Activation of β 3 adrenergic receptor decreases DNA synthesis in human skin fibroblasts via cyclic GMP/nitric oxide pathway

Background: Evidences have shown that β 1 and β 2 adrenoceptors co-exist in human fibroblasts, but it is not yet clear the functional expression of β 3 adrenoceptor in these cells. The aim of this study was to investigate the expression and biological effect of β 3 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 (β 3 agonist) on cell DNA synthesis, radioligand binding assay, cyclic GMP and cyclic AMP accumulation and nitric oxide synthase (NOS) activity were evaluated. Results: 3 H-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 β 3 adrenoceptor subtypes. ZD 7114 stimulation of β 3 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 β 3 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 β 3 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: β 3 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.Fil: Furlán, César. Universidad de Buenos Aires; ArgentinaFil: Sterin Borda, Leonor. Universidad de Buenos Aires; ArgentinaFil: Borda, Enri Santiago. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay; Argentina. Universidad de Buenos Aires. Facultad de Odontología; Argentin

Autoantibodies against Muscarinic Acetylcholine Receptor on Exocrine Glands in Sjögren Syndrome

These investigations demonstrate that serum antibodies against muscarinic acetylcholine receptors (mAChR) in primary Sjögren syndrome (pSS) and associated Sjögren syndrome (aSS) bind and activate both cholinergic receptors of M3 in salivary gland and M1 in neonatal myocardium and in the cerebral frontal cortex area subtypes; triggering the production of the second messengers and proinflammatory mediators related to mAChR activation. In this way the cholinergic autoantibodies damages these receptors, which thus starts acting as an antigen. On this basis M3 and M1 mAChR IgG can be considered new markers of pSS/aSS allowing the differentiation between dry eye and mouth of autoimmune and non-autoimmune nature. Given that cholinergic autoantibodies also deregulate the parasympathetic system of the target organs, they can also be seen as a new factor contributing to the etiopathology of the syndrome.Fil: Reina, Silvia Lorena. Universidad de Buenos Aires. Facultad de Odontología. Cátedra de Farmacología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Borda, Enri Santiago. Universidad de Buenos Aires. Facultad de Odontología. Cátedra de Farmacología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentin

Anti-M 3 peptide IgG from Sjögren's syndrome triggers apoptosis in A253 cells

Primary Sjögren´s syndrome (pSS) is an autoimmune disease that targets salivary and lachrymal glands, characterized by anti-cholinergic autoantibodies directed against the M3 muscarinic acetylcholine receptor (mAChR). The aim of this work was to evaluate if cholinergic autoantibodies contained in IgG purified from Sjögren sera could trigger apoptosis of A253 cell line. We also determined if caspase-3 and matrix metalloproteinase 3 (MMP-3) are involved in the induction of A253 cell death. Our results demonstrated that anti-cholinergic autoantibodies stimulate apoptosis and inositol phosphate (InsP) accumulation accompanied by caspase-3 activation and MMP-3 production. All of these effects were blunted by atropine and J104794, indicating that M3 mAChRs are impacted by the anti-cholinergic autoantibodies. The intracellular pathway leading to autoantibody-induced biological effects involves phospholipase C (PLC), calcium/calmodulin (CaM) and extracellular calcium as demonstrated by treatment with U-73122, W-7, verapamil, BAPTA and BAPTA-AM, all of which blocked the effects of the anti-cholinergic autoantibodies. In conclusion, anti-cholinergic autoantibodies in IgG purified from pSS patient´s sera mediates apoptosis of the A253 cell line in an InsP, caspase-3 and MMP-3 dependent manner.Fil: Reina, Silvia Lorena. Universidad de Buenos Aires. Facultad de Odontología. Cátedra de Farmacología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay; ArgentinaFil: Sterin Borda, Leonor. Universidad de Buenos Aires. Facultad de Odontología. Cátedra de Farmacología; ArgentinaFil: Borda, Enri Santiago. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay; Argentina. Universidad de Buenos Aires. Facultad de Odontología. Cátedra de Farmacología; Argentin

Modulation of c-Jun NH2-Terminal (JNK) by Cholinergic Autoantibodies from Patients with Sjögren’s Syndrome

Background: We wanted to determine (via an immunopharmacological approach) whether the c-Jun NH2 terminal kinase (JNK) cascade is phosphorylated in the submandibular gland by carbachol and cholinergic autoantibodies (IgG) present in the sera of patients with primary Sjögren’s syndrome (pSS) by interaction and activation of salivary gland muscarinic acetylcholine receptors (mAChRs). Methods: The JNK, PGE2 and NOS assays were measured in rat sub- mandibular gland with pSS IgG and carbachol alone or in the presence of different blocker agents. Results: pSS IgG- activated M3 mAChRs stimulated JNK phosphorylation whereas the activation of M1 mAChRs by carbachol stimulated JNK phosphorylation involving calcium-activated mechanism. The intracellular pathway leading to pSS IgG-induced biological effects on JNK activity involved activation of protein kinase C (PKC), inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) enzymes. Also, activation of COX-2 and COX-1 by pSS IgG and carbachol-induced PGE2 generation were involved. Conclusion: These results may contribute to better understanding the modulatory role of JNK enzymes by cholinergic autoantibodies from pSS patients acting on mAChR in rat submandibular gland.Fil: Borda, Enri Santiago. Universidad de Buenos Aires. Facultad de Odontología. Cátedra de Farmacología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay; ArgentinaFil: Passafaro, Daniela. Universidad de Buenos Aires. Facultad de Odontología. Cátedra de Farmacología; ArgentinaFil: Reina, Silvia. Universidad de Buenos Aires. Facultad de Odontología. Cátedra de Farmacología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay; ArgentinaFil: Sterin Borda, Leonor. Universidad de Buenos Aires. Facultad de Odontología. Cátedra de Farmacología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay; Argentin

Involvement of the endogenous nitric oxide signalling system in bradykinin receptor activation in rat submandibular salivary gland

Biochemical signalling events coupled to the bradykinin B2-receptor subtype, related to nitric oxide and prostaglandin E2 generation were studied in rat submandibular gland. Bradykinin stimulation of the B2-receptor triggered activation of phosphoinositide turnover, translocation of protein kinase C, stimulation of nitric oxide synthase activity, increased production of cGMP and release of prostaglandin E2. Bradykinin stimulation of nitric oxide synthase and cGMP production was blunted by agents able to interfere with calcium/calmodulin and phospholipase C activities, while a protein kinase C inhibitor was able to stimulate bradykinin action. Moreover, a specific B2-bradykinin antagonist of the reversible nitric oxide synthase inhibitor abrogated the bradykinin stimulation of nitric oxide synthase activity, cGMP accumulation and prostaglandin E2 generation. Furthermore, a specific inhibitor of phospholipase A2 blocked the bradykinin-induced prostaglandin E2 release. These results suggest that apart, from the direct effect of bradykinin as an inducer of vasopermeability, it also appears to be a vasoactive chemical mediator that triggers, through release of prostaglandin E2, a feedback mechanism that induces a protective adaptation of the gland, modulating the course of inflammation. (C) 2000 Elsevier Science Ltd.Fil: Genaro, Ana Maria. Universidad de Buenos Aires. Facultad de Odontología. Cátedra de Farmacología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Stranieri, Graciela M.. Universidad de Buenos Aires. Facultad de Odontología. Cátedra de Farmacología; ArgentinaFil: Borda, Enri Santiago. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad de Buenos Aires. Facultad de Odontología. Cátedra de Farmacología; Argentin

Role of M3 Muscarinic Acethylcholine Receptor Antibodies as a New Marker in Primary Sjögren Syndrome

Aims: This paper investigates the presence of M3 muscarinic acetylcholine receptor autoantibody present in the serum of patients with primary Sjögren syndrome (pSS). Main methods: We detected the levels of M3mAChR peptide IgG, PGE2, IL-1β in serum of SS patients using the enzyme-linked immune sorbent assay (ELISA). To measure the quantity of nitrite/nitrate, we used Griess reagent system. Key findings: Titres of M3mAChR antibody in sera from SS patients are significantly enhanced compared to healthy subjects (control). The enhancement of these autoantibodies is accompanied by the increase of the levels of PGE2, IL-1β and nitrite/nitrate in serum. Under in vitro conditions, the synthetic human M3 peptide impaires the increment of M3mAChR antibody but not that of nati-Ro/SSA antibody. In positive anti-Ro/SSA antibody patients, the increment of M3mAChR peptide IgG and the measured pro-inflammatory substances is related. Significance: On this basis, anti M3mAChR peptide IgG can be said to act as a modulator of the immune system and to play a role in the host-chronic increment of proinflammatory substances in SS patients with positive Ro/SSA antibody. This association between the antibody and the pathogenesis of SS disease may result in useful predicting SS.Fil: Reina, Silvia Lorena. Universidad Catolica de Las Misiones; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Pisoni, Cecilia. Centro de Educaciones Médicas e Investigación Clínica ; ArgentinaFil: González Arana, Roberto. Centro de Educaciones Médicas e Investigación Clínica ; ArgentinaFil: Ganzinelli, Sabrina Belen. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad de Buenos Aires. Facultad de Odontología. Cátedra de Farmacología; ArgentinaFil: Borda, Enri Santiago. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad de Buenos Aires. Facultad de Odontología. Cátedra de Farmacología; Argentin

Role of Anti Muscarinic Acetylcholine IgA and anti autoantibodies in whole saliva from Primary Sjögren’s syndrome patients

Aims: The purpose of this report is to describe saliva IgA antibody against M3 muscarinic acetylcholine receptors (mAChRs) and anti-Ro autoantibodies in patients with primary Sjögren’s syndrome (pSS). In addition we want to clarify if this antibody anti-Ro is or not related to the presence of anti-IgA M3 mAChRs autoantibodies in whole saliva of pSS patients. Methods: Whole saliva samples were collected from healthy volunteers (n=30), patients with pSS anti-Ro positive (n=60) and patients with pSS anti-Ro negative (n=30). Saliva IgA patients and healthy subjects were tested by ELISA recognized the synthetic 25-mer peptide corresponding to the extracellular loop of the human M3 mAChRs. Also, concentration of nitrite/nitrate was determined by ELISA. Results: Optical density values for saliva IgA from pSS anti-Ro positive are significantly higher than those from IgA anti-Ro negative patients and IgA from normal subjects. These molecular interactions between IgA and human M3 mAChR synthetic peptide increased in optical density values compared with IgA from pSS anti-Ro negative and healthy subjects when M3 mAChR synthetic peptide was used as coating antigen. The specificity of this reaction was assessed by the ability of the M3 synthetic peptide (1x10-5 M) to inhibit the action when whole saliva was incubated previously with the M3 synthetic peptide for 40 min at 37ºC and then added together in the microtiter plates. On the other hand, the concentration of nitrate/nitrite in whole saliva was significantly decreased in pSS anti-Ro positive patients in comparison with those from IgA anti-Ro negative patients and healthy subjects. Conclusions: Patients presenting in saliva anti IgA anti-Ro positive are statistically significant in optical density values than those IgA from anti-Ro negative patients and healthy individuals. Also, the hypofunction of the salivary glands is associated with significant decrement of nitrate/nitrites levels in the saliva in pSS-Ro positive without any changes in pSS-Ro negative and healthy subjects.Fil: Borda, Enri Santiago. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad de Buenos Aires. Facultad de Odontología. Cátedra de Farmacología; ArgentinaFil: Reina, Silvia Lorena. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad de Buenos Aires. Facultad de Odontología. Cátedra de Farmacología; ArgentinaFil: Ganzinelli, Sabrina Belen. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad de Buenos Aires. Facultad de Odontología. Cátedra de Farmacología; Argentin

Salivary inflammatory mediators and metalloproteinase 3 in patients with chronic severe periodontitis before and after periodontal phase I therapy

The role of IL-1β, PGE2 and MMP-3 in the pathogenesis of periodontal disease is well researched. This study aimed to asses and compared the salivary IL-1β, PGE2 and MMP-3 levels in patients with untreated chronic severe periodontitis and those treated with periodontal phase I therapy and periodontally healthy individuals as controls, in relationship to the presence of salivary anti-β1 IgA.Fil: Reina, Silvia Lorena. Universidad de Buenos Aires. Facultad de Odontología. Cátedra de Farmacología; Argentina; Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay; Argentina;Fil: Hoyos, Fernando. Universidad de Buenos Aires. Facultad de Odontología; Argentina;Fil: Carranza, Nelson. Universidad de Buenos Aires. Facultad de Odontología; Argentina;Fil: Borda, Enri Santiago. Universidad de Buenos Aires. Facultad de Odontología. Cátedra de Farmacología; Argentina; Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay; Argentina

Anti-M3 muscarinic acetylcholine receptor antibodies in systemic lupus erythematosus

Background: Evidences have shown that anti-M3 muscarinic acetylcholine receptor IgG (anti-M3 mAChR IgG) are clinically useful autoantibody that exert a cholinergic pharmacologic effect binding and interacting with M3 mAChR at the level of exocrine gland (salivary and ocular). Aims: The aim of this study was to determine the associations between serum level of anti-M3 mAChR IgG in patients with systemic lupus erythematosus (SLE) and other autoantibodies, serum prostaglandin E2 (PGE2), and clinical manifestations. Methods: Serum autoantibodies against M3 mAChR synthetic peptide were measured by enzyme-linked immuno absorbent assay (ELISA) using, as an antigen, a 25-mer peptide K-R-T-V-P-D-N-Q-C-F-I-Q-F-L-S-N-P-A-V-T-F-G-T-A-I corresponding to the amino acid sequence of the second extracellular loop of the human M3 mAChR. Serum levels of antinuclear antibodies (ANA), anti-Smith (Sm) antibodies, anti-phospholipid (APL) antibodies, and PGE2 were determined by ELISA in patients with SLE. Results: We found significantly enhanced titers of anti-M3 mAChR IgG in sera from SLE patients compared with healthy individuals (control). In addition, serum levels of PGE2 were significantly higher in SLE patients than in control patients and were significantly higher in active than in non-active SLE. No correlation was found with other autoantibodies present in SLE. By contrast, a positive correlation was found between anti-M3 mAChR IgG and PGE2 serum levels in SLE. Conclusions: As anti-M3 mAChR antibodies present in the sera of SLE patients may be another factor in the pathogenesis of this disease, and the increment of PGE2 in the sera of SLE has a modulatory action on the inflammatory process, suggesting that the presence of these autoantibodies against M3 mAChR may contribute to sustained immune deregulation and the strong inflammatory component observed in SLE.Fil: Reina, Silvia Lorena. Universidad de Buenos Aires. Facultad de Odontología. Cátedra de Farmacología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Pisoni, Cecilia. Centro de Educación Médica e Investigaciones Clínicas “Norberto Quirno”; ArgentinaFil: Eimon, Alicia. Centro de Educación Médica e Investigaciones Clínicas “Norberto Quirno”; ArgentinaFil: Carrizo, Carolina. Centro de Educación Médica e Investigaciones Clínicas “Norberto Quirno”; ArgentinaFil: Arana, Roberto. Centro de Educación Médica e Investigaciones Clínicas “Norberto Quirno”; ArgentinaFil: Borda, Enri Santiago. Universidad de Buenos Aires. Facultad de Odontología. Cátedra de Farmacología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentin

Changes in Cyclooxygenase-2’s Expression, and PGE2 ’s and 6-keto-PGF1α’s Levels in the Presence of the Muscarinic Acethylcholine Receptor Antibody in Primary Sjögren Syndrome

AbstractAims: This paper investigates the action of M3 muscarinic acetylcholine receptor antibody present in serum from patients with Sjögren syndrome (SS). Methods: Enzyme-linked immunoabsorbent assay (ELISA) was performed in the presence or absence of different enzymatic and specific receptors?antagonist drugs. The levels and the generation of PGE2, 6-keto-PGF1α and cyclic AMP (cAMP) in rat submandibular gland acini?s preparations and in serum from pSS patients were measured in the presence of pSS IgG anti M3 peptide. COX-2 mRNA gene?s expression at Real Time PCR was done in acini?s preparations from rat submandibular gland in the presence of the autoantibodies alone or once previously incubated with different inhibitors.Results: In this study, we show that the activation of M3 mAChR of rat submandibular gland acini?s preparation triggers an increment both in the production of COX-2 mRNA gene?s expression and in the production of PGE2 and 6-keto-PGF1α. These phenomena are accompanied by an increment in the production of cAMP in the acini?s preparation and do not affect COX-1 mRNA?s levels. Both prostanoids are augmented in the sera of pSS patients as compared with healthy individuals.Conclusions: The present study suggests a complex interplay between different factors involved in adaptativa autoimmunity in pSS patients at the level of exocrine glands. The presence of pSS IgG anti M3 peptide, the enhancement of COX-2 mRNA gene?s expression and the increment in the generation of PGE2 and 6-keto-PGF1α abolished by M3 specific cholinergic antagonist, could provide evidence of a link between autoimmunity and the submandibular gland parasympathetic system in the course of Sjögren?s syndrome. This evidence is further supported by an increment in the production of AMP cyclic nucleotide (cAMP), and the subsequent induction of desensitization, internalization and/or intracellular degradation of the glandular M3 mAChR displayed by the cholinergic autoantibody. All of these statements cited above, are responsible for xerostomy, xerophthalmia and other parasympathetic symptoms observed in SS patients.Fil: Reina, Silvia Lorena. Universidad de Buenos Aires. Facultad de Odontología. Cátedra de Farmacología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Pisoni, Cecilia. Centro de Educación Médica e Investigaciones Clínicas "Norberto Quirno"; ArgentinaFil: Eimon, Alicia. Centro de Educación Médica e Investigaciones Clínicas "Norberto Quirno"; ArgentinaFil: Carrizo, Carolina. Centro de Educación Médica e Investigaciones Clínicas "Norberto Quirno"; ArgentinaFil: Ganzinelli, Sabrina Belen. Universidad de Buenos Aires. Facultad de Odontología. Cátedra de Farmacología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Arana, Roberto. Centro de Educación Médica e Investigaciones Clínicas "Norberto Quirno"; ArgentinaFil: Borda, Enri Santiago. Universidad de Buenos Aires. Facultad de Odontología. Cátedra de Farmacología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentin