[Increased autoantibody production against AT(1)-receptors and alpha(1)-adrenergic receptors in hypertensive patients].

OBJECTIVE: To observe autoantibodies production against AT(1)-receptors and alpha(1)-adrenergic receptors and association to risk factors, such as sex, age, family history, course of hypertension and other cardiovascular diseases in hypertensive patients. METHODS: A total of 690 patients with essential hypertension admitted to our hospital were selected and autoantibodies against AT(1)-receptors and alpha(1)-adrenergic receptors were detected by ELISA. Multiple logistic regression analysis was performed based on obtained data. RESULTS: Positive rates for antibody against AT(1)-receptors and alpha(1)-adrenergic receptors were 47.1% (325/690) and 36.4% (251/690) respectively in this group of patients. Duration of hypertension history was significantly longer in the antibody against AT(1)-receptors and alpha(1)-adrenergic receptors positive groups [(9.3 +/- 11.0) year, (9.9 +/- 11.1) year] compared to the negative groups [(7.3 +/- 9.3) year, (7.2 +/- 9.5) year, all P < 0.01]. The ratio of family history with hypertension was also significantly higher in antibody positive groups than negative ones (47.69% vs 39.18%, P < 0.01). Regression analysis demonstrated that 5 risk factors were related to positive production of autoantibody against AT(1)-receptors including female gender, age, family history, duration of hypertension history and diabetes. However, just age, family history, duration of hypertension history were main factors responsible to the production of autoantibody against alpha(1)-adrenergic receptors (all P < 0.05). CONCLUSION: The environmental and genetic factors contributed to the autoantibody production in patients with essential hypertension.

The alpha-adrenoceptor mediation of the immunomodulatory effects of electroacupuncture in DNP-KLH immunized mice.

Our previous study demonstrated that successive electroacupuncture (EA) at ST36 acupoint reduces IgE production in BALB/c mice immunized with 2,4-dinitrophenylated keyhole limpet protein (DNP-KLH) by suppression of the Th2 cell lineage development. Here, we report that pretreatment of phentolamine (alpha-adrenoceptor antagonist, 10mg/kg, i.p.) completely blocks the EA-induced suppression of antigen-specific and total IgE levels in serum and IL-4 production in anti-CD3 mAb-activated splenocytes in DNP-KLH immunized mice. The results suggest that alpha-adrenoceptor play an important role in mediating the suppressive effects of EA on IgE production and Th2 cell response in DNP-KLH immunized mice.

Commercially available antibodies directed against α-adrenergic receptor subtypes and other G protein-coupled receptors with acceptable selectivity in flow cytometry experiments.

Several previous reports suggested that many commercially available antibodies directed against G protein-coupled receptors (GPCR) lack sufficient selectivity. Accordingly, it has been proposed that receptor antibodies should be validated by at least one of several criteria, such as testing tissues or cells after knockout or silencing of the corresponding gene. Here, we tested whether 12 commercially available antibodies directed against α-adrenergic receptor (AR) subtypes (α1A/B/D, α2A/B/C), atypical chemokine receptor 3 (ACKR3), and vasopressin receptor 1A (AVPR1A) suffice these criteria. We detected in flow cytometry experiments with human vascular smooth muscle cells that the fluorescence signals from each of these antibodies were reduced by 46 ± 10 %-91 ± 2 % in cells treated with commercially available small interfering RNA (siRNA) specific for each receptor, as compared with cells that were incubated with non-targeting siRNA. The tested antibodies included anti-ACKR3 (R&D Systems, mab42273), for which specificity has previously been demonstrated. Staining with this antibody resulted in 72 ± 5 % reduction of the fluorescence signal after ACKR3 siRNA treatment. Furthermore, staining with anti-α1A-AR (Santa Cruz, sc1477) and anti-ACKR3 (Abcam, ab38089), which have previously been reported to be non-specific, resulted in 70 ± 19 % and 80 ± 4 % loss of the fluorescence signal after α1A-AR and ACKR3 siRNA treatment, respectively. Our findings demonstrate that the tested antibodies show reasonable selectivity for their receptor target under our experimental conditions. Furthermore, our observations suggest that the selectivity of GPCR antibodies depends on the method for which the antibody is employed, the species from which cells/tissues are obtained, and on the type of specimens (cell, tissue/cell homogenate, or section) tested.

Anaphylatoxin C5a modulation of an alpha-adrenergic receptor system in the rat hypothalamus.

C5a anaphylatoxin injected via implanted cannulae into the perifornical region of the hypothalamus stimulated eating in sated rats. C5a also attenuated carbamyl choline-induced drinking, and carbamyl choline inhibited C5a-induced eating, a mutual inhibition characteristic of the adrenergic-cholinergic interactions at this site. The increased food intake induced by C5a was also reversed by phentolamine, an alpha-adrenergic antagonist. Granulocytes infiltrating as a result of C5a-mediated leukotaxis did not arrive at the site in time to influence C5a activity. We propose that C5a in some way activates an alpha-adrenergic receptor system in the hypothalamus, and that anaphylatoxins could mediate neuropsychiatric symptoms sometimes associated with immune complex diseases affecting the central nervous system.

Salivary secretion of immunoglobulin A by submandibular glands in response to autonomimetic infusions in anaesthetised rats.

Salivary secretion of immunoglobulin A (lgA) by submandibular glands is increased by stimuli from autonomic nerves. Since it is unclear which specific autonomic receptors transduce such stimuli, we have infused autonomimetics intravenously and compared secretion of fluid, IgA and stored proteins (peroxidase and total protein) with secretory responses during electrical stimulation of the parasympathetic nerve supply in anaesthetized rats. The greatest secretion of IgA was evoked by the alpha-adrenoceptor agonist phenylephrine and this was reduced by the beta-adrenoceptor blocking drug propranolol. The secretion of fluid or proteins but not IgA was increased with frequency of nerve stimulation and dose of methacholine (cholinergic), isoprenaline (beta-adrenergic) or phenylephrine (alpha-adrenergic).

Effects of D2-dopamine and alpha-adrenoceptor antagonists in stress induced changes on immune responsiveness of mice.

The involvement of catecholamine receptors (alpha-adrenergic, D2-dopamine (DA)) was investigated in restraint stress influenced immune responses with concomitant changes of G-protein signal transduction. Impairment of the spleen morphology, TH1/TH2 cytokine network and natural killer (NK) cell function was observed. In vivo administration of specific antagonists prior to restraint stress reversed the immunosuppression. These findings demonstrate that D2-type dopaminergic mechanism represents the dominant component in regulation of Galphas/Galphai(1,2)/Galphaq/11-protein signal transduction and contribute to cell responses at postreceptor level of both, central nervous and immune systems. G-protein-coupled receptors (GPCRs) can modulate cytokine production and may play a regulatory role in immune effector mechanisms.

Prolonged alpha-adrenergic stimulation causes changes in leukocyte distribution and lymphocyte apoptosis in the rat.

We have previously shown in the rat model that acutely or chronically increased peripheral catecholamines lead to suppression of lymphocyte responsiveness via alpha(2)-adrenoceptor activation. Here we investigated the effects of alpha-adrenergic treatment on total leukocyte numbers and proportions of leukocyte subsets in peripheral blood and lymphoid tissues. It was found that a 12-h treatment with subcutaneously implanted tablets, one containing norepinephrine (NE) and one propranolol, leads to an increase in total blood leukocyte counts, due to a pronounced increase in granulocytes. In contrast, the numbers of all classes of lymphocytes other than NK cells were decreased. This decrease in blood lymphocytes is apparently not due to redistribution, since in the thymus, spleen, mesenteric and peripheral lymph nodes, the total numbers of lymphocytes were decreased as well, without any changes in subpopulations. Analogous results were obtained with rats adrenalectomized before the catecholamine treatment. Animals that received the alpha-adrenergic treatment displayed significantly more apoptotic cells in the lymphoid organs, as determined by the TUNEL technique. In the spleen, the enhanced rate of apoptosis was confined to the white pulp; red pulp areas exhibited significantly fewer apoptotic cells. Thus, an increased alpha-adrenergic tone in rats led to a general loss of lymphocytes due to lymphocyte directed apoptosis that was independent of glucocorticoids.

The frequency of occurrence of anti-cardiac receptor autoantibodies and their correlation with clinical manifestation in patients with hypertrophic cardiomyopathy.

The purpose of this study was to investigate the frequency of occurrence of autoantibodies against G-protein coupled cardiovascular receptors and their relation to the clinical manifestation of hypertrophic cardiomyopathy (HCM). Autoantibodies against beta1-receptors, Muscarin-2-receptors, Angiotensin-II-receptor subtype 1 and alpha1-receptors were determined with ELISA in 52 patients with HCM (37 male, 15 female, mean age 55 +/- 15 years) and 40 healthy, age and sex matched controls. The clinical characterization of the HCM-patients included ECG, 24-h Holter, and echocardiography. The results showed that there is no significant difference in the frequency of a single autoantibody between HCM-patients and controls. However, if the number of patients who have autoantibodies against beta1-receptors and/or Muscarin-2-receptors were counted together, there are significantly more autoantibodies in HCM compared to controls (11 vs. 2, p = 0.035). Analysis of clinical data from this pooled group of patients showed that in patients with autoantibodies, heart rate variability (HRV), ultra low frequency (ULF) and very low frequency (VLF) were decreased (HRV by 20%, ULF by 50%, and VLF by 46%, p < 0.008) whereas the QTc-interval was increased by 8% (p < 0.02 each). The ratio of septal to posterior wall thickness was increased by 23% (p = 0.05), and the preejection period was prolonged by 46% in patients with autoantibodies (p < 0.001). These results suggest that the existence of these autoantibodies could be associated with an advanced stage or a severe manifestation of HCM.

Autoantibodies against AT1-receptor and alpha1-adrenergic receptor in patients with hypertension.

This study will explore the autoantibodies against AT1-receptor and alpha1-adrenergic receptor in patients with hypertension. Forty normotensives and 194 patients with hypertension were recruited for participation in this study. All patients accepted systemic combination drug treatment for antihypertension. According to the treatment results and the definition of refractory hypertension, the patients were divided into two groups: a refractory hypertension group and a non-refractory hypertension group. The epitope of the 2nd extracellular loop of type 1 angiotensin (AT1) receptor and alpha1-adrenergic receptor were synthesized and used as antigens to screen the autoantibodies against AT1-receptor and alpha1-adrenergic receptor by ELISA. The plasma renin activity and concentration of angiotensin II and catecholamine were also examined. The positive rates of the autoantibodies against AT1-receptor and alpha1-adrenergic receptor in patients with hypertension, 26.8% (52/194) and 25.3% (49/194), respectively, were higher than those in normotensives (7.5% and 5%)(p < 0.01). Further investigation showed that the frequencies of the autoantibodies against AT1-receptor and alpha1-adrenergic receptor in patients with refractory hypertension, 42.9% (42/98) and 36.7% (36/98), respectively, were higher than those in patients with non-refractory hypertension under systematic treatment (10.4% and 13.5%)(p < 0.01). The levels of circulating angiotensin II, catecholamine, proteinuria and serum creatine were also higher in the refractory hypertension group than in the non-refractory hypertension group. The findings showed that the frequencies of autoantibodies against AT1-receptor and alpha1-adrenergic receptor were higher in patients with hypertension, particularly in those with refractory hypertension, and that these autoantibodies might play a role in the pathogenesis of hypertension.

Monoclonal antibodies to rat brain alpha-adrenoceptors.

We have developed three hybridomas that produce monoclonal antibodies to rat cerebral alpha-adrenoceptors. Splenic lymphocytes from BALB/c mice immunized with unpurified digitonin-solubilized alpha-adrenoceptors were fused with the mouse myeloma line P3-x 63-Ag 8.653 to yield hybridoma cultures producing alpha-adrenoceptor monoclonal antibodies of the IgG class. However, these antibodies inhibited both alpha 1 and alpha 2 ligand binding suggesting that some molecular homology exists between alpha 1- and alpha 2-adrenoceptors in the rat brain.

Involvement of mu-opioid receptors and alpha-adrenoceptors in the immunomodulatory effects of dihydroetorphine.

The present study investigated the effects of acutely administered dihydroetorphine on mitogen-stimulated lymphocyte proliferation and lymphokine production in mice. These immune functions were significantly suppressed by dihydroetorphine at 24 microg/kg and 128 microg/kg in a dose-dependent fashion. This study further examined the involvement of micro-opioid receptors and alpha-adrenoceptors in the immunomodulatory effects of dihydroetorphine. The micro-opioid receptor antagonist, naloxone (4 mg/kg), and alpha-adrenoceptor antagonist, phentolamine (10 mg/kg), but not the beta-adrenoceptor antagonist, propranolol (10 mg/kg), effectively blocked dihydroetorphine-induced suppression of splenic lymphocyte proliferation and lymphokine production. These results demonstrate that dihydroetorphine has significant immunosuppressive effects in mice and the mechanisms of these effects may lie in its interactions with opioid receptors and adrenergic pathways.

Mutual modulation between norepinephrine and nitric oxide in haemocytes during the mollusc immune response.

Nitric oxide (NO) is one of the most important immune molecules in innate immunity of invertebrates, and it can be regulated by norepinephrine in ascidian haemocytes. In the present study, the mutual modulation and underlying mechanism between norepinephrine and NO were explored in haemocytes of the scallop Chlamys farreri. After lipopolysaccharide stimulation, NO production increased to a significant level at 24 h, and norepinephrine concentration rose to remarkable levels at 3 h and 12~48 h. A significant decrease of NO production was observed in the haemocytes concomitantly stimulated with lipopolysaccharide and α-adrenoceptor agonist, while a dramatic increase of NO production was observed in the haemocytes incubated with lipopolysaccharide and ß-adrenoceptor agonist. Meanwhile, the concentration of cyclic adenosine monophosphate (cAMP) decreased significantly in the haemocytes treated by lipopolysaccharide and α/ß-adrenoceptor agonist, while the content of Ca(2+) was elevated in those triggered by lipopolysaccharide and ß-adrenoceptor agonist. When the haemocytes was incubated with NO donor, norepinephrine concentration was significantly enhanced during 1~24 h. Collectively, these results suggested that norepinephrine exerted varied effects on NO production at different immune stages via a novel α/ß-adrenoceptor-cAMP/Ca(2+) regulatory pattern, and NO might have a feedback effect on the synthesis of norepinephrine in the scallop haemocytes.

Glucocorticoid-catecholamine interplay within the composite thymopoietic regulatory network.

This paper highlights the multiple putative thymic and extrathymic points of intersection and interaction between glucocorticoids (GCs) and catecholamines (CAs)--the end-point mediators of the major routes of communication between the brain and the immune system--in the context of intricate thymic T cell-developmental tuning. More specifically, we discuss in detail findings indicating that adrenal GCs can influence thymopoiesis by adjusting directly and/or indirectly (through modulation of pituitary and local ACTH synthesis) not only thymic GC synthesis, in a cell type-specific manner, but also thymic CA bioavailability (via altering CA outflow from sympathetic nerve endings and local CA synthesis), ß and α(1) -adrenoceptor (AR) expression, and/or AR-mediated intracellular signal transduction in thymic cells. In addition, this short review points to GC- and CA-sensitive stages along the multistep T cell-developmental journey and the possible effects of altered GC, and consequently CA signaling, on thymopoietic efficiency.

Potential relevance of alpha(1)-adrenergic receptor autoantibodies in refractory hypertension.

BACKGROUND: Agonistic autoantibodies directed at the alpha(1)-adrenergic receptor (alpha(1)-AAB) have been described in patients with hypertension. We implied earlier that alpha(1)-AAB might have a mechanistic role and could represent a therapeutic target. METHODOLOGY/PRINCIPAL FINDINGS: To pursue the issue, we performed clinical and basic studies. We observed that 41 of 81 patients with refractory hypertension had alpha(1)-AAB; after immunoadsorption blood pressure was significantly reduced in these patients. Rabbits were immunized to generate alpha(1)-adrenergic receptor antibodies (alpha(1)-AB). Patient alpha(1)-AAB and rabbit alpha(1)-AB were purified using affinity chromatography and characterized both by epitope mapping and surface plasmon resonance measurements. Neonatal rat cardiomyocytes, rat vascular smooth muscle cells (VSMC), and Chinese hamster ovary cells transfected with the human alpha(1A)-adrenergic receptor were incubated with patient alpha(1)-AAB and rabbit alpha(1)-AB and the activation of signal transduction pathways was investigated by Western blot, confocal laser scanning microscopy, and gene expression. We found that phospholipase A2 group IIA (PLA2-IIA) and L-type calcium channel (Cacna1c) genes were upregulated in cardiomyocytes and VSMC after stimulation with both purified antibodies. We showed that patient alpha(1)-AAB and rabbit alpha(1)-AB result in protein kinase C alpha activation and transient extracellular-related kinase (EKR1/2) phosphorylation. Finally, we showed that the antibodies exert acute effects on intracellular Ca(2+) in cardiomyocytes and induce mesentery artery segment contraction. CONCLUSIONS/SIGNIFICANCE: Patient alpha(1)-AAB and rabbit alpha(1)-AB can induce signaling pathways important for hypertension and cardiac remodeling. Our data provide evidence for a potential clinical relevance for alpha(1)-AAB in hypertensive patients, and the notion of immunity as a possible cause of hypertension.

Functional autoimmune epitope on alpha 1-adrenergic receptors in patients with malignant hypertension.

Because of the growing evidence that hypertensive disease is accompanied by immunological dysfunction, we have investigated autoimmunity in patients with malignant hypertension. Peptides corresponding to the sequence of the second extracellular loops of the human alpha 1-adrenergic receptor and the M2-muscarinic receptor were used as antigens in an ELISA. Serum from 4 (12%) of 33 healthy controls, 3 (20%) of 15 patients with malignant essential hypertension, and 7 (64%) of 11 with secondary hypertension showed positive responses in the ELISA for the alpha 1-adrenergic receptor peptide. Positive responses were significantly more common among the patients with secondary hypertension than in the other two groups (p < 0.01). By contrast, no autoantibodies against the M2-muscarinic receptor peptide were detected in either hypertensive group. Autoantibodies against the alpha 1-adrenergic receptor, affinity-purified from patients with positive responses, specifically recognised bands with molecular masses of 68, 40, and 37 kDa on immunoblotted membrane proteins of rat ventricles. The patients' autoantibodies caused a decrease in tritiated prazosin binding sites and an increase in heart beating frequency of neonatal cultured rat cardiomyocytes; antibodies purified from the controls had no effect. Circulating autoantibodies against the alpha 1-adrenergic receptor are present in a subgroup of patients with malignant hypertension. These autoantibodies have pharmacological activity in vitro, which suggests that they may be involved in the pathogenesis of malignant hypertension.

The effects of alpha and beta adrenergic agents on spleen cell antigen binding in four amphibian species.

Adults of two urodele amphibian species (Triturus cristatus carnifex and Cynops hongkongensis) and two anuran species (Rana temporaria and Xenopus laevis laevis) were immunized with a 25% suspension of sheep or horse erythrocytes. After eight or 14 days, splenic lymphocytes were removed, and their specific red cell-binding capacities tested by immunocytoadherence. Antigen-binding cells were classified as high-dose nonsecretory (S-) or secretory (S+), according to whether they bound a single layer or several layers or erythrocytes. The stimulation of both alpha and beta adrenoreceptors reduced the numbers of S+ rosettes formed by Triturus and Cynops lymphocytes, whereas a beta agonist increased and an alpha agonist decreased S+ rosette formation by Rana and Xenopus splenic lymphocytes. These effects were blocked by alpha and beta adrenoreceptor antagonists. Low-dose immunization of Xenopus with a 0.0025% suspension of sheep erythrocytes gave a minimal number of S+ rosettes two and eight days after immunization, and beta adrenoreceptor stimulation had no effect on antigen binding. These results are discussed in terms of the distribution of alpha and beta adrenoreceptors in amphibians and possible relationships between S+ and high-dose S- antigen-binding cells, and support the view that functional lymphocyte heterogeneity exists in these lower vertebrates.

Tachyphylaxis to inhaled isoproterenol and the effect of methylprednisolone in dogs.

Acute tachyphylaxis can be induced to inhaled isoproterenol (ISO) in anesthetized, closed-chested mongrel dogs. The responsiveness to ISO measured as the percent reduction in methacholine-induced bronchoconstriction was decreased significantly (p less than 0.01) after a 1-hr period of repeated ISO inhalations (ISO-loading). Intravenous administration of methylprednisolone (1 mg/kg) reversed the decrease in responsiveness to ISO.

Recent advances in the purification of the alpha 1-adrenoceptor.

In order to purify the alpha-adrenergic receptor labeled with [3H]POB, antibodies against the irreversible alpha-adrenergic antagonist, phenoxybenzamine (POB), have been raised in rabbits immunized with POB conjugated to immunoglobulin. The antigen was prepared by first reacting POB with the sulfhydryl group of cysteine, after which the POB complex was coupled to thyroglobulin in the presence of water soluble carbodimide. POB-specific antibodies were first detected 10 days after the first booster injection. These antibodies display a high degree of specificity. Slight cross-reactivity occurred only with compounds structurally related to POB, such as dibenamine and nitrogen mustards, while catecholamines and alpha-adrenergic compounds not structurally related to POB did not cross-react, even when present in 100,000-fold excess. Moreover, the antibodies are still capable of recognizing POB once bound to alpha-adrenergic receptor. When membranes, prelabeled with [3H]POB, are solubilized with digitonin and incubated with anti-POB antiserum, the radioactivity can be specifically precipitated upon addition of goat anti-rabbit gamma-globulin serum. Thus these antibodies may be a useful tool in the purification of the alpha-adrenergic receptor of rat liver covalently linked to POB.

Characterization of alpha 2-adrenergic receptor subtype-specific antibodies.

Subtypes of alpha 2-adrenergic receptors have been defined pharmacologically in a variety of mammalian tissues. The alpha 2A, alpha 2B, alpha 2C, and most recently alpha 2D subtypes have been characterized by their affinities for selective receptor antagonists and agonists. The genes that may encode the alpha 2A, alpha 2B, and alpha 2C subtypes have been identified in human and rat. In human these genes are termed alpha 2-C10, alpha 2-C2, and alpha 2-C4, respectively, based on their chromosomal localization, whereas three genes, designated RG20 alpha 2, RNG alpha 2, and RG10 alpha 2, are thought to be the corresponding rat homologues. These assignments were based on the pharmacology of the cloned receptor genes expressed in transfected cells and on the detection of homologous mRNAs by Northern blot analyses in cell lines or tissues with pharmacologically defined alpha 2-adrenergic receptors. However, the subtype assignment of cloned genes has not been fully resolved by these means. To help clarify the subtype assignment, we have raised antibodies against sequences from the divergent third intracellular loop of the human and rat alpha 2-adrenergic receptors. These antibodies were found to be subtype specific in immunoprecipitating either the cloned receptors expressed by DNA transfection or the pharmacologically defined receptors prepared from various tissues. Our immunological data corroborate the assignments of alpha 2-C2/RNG alpha 2 as encoding the alpha 2B subtype in NG108-15 cells and rat neonatal lung and of alpha 2-C4/RG10 alpha 2 as encoding the alpha 2C subtype in opossum kidney cells. Furthermore, antibodies against alpha 2-C10 and RG20 alpha 2 but not alpha 2-C2/RNG alpha 2 or alpha 2-C4/RG10 alpha 2 were both found to recognize alpha 2-adrenergic receptors expressed in rat submaxillary glands and in bovine pineal gland, two tissues with alpha 2D pharmacology. Because three genes were identified in the rat and human genome, these data suggest that the pharmacologically defined "alpha 2D receptor" is genetically of the alpha 2A subtype.