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


METHODS
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.
De Couto Pita
A
A
Pharmacology Unit, School of Dentistry, University of Buenos Aires, Buenos Aires, Argentina.
Passafaro
D
D
Ganzinelli
S
S
Borda
E
E
Sterin-Borda
L
L
D000704:Analysis of Variance; D000818:Animals; D003782:Dental Pulp / Q000187:drug effects / Q000201:enzymology; D008297:Male; D018721:Muscarinic Agonists / Q000627:therapeutic use; D009569:Nitric Oxide / Q000378:metabolism; D052247:Nitric Oxide Synthase Type II / Q000235:genetics / Q000378:metabolism*; D010862:Pilocarpine / Q000627:therapeutic use; D011487:Protein Conformation; D011671:Pulpitis / Q000188:drug therapy / Q000201:enzymology*; D012333:RNA, Messenger / Q000032:analysis; D051381:Rats; D017208:Rats, Wistar; D011976:Receptors, Muscarinic / Q000187:drug effects / Q000378:metabolism*; D015398:Signal Transduction / Q000502:physiology*; D018709:Statistics, Nonparametric
D018721:Muscarinic Agonists; D012333:RNA, Messenger; D011976:Receptors, Muscarinic; D010862:Pilocarpine; D009569:Nitric Oxide; D052247:Nitric Oxide Synthase Type II