Elevated IL-1ß levels in anti-Ro/SSA connective tissue diseases patients with prolonged corrected QTc interval.

OBJECTIVES: Patients with systemic lupus erythematosus (SLE) and primary Sjögren's syndrome (pSS) have increased IL-1ß levels. IL-1ß and other pro-inflammatory cytokines have a modulating activity on cardiac ion channels and have been associated with increased arrhythmic risk in rheumatoid arthritis patients. Likewise, adult patients with connective tissue diseases (CTDs) may have prolonged QTc intervals associated with the presence of anti-Ro/SSA antibodies. Our objective was to evaluate the presence of serum IL-1ß in subjects with CTDs, in relation to the presence of anti-Ro/SSA antibodies and QTc interval duration. METHODS: 12-lead electrocardiograms (ECG) were performed and blood was withdrawn, measuring electrolytes, IL-1ß anti-Ro/SSA antibodies by ELISA in 73 patients with CTDs. RESULTS: 55 patients were anti-Ro/SSA positive and 18 were anti-Ro/SSA negative. Patients with anti-Ro/SSA positive antibodies had a significantly greater median IL-1ß serum level: 7.29 (range: 0.17-17.3 pg/ml) compared to patients with anti-Ro/SSA negative antibodies whose median was: 1.67 (range 0.55-4.12 pg/ml) p<0.001. The mean QTc interval values obtained in both groups were not significantly different (417.7±23.1 vs. 414.7±21.2, p=0.63). The QTc interval was prolonged in 11 (20%) patients, who were all anti-Ro/SSA positive versus 0 (0 %) in anti-Ro/SSA negative patients p=0.05. Median IL-1ß levels were: 8.7 (range: 2.69-15.1 pg/ml) in patients with prolonged QTc interval versus median: 5.0 (range: 0.17-17.3 pg/ml) in those with normal QTc interval values (<440ms) p=0.006. CONCLUSIONS: IL-1ß is elevated in patients with CTDs that have both anti-Ro/SSA antibodies and prolonged QTc intervals.

Action of anti-M3muscarinic acetylcholine receptor IgG of primary Sjögren's syndrome on the enzymatic antioxidant system in rat submandibular gland.

BACKGROUND: We demonstrate that serum immunoglobulin G (IgG) directed against glandular M3 muscarinic acetylcholine receptors (M3mAChR) and pilocarpine triggers the increment of superoxide dismutase (SOD) and catalase (CAT) and the production of nitric oxide (NO) and prostaglandin E2(PGE2). METHODS: Enzyme-linked immunosorbent assay (ELISA) was performed in the presence of the human M2mAChR synthetic peptide as antigen to detect in serum of pSS patients the autoantibodies. Further, SOD and CAT specific activity and NO were determined chemically in the presence of anti-M3mAChR IgG and pilocarpine. The level of PGE2generation in the presence of autoantibody and pilocarpine was determined by ELISA. RESULTS: An association between anti-M2mAChR autoantibodies and pilocarpine given the increment of the specific activity of SOD and CAT in the serum of pSS patients and in the rat submandibular gland was observed. As a result of this action, M3synthetic peptide and atropine abrogated the stimulatory action. The L-type calcium channel, calcium/calmodulin complex and COX-2 inhibitors selectively blocked the increment of the specific activity of SOD and CAT in the rat submandibular gland. An increased production of NO and PGE2by the cholinergic autoantibody and pilocarpine was also detected. CONCLUSION: On the basis of these results, the increment of the specific activity of SOD and CAT in pSS patients as compared to control healthy individuals may be seen as a defensive reaction to the increment of the amount of ROS, which becoming uncontrollable, leads to irreversible cellular and tissue damage.

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 M(3) 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 M(3) 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.

ß1-Adrenoceptor antibody-induced increase in soluble CD40 ligand release in chronic periodontitis patients: role of prostaglandin E(2).

In this paper, we demonstrate that circulating antibodies from chronic periodontitis patients reacting with atrial ß(1)-adrenoceptors (ß(1)-ARs) act as an inducer of soluble CD40 ligand (sCD40L) release and prostaglandin E(2) (PGE(2)) generation. By enzyme-linked immunosorbent assay using ß(1) synthetic peptide (with an amino acid sequence identical to the second loop of human myocardial ß(1)-ARs) as a coating antigen, we demonstrated reactivity against the second extracellular loop on human myocardial ß(1)-ARs. This autoantibody present in the serum of chronic periodontitis patients was significantly correlated with the release of sCD40L and PGE(2). The release of sCD40L was blunted by atenolol, SP600125 and ß(1) synthetic peptide, and PGE(2) generation was inhibited by DuP 697 and slightly by FR122049. The effects of the antibody incubated with isolated rat atria upregulated sCD40L release with an increase of PGE(2) production and c-Jun N-terminal kinase phosphorylation. These results indicate that in chronic periodontitis patients, there is a positive association between sCD40L release and PGE(2) generation via the action of ß(1)-AR antibodies.

Role of anti-ß1 adrenergic antibodies from patients with periodontitis in cardiac dysfunction.

BACKGROUND: The presence of serum autoantibodies against ß(1) adrenoreceptors (ß(1)-ARs) in human gingival fibroblast from patients with periodontitis inhibits primary cell-specific growth and induces over-expression of pro-inflammatory mediators. Serum ß(1)-AR autoantibodies from patients with periodontitis react with myocardium and modify cardiac contractility. The relationship between the presence of serum ß(1)-AR autoantibodies and alterations in heart rate variability (HRV) was also studied. METHODS: An enzyme-linked immunosorbent assay (ELISA) using cardiac and gingival fibroblast membranes or synthetic peptides corresponding to the second extracellular loop of human ß(1)-AR was used to detect serum autoantibodies. The HRV was assessed from RR interval files generated from 22:00 to 08:00 hours. The autoantibody effects on contractility were measured on spontaneous rat isolated atria. RESULTS: Circulating autoantibodies from 36 patients with periodontitis and 20 healthy individuals (controls) interacted with fibroblasts, the cardiac surface, and ß(1)-AR synthetic peptides. The distributions of serum antibodies against gingival and myocardium membranes and ß(1)-AR synthetic peptide were 88.8%, 77.7%, and 92.8%, respectively. Moreover, 88.5% of patients with periodontitis whose sera were positive against ß(1)-AR synthetic peptide had decreased HRV. The corresponding affinity-purified anti-ß(1)-AR peptide IgG displayed partial agonist-like activity modifying the isolated atria contractility. CONCLUSION: This manuscript describes that patients with periodontitis showed increased levels of serum IgG with reactive activity against ß(1)-AR. Those patients demonstrated decrease in heart rate, and IgG derived from their sera induced aberrant contractility of heart atrium. We propose that periodontitis increases the risk of cardiovascular diseases, although it increases anti-ß(1)-AR autoantibody that alters myocardial contractility.

Atorvastatin inhibits the inflammatory response caused by anti-M(3) peptide IgG in patients with primary Sjögren's syndrome.

Experimental and clinical investigations have revealed that statins can down-regulate acute and chronic inflammatory processes. Whether statins express anti-inflammatory activities in the salivary glands in patients with primary Sjögren's syndrome (pSS) is not known. The in vitro and in vivo effect of atorvastatin on rat submandibular gland treated with anti-M(3) peptide IgG purified from SS patients was studied. The anti-inflammatory effects of atorvastatin were assessed by measuring the levels of IL-1ß, PGE(2) and MMP-3 by ELISA. Atorvastatin inhibited the increase in the production of IL-1ß, PGE(2) and MMP-3 in submandibular glands treated with anti-M(3) peptide IgG. A positive correlation between IL-1ß production with accumulation of PGE(2) and MMP-3 was observed. Rats pre-treated orally with atorvastatin (30 mg kg(-1)) or vehicle (phosphate-buffered solution) once a day for three consecutive days impaired the increment in the production of IL-1ß, PGE(2) and MMP-3 in the submandibular gland in the presence of anti-M(3) peptide IgG. In conclusion, the anti-inflammatory effects of atorvastatin are dependent upon inhibition of production of a pro-inflammatory cytokine (IL-1ß) and pro-inflammatory mediators such as PGE(2) and MMP-3. These data suggest that atorvastatin may constitute an anti-inflammatory effect in SS.

Lidocaine-induced apoptosis of gingival fibroblasts: participation of cAMP and PKC activity.

Local anaesthetics are drugs that prevent or relieve pain by interrupting nervous conduction and are the most commonly used drugs in dentistry. Their main targets of action are voltage-dependent Na+ channels. The Na+ channel is modulated by phosphorylation of two enzymes: PKA (protein kinase A) and PKC (protein kinase C). We studied the ability of lidocaine to modulate programmed cell death of human gingival fibroblasts and the mechanisms involved in this process. Lidocaine (10-5 to 10-7 M) stimulated apoptosis in primary cultures and the caspase-3 activity in a concentration-dependent manner. The stimulatory effect of lidocaine on apoptosis was attenuated in the presence of HA 1004 (PKA inhibitor) and stimulated by staurosporine and Go 6976 (PKC inhibitors). Lidocaine-induced apoptotic nuclei correlated positively with cAMP accumulation and negatively with PKC activity. These results show that lidocaine promotes apoptosis in human gingival fibroblasts at concentrations used for local anaesthesia. The mechanism involves PKA stimulation and PKC inhibition, which in turn stimulates caspase-3 and leads to programmed cell death.

Muscarinic cholinoceptor activation by pilocarpine triggers apoptosis in human skin fibroblast cells.

The aim of the present work was to examine the role of muscarinic acetylcholine receptors (mAChRs) on apoptosis in human skin fibroblast cells. Neonatal human skin fibroblast cultures were stimulated with pilocarpine in the presence or absence of specific antagonists. Pilocarpine stimulates apoptosis, total inositol phosphates (InsP) accumulation and nitric oxide synthase (NOS) activity. All these effects were inhibited by atropine, mustard hydrochloride (4-DAMP) and pirenzepine, indicating that M(1) and M(3) mAChRs are implicated in pilocarpine action. Pilocarpine apoptotic action is accompanied by caspase-3 and JNK activation. The intracellular pathway leading to pilocarpine-induced biological effects involved phospholipase C, calcium/calmodulin and extracellular calcium as U-73122, W-7, verapamil, BAPTA and BAPTA-AM blocked pilocarpine effects. L-NMMA, a NOS inhibitor, had no effect, indicating that the enzyme does not participate in the apoptosis phenomenon. These results may contribute to a better understanding of the modulatory role of the parasympathetic muscarinic system on the apoptotic human skin fibroblast process.

Autoantibodies from schizophrenia patients induce cerebral cox-1/iNOS mRNA expression with NO/PGE2/MMP-3 production.

We demonstrated that circulating antibodies from schizophrenia patients, which interact with cerebral M1 muscarinic acetylcholine receptors (M1 mAChRs), trigger production of nitric oxide (NO), prostaglandin E2 (PGE2) and matrix metalloproteinase-3 (MMP-3), and act as inducers of cyclooxygenase-1 (cox-1) and inducible nitric oxide synthase (iNOS) mRNA expression in the rat frontal cortex. The corresponding affinity-purified anti-M1 peptide IgG from schizophrenia patients, while stimulating cerebral M1 mAChRs, increases NOS activity, PGE2 and MMP-3 production associated with iNOS over-activity and mRNA expression. Moreover, PGE2 and MMP-3 production is the result of cox-1 expression and activity. All these effects were inhibited by pirenzepine or haloperidol and mimicked the action of the authentic mAChR agonist. Concurrent analysis of the effects of iNOS, phospholipase C, protein kinase C and calcium/calmodulin inhibition showed that antibody up-regulation of NOS activity, PGE2 and MMP-3 production is under the control of the endogenous NO signalling system. These results provide evidence of the role that cholinergic receptor antibodies play in the development of cerebral inflammation, which shows that an antibody that interacts with cerebral mAChRs can induce expression of pro-inflammatory mediators, and support the participation of an autoimmune process in a particular group of chronic schizophrenia patients.

Beta-adrenergic-induced CD40 overexpression on gingival fibroblasts: role of PGE2.

CD40, a member of the tumour necrosis factor-alpha receptor family, is constitutively expressed by cells of haematopoietic and non-haematopoietic origin, including fibroblasts. Signalling through this receptor molecule regulates inflammatory mediator secretion by many cell types. The work has been performed in healthy subjects and the authors studied, by cellular culture, flow cytometric analysis and ELISA assay, the expression of CD40 and PGE2 (prostaglandin E2) generation on gingival fibroblasts stimulated by beta-AR (beta-adrenoceptor) agonists. Herein, the authors demonstrate that beta-AR subtype activation via their own specific agonists markedly increased CD40 expression on human gingival fibroblasts. This effect was prevented by beta-AR subtype-specific antagonists. In addition, gingival fibroblast beta-AR stimulation resulted in an increase in PGE2 generation. The inhibition of PLA2 (phospholipase A2) and COX-1 (cyclo-oxygenase-1) but not COX-2 impaired beta-AR increase of PGE2, an effect that was restored by the addition of low concentrations of PGE2, suggesting that PGE2 generation is implicated in the mechanism underlying beta-AR-agonist-mediated CD40 overexpression. Our work has revealed an endogenous beta-AR mediator network involving gingival fibroblasts.

Cholinergic autoantibodies from primary Sjögren's syndrome modulate submandibular gland Na+/K+-ATPase activity via prostaglandin E2 and cyclic AMP.

We demonstrate that patients with primary Sjögren's syndrome (pSS) produce functional IgG autoantibodies that interact with the glandular M(3) muscarinic acetylcholine receptors (mAChRs). These autoantibodies act as a partial muscarinic agonist, increasing prostaglandin E(2) (PGE(2)) and cyclic AMP production through modifying Na(+)/K(+)-ATPase activity, but also interfere with the secretory effect of the parasympathetic neurotransmitter. The IgG from patients with pSS has two effects on the submandibular gland. On the one hand, it may act as an inducer of the proinflammatory molecule (PGE(2)) that, in turn, inhibits Na(+)/K(+)-ATPase activity. On the other hand, it plays a role in the pathogenesis of dry mouth, abolishing the Na(+)/K(+)-ATPase inhibition and the net K(+) efflux stimulation of the salivary gland in response to the authentic agonist pilocarpine, decreasing salivary fluid production.

Increased leukotriene concentration in submandibular glands from rats with experimental periodontitis.

OBJECTIVE AND DESIGN: In the present study, we investigated the relation between the inflammatory mediators such as nitric oxide, prostaglandins, and cysteinyl-leukotrienes with mucin release and the sympathetic system in submandibular glands from rats with experimental periodontitis. MATERIALS OR SUBJECTS: Submandibular glands from rats with experimental periodontitis. TREATMENT: For the first experiment, rats were treated with hydrocortisone sc, 1 mg/kg for 3 days. All other experiments were carried out in isolated submandibular glands from untreated rats. Submandibular glands were treated with cysteinyl-leukotrienes, isoproterenol, NDGA, FPL 55712, L-NMMA, Nio, Nz, AMG, indomethacin, DuP 697 and atenolol. METHODS: Nitric oxide synthase activity, prostaglandin and cysteinyl-leukotriene productions and mucin secretion were determined. The Newman-Keuls statistical test was applied after analysis of variance. RESULTS: In rats with periodontitis hydrocortisone-induced a 36.6% (P < 0.05) decrease in mucin release. Only cysteinyl-leukotriene production was increased in rats with ligature (79.2%, P < 0.001). Either the inhibition of cysteinyl-leukotriene production or the block of leukotriene receptor abolished the increase in mucin secretion by 25.6% (P < 0.05) and 37% (P < 0.01), respectively, in glands from rats with ligature. On the other hand, the presence of cysteinyl-leukotrienes in the incubation medium induced mucin release from submandibular glands. Atenolol diminished by 24% (P < 0.05), the increase in cysteinyl-leukotrienes observed in rats with periodontitis. Besides, isoproterenol induced cysteinyl-leukotriene production in both groups. CONCLUSION: In submandibular glands from rats with periodontitis, the increment in mucin release and cysteinyl-leukotrienes production are related events and both are associated with the sympathetic system.

Beta-adrenoceptor alterations coupled with secretory response and experimental periodontitis in rat submandibular glands.

In this paper we have studied the influence of a well-established rat model of periodontitis on resting and adrenergic-stimulated mucin secretion from rat submandibular glands. The selective beta(1)-receptor subtype agonist, dobutamine, induced mucin secretion while the selective beta(2)-, alpha(1)- and alpha(2)-agonists, soterenol, phenylephrine and clonidine, respectively, did not. In rats subjected to ligature-induced periodontitis mucin release, under unstimulated conditions (basal values), was significantly increased. This increment was abolished in the presence of propranolol and atenolol. Isoproterenol, concentration-dependent, increased mucin release in control and in ligature-induced periodontitis rats. Maximal effect of isoproterenol was decreased in rats with ligature while EC(50) was increased. Neither, the inhibition of NOS by l-NMMA nor the inhibition of COX by indomethacin could revert the effect of ligature on mucin release under unstimulated and isoproterenol-stimulated conditions. The inhibition of adenylyl cyclase by SQ 22536 resulted in a right shift of isoproterenol concentration-response curves in both groups, control and with ligature and returned basal values of rats with ligature to control ones. beta-Receptor population was decreased in submandibular gland membranes from rats with ligature without changes in affinity. Potencies of the beta-receptor antagonists in the competition studies were similar in both groups under study, control and with ligature. We conclude that in rats subjected to ligature-induced periodontitis unstimulated mucin secretion is increased. The increment seems to be due to an activation of the sympathetic system since it is inhibited by the beta-adrenoceptors antagonists and by the inhibition of the adenylyl cyclase. We can speculate that inflammatory mediators from the experimental periodontitis could be involved in the mechanism underlying the activation of the sympathetic system.

Differential regulation on human skin fibroblast by alpha1 adrenergic receptor subtypes.

Alpha 1 adrenoceptor (alpha1-AR) regulation of DNA synthesis was studied in human neonatal foreskin fibroblast. Saturation assay with a specific radioligand for alpha1 adrenergic [3H]-prazosin revealed two saturated and specific binding sites with high or low affinity. Competitive binding assay with different antagonist subtypes, defined pharmacologically three major types of alpha1-AR. The alpha1-AR agonists (from 1x10(-10) to 1x10(-4) M) triggered a biphasic action on DNA synthesis reaching maximal stimulation at 1x10(-9) M and maximal inhibition at 1x10(-6) M. Prazosin, abolished the stimulatory (pA2: 9.24) and inhibitory (pA2: 8.80) actions of alpha1-AR agonists. The alpha1-AR stimulation resulted in the activation of phosphoinositide turnover (InsP) via phospholipase C (PLC) involving calcium/calmodulin (CaM) and nitric oxide synthase (NOS) that correlates with the DNA synthesis increment; whereas the inhibition resulted in a decrease of cyclic AMP (cAMP) accumulation via adenylate cyclase inhibition. The potency displayed by the specific antagonists tested in binding, DNA synthesis, InsP and NOS at low agonist concentration suggests that they can be elicited by the activation of the same receptor (alpha1B-AR subtype); while the decrement in DNA synthesis and cAMP at high concentration account by the activation of alpha1D-AR coupled to Gi protein. Non-functional alpha1A-AR in neonatal human foreskin fibroblast was observed. Results suggest that the expression of alpha1-AR subtypes on human skin fibroblast may differentially activate signaling pathways that modulate physiological response of the cells.

Anti-brain cholinergic auto antibodies from primary Sjögren syndrome sera modify simultaneously cerebral nitric oxide and prostaglandin biosynthesis.

The presence of circulating antibodies from primary Sjögren Syndrome (pSS) patients enable to interact with rat cerebral frontal cortex by activating muscarinic acetylcholine receptors (mAChR). ELISA assay for PGE2 generation, nitric oxide synthase (NOS) activity was measured in cerebral frontal cortex slices by production of [U-14C]-citruline and mRNA isolation/quantitative PCR for COX-1 and COX-2 gene expression were carried out. By ELISA assay, it was shown that IgG from pSS patients reacted to cerebral frontal cortex cell surface and with human M1 and M3 mAChR. Beside pSS IgG displayed an agonistic-like activity stimulating NOS activity and PGE2 production associated with an increased COX-1 mRNA gene expression, without affecting COX-2 mRNA levels. Inhibition of phospholipase A2 (PLA2) and NOS prevented pSS IgG effects upon both PGE2 production and COX-1 mRNA levels. The results support the notion that serum IgG auto antibodies in pSS patients target cerebral mAChR may have pathogenic role in immune neuroinflammation and on cognitive dysfunction present in pSS patients.

Signaling pathways involved in pilocarpine-induced mucin secretion in rat submandibular glands.

We have studied the signaling pathways involved in pilocarpine-induced mucin release in rat submandibular slices. Pilocarpine produced a significant increment of PGE2 levels and a positive (r=0.8870) and significant (p=0.0077) correlation between PGE2 production and mucin released was determined. The participation of PGE2 was confirmed by the use of indomethacin (indo) and of acetyl salicylic acid (ASA), cyclooxygenase inhibitors, which inhibited pilocarpine-induced mucin release. The muscarinic receptors involved in the regulation of mucin release were identified as M1 and M4 by the use of the selective acetylcholine receptors (mAChR) antagonists, pirenzepine, AF-DX 116, 4-DAMP and tropicamide. The secretory process was dependent on both, intracellular and extracellular calcium pools since it was inhibited by thapsigargin and verapamil. Cyclic AMP, nitric oxide synthase and PKC also participated in pilocarpine-induced mucin release. It is concluded that pilocarpine, by activation the M1 and M4 mAChR subtypes induces an increase of intracellular Ca2+ concentration ([Ca2+]I) and elevates cAMP levels, which in turn stimulates COX, PKC and NOS and promotes mucin exocytosis. PGE2 released induces cAMP accumulation which, together with PKC are involved in the PGE2 increased Ca2+/cAMP-regulated exocytosis. Thus, cAMP accumulation induced by cholinergic stimulation is, in part, the result of PGE2 production.

Nitric oxide synthase and PGE2 reciprocal interactions in rat dental pulp: cholinoceptor modulation.

In this study we determined the effect of cholinoceptor agonist pilocarpine on the stimulation of nitric oxide synthase (NOS) and on prostaglandin E2 (PGE2) generation upon rat dental pulp. By reverse transcriptase/polymerase chain reaction (RT-PCR) we identified several products corresponding to m1, m2, m3, and m4 muscarinic acetylcholine receptors (mAChRs). The stimulation of M1, M2, M3, and M4 mAChRs by pilocarpine increases NOS activity and PGE2 generation. There is a correlation (correlation coefficient=0.05) between NOS activity and PGE2 generation through the activation of phosphoinositide by phospholipase C (PLC), phospholipase A2 (PLA2), and cyclooxygenase 1 (COX-1). Exogenous PGE2 restored NOS activity inhibited by indomenthacin (INDO), whereas nitric oxide (NO) donor restored PGE2 generation inhibited by NG-methyl-L-arginine acetate salt (L-NMMA). These data indicate that both NO and PGE2 interact with their own respective biosynthetic pathways modulating NOS and COX activities. Results could contribute to understanding the involvement of NO and PGE2 in healthy dental pulp given that cellular signals through the parasympathetic system modulate the function of the dentin-pulp complex.

Levels of anti-M2 and anti-beta1 autoantibodies do not correlate with the degree of heart dysfunction in Chagas' heart disease.

Chronic chagasic cardiomyopathy (CCC) is characterized mainly by a dilated cardiomyopathy complicated by frequent and complex ventricular arrhythmias and/or conduction defects. The aim of the present study was to evaluate functional implications of neurotransmitter receptor autoantibodies in vivo. Sera from chagasic patients were used to measure the level of autoantibodies to peptide fragments from the M2 cholinergic and beta1 adrenergic receptors. Optical density values and the frequency of anti-M2 and anti-beta1 antibodies were significantly higher in the indeterminate form and in CCC patients than in normal individuals. There was no correlation between levels of autoantibodies and clinical parameters of ventricular dysfunction, as assessed by echocardiography. Patients presenting with chronotropic insufficiency in exercise test had higher levels of anti-M2 but not anti-beta1 autoantibodies. Although anti-M2 and anti-beta1 antibodies do not appear to play a role in the pathophysiology of the heart failure that accompanies severe CCC, anti-M2 cholinergic autoantibodies may contribute to the pathogenesis of Chagas' disease dysautonomia.

Signal transduction underlying carbachol-induced PGE2 generation and cox-1 mRNA expression of rat brain.

In this paper we have determined the different signal pathways involved in M(1) and M(3) muscarinic acetylcholine receptor (mAChR) dependent stimulation of cyclo-oxygenase 1 (cox-1) mRNA gene expression and PGE(2) production on rat cerebral frontal cortex. Carbachol stimulation of M(1) and M(3) mAChR exerts an increase in cox-1 mRNA gene expression without affecting cox-2 mRNA expression and increased PGE(2) generation. Besides, increased phosphoinositide (PI) turnover and stimulation of nitric oxide synthase (NOS) and cyclic GMP (cGMP) production. Inhibitors of phospholipase A(2) (PLA(2)), COX and phospholipase C (PLC), calcium/calmodulin (CaM), NOS and soluble guanylate cyclase prevent the carbachol effect. These results suggest that carbachol-activation of M(1) and M(3) mAChR increased PGE(2) release associated with an increased expression of cox-1 and NO-cGMP production. The mechanism appears to occur directly to PLC stimulation and indirectly to PLA(2) activation. These results may contribute to understand the effects and side effect of non-steroidal anti-inflammatory drugs in patients with cerebral degenerative diseases.

Differential CB1 and CB2 cannabinoid receptor-inotropic response of rat isolated atria: endogenous signal transduction pathways.

In this study, we have determined the contractile effects of CB1 and CB2 cannabinoid receptor activation on rat isolated atria and the different signaling pathways involved. Anandamide did not has significantly effect on atria contractility, however, the treatment with both CB1 (AM251) or CB2 (AM630) receptor antagonists, the endocannabinoids triggered stimulation or inhibition on contractility respectively. The ACEA stimulation of CB1 receptor exerted decrease on contractility, that significantly correlated with the decrement of cAMP and the stimulation of nitric oxide synthase (NOS) and the accumulation of cyclic GMP (cGMP). On the contrary, JWH 015 stimulation of CB2 receptor triggered positive contractile response that significantly correlated with the increase cAMP production. The inhibiton of adenylate cyclase activity impaired the JWH 015 activation of CB1 receptor induced positive contractile effect, while inhibitors of phospholipase C (PLC), NOS and soluble nitric oxide (NO)-sensitive guanylate cyclase blocked the dose-response curves of ACEA on contractility. Those inhibitors also attenuated the CB1 receptor-dependent increase in activation of NOS and cGMP accumulation. These results suggest that CB2 receptor agonist mediated positive contractile effect associated with increased production on cAMP while CB1 receptor agonist mediated decrease on contractility associated with decreased cAMP accumulation and increase production of NO and cGMP; that occur secondarily to stimulation of PLC, NOS and soluble guanylate cyclase. Data give pharmacological evidence for the existence of functional CB1 and CB2 cannabinoid receptors in rat isolated atria and may contribute to a better understanding the effects of cannabinoids in the cardiovascular system.