Differential signalling pathways involved in cholinoceptor‐dependent stimulation of nitric oxide isoforms in dental pulp

Abstract

AIM: To investigate the role of muscarinic acetylcholine receptor (mAChR) subtype activity in the regulation of endothelial- (e) and neuronal- (n) nitric oxide synthase (NOS) expression and activity. METHODOLOGY: Rat dental pulp tissue was used throughout the study. The e-nos and n-nos mRNA levels were specifically measured using reverse transcriptase polymerase chain reaction procedures that involve simultaneous co-amplification of both target cDNA and a reference template with a single set of primers. NOS activity was measured by the production of [U-(14)C]-citrulline from [U-(14)C]-arginine. RESULTS: Stimulation of M(1)/M(2) and M(3)/M(4) mAChRs with pilocarpine caused an increase in e-nos and n-nos mRNA levels and NOS activity in the dental pulp. The specific mAChR subtype antagonists, L-NMMA, l-NIO and N(2)-propyl-L-arginine but not aminoguanidine attenuated all these effects. Inhibitors of phospholipase C (PLC), protein kinase C (PKC) and calcium/calmodulin (CaM) prevented the pilocarpine-dependent increase in n-nos and e-nos mRNA levels and NOS activity. CONCLUSIONS: Activation of mAChR subtypes stimulated NOS activity by increasing the production of NO through e-nos and n-nos gene expression and NOS activity. The mechanism appears to occur secondarily to stimulation of CaM and PKC enzymatic activity.