Leonor Sterin-Borda

Circulating antibodies against rat parotid gland M3 muscarinic receptors in primary Sjögren's syndrome.

In this study we demonstrate that IgG present in the sera of patients with primary Sjögrens syndrome (PSS) could bind and activate muscarinic acetylcholine receptors (mAChRs) of rat parotid gland. These antibodies were able to inhibit in a non‐competitive manner the binding of 3H‐quinuclidinyl benzilate (QNB) to  mAChRs of purified rat parotid gland membranes. Moreover, IgG from PSS could modify biological effects mediated by  mAChR activation; i.e. decrease cAMP, increase phosphoinositide turnover without affecting cGMP. Atropine and 4‐DAMP blocked all of these effects, and carbachol mimicked them, confirming the M3 subtype  mAChRs mediated PSS IgG action. Neither binding nor biological effect were obtained with IgG from sera of normal women. The prevalence of cholinergic antibody was 100% in PSS, and was independent of Ro/SS‐A and La/SS‐B antibodies. It could be concluded that antibody against  mAChRs may be another serum factor to be considered in the pathophysiology of the development of PSS.

Mitogenic effect of erythropoietin on neonatal rat cardiomyocytes: Signal transduction pathways

The mitogenic effect of recombinant human erythropoietin (rHuEpo) on primary cultures of neonatal rat cardiac myocytes was observed. rHuEpo triggered a dose‐dependent increase in myocyte proliferation. The hormone effect over optimally grown control culture 1 day after addition was maximum with 0.5 U/ml and was inhibited with anti‐rHuEpo. Inhibitors of enzymatic pathways known to be involved in the cytokines intracellular mechanism such as genistein (tyrosine kinase inhibitor), 2‐nitro‐4‐carboxiphenyl‐N,N‐diphenylcarbamate (phospholipase C [PLC] inhibitor), and 1‐(5‐isoquinolinylsufonyl)‐2‐methyl‐piperazine (protein kinase C [PKC] inhibitor) prevented the mitogenic action of rHuEpo. Also the inhibition of Na+‐K+‐ATPase activity by ouabain blunted the stimulatory action of rHuEpo on cell proliferation. The mitogenic action of the hormone was correlated with cardiac membrane paranitrophenilphosphatase (pNPPase) and PKC activity, since concentrations of rHuEpo that stimulate DNA synthesis increased pNPPase and PKC activity. Moreover, the enzymatic inhibition of tyrosine kinase, PLC, and PKC attenuated the stimulatory action of rHuEpo upon cardiac pNPPase activity. In this paper we demonstrate a non‐hematopoietic action of rHuEpo showing both mitogenic and enzymatic effect upon primary myocyte cell culture and on pNPPase activity of neonatal rat heart. These effects are related to the capacity of rHuEpo to stimulate Na+‐K+‐ATPase activity and appear to be secondary to the activation of tyrosine kinase and PKC, indicating that in the rHuEpo mediated mitogenic action on cardiomyocytes involves the activation of the same enzymatic pathways that have been described by other cytokines in different tissues.

Antibodies against cerebral M1 cholinergic muscarinic receptor from schizophrenic patients: molecular interaction.

We demonstrated the presence of circulating Abs from schizophrenic patients able to interact with cerebral frontal cortex-activating muscarinic acetylcholine receptors (mAChR). Sera and purified IgG from 21 paranoid schizophrenic and 25 age-matched normal subjects were studied by indirect immunofluorescence, flow cytometry, immunoblotting, dot blot, ELISA, and radioligand competition assays. Rat cerebral frontal cortex membranes and/or a synthetic peptide, with an amino acid sequence identical with that of human M1 mAChR, were used as Ags. By indirect immunofluorescence and flow cytometry procedures, we proved that serum-purified IgG fraction from schizophrenic patients reacted to neural cell surfaces from rat cerebral frontal cortex. The same Abs were able to inhibit the binding of the specific M1 mAChR radioligand [3H]pirenzepine. Immunoblotting experiments showed that IgG from schizophrenic patients revealed a band with a molecular mass coincident to that labeled by an anti-M1 mAChR Ab. Using synthetic peptide for dot blot and ELISA, we demonstrated that these Abs reacted against the second extracellular loop of human cerebral M1 mAChR. Also, the corresponding affinity-purified antipeptide Ab displayed an agonistic-like activity associated to specific receptor activation, increasing cyclic GMP production and inositol phosphate accumulation, and protein kinase C translocation. This paper gave support to the participation of an autoimmune process in schizophrenia.

Antibodies against astrocyte M1 and M2 muscarinic cholinoceptor from schizophrenic patients' sera.

We demonstrated the presence of circulating antibodies from schizophrenic patients able to interact with cultured astrocytes activating muscarinic acetylcholine receptors (mAChRs). Sera and purified IgG from 15 paranoid schizophrenic and 15 age‐matched normal subjects were studied by indirect immunofluorescence (IFI), flow cytometry, dot blot, enzyme immunoassay (ELISA), and radioligand competition assays. Astrocyte membranes and/or a synthetic peptide, with identical amino acid sequence of human M1 and M2 mAChR, were used as antigens. By IFI and flow cytometry procedures, we proved that serum purified IgG fraction from schizophrenic patients, reacted to astrocyte cell surface. The same antibodies were able to inhibit the binding of the specific mAChR radioligand 3H‐QNB. Using synthetic peptide for dot blot and ELISA, we demonstrated that these antibodies reacted against the second extracellular loop of human cerebral M1 and M2 mAChR. Also, the corresponding affinity‐purified antipeptide antibody displayed an agonistic‐like activity associated to specific M1 and M2 mAChR activation, increasing inositol phosphates accumulation and decreasing cyclic AMP production, respectively. This article gives support to the participation of an autoimmune process in schizophrenia disease.

Novel insight into the mechanisms involved in the regulation of the m1 muscarinic receptor, iNOS and nNOS mRNA levels.

In this paper we have determined the different signaling pathways involved in M(1) muscarinic acetylcholine receptor (mAChR)-dependent stimulation of m1 mAChRs, neural and inducible isoforms of nitric oxide synthase (nNOS and iNOS)-mRNA gene expression of rat frontal cortex. Carbachol-stimulation of M(1) mAChRs exerts an increase in m1 mAChR-mRNA, activation of phosphoinositide (PI) turnover, translocation of protein kinase C (PKC) and stimulation of NOS activity. Inhibitors of phospholipase C (PLC), calcium/calmodulin and NOS, but not guanylate cyclase, prevent the carbachol-dependent increase of m1 mAChR-mRNA levels. These inhibitors also attenuate the muscarinic receptor-dependent increase in nNOS and iNOS mRNA levels. These results suggest that carbachol-activation of M(1) mAChRs increases m1 mAChR, nNOS and iNOS mRNA levels associated with increased production of nitric oxide (NO). The mechanism appears to occur secondarily to stimulation of PI turnover via PLC activation. This in turn, triggers a cascade reaction involving calcium/calmodulin and PKC, leading to activation of NOS. On the basis of our results, the activation of M(1) mAChRs appears to induce nNOS and iNOS expression and, reciprocally, the activator of NOS up-regulates m1 mAChR gene expression. These results may contribute to a better understanding of the effects and side effects of cholinomimetic treatment in patients with neurodegenerative diseases.

Alterations in cardiac beta-adrenergic receptors in chagasic mice and their association with circulating beta-adrenoceptor-related autoantibodies

OBJECTIVE Cardiac tissue from chagasic mice was studied to evaluate the expression and biological activity of beta-adrenoceptors in association with circulating beta-adrenoceptor-related autoantibodies. METHODS BALB/c inbred mice that were either treated or not treated with atenolol (2.5 mg/kg) and infected or not infected with 1 x 10(4) trypomastigotes (CA-1 strain) were sacrificed weekly up to week nine. Morphological, binding and contractility studies were performed on the four different groups of animals. The effect of their serum antibodies was also assayed in binding and contractility studies on normal heart preparations. RESULTS Hearts from chagasic myocarditis mice showed a beta-adrenoceptor-related dysfunction, with a decrease in heart contractility, impaired response to exogenous beta-adrenoceptor agonist and a significant reduction in beta-adrenergic binding sites. Those effects were maximum at eight-nine weeks post-infection and were improved by treating infected mice with atenolol. In addition, serum or IgG from chagasic myocarditis mice was capable of interacting with cardiac beta-adrenoceptors, reducing the number of binding sites and inhibiting the contractile response to exogenous norepinephrine. IgG effects that were observed in normal myocardium, were highest in sera from mice eight-nine weeks post-infection and correlate with the degree of myocarditis. Moreover, chagasic autoantibodies from infected mice recognized a peptide corresponding to the sequence of the second extracellular loop of the human beta 1-adrenoceptor. CONCLUSIONS (1) The development of alterations in beta-adrenergic receptors, related to cardiac dysfunction, may be associated with the presence of circulating antibodies against these receptors and (2) it is possible that the chronic deposits of these autoantibodies in cardiac beta-adrenoceptors could lead to a progressive blockade with sympathetic denervation, a phenomenon that has been described in the course of chagasic myocarditis.

Autoantibodies against cerebral muscarinic cholinoceptors in Sjögren syndrome: functional and pathological implications

Previous studies have demonstrated that antibodies against muscarinic acetylcholine receptors (mAChRs) from exocrine glands, correlates with Sjögren syndrome (SS) in the majority of patients. The aim of the present investigation was to establish if serum IgG antibodies present in SS interacts with cerebral mAChRs. Results show that anti-cerebral IgG are present in the sera of 40% SS patients studied. Autoantibodies were able to interact with mAChRs of cerebral frontal cortex membranes inhibiting the [(3)H]QNB binding to its specific receptor. Moreover, tested by ELISA and dot blot they recognized the synthetic peptides corresponding to the second extracellular loop of human M(1) and M(3) mAChR. In addition, the corresponding affinity-purified anti-M(1) and anti-M(3) peptide IgGs displayed an agonistic activity, stimulating phosphoinositide hydrolysis. The results support the notion that serum IgG autoantibodies in SS patients target cerebral mAChRs may have some role in the pathogenesis of higher cognitive dysfunction present in SS patients.

Activation of beta3 adrenergic receptor decreases DNA synthesis in human skin fibroblasts via cyclic GMP/nitric oxide pathway.

Background: Evidences have shown that β1 and β2 adrenoceptors co-exist in human fibroblasts, but it is not yet clear the functional expression of β3 adrenoceptor in these cells. The aim of this study was to investigate the expression and biological effect of β3 adrenoceptor activation in human skin fibroblast and the different signaling pathways involved in its effect. Methods: For this purpose in vitro cultures of human skin fibroblast were established from human foreskin and grown in Dulbecco’s modified Eagle’s medium. The effect of ZD 7114 (β3 agonist) on cell DNA synthesis, radioligand binding assay, cyclic GMP and cyclic AMP accumulation and nitric oxide synthase (NOS) activity were evaluated. Results: 3H-CGP binding to human fibroblast membranes was a saturable process to a single class of binding site. The equilibrium parameters were: Kd 20±3 pM and Bmax 222±19 fmol/mg protein. Ki values showed that these cells express a high number of β3adrenoceptor subtypes. ZD 7114 stimulation of β3 adrenoceptor exerts a concentration-dependent inhibition of DNA synthesis and cAMP accumulation with parallel increase in NOS activity that led to cGMP accumulation. All these effects were blocked by the β3 adrenoceptor antagonist (SR 59230A). The effect of ZD 7114 on DNA synthesis significantly correlated with its action either on cAMP or NOS-cGMP signaling system. Inhibitors of NOS activity and NO-sensitive guanylate cyclase prevented the inhibitory effect of ZD 7114 on DNA synthesis. In addition, the β3 adrenoceptor-dependent increase in cGMP and activation of NOS were blocked by the inhibition of phospholipase C (PLC), calcium/calmodulin (CaM), endothelial NOS activity and cGMP accumulation. Conclusions: β3 adrenoceptor activation exerts inhibitory effect on human fibroblast DNA synthesis as a result of the activation of NO-cGMP pathway and the inhibition of adenylate cyclase activity. The mechanism appears to occurs secondarily to stimulation of PLC and CaM. This in turn triggers cascade reaction leading to increase production of NO-cGMP with decrease in cAMP accumulation.

Differences in the regulatory mechanism of amylase release by rat parotid and submandibular glands

It is not known whether the mechanisms involved in amylase release in submandibular and parotid glands are similar. Here, the participation of different signalling pathways in amylase release by the parotid and submandibular glands of the male rat was compared by studying the secretory response after beta-adrenergic stimulation. The beta-adrenergic agonist isoproterenol induced an increase of cAMP in both salivary glands, but while in the parotid it triggered amylase release, in the submandibular it was unable to increase amylase secretion. Parotid amylase release was dependent on adenylate cyclase activation, as SQ-22536 inhibited the secretory effect. In contrast, submandibular amylase secretion did not depend on the intracellular concentration of cAMP, as SQ-22536 did not modify its secretory response. Moreover, other activators of adenylate cyclase, such as forskolin and prostaglandin E2, also failed to modify amylase release by the submandibular gland. Neither ionophores nor calcium-blocking agents, as well as calcium-calmodulin and nitric oxide synthase inhibitors, were effective in modifying basal amylase release by the submandibular gland. However, the disruption of microfilaments with cytochalasin B, but not the disruption of microtubules with colchicine, prevented amylase release in that gland. It is concluded that amylase exocytosis in the submandibular gland is a constitutive non-regulated phenomenon, as it is independent of extracellular or intracellular signals. It depends only on the integrity of the microfilaments, probably used by the vesicles to travel from the Golgi apparatus to the plasma membrane.

Chagasic IgG binds and interacts with cardiac beta adrenoceptor-coupled adenylate cyclase system

It has been previously shown that sera from chagasic patients have an antibody specific for beta adrenoceptors, independently of other tissue-reactive antibodies as the EVI (endocardium, blood vessels, interstitium) system, and it is highly specific to other heart diseases. In this paper we demonstrate that the IgG present in chagasic sera was able to bind to beta adrenoceptors of the heart and also to interact with the membrane bound adenylate cyclase complex, inducing stimulation of enzymatic activity. Moreover, this antibody stimulated contractile activity of guinea pig myocardium, that could be blocked by a specific beta adrenoceptor antagonist. Chagasic IgG inhibited the binding of (-)-(3H)-dyhidroalprenolol to a beta-adrenergic receptor of purified guinea pig myocardial membranes behaving as non-competitive inhibitor. This IgG also exerted a non-competitive inhibition upon the mechanical effect of exogenous norepinephrine.