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An anti-β1-adrenergic antibody from the sera of periodontitis patients (anti-β1-AR IgG) against the second extracellular loop of the human β1-adrenoceptor (β1-AR) has been shown to cause rat atria apoptosis. The anti-β1-AR IgG binds and activates atria β1-AR, increasing the intracellular calcium concentration, which, in turn, activates caspases-3, -8, and -9. The β1-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-β1-AR IgG, with increased generation of PGE2. Comparatively, xamoterol, an authentic β1-AR agonist, mimicked the autoantibody effect on rat atria β1-AR apoptosis. Our results suggest that autoantibodies from the sera of periodontitis patients bind and interact with rat atria β1-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 β1-AR, which could lead to heart failure. These results suggest an important link between periodontitis and cardiovascular disease. Fil: Reina, Silvia Lorena. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad de Buenos Aires. Facultad de Odontología. Cátedra de Farmacología; Argentina Fil: Ganzinelli, Sabrina Belen. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad de Buenos Aires. Facultad de Odontología. Cátedra de Farmacología; Argentina Fil: Sterin, Leonor Josefina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad de Buenos Aires. Facultad de Odontología. Cátedra de Farmacología; Argentina Fil: Borda, Enri Santiago. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad de Buenos Aires. Facultad de Odontología. Cátedra de Farmacología; Argentina

In this paper, we demonstrate that circulating antibodies from chronic periodontitis patients reacting with atrial β(1)-adrenoceptors (β(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 β(1) synthetic peptide (with an amino acid sequence identical to the second loop of human myocardial β(1)-ARs) as a coating antigen, we demonstrated reactivity against the second extracellular loop on human myocardial β(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 β(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 β(1)-AR antibodies.

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.

J Oral Pathol Med (2012) 41: 242–248 Background: The presence of serum autoantibodies against β1 adrenoreceptors (β1-ARs) in human gingival fibroblast from patients with periodontitis inhibits primary cell-specific growth and induces over-expression of pro-inflammatory mediators. Serum β1-AR autoantibodies from patients with periodontitis react with myocardium and modify cardiac contractility. The relationship between the presence of serum β1-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 β1-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 β1-AR synthetic peptides. The distributions of serum antibodies against gingival and myocardium membranes and β1-AR synthetic peptide were 88.8%, 77.7%, and 92.8%, respectively. Moreover, 88.5% of patients with periodontitis whose sera were positive against β1-AR synthetic peptide had decreased HRV. The corresponding affinity-purified anti-β1-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 β1-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-β1-AR autoantibody that alters myocardial contractility.

The presence of serum autoantibodies in periodontitis (P) patients against β1-adrenoceptor (β1-AR), using cardiac membranes or a synthetic β1-AR peptide corresponding to the second extracellular loop of human β1-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-β1-AR IgG titters correlated with the decrease in HRV of the same patients and the anti-β1-AR peptide IgG displayed partial agonist-like activity and modified the contractility of isolated atria, produced cyclic nucleotides, and inhibited the β1-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-β1-AR autoantibodies in alteration of myocardial contractility.

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 Sjogren’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.

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 E2 (PGE2), 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-14C]-citrulline from [U-14C]-arginine. PGE2 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, PGE2, and MMP-3 production associated with iNOS overactivity. Moreover, PGE2 and MMP-3 production were the result of cox-2 expression. Pilocarpine (5 × 10-11 mol/L to 5 × 10-9 mol/L), acting on mAChRs, triggered a negative effect on NOS activity, PGE2, 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, PGE2, and MMP-3 by acting on mAChR mutation induced by pulpitis might be useful therapeutically as a local treatment. Fil: de Couto Pita, Alejandra Karina. Universidad de Buenos Aires. Facultad de Odontología. Cátedra de Farmacología; 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 Fil: Ganzinelli, Sabrina Belen. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay; Argentina. Universidad de Buenos Aires. Facultad de Odontología. Cátedra de Farmacología; Argentina Fil: Passafaro, Daniela. Universidad de Buenos Aires. Facultad de Odontología. Cátedra de Farmacología; Argentina Fil: Sterin, Leonor Josefina. Universidad de Buenos Aires. Facultad de Odontología. Cátedra de Farmacología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay; Argentina

Langerhans cell histiocytosis (LCH) is a rare disorder mainly of children, whose pathogenesis is still unknown. Some studies have demonstrated that LCH lesions produce different cytokines abnormally that may be relevant to the pathogenesis of the disease. The purpose of this study was to investigate interleukin-1 beta (IL-1 beta) and prostaglandin E2 (PGE(2)) levels in saliva from children with different clinical subtypes of LCH. We studied 29 children with LCH: seven unifocal (Group I), seven multifocal (Group II), 15 multisystemic (Group III) and 12 healthy volunteers (Group IV). Salivary IL-1 beta and PGE(2) levels were significantly higher in LCH than in normal children. A multi-comparison test showed significantly (P < 0.001) higher levels of both IL-1 beta and PGE(2) in saliva from Group III compared with Groups II and I. A significant correlation (r = 0.05) between IL-1 beta and PGE(2) concentrations in saliva from each group was determined. Our findings demonstrated an association between high concentrations of salivary IL-1 beta and PGE(2) and advanced stages of the disease. This allows us to suggest that the abnormal amount of these factors in saliva may serve as a risk marker for disease progression.

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.

Alpha 1 adrenoceptor (α 1 -AR) regulation of DNA synthesis was studied in human neonatal foreskin fibroblast. Saturation assay with a specific radioligand for α 1 adrenergic [ 3 H]-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 α 1 -AR. The α 1 -AR agonists (from 1 × 10 −10 to 1 × 10 −4 M) triggered a biphasic action on DNA synthesis reaching maximal stimulation at 1 × 10 −9 M and maximal inhibition at 1 × 10 −6 M. Prazosin, abolished the stimulatory (pA 2 : 9.24) and inhibitory (pA 2 : 8.80) actions of α 1 -AR agonists. The α 1 -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 (α 1B -AR subtype); while the decrement in DNA synthesis and cAMP at high concentration account by the activation of α 1D -AR coupled to G i protein. Non-functional α 1A -AR in neonatal human foreskin fibroblast was observed. Results suggest that the expression of α 1 -AR subtypes on human skin fibroblast may differentially activate signaling pathways that modulate physiological response of the cells.

Background and purpose: Agonists of the M2 muscarinic acetylcholine receptor (mAChR) increase mRNA for this receptor and mRNA for endothelial and neuronal isoforms of NO synthase (eNOS or nNOS). Here we examine the different signalling pathways involved in such events in rat cardiac atria. Experimental approach: In isolated atria, the effects of carbachol on mRNA for M2 receptors, eNOS and nNOS were measured along with changes in phosphoinositide (PI) turnover, translocation of protein kinase C (PKC), NOS activity and atrial contractility. Key Results: Carbachol increased mRNA for M2 receptors, activation of PI turnover, translocation of PKC and NOS activity and decreased atrial contractility. Inhibitors of phospholipase C (PLC), calcium/calmodulin (CaM), NOS and PKC prevented the carbachol-dependent increase in mRNA for M2 receptors. These inhibitors also attenuated the carbachol induced increase in nNOS- and eNOS-mRNA levels. Inhibition of nNOS shifted the dose response curve of carbachol on contractility to the right, whereas inhibition of eNOS shifted it to the left. Conclusions and implications: From our results, activation of M2 receptors induced nNOS and eNOS expression and activation of NOS up-regulated M2 receptor gene expression. The signalling pathways involved included stimulation of PI turnover via PLC activation, CaM and PKC. nNOS and eNOS mediated opposing effects on the negative inotropic effect in atria, induced by stimulation of M2 receptors. These results may contribute to a better understanding of the effects and side effects of cholinomimetic treatment in patients with cardiac neuromyopathy. British Journal of Pharmacology (2007) 151, 175–185. doi:10.1038/sj.bjp.0707180

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.

1 The aim of the present work was to examine the role of muscarinic acetylcholine receptors (mAChR) on DNA synthesis and CD40 expression in human fibroblast cells. Neonatal human skin fibroblast cultures were stimulated with carbachol in presence or absence of specific antagonists and the following parameters were measured: identification of mAChR subtypes, DNA synthesis, inositol phosphates (InsP) production and CD40 expression. 2 Human fibroblasts express mAChR with Kd 0.47 ± 0.11 nm and Bmax 236 ± 22 fmol mg protein-1. Carbachol stimulates DNA synthesis, InsP and the expression of CD40. All these effects were inhibited by atropine, mustard hydrochloride (4-DAMP) and pirenzepine but not by AF-DX 116 and tropicamide, indicating that M3 and M1 mAChR are implicated in carbachol action. The relative Ki of the antagonists obtained by competition binding assay was in parallel to the relative potency for blocking both carbachol-stimulated InsP accumulation and DNA synthesis. 3 The intracellular pathway leading to carbachol-induced biological effects involved phospholipase C and calcium/calmodulin, as U-73122 and trifluoroperazine blocked carbachol effects, respectively. Calphostin C, a protein kinase C inhibitor, had no effect, indicating that this enzyme does not participate in the system. 4 These results may contribute to a better understanding of the modulatory role of the parasympathetic muscarinic system on normal human fibroblast function. Fil: Casanova, M.. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina Fil: Furlán, C.. Universidad de Buenos Aires. Facultad de Odontología; Argentina Fil: Sterin, Leonor Josefina. Universidad de Buenos Aires. Facultad de Odontología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay; Argentina Fil: Borda, Enri Santiago. Universidad de Buenos Aires. Facultad de Odontología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay; Argentina

SUMMARY 1 In the present study, we examined whether cannabinoid receptor expression and the effects of receptor stimulation vary as a function of gonadal status in a peripheral tissue, namely the male rat parotid gland. Four groups of male rats were studied: gonadal intact, castrated, castrated testosterone (1 mg/100 g bodyweight) treated and gonadal intact testosterone treated. 2 The results showed that the density of CB1 receptors decreased after castration and that receptor density was restored to control values after testosterone treatment. This decrement was associated with a decrease of anandamide (10-10 to 10-5 mol/L)-induced cAMP accumulation and amylase release without changes in the anandamide-induced inhibition of Na+/K+-ATPase activity. 3 Castration did not modify either the subtype of cannabinoid receptor involved in the actions of anandamide or drug affinity for the receptor. 4 The mechanism underlying anandamide-induced cAMP accumulation, amylase release and inhibition of Na+/K+-ATPase activity, namely through the activation of adenylyl cyclase, was the same in control and castrated rats. 5 Basal cAMP accumulation, amylase release and Na+/K+-ATPase activity were not altered by castration. 6 Castration had no effect on the concentration of total protein. 7 It can be concluded that CB1 cannabinoid receptor expression is regulated by testosterone in male rat parotid gland and this has functional implications for cAMP accumulation and 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. Methods: Amylase release, binding and cyclic adenosine monophosphate (cAMP) assays, inositol phosphates (IPs) production and nitric oxide synthase (NOS) activity in the presence of cyclopentyl-1,3-dipropylxanthine (CPA) alone or in the presence of different inhibitory drugs were performed. Results: The binding parameters of specific A1 antagonist [3H]-cyclopentyl 1,3-dipropilxanthine ([3H]-DPCPX) in parotid gland membranes show a population of high affinity sites with Kd (nm) 0.53 ± 0.06 and Bmax (fmol mg−1 protein) 122.6 ± 10.2. CPA stimulation of A1 receptor exerts an increase in amylase release, IPs accumulation, cAMP production and NOS activity. All these A1 agonist effects were blocked by the A1 receptor antagonist DPCPX. Inhibitors of phospholipase C (PLC), calcium/calmodulin (CaM), protein kinase C (PKC), and adenylate cyclase, but not NOS, activities attenuated the CPA stimulatory effect on amylase release. The effect of CPA on amylase release significantly correlated with its action either on cAMP or on IPs accumulation. Conclusion: These results suggest that CPA activation of parotid gland A1 receptor induces a stimulatory effect on amylase release associated with increased production of cAMP and IPs accumulation. The mechanism appears to occur secondarily to stimulation of phosphoinositide turnover via PLC activation. This, in turn, triggers cascade reactions involving CaM and PKC. The CPA stimulation of NOS does not appear to participate in amylase release.

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.

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

We demonstrated the presence of circulating antibodies from Sjögren syndrome (SS) patients enable to interact with rat cerebral frontal cortex by activating muscarinic acetylcholine receptors (mAChRs). IgG from SS and IgG from normal subjects were studied by flow cytometry, enzyme immunoassay (ELISA), and radioligand binding assays. By flow cytometric and ELISA procedures, it was shown that IgG from SS patients reacted to cerebral frontal cortex cell surface. SS IgG was able to interact with mAChR by inhibiting 3H-QNB binding to its specific receptor. Besides, SS IgG displayed an agonistic-like activity associated to specific M1 and M3 mAChR activation, increasing nitric oxide synthase (NOS) isoform activities. Neuronal (n) and endothelial (e) NOS-mRNA gene expression of rat frontal cortex is induced by SS IgG. This article supports the participation of humoral immune alterations in SS, resulting in central parasympathetic functional deregulation. These antibodies alter mAChR activation, NOS activity, and eNOS and nNOS gene expression.

Cholinergic regulation of baker's yeast cell phagocytosis in rat cultured astrocytes was studied. Phagocytic activity was reduced by 1 x 10(-5) M of atropine or pirenzepine, but not by AF-DX116 or 4-DAMP. In addition, carbachol stimulated phagocytosis in a dose-dependent manner. Furthermore, only 1 x 10(-5)M of atropine, pirenzepine and 4-DAMP significantly reduced enhanced activity induced by 1 x 10(-7)M carbachol. It was also observed that L-NMMA, staurosporine, or U-73122, reduced phagocytosis activity while TFP failed to do so. Nitrite levels in astrocyte supernatants increased after baker's yeast cells were incorporated to astrocyte cultures, correlating with enhanced phagocytosis induced by carbachol stimulation, and were reduced by 1 x 10(-5) M of atropine, pirenzepine or aminopiridine, but not by AF-DX116 or 4-DAMP. Enhanced NO production triggered by astrocyte phagocytosis may have pathological consequences.

In this study, we investigated the different signalling pathways involved in muscarinic acetylcholine M(3) receptor-dependent modulation of Na(+)-K(+)-ATPase in parotid glands from normal and castrated rats. Carbachol inhibited the enzyme activity in parotid glands from control rats while it stimulated the enzyme activity in castrated rats. The inhibition of Ca(2+) calmodulin by trifluoperazine abolished the inhibitory effect of carbachol in control rats, while the inhibition of protein kinase C by staurosporine stimulated Na(+)-K(+)-ATPase. In castrated rats, trifluoperazine inhibited the carbachol-stimulant effect while staurosporine had no effect. Results indicate that in control glands the activation of a phospholipid-Ca(2+) calmodulin-dependent protein kinase C is responsible for the inhibitory effect of carbachol on Na(+)-K(+)-ATPase activity. In castrated rats, the activation of the enzyme by carbachol is regulated by its Ca(2+) calmodulin-stimulating action, and not by activation of protein kinase C. The activation of the Na(+)-K(+)-ATPase observed in castrated rats resulted in a decrease in carbachol-induced net K(+) efflux and thereby could decrease salivary fluid production.

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.

We demonstrated the presence of circulating antibodies from schizophrenic patients able to interact with cultured astrocytes activating muscarinic acetylcholine receptors (mAChRs). Sera and purified IgG from 15 paranoid schizophrenic and 15 age-matched normal subjects were studied by indirect immunofluorescence (IFI), flow cytometry, dot blot, enzyme immunoassay (ELISA), and radioligand competition assays. Astrocyte membranes and/or a synthetic peptide, with identical amino acid sequence of human M(1) and M(2) mAChR, were used as antigens. By IFI and flow cytometry procedures, we proved that serum purified IgG fraction from schizophrenic patients, reacted to astrocyte cell surface. The same antibodies were able to inhibit the binding of the specific mAChR radioligand (3)H-QNB. Using synthetic peptide for dot blot and ELISA, we demonstrated that these antibodies reacted against the second extracellular loop of human cerebral M(1) and M(2) mAChR. Also, the corresponding affinity-purified antipeptide antibody displayed an agonistic-like activity associated to specific M(1) and M(2) mAChR activation, increasing inositol phosphates accumulation and decreasing cyclic AMP production, respectively. This article gives support to the participation of an autoimmune process in schizophrenia disease.

The goal of this study was to investigate whether cruzipain, a Trypanosoma cruzi immunodominant antigen, was able to induce antibodies reactive to the cardiac M(2) muscarinic acetylcholine receptor (M(2) mAChR). Immunization with cruzipain that was devoid of enzyme activity triggered IgG antibodies against cardiac M(2) mAChR. By radioligand competition assay we proved that the anti-cruzipain IgG fraction, purified from serum, inhibited binding of the specific M(2) mAChR radioligand [(3)H]quinuclidinyl benzilate. We also demonstrated that anti-cruzipain IgG reacted against the second extracellular loop of the M(2) mAChR. The corresponding affinity-purified serum anti-M(2)e2 IgG (reacting against a synthetic peptide corresponding to this loop in humans) displayed agonist-like activity associated with specific M(2) mAChR activation - increase of cGMP, inositol phosphate accumulation and nitric oxide synthase activity - triggering a decrease in myocardial contractility. Moreover, the same IgG fraction decreased heart frequency, related to inhibition of adenylate cyclase activity. These results imply that cruzipain plays a role in the production of antibodies against M(2) mAChR, which have been related to the pathogenesis of dysautonomic syndrome described in Chagas' disease.

Summary. Mice myocardia, in which plasma erythropoietin (EPO) concentrations were modified in response to different experimental conditions, were studied to evaluate contractility (dF/dt). CF1 mice were randomly separated into four main groups: group I, normocythaemic normoxic; group II-a, normocythaemic intermittently exposed to hypobaria for 72 h; group II-b, normocythaemic intermittently exposed to hypobaria for 3 weeks; group III, hypertransfused polycythaemic exposed to 72 h hypobaria; and group IV, hypertransfused polycythaemic maintained in normobaric air. Plasma EPO, contractile studies and binding assays were performed. The dF/dt was significantly higher in group II-a than in group I and group II-b; but in groups III and IV, the dF/dt was reduced. The toxic action of ouabain was reduced and delayed in its onset, accompanied by increased numbers of 3H-ouabain binding sites in group II-a. Contractility was positively correlated with plasma EPO (pEPO) in the different groups. Treating group I with recombinant human (rHu)-EPO enhanced contractility while treating group II-a with a monoclonal anti-EPO decreased the dF/dt. The inhibition of enzymatic pathway(s) known to participate in the cytokines signal transduction, decreased the basal dF/dt values on atria from group II-a and on group I atria treated with rHu-EPO. The results demonstrated: (1) a cardiac non-haematopoietic effect of EPO; (2) that mice in which the pEPO concentration increased showed improvement in contractility and in the therapeutic action of ouabain; and (3) it is possible that EPO may act as a cardioprotective agent by modulating the cardiac Na+–K+ pump.

Atria from mice fed a selenium-deficient (Se−) diet have a diminished β-adrenoceptor-inotropic cardiac response to isoproterenol or norepinephrine compared with atria from mice fed the same diet supplemented with 0.2 mg/kg Se as sodium selenite (Se+). This diminished response could be reversed by feeding Se− mice the Se+ diet for 1 wk or by pretreatment with nitric oxide synthase (NOS) inhibitors such as N G-monomethyl-l-arginine or aminopyridine. Elevated serum concentrations of nitrite/nitrate as well as a threefold increase in the atrial NOS activity were seen in the Se− versus Se+ mice. Western blotting and indirect immunofluorescence indicated an enhanced expression of inducible NOS in hearts from Se− mice. Increased expression and activity of NOS and increased nitrite/nitrate levels from Se− mice correlated with an impaired response to β-adrenoceptor inotropic cardiac stimulation. Elevated nitric oxide levels may account for some of the pathophysiological effects of Se deficiency on the heart.

L. Sterin-Borda, L. Joensen, C. Bayo-Hanza, M. Esteva and E. Borda, Therapeutic Use of Muscarinic Acetylcholine Receptor Peptide to Prevent Mice Chagasic Cardiac Dysfunction. Journal of Molecular and Cellular Cardiology (2002) 34 , 1645–1654. Therapeutic use of a peptide corresponding to the aminoacid sequence of the second extracellular loop of human M 2 muscarinic acetylcholine receptor (M 2 mAChR peptide) was studied. Expression and biological activity of M 2 mAChR in association with circulating M 2 mAChR-related antibodies in cardiac tissue from chagasic mice were evaluated. Mice infected or not with trypomastigotes Tulahuen strain either treated or not treated with M 2 mAChR peptide were sacrificed at 8–9 weeks post-infection. Morphological, binding and contractility studies were performed on all animal groups. Hearts from infected mice showed a mAChR-related dysfunction, with a decrease in heart contractility, impaired response to exogenous mAChR agonist (carbachol) and a significant reduction of mAChR binding sites. Treating infected mice with M 2 mAChR peptide reversed those effects. Moreover, autoantibodies from infected mice recognized the M 2 mAChR peptide. In addition, serum from infected mice and the corresponding affinity purified IgG was capable of interacting with cardiac mAChR, reducing the number of binding sites and inhibiting the contractile response to exogenous agonist. In conclusion, (1) the development of alterations in mAChR related to cardiac dysfunction, may be associated with the presence of circulating antibodies against these receptors and (2) the chronic treatment with M 2 mAChR peptide prevented infected mice heart dysfunction. The mechanism could be explained by the ability of the M 2 mAChR peptide to inhibit the chronic interaction of autoantibodies specific to mAChR. The implication of M 2 mAChR peptide treatment in the host's immune response is discussed.

We studied β-adrenergic and muscarinic cholinergic receptor (MR) expression and proliferative response in lymphocytes from animals under chronic mild stress (CMS) model of depression (CMS animals). Animals were subjected to CMS (periods of food or water deprivation, changes in lighting conditions, tilted cage, etc.) for 12 weeks. CMS lymphocytes showed an altered mitogen-induced proliferation. CMS-B and -T lymphocytes showed an increment on β-adrenoceptor number and on intracellular responses to a β-agonist. CMS-T cells showed higher MR expression and lower cGMP responses than normal lymphocytes. MR were not detectable in normal B cells while CMS-B cells showed both MR expression and cGMP response. Beta and muscarinic stimulation influenced lymphocyte proliferative responses, in accordance with cAMP and cGMP responses. After 12 weeks of the CMS procedure, animals were treated with fluoxetine while the CMS procedure continued. Fluoxetine treatment reverted the alterations induced by CMS. These findings suggest a possible mechanism for the immune alterations found in depressive disorders and for the effect of fluoxetine treatment on immune response.

It is known that nitric oxide modulates the prostaglandin generation. However, little is known about the regulatory action of prostaglandin on nitric oxide production. There is a molecular cross-talk between nitric oxide and prostaglandin. Here, we examined biochemical signalling pathways coupled to the prostaglandin E 2 (PGE 2 ) receptor related to nitric oxide synthase stimulation in rat submandibular gland. PGE 2 through the stimulation of its own receptor, triggered activation of phosphoinositide turnover (IPs), translocation of protein kinase C (PKC), stimulation of nitric oxide synthase activity (NOS) and increased production of cyclic GMP (cGMP). PGE 2 stimulation of NOS and cGMP production was blunted by agents interfering with calcium influx, calcium/calmodulin and phospholipase C (PLC) activities; while PKC inhibitor was able to stimulate PGE 2 effects. PGE 2 did not evoke amylase release, indicating that NOS/ cGMP pathway were not associated with this enzyme secretion. Our results suggest that this prostanoid could act as vasoactive chemical mediator through its ability to activate NOS–cGMP pathway via own gland membrane receptor.

In this study we have determined the different signalling pathways involved in adenosine A1-receptor (A1-receptor)-dependent inhibition of contractility in rat isolated atria. N-cyclopentyladenosine (CPA) stimulation of A1-receptor exerts: negative inotropic response, inositol phosphates accumulation, stimulation of nitric oxide synthase (NOS), increased production of nitric oxide (NO) and cyclic GMP. Inhibitors of phospholipase C (PLC), protein kinase C (PKC), calcium/calmodulin, NOS and guanylate cyclase shifted the dose-response curve of CPA on contractility to the right. Those inhibitors also attenuated the A1-receptor-dependent increase in cyclic GMP and activation of NOS. These results suggest that CPA activation of A1-receptors exerts a negative inotropic effect associated with increased production of nitric oxide and cyclic GMP. The mechanism appears to occur secondarily to stimulation of phosphoinositide turnover via PLC activation. This, in turn, triggers cascade reactions involving calcium/calmodulin and PKC, leading to activation of NOS and soluble guanylate cyclase. British Journal of Pharmacology (2002) 135, 444–450; doi:10.1038/sj.bjp.0704487

Background: Histamine is released from mast cells by immunologic and non-immunologic stimuli during salivary gland inflammation, regulating salivary secretion. The receptor-secretory mechanism has not been studied in detail.Aims: The studies reported were directed toward elucidating signal transduction/second messenger pathways within the rat submandibular gland associated with 2-thiazolylethylamine (ThEA)-induced H1-receptor responses.Materials and methods: To assess the H1receptor subtype expression in the rat submandibular gland, a radioligand binding assay was performed. The study also included inositolphosphates and cyclic GMP accumulation, protein kinase C and nitric oxide synthase activities, and amylase release.Results: The histamine H1receptor subtype is expressed on the rat submandibular gland with high-affinity binding sites. The ThEA effect was associated with activation of phosphoinositide-specific phospholipase C, translocation of protein kinase C, stimulation of nitric oxide synthase activity and increased production of cyclic GMP. ThEA stimulation of nitric oxide synthase and cyclic GMP was blunted by agents able to interfere with calcium movilization, while a protein kinase C inhibitor was able to stimulate ThEA action. On the other hand, ThEA stimulation evoked amylase release via the H1receptor but was not followed by the L-arginine/nitric oxide pathway activation.Conclusions: These results suggest that, apart from the effect of ThEA on amylase release, it also appears to be a vasoactive chemical mediator that triggers vasodilatation, modulating the course of inflammation.

BACKGROUND AND AIMS—Gastrointestinal disorders is one of the clinical manifestations of chronic Chagas' disease. The pathogenesis seems to be associated with autonomic dysfunction. Here, we consider the muscarinic cholinoceptor mediated alteration in distal colon function in chagasic megacolon. PATIENTS—Patients were divided into four groups: group I, chronic chagasic patients with megacolon; group II, chronic chagasic patients without megacolon; group III, non-chagasic patients with megacolon; and group IV, normal healthy volunteers (control). METHODS—Binding assay and immunoblot of cholinoceptors from human and rat colon and enzyme immunoassay (ELISA) using a synthetic 24mer peptide corresponding to the second extracellular loop of human M2 muscarinic acetylcholine receptors (mAChR) were used to detect the presence of serum antibodies. The effect of antibodies on basal tone and 3',5'-cyclic monophosphate (cAMP) production of human and rat distal colon strips were also tested. RESULTS—Group I but not the other groups had circulating antibodies capable of interacting with human colon activating M2 mAChR, as they competed with binding of specific radioligand to mAChR and interacted with the second extracellular loop of human M2 mAChR. Moreover, affinity purified anti-M2 peptide IgG from group I, in common with monoclonal antihuman M2 mAChR, recognised bands with a molecular weight corresponding to colon mAChR. This antibody also displayed an agonist-like activity, increasing basal tone and decreasing cAMP accumulation. Both effects were blunted by AF-DX 116 and neutralised by the synthetic peptide. CONCLUSIONS—In chagasic patients with megacolon there are antibodies that can recognise and activate M2 mAChR. The implications of these autoantibodies in the pathogenesis of chagasic megacolon is discussed. Keywords: chagasic megacolon; acetylcholine receptor; antibodies; colon contractility

Isolated congenital heart block may be associated with autoimmune disorder such as Sjögren Syndrome and systemic lupus erythematosus. In this work we demonstrate circulating autoantibodies against neonatal heart M1 muscarinic acetylcholine receptor (mAChR) in the sera of children with congenital heart block. This antibody were able to react with the second extracellular loop of the human M1 mAChR as demonstrated using a synthetic peptide in enzyme immune assay and binding assay. Affinity purified anti-peptide IgG as well as total IgG from children with congenital heart block, interfered with the specific radioligand occupancy from neonatal heart M1 mAChR, interacting irreversibly. The antipeptide antibodies also displayed an 'agonist-like' activity, i.e. decreased contractility, activated nitric oxide synthase activity and increased production of cyclic GMP. All of these effects were selectively blunted by pirenzepine and neutralized by the synthetic M1 peptide. Both binding and biological effects were obtained using neonatal rat heart instead adult heart and were independent of Ro/SS-A and La/SS-B antibodies and were also absent in the sera of normal children. A clinical relevance of these findings is demonstrated by a strong association between the existence of circulating M1 mAChR antipeptide antibodies and the presence of isolated congenital heart block, making these antibodies a proper marker of this disease.

beta-Adrenoceptor (betaAR) expression and function as well as its modulation via intracellular transduction signals, were analyzed on the T cell lymphoma BW5147. Independently to the kinetic of proliferation and relative to the number of receptors displayed in normal T lymphocytes, BW5147 cells displayed a decreased number of betaAR, uncoupled to adenylate cyclase, but coupled to protein kinase C stimulation. This last effect was impaired by a beta-antagonist and by blockers of the enzymatic pathways involved in T lymphocyte proliferation, inducing a recovery of betaAR sites. Down-regulation of betaAR would implicate the loss of a negative neuroimmune control mechanism for lymphocyte proliferation. The coupling of the remaining sites to a positive signal for cellular activation, would contribute to establish an hyperproliferative state.

In this work, we studied the effect of fluoxetine on human T-lymphocyte proliferation using optimal and suboptimal concanavalin A concentrations. In particular, we analyzed the influence of fluoxetine on the kinases that are involved in intracellular signalling after stimulation with mitogens. We found that fluoxetine promoted the Ca2+-mediated proteolysis of protein kinase C (PKC) and increased cyclic-AMP (cAMP) levels, thereby impairing lymphocyte proliferation, when optimal concanavalin A concentrations were used. In contrast, when suboptimal concanavalin A concentrations were used, fluoxetine only increased PKC translocation, without modifying cAMP levels, leading to T-cell proliferation. According to our results, fluoxetine has a dual effect on T-cell proliferation by modulating the PKC and protein kinase A pathways. This mechanism is thought to be mediated through Ca2+ mobilization.

L'haloperidol influe sur le blocage des recepteurs D 2 de la dopamine, mais il peut aussi interagir avec de multiples neurotransmetteurs (NT). Son action sur le renouvellement des phosphoinositides (PI) a ete examinee sur des preparations de cortex cerebral. L'haloperidol a induit une augmentation du taux d'inositol phosphates (IP), qui n'a ete diminuee que par le bloqueur des recepteurs adrenergiques α 1 , prazosine. L'effet maximal de l'haloperidol (E max ) a ete plus faible que celui de l'agoniste complet norepinephrine (NE). Les courbes dose-reponse tant pour la NE en presence de doses sous-maximales d'haloperidol que pour l'haloperidol en presence de doses E max de NE ont montre que l'haloperidol agit comme un agoniste partiel des recepteurs adrenergique α 1 cerebraux. L'effet de l'haloperidol sur l'hydrolyse des PI est vehicule par l'activation de la phospholipase C, etant donne que NCDC et U-73122 ont pu inhiber tant les actions de la NE que celles de l'haloperidol. De plus, le neuroleptique induit une activation directe des recepteurs adrenergiques α 1 , etant donne que ses actions n'ont pas ete modifiees par la cocaine et ont persiste malgre une denervation cerebrale chimique des rats au moyen de 6-hydroxytryptamine (6-OHDA). La possibilite que cette action agoniste sur les recepteurs adrenergiques α 1 fasse partie des effets secondaires de l'haloperidol est aussi discutee.

In addition to its effect on D2 dopamine receptor blockades, haloperidol is able to interact with multiple neurotransmitters (NTs). Its action on phosphoinositide (PI) turnover was studied on cerebral cortex preparations. It induces an increase in inositol phosphate (IP) accumulation, which was only blunted by the α1-adrenoceptor blocker prazosin. Haloperidol maximal effect (Emax) was less than the effect of the full agonist norepinephrine (NE), and dose-response curves for both NE in the presence of submaximal doses of haloperidol and haloperidol in the presence of Emax doses of NE showed that haloperidol behaves as a partial agonist of cerebral α1-adrenoceptors. Its effect on PI hydrolysis is mediated through phospholipase C activation, as 2-nitro-4-carboxyphenyl-N,N-diphenylcarbamate (NCDC) and 1-[6-([(17β)-3-methoxyestra-1,3,5(10)-trien-17-yl]amino)hexyl]-1H-pyrrole-2,5-dione) (U-73122) were able to abrogate both haloperidol and NE actions. Further, the typical neuroleptic exerts a direct activation of α1-adrenoceptors as its actions were not modified by cocaine and persisted in spite of chemical rat cerebral denervation with 6-hydroxydopamine (6-OHDA). The possibility that this agonistic action on α1-adrenoceptors would be involved in haloperidol side effects is also discussed.Key words: haloperidol, neuroleptics, α1-adrenoceptor, phosphoinositide hydrolysis, cerebral frontal cortex.

This study reports the expression of inducible nitric oxide synthase (NOS) in heart from autoimmune myocarditis mice associated with an alteration in their contractile behavior. By mean of the production of [U −14 C]citrulline from [U −14 C]arginine and immunoblot assay, the expression of iNOS was demonstrated in autoimmune atria that was normally absent. The iNOS activity decreased with administration of dexametasone and in mice treated with monoclonal anti-interferon- γ antibody (anti-IFN- γ mAb). The inhibitors of protein kinase C activity (staurosporine) but not calcium/calmodulin (trifluoperazine) attenuated the iNOS activity. Moreover, autoimmune atria presented contractile alterations (lower values of dF/d t than control). The in vivo treatment with inhibitors of NOS activity or anti-IFN- γ mAb or dexametasone improved the contractile activity of autoimmune atria with no change in the contractility of normal atria. The results suggest that the infiltrative cells in myocarditis heart have a potential role in cardiac dysfunction by production of IFN- γ and subsequent expression of iNOS, that in turn alter the contractile behavior of the heart. The data indicate that cytokines induced activation of L -arginine nitric oxide pathway in myocarditis atria leading to contractile dysfunction.

The present study was undertaken to analyse the effect of fluoxetine upon murine T-lymphocyte proliferation. We found that fluoxetine exerted a dual effect, which depended on the degree of lymphocyte activation: at mitogenic concentration (2 μg/mL) of concavalin A (Con A), we observed an inhibitory effect on cellular proliferation, whereas, on submitogenic Con A concentration (1 μg/mL), fluoxetine stimulated the cellular response. Given these facts, we studied PKC activation and calcium mobilisation in both stimulatory and inhibitory effects of fluoxetine on T-cell proliferation. We observed that fluoxetine increased PKC translocation obtained with 1 μg/mL Con A concentration, whereas PKC was degraded when 2 μg/mL was used. This mechanism is thought to be mediated by calcium mobilisation. According to our results, fluoxetine seemed to modulate calcium influx, which, in turn, would influence PKC translocation, modulating the immune response.

Biochemical signalling events coupled to muscarinic cholinergic receptors (mAChR), specifically those related to nitric oxide (NO) production, were studied on rat cerebral frontal cortex. The mAChR agonist carbachol was found to exert a specific biphasic action on NO synthase (NOS) activity: low doses ranging between 10(-9) M to 10(-7) M lead to NOS activation while higher doses (10(-6) M) inhibited enzymatic activity. Carbachol stimulatory action was blunted by agents that interfere with calcium-calmodulin while a protein kinase (PKC) inhibitor, staurosporine was able to abrogate the inhibitory effect. Moreover, PKC activity showed maximum translocation to cerebral frontal cortex membranes with carbachol concentrations that inhibited NO production. Products from phosphoinosite (PI) hydrolysis are involved in these actions as carbachol was found to increase PI turnover in a dose dependent manner. These results would serve as an example of cross-talk between both enzymatic pathways.

We examined some of the signalling events in the negative modulation of isoproterenol-induced stimulation of contractility in rat isolated atria. Isoproterenol-mediated positive inotropic response is accompanied by the stimulation of nitric oxide synthase (NOS) and an increase in the production of cyclic GMP (cGMP). Inhibition of NOS and guanylate cyclase increased the dose-response curve of isoproterenol on contractility. Inhibitors of calcium flux or calcium calmodulin, but not of protein kinase C, abrogated these mechanisms. The existence of a modulatory negative inotropic–cyclic GMP–mediated mechanism limiting the effect of β-adrenergic stimulation in myocardium is discussed.

SUMMARY IgA was obtained from HIV-infected haemophilic patients and the intracellular signals triggered by its reaction with isolated rat intestinal strips were studied. HTV+ IgA stained intestinal microvilli with a granular immunofluorescence pattern and bound to the muscarinic acetylcholine receptor (mAChR), displacing the specific muscarinic cholinergic antagonist QNB in a non-competitive manner. It triggered the signals that are the consequence of mAChR stimulation in die intestine. Thus, it decreased cAMP synthesis and increased guanosine 3′:5′-cyclic monophosphate (cGMP) formation and phosphoinositide (PI) turnover of the intestine. In addition, it stimulated prostaglandin E2 (PGE2) synthesis by intestinal strips. Through its effect on PGE2 synthesis, HIV+ IgA could have a dual action. On the one hand, it could enhance immunosuppression at a local level, favouring pathogen growth and subsequent intestinal dysfunction. On the other hand, PGE2 could directly increase intestinal motility and electrolyte/fluid loss. Both effects could be involved in intestinal damage in AIDS.

SUMMARY Human and experimental Chagas' disease causes peripheral nervous system damage involving neuromuscular transmission alterations at the neuromuscular junction. Additionally, autoantibodies directed to peripheral nerves and sarcolemmal proteins of skeletal muscle have been described. In this work, we analyse the ability of serum immunoglobulin factors associated with human chagasic infection to bind the affinity-purified nicotinic acetylcholine receptor (nAChR) from electric organs of Discopyge tschudii and to identify the receptor subunits involved in the interaction. The frequency of serum anti-nAChR reactivity assayed by dot-blot was higher in seropositive chagasic patients than in uninfected subjects. Purified IgG obtained from chagasic patients immunoprecipitated a significantly higher fraction of the solubilized nAChR than normal IgG. Furthermore, immunoblotting assays indicated that α and β are the main subunits involved in the interaction. Chagasic IgG was able to inhibit the binding of α-bungarotoxin to the receptor in a concentration-dependent manner, confirming the contribution of the α-subunit in the autoantibody-receptor interaction. The presence of anti-nAChR antibodies was detected in 73% of chagasic patients with impairment of neuromuscular transmission in conventional electromyographical studies, indicating a strong association between seropositive reactivity against nAChR and electromyographical abnormalities in chagasic patients. The chronic binding of these autoantibodies to the nAChR could induce a decrease in the population of functional nAChRs at the neuromuscular junction and consequently contribute to the electrophysiological neuromuscular alterations described in the course of chronic Chagas' disease.

Taking into account that the activation of different subtypes of ileal muscarinic acetylcholine receptors (mAChR) regulate gut functions such as tone, motility, and electrolyte secretion, we characterized the expression of mAChR in ileal-purified membranes. We also studied intracellular signals triggered by mAChR activation. Binding parameters obtained from saturation assays with the nonselective tritiated muscarinic antagonist, quinuclidynil benzilate ([3H]-QNB), were maximal number of binding sites (Bmax): 30 ± 2 fmol/mg prot and dissociation constant (Kd): 0.2 ± 0.03 nM. The competitive inhibition of [3H]-QNB specific binding by various nonlabelled muscarinic antagonists was measured and the rank order of potency was: atropine (ATROP) > 4-DAMP > AF-DX 116 > pirenzepine (PZ). The activation of mAChR by carbachol (CARB) increased ileal motility in a concentration-dependent manner (EC50 2 × 10−7 M). The antagonists' order of potency to displace dose-response curve of CARB was: ATROP > 4-DAMP > AF-DX116 > PZ. Optimal concentration of CARB on ileal strips increased phosphoinositide turnover and cGMP levels by activating m3 receptor subtype and decreased isoproterenol (ISO) stimulated levels of cAMP due to M2 receptor activation. We can conclude that the activation of different mAchR subtypes triggers different intracellular signals that could regulate intestinal tone and motility.

The possible role of altered humoral immune response in the pathogenesis of the chronic chagasic cardioneuromyopathy was examined by analyzing the interaction of IgG from T. cruzi infected patients with cardiac muscarinic acetylcholine receptors (mAChR). Human chagasic IgG by activating cardiac M2 mAChR, simulated the agonist actions triggering negative inotropic effect, inositol phosphate accumulation, nitric oxide synthase stimulation and increased production of cyclic GMP. Inhibitors of phospholipase C, protein kinase C, calcium/calmodulin, nitric oxide synthase and guanylate cyclase activities; prevented chagasic IgG effects on signaling pathways involved in M2 mAChR activation. In addition, sodium nitroprusside or 8-bromo cyclic GMP, mimicked the chagasic IgG effect associated with cholinergic-mediated cellular transmembrane signals. Moreover, these chagasic IgG immunoprecipitated the mAChRs solubilized from cardiac membranes. By means of SDS-PAGE and immunoblotting analysis, chagasic sera recognized a band of 70-75 kDa. The major protein recognized by chagasic IgG had an Rf coincident with the peak of [3H] propylbenzilylcholine mustard with an apparent molecular weight similar to that of mAChRs, which disappeared in the presence of atropine. The specificity of this interaction was checked by immunoprecipitation of rat cardiac mAChR and immunoblotting of pure human M2 mAChRs. Chronic interaction of chagasic IgG with myocardial mAChRs, behaving as a muscarinic agonist, might lead to cell dysfunction or tissue damage. Also, these antibodies could produce desensitization, internalization or degradation of mAChRs; explaining the progressive blockade of mAChRs in myocardium with parasympathetic denervation, a phenomenon that has been described in the course of Chagas' cardioneuromyopathy.