Increase nitric oxide synthase activity in parotid glands from rats with experimental periodontitis.

OBJECTIVE: In this study we investigated the activity of the nitric oxide synthase (NOS) in parotid glands from rats with experimental periodontitis and controls. METHODS: Periodontitis was produced by a ligature placed around the cervix of the two lower first molar. Experiments were carried out 22 days after the ligature. RESULTS: Ligation caused an increase in parotid NOS activity. The selective blocker of the inducible isoform of the enzyme partially inhibited its activity in parotid glands from rat with ligature. In controls, the activity was partially inhibited by the antagonists of the selective neural and endothelial isoforms. NOS activity in rats with ligature was cyclic adenosine monophosphate (cAMP)-dependent while in controls it was calcium-dependent. Prostaglandin E2 concentration was increased in parotid gland from rats with ligature. The inhibitor of prostaglandin production, FR 122047, diminished both, prostaglandin production and NOS activity. In rats with ligature unstimulated amylase released is increased. Both, prostaglandin and NOS were involved in the increment of amylase release. CONCLUSION: It can be concluded that in parotid glands from ligated rats, prostaglandin E2 production is increased and, through cAMP accumulation, activates the inducible NOS isoform. The increment of nitric oxide production participates in the increase in basal amylase release.

Acquisition of flavour preferences in pigs through interactions with conspecifics that had previously consumed flavoured protein solutions.

It is known that pigs can acquire flavour preferences by brief social interactions with conspecifics that previously consumed a flavoured solid feed. However, there is no information about whether a flavoured solution could support flavour preferences through social transmission. Ninety-six pigs (49 days old) were housed in 12 pens (8 pigs/pen). Four animals per pen were randomly selected to act as observers and four as demonstrators. Demonstrator animals were temporarily moved to an empty pen where a protein solution was offered (porcine digestive peptides (PDPs), 4% weight/volume) with the addition of 0.075% aniseed (six pens) or garlic (six pens) powdered artificial flavours for 30 min. Afterwards, demonstrators were returned to interact with observer animals for 30 min. A choice test (30 min) between aniseed and garlic PDP was performed for each observer group after the interaction. Observers showed a higher intake of solutions previously consumed by their demonstrator conspecifics (648 v. 468 ml; SEM 61.36, P < 0.05). As with flavoured solid feeds, protein solutions containing artificial flavours can create preferences in pigs for those flavours through social transmission from conspecifics.

Autoantibodies to beta1-adrenoceptors in human chronic periodontitis induce overexpression of fibroblast CD40 and trigger prostaglandin E2 generation.

BACKGROUND AND OBJECTIVE: Autoimmune mechanisms may contribute to the pathogenesis of periodontal disease. Autoantibodies with the potential to bind and activate beta(1)-adrenoceptors (beta(1)-AR) of human gingival fibroblasts were studied to provide evidence of altered humoral immune response in chronic periodontal disease. MATERIAL AND METHODS: Flow cytometry and enzyme-linked immunosorbent assay using cell culture-adherent gingival fibroblasts and/or their purified membranes and/or a synthetic peptide corresponding to the second extracellular loop of human beta(1)-AR were used to detect serum antibodies. The effects of antibodies from chronic periodontal disease patients on PGE(2) generation and CD40 expression were also tested. RESULTS: Circulating immunoglobulin G (IgG) from chronic periodontal disease patients (but not from normal individuals) interacted with the fibroblast surface, activating beta(1)-AR. Atenolol or CGP 20712 (beta 1-AR antagonists) and beta(1) synthetic peptide inhibited the interaction of IgG with beta(1)-AR. Immunoglobulin G from chronic periodontal disease patients also displayed agonist-like activity associated with specific beta(1)-AR activation, increasing PGE(2) generation and CD40 overexpression. The corresponding affinity-purified anti-beta(1)-AR peptide IgG mimicked these effects. Both effects were prevented by inhibition of cyclo-oxygenase. CONCLUSION: This article supports the participation of humoral immune alterations in chronic periodontal disease resulting in postsynaptic functional deregulation. Overproduction of proinflammatory mediators (PGE(2) and CD40 expression) is induced as a consequence of antibody-beta(1)-AR interaction. The PGE(2)-CD40-IgG axis may play a part in the pathophysiological mechanisms underlying the inflammatory process in chronic periodontal disease.

Differential cholinoceptor modulation of nitric oxide isoforms in experimentally-induced inflammation of dental pulp tissue.

AIM: The aim of the study was to investigate the role of muscarinic acetylcholine receptor (mAChR) activity in the regulation of endothelial (e), neuronal (n) and inducible (i) nitric oxide synthase (NOS) activity and expression in experimentally induced inflammation of rat dental pulp tissue. METHODOLOGY: 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 tissues were used as controls. NOS activity was measured by the production of [U-(14)C]-citrulline from [U-(14)C]-arginine. Nitrite/nitrate assay was evaluated by the conversion of nitrate to nitrite in the presence of nicotinamide adenine dinucleotide phosphate. i-nos, e-nos and n-nos mRNA levels were measured using reverse-transcriptase polymerase chain reaction by co-amplification of target cDNA with a single set of primers. RESULTS: Application of LPS to the pulp increased NOS activity and nitrate production (P < 0.001), generated by iNOS over-activity and expression. Pilocarpine acting on mAChRs triggered a biphasic action on NOS activity and NO accumulation. At low concentrations, pilocarpine induced a negative effect associated with a decrease in i-nos mRNA level, whilst at high concentration, it produced a positive effect associated with increased e-nos and n-nos mRNA levels. In control pulp tissue, only the positive effect of pilocarpine was observed. CONCLUSIONS: Irreversible pulpitis changes mAChR conformation increasing its efficiency of coupling to transducing molecules that in turn induce activate iNOS. The capacity of pilocarpine to prevent NO accumulation and iNOS activity, by acting on mAChR mutation induced by pulpitis, might be useful therapeutically as a local treatment.

Changes in floral biology and inbreeding depression in native and invaded regions of Datura stramonium.

Plant populations invading new environments might compromise their fitness contribution to the next generation, because of the lack of native specialist pollinators and/or potential mates. Thus, changes in plant mating system and traits linked to it are expected in populations colonising new environments where selection would favour selfing and floral traits that maximise reproductive output. To test this, we studied native (Mexico) and non-native (Spain) populations of the obligate sexual reproducing annual weed Datura stramonium. Flower size, herkogamy, total number of seeds per plant, number of visits by and type of pollinators, and inbreeding depression were assessed in native and non-native populations. Finally, we measured phenotypic selection on corolla size and herkogamy in each population. Flower size and herkogamy showed wide and similar variation in both ranges. However, the largest average flower size was found in one non-native population whereas the highest average positive herkogamy was detected in one native population. On average, flowers in the native range received more visits by pollinators. Hawkmoths were the main visitors in the native populations while only bees were observed visiting flowers in Spain's populations. Only in the native range was inbreeding depression detected. Selection to reduce herkogamy was found only in one native population. Absence of both inbreeding depression and selection on floral traits suggest a change in mating system of D. stramonium in a new range where generalist pollinators may be promoting high reproductive success. Selection against deleterious alleles might explain the reduction of inbreeding depression, promoting the evolution of selfing.

Mechanisms involved in the regulation of mRNA for M2 muscarinic acetylcholine receptors and endothelial and neuronal NO synthases in rat atria.

BACKGROUND AND PURPOSE: Agonists of the M(2) 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 M(2) 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 M(2) 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 M(2) 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 M(2) receptors induced nNOS and eNOS expression and activation of NOS up-regulated M(2) 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 M(2) receptors. These results may contribute to a better understanding of the effects and side effects of cholinomimetic treatment in patients with cardiac neuromyopathy.

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 PGE(2) production. Our findings indicated that pSS IgG-stimulating M(3), M(4), and M(1) cholinoreceptors exerted an increase in COX-2 mRNA without affecting COX-1 mRNA expression and increased PGE(2) production. Inhibitors of phospholipase A(2), COX- s, L-type calcium channel currents, and Ca(2+)-ATPase from sarcoplasmic reticulum prevented the pSS IgG effect on PGE(2) production. An ionophore of calcium mimicked pSS IgG action, suggesting a crucial role of calcium homeostasis in the cholinoreceptor-stimulated increase in PGE(2) production. Moreover, the amounts of PGE(2) 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.

Signaling pathways leading to prostaglandin E(2) production by rat cerebral frontal cortex.

In this paper, we have determined the effect of both muscarinic acetylcholine receptor (mAChR) and exogenous prostaglandin E(2) (PGE(2)) on PGE(2) production and cyclooxygenases (COX) mRNA gene expression on rat cerebral frontal cortex. Carbachol and PGE(2) increase endogenous PGE(2) production and the COX-1 mRNA levels by activation of PLA(2)s. The COX-1 and COX-2 activity participated in the production of PGE(2) triggered by exogenous PGE(2). While in carbachol-PGE(2) only COX-1 activity is affected. The specific inhibition of PGE(2) receptor was able to impair the increase of endogenous PGE(2) production triggered by both carbachol and exogenous PGE(2). These results suggest that carbachol-activation mAChR increased PGE(2) production that in turn interacting with its own receptor triggers an additional production of PGE(2). Both mechanisms appear to occur by using PLA(2) signaling system. This data should be able to contribute to understand the involvement of PGE(2) in normal brain function and its participation in neuroinflammatory processes.

Muscarinic cholinoceptor activation modulates DNA synthesis and CD40 expression in fibroblast cells.

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.

The use of porcine digestible peptides and their continuity effect in nursery pigs.

A total of 552 entire male and female nursery pigs were selected to be used in 2 different experiments that aimed to study if milk ingredients can be replaced by highly preferred protein sources (Exp. 1) and if pre- and postnatal exposure of those protein ingredients through the maternal diet may increase pig performance (Exp. 2). In Exp. 1, 240 pigs were separated after weaning (28 d) into 2 groups depending on the presence of lactose in their diets. Pigs ( =120) fed diets with the precence of lactose (lactose +) were given prestarter (0-14 d) and starter (15-33 d) diets with 142 and 50 g/kg of sweet milk whey, respectively; the lactose-free group ( = 120) was offered an isoenergetic diet with 20 g/kg of porcine-digestible peptides (PDP; Palbio 62SP; Bioibérica S.A., Palafolls, Spain) and wheat replacing sweet milk whey. Choice and 1-feeder tests were performed in another group of animals ( = 72) to evaluate the preference and acceptance for both diets. Pigs preferred ( = 0.039) the lactose+ over the lactose-free diet after a 30-min choice test and consumed more ( = 0.001) lactose+ than lactose-free diet in a 1-feeder test. However, no difference ( > 0.467) in performance was observed between groups for the entire nursery period. In Exp. 2, 120 animals were obtained from sows that, during late gestation (14 d) and lactation (28 d), were fed diets containing 20 g/kg of PDP and another 120 animals were obtained from sows fed an isoenergetic diet without PDP inclusion. Placenta samples were collected at farrowing to assess the volatile compounds present in the placental fluid of sows. After weaning, all pigs received a feed containing 20 g/kg of PDP in the prestarter and starter diets. A principal components analysis of the total volatile compounds showed the exclusive presence of sulfur-containing compounds and a higher presence of terpene compounds in the placental fluid of PDP-supplemented sows. In addition, pigs coming from sows fed diets supplemented with PDP tended to show a higher ADFI ( = 0.07) and ADG ( = 0.06) than did pigs coming from control sows during the 15 to 33 d after-weaning period. These results suggest that dietary incorporation of sweet milk whey may be replaced by a specific protein source without affecting performance of pigs after weaning. However, more experiments are needed to elucidate the mechanism for the sow's diets' influence over pig's performance.

Intracellular signals coupled to different rat ileal muscarinic receptor subtypes.

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 x 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 ml 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.

Increases in cyclic AMP levels couple to H1 receptors in atria from autoimmune myocarditis mice.

We have previously shown that myocardium from experimental autoimmune myocarditis expresses H1 receptors not present in normal mice heart. ThEA acting via H1 receptors, augments cyclic AMP production in atria from autoimmune myocarditis mice without any effect on atria from control mice. Addition of mepyramine before ThEA caused cyclic AMP levels to fall to a level similar to basal, confirming the H1 receptor participation. Histamine at low concentrations mimicked the ThEA action on H1 receptor-stimulation of cyclic AMP production by autoimmune myocardium. The fact that the inhibition of phospholipase C blocked the cyclic AMP stimulation by ThEA, supports the assumption that this action is secondary to receptor-mediated hydrolysis of phosphoinositides, generating some oxidative metabolites (IP3-DAG), which in turn may be responsible for the cyclic AMP effect. So, the inhibition of protein kinase C and calcium/calmodulin partially prevented the stimulatory action of ThEA on cyclic AMP levels in autoimmune myocardium, suggesting that both pathways are implicated in this effect. Data shows that the stimulation of H1 receptors by specific agonist in atria from autoimmune myocarditis mice, augments the cyclic AMP, requiring the hydrolysis of phosphoinositide cycle. The role of this cyclic AMP augmentation in myocardium from autoimmune myocarditis mice, will provide a basis to assess the role of this second messenger as an important factor in the regulation and/or modulation of the physiological behaviour of the heart in the course of autoimmune myocarditis.

Role of nitric oxide in cardiac beta-adrenoceptor-inotropic response.

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 beta-adrenergic stimulation in myocardium is discussed.

Neuronal nitric oxide synthase activity in rat urinary bladder detrusor: participation in M3 and M4 muscarinic receptor function.

1. The aim of this paper was to determine the different signalling cascades involved in contraction of the rat urinary bladder detrusor muscle mediated via muscarinic acetylcholine receptors (muscarinic AChR). Contractile responses, phosphoinositides (IPs) accumulation, nitric oxide synthase (NOS) activity and cyclic GMP (cGMP) production were measured to determine the reactions associated with the effect of cholinergic agonist carbachol. The specific muscarinic AChR subtype antagonists and different inhibitors of the enzymatic pathways involved in muscarinic receptor-dependent activation of NOS and cGMP were tested. 2. Carbachol stimulation of M(3) and M(4) muscarinic AChR increased contractility, IPs accumulation, NOS activity and cGMP production. All of these effects were selectively blunted by 4-DAMP and tropicamide, M(3) and M(4) antagonists respectively. 3. The inhibitors of phospholipase C (PLC), calcium/calmodulin (CaM), neuronal NOS (nNOS) and soluble guanylate cyclase, but not of protein kinase C and endothelial NOS (eNOS), inhibited the carbachol action on detrusor contractility. These inhibitors also attenuated the muscarinic receptor-dependent increase in cGMP and activation of NOS. 4. In addition, sodium nitroprusside and 8-bromo-cGMP, induced negative relaxant effect. 5. The results obtained suggest that carbachol activation of M(3) and M(4) muscarinic AChRs, exerts a contractile effect on rat detrusor that is accompanied by an increased production of cGMP and nNOS activity. The mechanism appears to occur secondarily to stimulation of IPs turnover via PLC activation. This in turn, triggers cascade reactions involving CaM, leading to activation of nNOS and soluble guanylate cyclase. They, in turn, exert a modulator inhibitory cGMP-mediated mechanism limiting the effect of muscarinic AChR stimulation of the bladder.

Alterations in cardiac beta-adrenergic receptors in chagasic mice and their association with circulating beta-adrenoceptor-related autoantibodies.

OBJECTIVE: Cardiac tissue from chagasic mice was studied to evaluate the expression and biological activity of beta-adrenoceptors in association with circulating beta-adrenoceptor-related autoantibodies. METHODS: BALB/c inbred mice that were either treated or not treated with atenolol (2.5 mg/kg) and infected or not infected with 1 x 10(4) trypomastigotes (CA-1 strain) were sacrificed weekly up to week nine. Morphological, binding and contractility studies were performed on the four different groups of animals. The effect of their serum antibodies was also assayed in binding and contractility studies on normal heart preparations. RESULTS: Hearts from chagasic myocarditis mice showed a beta-adrenoceptor-related dysfunction, with a decrease in heart contractility, impaired response to exogenous beta-adrenoceptor agonist and a significant reduction in beta-adrenergic binding sites. Those effects were maximum at eight-nine weeks post-infection and were improved by treating infected mice with atenolol. In addition, serum or IgG from chagasic myocarditis mice was capable of interacting with cardiac beta-adrenoceptors, reducing the number of binding sites and inhibiting the contractile response to exogenous norepinephrine. IgG effects that were observed in normal myocardium, were highest in sera from mice eight-nine weeks post-infection and correlate with the degree of myocarditis. Moreover, chagasic autoantibodies from infected mice recognized a peptide corresponding to the sequence of the second extracellular loop of the human beta 1-adrenoceptor. CONCLUSIONS: (1) The development of alterations in beta-adrenergic receptors, related to cardiac dysfunction, may be associated with the presence of circulating antibodies against these receptors and (2) it is possible that the chronic deposits of these autoantibodies in cardiac beta-adrenoceptors could lead to a progressive blockade with sympathetic denervation, a phenomenon that has been described in the course of chagasic myocarditis.

Tyrosine kinase regulatory action on ileal muscarinic effects of IFN-gamma.

Interferon-gamma (IFN-gamma) is a pleiotropic cytokine that has a large number of immunologic and nonimmunologic functions. We have described that IFN-gamma could activate muscarinic cholinergic receptors (mAchR) of rat intestine, stimulating ileal motility. We also observed that mAchR activation induced inhibition of cAMP levels and stimulation of cGMP formation. The objectives of our work were to clarify the signal transduction pathways involved in regulation of ileal motility through mAchR activation by IFN-gamma. Our results demonstrate that this cytokine produces an ileal cholinergic response through tyrosine kinase activity. The activation of tyrosine kinase mediates ileal contractility, phosphoinositide hydrolysis by phospholipase C, nitric oxide synthase via protein kinase C, and cGMP synthesis. The increment in ileal motility is probably due to hyperproduction of prostaglandin E2 (PGE2) by ileal tissue. This prostanoid is an important mediator because it stimulates ileal motility. We conclude that IFN-gamma not only immunomodulates the gut microenvironment but also exerts a local nonimmunologic regulation on intestinal motility.

Trypanosoma cruzi attachment to lymphocyte muscarinic cholinergic and beta adrenergic receptors modulates intracellular signal transduction.

Plasma membrane vesicles of Trypanosoma cruzi (PMVs) formed saturation binding isotherms with naive murine T lymphocytes. Parasite membrane attachment to the muscarinic cholinergic receptors of Lyt 2.2+T cells (suppressor cells) resulted in the synthesis of cGMP, attenuation of cAMP levels and in the secretion of prostaglandin E2, an immunoregulator effector substance. These T suppressor cell signals were blunted by atropine and by monospecific antibody against T. cruzi surface epitopes. The interaction of T. cruzi PMVs with the beta adrenergic receptors of Lyt L3T4+T cells (helper cells) resulted in the synthesis of cAMP and in the attenuation of cGMP levels. T helper cells did not secrete prostaglandin E2 when T. cruzi PMVs were added to this system. These T helper cell signals were blunted by propranolol and by monospecific antibody against T. cruzi surface epitopes. The interaction of T. cruzi with T lymphocytes may result, therefore, in the down-regulation of the immune response induced by prostaglandin E2 T suppressor cell secretion and by cAMP inhibition of proliferation of T helper cells.

Expression of alpha-adrenoceptors in a human transformed lymphoblastoid cell line.

The presence of alpha-adrenergic receptors (absent in normal lymphocytes) has been demonstrated in transformed human lymphocytes of the Raji cell line. Binding properties of beta-adrenergic receptors were similar to those reported for normal lymphocytes. A single population of alpha 2-adrenergic receptors was characterized in intact Raji lymphoblasts by binding and saturation assays with the alpha 2-adrenergic antagonist yohimbine. Competition curves with [3H]yohimbine indicate the presence of typical alpha 2-adrenoceptors. Reaction of Raji with the alpha 2-adrenergic agonist clonidine (10(-6) M) stimulated their growth rate. In contrast, the alpha 1-adrenergic agonist methoxamine (10(-6) M) had no effect. Previous work indicates that Raji can actively produce thromboxanes (TX) and that these decreased atrium contractility. In agreement with these results and with the binding studies, it is now shown that clonidine stimulation enhanced the negative inotropic effects of Raji on isolated rat atria. This reaction was prevented by incubation of Raji with yohimbine (10(-6) M) but not with the alpha 1-adrenergic antagonist prazosin (10(-6) M) or the beta-adrenergic antagonist propranolol (10(-7) M). The biologic effect of Raji on rat atria was probably due to production of cyclooxygenase metabolites of arachidonic acid, because it was blocked by preincubation of the cells with the cyclooxygenase inhibitors indomethacin (10(-6) M) and aspirin (10(-4) M) or the thromboxane synthetase inhibitors nictindol (10(-5) M) and imidazole (10(-4) M).(ABSTRACT TRUNCATED AT 250 WORDS)

Cholinergic response of isolated rat atria to recombinant rat interferon-gamma.

Addition of recombinant rat interferon-gamma (IFN-gamma) to beating rat atria decreased the contractile strength in a dose-dependent manner. The effect was specific of IFN-gamma since it was abrogated by monoclonal anti-rat IFN-gamma. It required the activation of the cholinergic system of the heart as inhibition of both nicotinic (10(-7) M hexametonium) and muscarinic cholinoceptors (10(-7) M atropine) prevented the reaction. Hemicholinium (2 x 10(-5) M) and tetrodotoxin (5 x 10(-7) M) also reduced the response. Likewise, IFN-gamma potentiated the action of the muscarinic agonist carbachol. IFN-gamma simulated the biological effect of cholinergic agonists because: (a) it increased cGMP formation; (b) it decreased cAMP formation; and (c) it reduced heart contractility at doses that can be considered physiologic. IFN-gamma also modified the muscarinic receptor by interfering with the binding of the radiolabelled antagonist quinuclidinyl benzilate [( 3H]QNB). It is suggested that IFN-gamma binding to IFN-gamma receptors in the heart may lead to a cholinergic response by interaction of both receptor systems on the surface of atrial cells.

Muscarinic acetylcholine receptor antibodies as a new marker of dry eye Sjögren syndrome.

PURPOSE: The authors investigated whether circulating autoantibodies against M(3) muscarinic acetylcholine receptors (mAChRs) could be a new marker for diagnosis for primary and secondary Sjögren syndrome (SS) dry eye. METHODS: Enzyme-linked immunosorbent assay (ELISA) using both rat exorbital lacrimal gland acinar cell membranes and synthetic 25-mer peptide as antigens was used to determine autoantibodies against acinar cells and M(3) mAChRs. Also, nitric oxide synthase (NOS) activity was assessed to determine the biological effect of these autoantibodies in relation to the M(3) mAChR. RESULTS: Sera from dry eye primary SS (pSS) or secondary SS (sSS) patients tested by ELISA recognized membrane lacrimal gland acinar cells antigens and the synthetic 25-mer peptide, corresponding to the second extracellular loop of human M(3) mAChRs. Moreover, the IgG fraction and the corresponding affinity-purified anti-M(3) peptide autoantibodies from the same patients were able to activate NOS coupled to lacrimal gland M(3) mAChRs. As controls, IgG and sera from women without dry eye with or without rheumatoid arthritis and from normal control subjects gave negative results on ELISA and biological assay; thus demonstrating the specificity of the reaction. CONCLUSIONS: Autoantibodies against mAChR may be considered among the serum factors implicated in the pathophysiology of the development of pSS dry eyes and could be a new marker to differentiate SS dry eyes from non-SS dry eyes.