibited convincing target specificity in immunoblotting with Sc1472 apparently being least unsuitable. In immunocytochemistry, LSA4198 and Sc1472 appeared most promising, exhibiting at least some degree of specificity. As these two antibodies have been raised against different epitopes (N- and C-terminus of the receptor, respectively), we propose that concordant staining by both antibodies provides the most convincing evidence for ß(3)-adrenoceptor labelling in cyto- or histochemistry studies.

Immunoexpression of adrenergic receptors in detrusor from patients with prune belly syndrome: a digital quantification.

INTRODUCTION: Prune belly syndrome (PBS) presents with large-capacity bladders, high compliance and post-void residual volumes. Operative and conservative treatments are controversial. When histologically compared to normal bladder, bladder outlet obstruction results in an up- or down-regulation of adrenoceptors. Our goal was to study the immunoexpression of adrenoceptors in detrusor from patients with PBS. MATERIALS AND METHODS: Bladder domes from PBS patients (n=14) were studied (PBG). For normal controls, bladder specimens were obtained at adult surgery (n=13) (CG1) and at child autopsy (n=5) (CG2). Staining was performed using antibodies to alpha1a, alpha1b, alpha1d and beta3 adrenoceptors. Five to 10 images were captured on an optic microscope with a digital camera and analysed with Photoshop. The immunocyhistochemical index with arbitrary units was calculated and compared. RESULTS: Mean age was 1.28, 64 and 1.41 years for PBG, CG1 and CG2, respectively. The immunohistochemical index with arbitrary units of alpha1a receptors was 0.06 in PBG, 0.16 in CG1 and 0.14 in CG2 (p=0.008); of alpha1b 0.06, 0.06 and 0.07 (p=0.781); and of alpha1d 0.04, 0.04 and 0.05 (p=0.618). Regarding beta3 the respective values were 0.07, 0.14 and 0.10 (p=0.378). CONCLUSION: Our results show a decrease in alpha1a-adrenoceptor immunostaining intensity in detrusor from children with PBS. Further in vitro studies are needed to determine whether these observations are physiologically significant.

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.

Stimulation of the ADRB3 adrenergic receptor induces relaxation of human placental arteries: influence of preeclampsia.

Preeclampsia, which complicates 3-8% of pregnancies, is one of the leading causes of neonatal morbidity and mortality. Its pathophysiology remains unclear. The aim of the present study was to investigate the presence and the role of beta2- and beta2-adrenergic receptors (ADRB2 and ADRB3, respectively) in human placental arteries and to assess the influence of preeclampsia on ADRB responsiveness. SR 59119A, salbutamol, and isoproterenol (ADRB3, ADRB2, and nonselective ADRB agonists, respectively) induced a concentration-dependent relaxation of placental artery rings obtained from women with uncomplicated or preeclamptic pregnancies. SR 59119A-induced relaxation was unaffected by the blockade of ADRB1 and ADRB2 by 0.1 microM propranolol but was significantly decreased by the blockade of ADRB1, ADRB2, and ADRB3 by 10 microM propranolol. Both SR 59119A and salbutamol were associated with a significant increase in cAMP production that was significantly inhibited by pretreatment with 0.1 microM propranolol only for salbutamol. SR 59119A-induced relaxation (E(max) = 28% +/- 5% vs. 45% +/- 4%, respectively) and cAMP production (2.7 +/- 0.5 vs. 4.9 +/- 0.4 pmol/mg of protein, respectively; P < 0.01) were decreased in arteries obtained from preeclamptic compared to normotensive women. Both ADRB2 and ADRB3 transcripts were expressed at the same level between arteries from normotensive and preeclamptic women. Western blot analysis, however, revealed a decreased expression of the ADRB3 immunoreactive protein in arteries from preeclamptic compared to normotensive women. We suggest the presence of functional ADRB2 and ADRB3 in human placental arteries. Even if preeclampsia is associated with an impairment of the ADRB3 responsiveness, ADRB3 agonists may have future pharmaceutical implications in the management of pregnancy-related disorders.

[Distribution and property of anti-beta3-adrenoceptor autoantibody in patients with heart failure].

OBJECTIVE: To investigate the biological effects of anti-beta(3) adrenoceptor (beta(3)-AR) autoantibody in the serum of patients with heart failure, which may contribute to a new therapeutic clue for heart failure. METHODS: The synthetic peptide of the second extracellular loop of the beta(3)-AR was used as the antigen to screen sera of patients with heart failure and of healthy controls by using enzyme-linked immunosorbent assay. IgG in the patients group of positive autoantibody sera was prepared by using a MabTrap Kit (Amersham) following the manufacturer's instructions. The effects of IgG per each group both on contractile response of adult isolated cardiomyocytes and on beating frequency of cultured neonatal rat cardiomyocytes were observed. RESULTS: The positive rate of anti-beta(3)-AR autoantibody was 26.7% (mean antibody titer: 1:43.27 +/- 2.71) or 11.0% (mean antibody titer: 1:14.59 +/- 1.61) in patients or healthy subjects, respectively P < 0.05. Compared with the control group, the autoantibody against beta(3)-AR from the patients group decreased cell shortening amplitude/cell shortening 3.84% +/- 0.33%, the velocity of shortening -0.47 microm/s +/- 0.07 microm/s and relengthening 0.17 microm/s +/- 0.02 microm/s in adult isolated cardiac myocytes, respectively. The autoantibody in the patients group decreased the beating rate in cultured neonatal rat cardiac myocytes by 47.1 beats/min +/- 8.11 beats/min, which could have a 6-hour continuance. This decreasing was not modified by Nadolol (beta(1)-AR and beta(2)-AR antagonist) in pretreating myocytes, but was nearly prevented by Bupranolol (nonselective beta-AR antagonist) or beta(3)-AR specific antigen. CONCLUSION: It seems reasonable to state that a high titer of the autoantibody against beta(3)-AR in the serum in patients with heart failure, which could have a negative inotropic and chronotropic effect, may be a part of pathophysiological mechanisms of heart failure.