RESUMO
The mechanism of pain in dentine hypersensitivity is poorly understood but proposed to result from the activation of dental sensory neurons in response to dentinal fluid movements. Odontoblasts have been suggested to contribute to thermal and mechanosensation in the tooth via expression of transient receptor potential (TRP) channels. However, a mechanism by which odontoblasts could modulate neuronal activity has not been demonstrated. In this study, we investigated functional TRP channel expression in human odontoblast-like cells and measured ATP release in response to TRP channel activation. Human immortalized dental pulp cells were driven toward an odontoblast phenotype by culture in conditioned media. Functional expression of TRP channels was determined with reverse transcription polymerase chain reaction and ratiometric calcium imaging with Fura-2. ATP release was measured using a luciferin-luciferase assay. Expression of mRNA for TRPA1, TRPV1, and TRPV4 but not TRPM8 was detected in odontoblasts by reverse transcription polymerase chain reaction. Expression of TRPV4 protein was detected by Western blotting and immunocytochemistry. The TRPA1 agonists allyl isothiocyanate and cinnamaldehyde and the TRPV4 agonist GSK1016790A caused a concentration-dependent increase in intracellular Ca(2+) concentration that was inhibited by the selective antagonists HC030031, AP18, and HC067047, respectively. In contrast, exposure to the TRPV1 agonist capsaicin or the TRPM8 agonist icilin had no effect on intracellular Ca(2+) concentration. Treatment with allyl isothiocyanate, cinnamaldehyde, or GSK1016790A caused an increase in ATP concentration in culture medium that was abolished by preincubation with TRP channel antagonists. These data demonstrate that activation of TRPA1 and TRPV4 channels in human odontoblast-like cells can stimulate ATP release. We were unable to confirm the presence of thermosensitive TRPV1 and TRPM8 that has previously been reported in odontoblasts.
Assuntos
Trifosfato de Adenosina/metabolismo , Canais de Cálcio/fisiologia , Proteínas do Tecido Nervoso/fisiologia , Nociceptores/fisiologia , Odontoblastos/fisiologia , Canais de Cátion TRPV/fisiologia , Canais de Potencial de Receptor Transitório/fisiologia , Acetanilidas/farmacologia , Acroleína/análogos & derivados , Acroleína/farmacologia , Sinalização do Cálcio/efeitos dos fármacos , Capsaicina/farmacologia , Técnicas de Cultura de Células , Linhagem Celular , Meios de Cultivo Condicionados , Polpa Dentária/citologia , Humanos , Isotiocianatos/farmacologia , Leucina/análogos & derivados , Leucina/farmacologia , Proteínas do Tecido Nervoso/agonistas , Proteínas do Tecido Nervoso/antagonistas & inibidores , Odontoblastos/metabolismo , Purinas/farmacologia , Pirimidinonas/farmacologia , Fármacos do Sistema Sensorial/farmacologia , Sulfonamidas/farmacologia , Canal de Cátion TRPA1 , Canais de Cátion TRPM/agonistas , Canais de Cátion TRPV/agonistas , Canais de Potencial de Receptor Transitório/agonistas , Canais de Potencial de Receptor Transitório/antagonistas & inibidoresRESUMO
Analgesic activity of methanol leaf extract of C. scandens obtained by column chromatography and its graded solvent fractions, was evaluated in mice using acetic acid-induced abdominal writhing and formalin-induced paw licking. The extract and fractions significantly inhibited abdominal writhing and two phases of formalin-induced paw licking in mice, indicating that antinociceptive activity may involve inhibition of pain by peripheral and central mechanisms.