Silvia Reina

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.

Anti-M3 muscarinic cholinergic autoantibodies from patients with primary Sjögren's syndrome trigger production of matrix metalloproteinase-3 (MMP-3) and prostaglandin E2 (PGE2) from the submandibular glands

BACKGROUND We demonstrated that serum immunoglobulin G (IgG) from patients with primary Sjögrens 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)).

Cholinoreceptor Autoantibodies in Sjögren Syndrome

Previous studies have demonstrated that antibodies against cholinoreceptors of exocrine glands correlate with dry mouth in persons with primary Sjögren syndrome (pSS). The aim of the present investigation was to establish if serum IgG antibodies (pSS IgG) were able to interact with cholinoreceptors in rat submandibular gland-dependent stimulation of cyclooxygenase 2 (COX-2) mRNA expression and PGE2 production. Our findings indicated that pSS IgG-stimulating M3, M4, and M1 cholinoreceptors exerted an increase in COX-2 mRNA without affecting COX-1 mRNA expression and increased PGE2 production. Inhibitors of phospholipase A2, COX- s, L-type calcium channel currents, and Ca2+-ATPase from sarcoplasmic reticulum prevented the pSS IgG effect on PGE2 production. An ionophore of calcium mimicked pSS IgG action, suggesting a crucial role of calcium homeostasis in the cholinoreceptor-stimulated increase in PGE2 production. Moreover, the amounts of PGE2 in saliva and in sera from persons with pSS were significantly higher than in pre- or post-menopausal women. These findings illustrate the importance of autoantibodies to cholinoreceptors in the generation of chronic inflammation of target tissues in SS.

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.

Pro-apoptotic effect of anti-β1-adrenergic receptor antibodies in periodontitis patients.

An anti-β(1)-adrenergic antibody from the sera of periodontitis patients (anti-β(1)-AR IgG) against the second extracellular loop of the human β(1)-adrenoceptor (β(1)-AR) has been shown to cause rat atria apoptosis. The anti-β(1)-AR IgG binds and activates atria β(1)-AR, increasing the intracellular calcium concentration, which, in turn, activates caspases-3, -8, and -9. The β(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-β(1)-AR IgG, with increased generation of PGE(2). Comparatively, xamoterol, an authentic β(1)-AR agonist, mimicked the autoantibody effect on rat atria β(1)-AR apoptosis. Our results suggest that autoantibodies from the sera of periodontitis patients bind and interact with rat atria β(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 β(1)-AR, which could lead to heart failure. These results suggest an important link between periodontitis and cardiovascular disease.

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 M1 and M3 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. J. Cell. Physiol. 222: 640–647, 2010.

Autoantibodies to the β 1 -Adrenoceptor from Patients with Periodontitis as a Risk Factor for Cardiac Dysfunction

The presence of serum autoantibodies in periodontitis (P) patients against β 1-adrenoceptor (β 1-AR), using cardiac membranes or a synthetic β 1-AR peptide corresponding to the second extracellular loop of human β 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-β 1-AR IgG titters correlated with the decrease in HRV of the same patients and the anti-β 1-AR peptide IgG displayed partial agonist-like activity and modified the contractility of isolated atria, produced cyclic nucleotides, and inhibited the β 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-β 1-AR autoantibodies in alteration of myocardial contractility.

Endogenous signalling system involved in parotid gland adenosine A1 receptor‐amylase release

Aim:  In this study, we have determined signalling pathways involved in adenosine A1 receptor (A1 receptor)‐dependent stimulation of amylase release in rat parotid gland.

Influence of lidocaine on ouabain-induced inotropic response in rat atria

In this paper we demonstrated that lidocaine broadens the therapeutic range of ouabain action having a protective effect on ouabain-induced toxicity on rat atria. The lidocaine effect on therapeutic ouabain action was associated with the increase in the sensitivity of Na(+)-K(+)-ATPase related to a decreased in the equilibrium dissociation constant (K(d)) of high affinity binding sites. Lidocaine suppressed the ouabain-induced tonotropic effect and arrhythmias, decreasing the number of low affinity binding sites (B(max)) without changes in K(d). Blockade of Na(+)-Ca(2+) exchange with KB-R7943 or dual Na(+)-Ca(2+) channel with flunarizine, mimicked lidocaine effect increasing ouabain therapeutic action, extending its concentration range tolerated, delaying the onset of contracture. Lidocaine itself triggered negative inotropic response at high concentration. This effect was increased in the presence of flunarizine and verapamil but not by the inhibition of calcium/calmodulin with W-7. The mechanism underlying the lidocaine-induced negative inotropic response, appears to be different that underlying the positive inotropic effect on ouabain action. This study provides evidence that lidocaine can interact with the same or similar binding sites for ouabain in rat atrial tissue, providing a protective effect on ouabain-induced changes in contractility. The contribution of Na(+)-Ca(2+) exchange and/or Ca(2+) overload on lidocaine effect is discussed.

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

Abstract Background: 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