3.1.4. DAG lipase is required for bradykinin-induced enhancing of capsaicin cells
The results above have shown that bradykinin-induced sensitisation of capsaicin-sensitive neurons is mediated by EP4 and cyclooxygenase. To investigate this further we looked at the DAG lipase branch of the PLC pathway since PLC activation results in the production of DAG which can then metabolised to arachidonic acid by cyclooxygenase enzymes, particularly the constitutively expressed isoform, COX-1. To confirm this, experiments were conducted to examine the effect of DAG lipase inhibitor, RHC-80267, on the bradykinin-induced enhancement of capsaicin-induced calcium responses in cultured DRG neurons.
Figure 5A representative traces showing the effect of the DAG lipase inhibitor RHC-80267 on capsaicin responses and its effect on bradykinin mediated sensitisation following bradykinin application. Capsaicin (80nm; 10seconds) was applied twice in absence and presence of BK (100nM; 3-minutes, bar) with second application in presence of BK and the DAG lipase inhibitor, RHC-80267 (20µM; 20minutes).
RHC-80267 (20µM; 20 min) attenuated the bradykinin-induced sensitisation of capsaicin-induced calcium responses (Fig 5B). The response ratio of the first and second capsaicin responses was 61.9± 4.9 % in the presence of (RHC-80267 +bradykinin) which was significantly reduced compared to the effect of bradykinin alone (115±11.8 %).(** P< 0.01, one-way ANOVA by Bonferroni’s multiple comparison tests).