PGE2 involvement in experimental infection with Trypanosoma cruzi subpopulations

PGE₂ involvement in experimental Trypanosoma cruzi infection depends on the lethal capacity of the parasite subpopulation used. Mice acutely infected with non-lethal K98 displayed an enhancement in PGE₂ serum levels during the acute period, while those infected with lethal T. cruzi subpopulations (RA or K98-2) showed levels not different from normal mice. The enhancement detected in K98 group could be related both to an increased number of CD8+ T cell number and to enhanced PGE₂ release per cell by CD8+; values of PGE₂ release by adherent cells were not altered in this group. Treatment with cyclooxygenase inhibitors enhanced mortality rates of mice infected with K98, and administration of 16,16-dimethyl PGE₂ (dPGE) reversed this effect. However, mice infected with RA did not reduce their mortality rates by administration of diverse doses of dPGE. These findings suggest that PGE₂ could play a role in resistance in mice infected with K98.     

Nitrogen fertilizer enhances vegetation establishment of a high-altitude machine-graded ski slope

Machine grading is frequently required to prepare the terrain when building high-altitude ski slopes in the Alps. However, this kind of disturbance alters the natural environment, destroying the vegetation and hampering its reestablishment. Thus, specific restoration plans are necessary to encourage the recovery of vegetation, which is already affected by different natural constraints in this harsh environment. One of the main critical factors affecting plant growth in high-altitude areas is the lack of available nitrogen (N) in the soil. In this context, the addition of a slow-release N fertilizer was carried out in an experimental revegetated ski slope between 2,800 and 2,900 m above sea level in the western Italian Alps. Both vegetation and soil were monitored during a 5-year period in order to test the effectiveness of N addition on the restoration process. Even if effects on soil carbon and N contents were negligible, vegetation was remarkably affected by the fertilization, since the total vegetation cover and the species richness significantly increased. Against the expectations, there was a remarkable increase in spontaneous forbs, rather than in most of the sown graminoids, which slightly varied during the experimental period. Actually, graminoids responded in different ways, mostly increasing (likewise forbs), but the slight decrease of the dominant Festuca nigrescens (Chewing's Fescue) masked their spread. This study confirms the noteworthy role of N in high-altitude alpine soils and consequently its importance to improve the restoration process of degraded ecosystems.     

3H-noradrenaline metabolism in the isolated epididymal and prostatic portions of the rat vas deferens

The spontaneous ³H-noradrenaline efflux from the isolated epididymal and prostatic portions of the rat vas deferens, was investigated. The spontaneous tritium efflux was higher in the epididymal portion than in the prostatic one from normal animals. Such differences were abolished after castration. On the other hand, acetylsalicylic acid enhanced the total tritium efflux only in the epididymal portion, whereas phentolamine and phenoxybenzamine increased the total tritium efflux from the two portions of the rat vas deferens in both experimental groups. The prostatic portion released a similar amount of ³H normetanephrine than the apididymal end, whereas other metabolic fractions (3,4-dihydroxyphenylglycol; 3,4-dihydroxymandelic acid and O-methylated deaminated metabolites) were smaller in the prostatic portion in comparison with the epididymal portion from control vas deferens. The results presented in the isolated rat vas deferens suggest the existence of a prostaglandin as well as an alpha adrenoceptive modulation of the spontaneous total tritium efflux.     

H(1)-Receptor activation triggers the endogenous nitric oxide signalling system in the rat submandibular gland

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 H(1)-receptor responses. MATERIALS AND METHODS: To assess the H(1) receptor 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 H(1) receptor 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 H1 receptor 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.     

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

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

Anti-M(3) peptide IgG from Sjögren's syndrome triggers apoptosis in A253 cells

Primary Sj?gren'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 Sj?gren 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.     

Circulating antibodies against neonatal cardiac muscarinic acetylcholine receptor in patients with Sjögren's syndrome

Isolated congenital heart block may be associated with Primary Sjogren's Syndrome. In this work we demonstrated that IgG present in the sera ofpatients with Primary Sjogren's Syndrome (PSS) could bind and activate muscarinic acetylcholine receptors of rat neonatal atria. These antibodies were able to inhibit in a irreversible manner the binding of ³H-QNB to muscarinic cholinergic receptors of purified rat atria membranes. Moreover, IgG from PSS individuals could modify biological effects mediated by muscarinic cholinoceptors activation, i.e. decrease contractility and cAMP and increase phosphoinositide turnover and cGMP. Atropine blocked all of these effects and carbachol mimicked them; confirming muscarinic cholinergic receptors-mediated PSS IgG action. Neither binding nor biological effect were obtained using adult instead of neonatal rat atria. IgG from sera of normal women were not effective in the studied system. The prevalence of cholinergic antibody was 100% in PSS and was independent of Ro/SS-A and La/SS-B antibodies. It could be concluded that antibody against muscarinic cholinergic receptors may be another serum factor to be considered in the pathophysiology of the development of congenital heart block.     

β‐Adrenergic‐induced CD40 overexpression on gingival fibroblasts: Role of PGE2

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

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.     

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

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