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.

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.