Differential signalling pathways involved in cholinoceptor-dependent stimulation of nitric oxide isoforms in dental pulp.
OBJECTIVE
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.


METHODS
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.
Sterin-Borda
L
L
Pharmacology Unit, School of Dentistry, University of Buenos Aires, and Argentine National Research Council, Buenos Aires, Argentina. enri@farmaco.odon.uba.ar
Furlan
C
C
Reina
S
S
Orman
B
B
Borda
E
E
D000818:Animals; D001120:Arginine / Q000494:pharmacology; D002147:Calmodulin / Q000037:antagonists & inhibitors; D003782:Dental Pulp / Q000201:enzymology / Q000378:metabolism*; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006146:Guanidines / Q000494:pharmacology; D008297:Male; D018721:Muscarinic Agonists / Q000494:pharmacology; D018377:Neurotransmitter Agents / Q000032:analysis*; D009569:Nitric Oxide / Q000032:analysis*; D019001:Nitric Oxide Synthase / Q000032:analysis* / Q000037:antagonists & inhibitors; D052248:Nitric Oxide Synthase Type I / Q000032:analysis / Q000037:antagonists & inhibitors; D052250:Nitric Oxide Synthase Type III / Q000032:analysis / Q000037:antagonists & inhibitors; D009952:Ornithine / Q000031:analogs & derivatives / Q000494:pharmacology; D010862:Pilocarpine / Q000494:pharmacology; D011493:Protein Kinase C / Q000037:antagonists & inhibitors; D051381:Rats; D017208:Rats, Wistar; D011976:Receptors, Muscarinic / Q000187:drug effects / Q000502:physiology*; D015398:Signal Transduction / Q000187:drug effects / Q000502:physiology*; D010738:Type C Phospholipases / Q000037:antagonists & inhibitors; D019323:omega-N-Methylarginine / Q000494:pharmacology
D002147:Calmodulin; D004791:Enzyme Inhibitors; D006146:Guanidines; D018721:Muscarinic Agonists; D018377:Neurotransmitter Agents; D011976:Receptors, Muscarinic; D010862:Pilocarpine; D019323:omega-N-Methylarginine; D009569:Nitric Oxide; C065027:N(G)-iminoethylornithine; D001120:Arginine; D009952:Ornithine; D019001:Nitric Oxide Synthase; D052248:Nitric Oxide Synthase Type I; D052250:Nitric Oxide Synthase Type III; D011493:Protein Kinase C; D010738:Type C Phospholipases; C004479:pimagedine