Pro-apoptotic effect of anti-ß1-adrenergic receptor antibodies in periodontitis patients.

An anti-ß(1)-adrenergic antibody from the sera of periodontitis patients (anti-ß(1)-AR IgG) against the second extracellular loop of the human ß(1)-adrenoceptor (ß(1)-AR) has been shown to cause rat atria apoptosis. The anti-ß(1)-AR IgG binds and activates atria ß(1)-AR, increasing the intracellular calcium concentration, which, in turn, activates caspases-3, -8, and -9. The ß(1)-AR and the post-receptor activation of calcium/calmodulin (CaM) lead to increased inducible nitric oxide synthase (iNOS) activity, with an increase in cyclic GMP (cGMP) accumulation as well as increased JNK phosphorylation and cyclic AMP (cAMP) production. We also observed an apoptotic effect of anti-ß(1)-AR IgG, with increased generation of PGE(2). Comparatively, xamoterol, an authentic ß(1)-AR agonist, mimicked the autoantibody effect on rat atria ß(1)-AR apoptosis. Our results suggest that autoantibodies from the sera of periodontitis patients bind and interact with rat atria ß(1)-AR, provoking apoptosis. This implicates a series of modulatory cardiac signaling events that could alter normal heart function and may occur with chronic stimulation of the atria ß(1)-AR, which could lead to heart failure. These results suggest an important link between periodontitis and cardiovascular disease.

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.

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.

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.

Cholinergic Autoantibodies from Primary Sjögren's Syndrome Inhibit Mucin Production via Phospholipase C and Cyclooxygenase-2 In the Rat Submandibular Gland.

BACKGROUND: Patients with primary Sjögren's syndrome (pSS) produce functional IgG against cholinoreceptor of exocrine glands modifying their activity. The aim of the present work was to demonstrate pSS IgG antibodies (pSS IgG) interacting with M(3) muscarinic acetylcholine receptors (mAChR) of rats submandibular glands that alter mucin release and production via phospholipase C (PLC) and cyclooxigenase-2 (COX-2) pathways. METHODS: Mucin release and production of prostaglandin E2 (PGE2), and total inositol phosphates (InsP) were measured in rat submandibular gland in the presence of pSS IgG auto antibodies. RESULTS: The auto antibodies interacting with M3 mAChR decreased mucin release and production through stimulation of PLC and COX-2. This stimulation leads to an incremental increase in InsP production and in PGE2 generation, inducing signalling through the prostaglandin membrane receptors subtype 2 (EP2). Moreover, the decrease in mucin production had negative correlation with PGE(2) generation and InsP accumulation. CONCLUSION: IgG in patients with pSS could play an important role in the pathoetiology of dry mouth, decreasing the salivary mucin through the production of proinflammatory substances and leading to the reduction in the protection of the oral tissues.

Autoantibodies to the ß(1)-Adrenoceptor from Patients with Periodontitis as a Risk Factor for Cardiac Dysfunction.

The presence of serum autoantibodies in periodontitis (P) patients against ß(1)-adrenoceptor (ß(1)-AR), using cardiac membranes or a synthetic ß(1)-AR peptide corresponding to the second extracellular loop of human ß(1)-AR as antigens, permit us to detect circulating antibody from 40 P patients but not in 20 normal individuals (control). Simultaneously, the P patients exhibited a decrease in HRV. Anti-ß(1)-AR IgG titters correlated with the decrease in HRV of the same patients and the anti-ß(1)-AR peptide IgG displayed partial agonist-like activity and modified the contractility of isolated atria, produced cyclic nucleotides, and inhibited the ß(1)-AR agonistic activity of isoproterenol. We demonstrated in this study an association between periodontitis infection and an increased risk of cardiac disease, thereby highlighting the role of anti-ß(1)-AR autoantibodies in alteration of myocardial contractility.

Enhancement of carbachol-induced amylase secretion in parotid glands from rats with experimental periodontitis.

OBJECTIVE: In a previous study we observed that parotid glands from rats with experimental periodontitis showed an increase in basal amylase release as a result of an increase in cAMP accumulation induced by PGE(2) production. The aim of this work was to study whether this change in amylase release influences the secretory effect of carbachol. DESIGN: Experimental periodontitis was induced through placing a black thread around the cervix of the two lower first molars. Experiments were done 22 days after ligature induced periodontitis. Amylase release was evaluated in vitro and determined using a colorimetric method which uses starch as substrate. RESULTS: The effect of carbachol was increased in parotid glands from periodontitis rats. The effect of 10(-6)M carbachol was inhibited by 4-DAMP (10(-6)M), U-73122 (5 × 10(-6)M) and trifluoperazine (5 × 10(-6)M) in both groups. No changes were observed in the binding sites and affinity in parotid membranes from rats with experimental periodontitis. The inhibition of the adenylyl cyclase and the cyclooxygenase induced a right shift of the carbachol concentration-response curve in periodontitis group whilst the opposite effect was observed in control group in the presence of db-cAMP and PGE(2). CONCLUSIONS: Parotid glands from rats with experimental periodontitis release more amylase in response to carbachol suggesting an interaction between Ca(2+) and cAMP in the fusion/exocytosis step of secretory vesicles.

Anti-M(3) muscarinic cholinergic autoantibodies from patients with primary Sjögren's syndrome trigger production of matrix metalloproteinase-3 (MMP-3) and prostaglandin E(2) (PGE(2)) from the submandibular glands.

BACKGROUND: We demonstrated that serum immunoglobulin G (IgG) from patients with primary Sjögren's syndrome (pSS), interacting with the second extracellular loop of human glandular M(3) muscarinic acetylcholine receptors (M(3) mAChR), trigger the production of matrix metalloproteinase-3 (MMP-3) and prostaglandin E(2) (PGE(2)). METHODS: Enzyme-linked immunosorbent assays (ELISAs) were performed in the presence of M(3) mAChR synthetic peptide as antigen to detect in serum the autoantibodies. Further, MMP-3 and PGE(2) production were determined in the presence of anti-M(3) mAChR autoantibodies. RESULTS: An association was observed between serum and anti-M(3) mAChR autoantibodies and serum levels of MMP-3 and PGE(2) in pSS patients. Thus, we established that serum anti-M(3) mAChR autoantibodies, MMP-3 and PGE(2) may be considered to be early markers of pSS associated with inflammation. Affinity-purified anti-M(3) mAChR peptide IgG from pSS patients, whilst stimulating salivary-gland M(3) mAChR, causes an increase in the level of MMP-3 and PGE(2) as a result of the activation of phospholipase A(2) (PLA(2)) and cyclooxygenase-2 (COX-2) (but not COX-1). CONCLUSIONS: These results provide a novel insight into the role that cholinoceptor antibodies play in the development of glandular inflammation. This is the first report showing that an antibody interacting with glandular mAChR can induce the production of pro-inflammatory mediators (MMP-3/PGE(2)).

Inflammation triggers constitutive activity and agonist-induced negative responses at M(3) muscarinic receptor in dental pulp.

The purpose of this study was to investigate whether the inflammation of rat dental pulp induces the muscarinic acetylcholine receptor (mAChR) constitutive receptor activity. Pulpitis was induced with bacterial lipolysaccharide in rat incisors dental pulp. Saturation assay with [(3)H]-quinuclidinyl benzilate ([(3)H] QNB), competitive binding with different mAChR antagonist subtypes, and nitric oxide synthase (NOS) activity were performed. A drastic change in expression and response to mAChR subtypes was observed in pulpitis. Inflamed pulp expressed high number of M(3) mAChR of high affinity, whereas the M(1) mAChR is the main subtype displayed in normal pulp. Consistent with the identification of the affinity constant (Ki) of M(3) and Ki of M(1) in both pulpitis and in normal pulps are the differences in the subtype functionality of these cells. In pulpitis, pilocarpine (1 × 10(-11) mol/L to 5 × 10(-9) mol/L) exerted an inhibitory action on NOS activity that was blocked by J 104129 fumarate (highest selective affinity to M(3) mAChR). In normal pulps, pilocarpine (1 × 10(-11) mol/L to 5 × 10(-9) mol/L) has no effect. NOS basal activity was 5.9 times as high in pulpitis as in the normal pulp as a result of the activation of inducible NOS. The irreversible pulpitis could induce a mAChR alteration, increasing the high-affinity receptor density and transduction-coupling efficiency of inducible NOS activity, leading to a spontaneously active conformation of the receptor. Pilocarpine acting as an inverse agonist might be useful therapeutically to prevent necrosis and subsequent loss of dental pulp.

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.

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.

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.

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.

Circulating beta(1) Adrenergic Autoantibodies from Patients with Chronic Periodontitis Interact with Gingival Fibroblasts.

OBJECTIVES: To demonstrate the presence of circulating autoantibodies (Abs) from patients with chronic periodontitis (CP) that interacted with human gingival fibroblast membranes activating beta(1) adrenoceptors (beta(1)-AR). METHODS: Sera and purified IgG from 25 patients with CP and 20 age-matched healthy subjects were studied by flow cytometry, ELISA and DNA synthesis. Human gingival fibroblast membranes and/or synthetic peptides with amino acid sequences identical to human beta(1)-AR were used as antigens. RESULTS: By flow cytometry and ELISA procedures, we proved that the serum-purified IgG fraction from patients with CP reacted with the fibroblast surface and to the beta(1) synthetic peptide. The corresponding affinity-purified anti-beta(1) peptide Abs displayed agonist-like activity associated with specific receptor activation, inhibiting the DNA synthesis of human gingival fibroblasts. CONCLUSIONS: This study demonstrates that beta(1)-AR autoantibodies are elevated in patients with CP. These autoantibodies were targeted to the fibroblasts, and specifically to the beta(1)-AR, and has receptor-like activity inhibiting DNA synthesis.

Experimental periodontitis induces a cAMP-dependent increase in amylase activity in parotid glands from male rats.

It is known that subjects with periodontitis show enhanced amylase concentration in saliva. Our purpose was to analyze the release of amylase in parotid glands from rats with experimental periodontitis and controls. We present evidence that periodontitis induces an increase in resting amylase activity and release without changes in isoproterenol-induced amylase secretion. Changes in amylase were reverted by the inhibition of the adenylyl cyclase by SQ 22536, the cyclooxygenase type 1 by FR 122047 and by blocking the vasoactive intestinal peptide (VIP) receptor with VIP 6-28. Parotid glands from rats with periodontitis showed an increase in cAMP levels that was also reverted in the presence of SQ 22536, FR 122047 and VIP 6-28. We concluded that both PGE(2) and VIP are produced in parotid glands from rats with periodontitis and, by activating their own receptors in acinar cells, induce cAMP accumulation leading to an increase in amylase basal secretion.

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.

Cholinoceptor modulation on nitric oxide regulates prostaglandin E(2) and metalloproteinase-3 production in experimentally induced inflammation of rat dental pulp.

The purpose of this study was to investigate the role of muscarinic acetylcholine receptor (mAChR) activity in the regulation of inducible nitric oxide synthase (iNOS) activity, prostaglandin E(2) (PGE(2)), and metalloproteinase-3 (MMP-3) in experimentally induced inflammation of rat incisors dental pulp. Inflammation was induced by application of bacterial lipopolysaccharide (LPS) to the pulp. Extirpated pulp tissue samples were incubated in saline solution until the various experiments were performed. Saline-treated pulp and healthy pulp were used as controls. NOS activity was measured by the production of [U-(14)C]-citrulline from [U-(14)C]-arginine. PGE(2) and MMP-3 production were evaluated by an enzyme-linked immunosorbent assay (ELISA) and cyclooxygenase (cox-1 and cox-2) messenger RNA levels were measured using a reverse-transcriptase polymerase chain reaction by coamplification of target complementary DNA with a single set of primers. The application of LPS to the pulp increased NOS activity, PGE(2), and MMP-3 production associated with iNOS overactivity. Moreover, PGE(2) and MMP-3 production were the result of cox-2 expression. Pilocarpine (5 x 10(-11) mol/L to 5 x 10(-9) mol/L), acting on mAChRs, triggered a negative effect on NOS activity, PGE(2), and MMP-3 production. In control pulp, no action of pilocarpine was observed. Pulpitis changed mAChR conformation, increasing its coupling efficiency to transducing molecules that in turn activate iNOS. The capacity of pilocarpine to prevent iNOS activity, PGE(2), and MMP-3 by acting on mAChR mutation induced by pulpitis might be useful therapeutically as a local treatment.

Chagasic antibodies induce cardiac COX-2/iNOS mRNA expression with PGE2/NO production.

We demonstrate that serum IgG in chagasic patients interacting with the second extracellular loop of human cardiac M(2) muscarinic acetylcholine receptors (M(2) mAChR) trigger the production of PGE(2) and NO, that in turn induces COX-2/iNOS mRNA expression. An association between serum anti-M(2) peptide IgG, anti-cardiac membrane IgG and PGE(2) levels (p<0.05) in chagasic dysautonomic patients was observed. Thus, we establish that serum anti-mAChR autoantibodies and PGE(2) might be considered as early markers of Chagas' associated dysautonomia. Affinity purified anti-M(2) peptide IgG from chagasic sera, while stimulating myocardial M(2) mAChR, it exerts an increase on PGE(2) generation and NOS activity, as well as COX-2/iNOS isoforms mRNA expression. The expression of these genes is related with phosphoinositides (PIs), cGMP accumulation and PKC activity. Inhibition of these enzymes shows that chagasic autoantibodies up-regulation of COX-2/iNOS mRNA level is under the control of endogenous iNO/cGMP signaling system. These results provide a novel insight into the role that cholinoceptor antibodies play in the development of myocardial inflammation. To our knowledge, there has been no previous report showing that an antibody interacting with heart mAChR can act as expression inducer of proinflammatory mediators.