Figure legends
Fig 1: Schematic diagram represent the drug application via U tube to DRG neurons.
Fig 2. A) Representative traces of capsaicin (Cap) induced calcium responses to 80nM capsaicin applied twice for 10 seconds with a 10 minute interval between each application. KCl (60mM) was applied at the end of the experiment to confirm cell viability. B)Representative traces showing the sensitisation effect of 500nm PGE2 on capsaicin elicited calcium responses. Capsaicin (80nM; 10seconds) was applied twice with a 10 minute interval between each application. The second capsaicin application was performed in presence of PGE2 (500nM; 3-minutes). C) Representative traces showing the sensitisation effect of bradykinin (100nM; 3minutes) on capsaicin-evoked calcium response in cultured DRG neurons. Capsaicin (80nM; 10seconds) was applied twice with 10 minutes interval between each application. Bradykinin (100nM; 3-minutes) was also present during the second capsaicin application. D) Histogram showing the mean ± SEM response ratio (where the second response expressed as a percentage of the first one) for 98 cells (6 coverslips) in control and 115 cells (8 coverslips) with PGE2 exposed conditions. These were significantly different (two-tailed t-test, *P< 0.05). E) Histogram showing the mean ± SEM response ratio (where the second response expressed as a percentage of the first one) for 98 cells (6 coverslips) in control and (101 cells; 6 coverslips) for the BK condition (*P< 0.05).
Fig 3. COX-1 but not COX-2 is required for bradykinin mediated modulation of capsaicin-sensitive cells. A) Representative traces show the effect of the COX-2 inhibitor (NS398; 10μM) on capsaicin responses and its effect on sensitisation following bradykinin application. Capsaicin (80nm; 10seconds) was applied twice in the absence and presence of BK (100nM; 3-minutes, bar) with second application and in presence of NS398. B) Representative traces show the effect of the COX-1 inhibitor (SC560; 100nM) on capsaicin responses and its effect on sensitisation following bradykinin application. Capsaicin (80nm; 10seconds) was applied twice in the absence and presence of BK (100nM; 3-minutes, bar) with second application and in presence of SC560. C) Histogram showing mean ±SEM response ratio for control (98 cells; 6 coverslips) and in presence of the BK (101 cells; 6 coverslips), then in the presence of the BK along with each inhibitor. The number in parentheses indicates sample size the dashed line represents the bradykinin response ratio, (*P < 0.05, one-way ANOVA followed by Bonferroni’s multiple comparison test).
Fig 4. A) Representative traces showing the effect of prostaglandin receptor antagonists applied as a cocktail on the capsaicin-induced calcium responses in cultured DRG neurons response, and their effect on bradykinin mediated sensitisation of this response. Capsaicin (80nm for 10 seconds) was applied twice with a 10 minutes interval between each application in absence and presence of BK (bar, 100nM for 3 minutes) with second application in presence of antagonist cocktail for 10minutes. B) Representative traces showing the effect of prostaglandin receptor antagonists (EP4 antagonist, GW627368 or the EP3 antagonist, L-798106) on the capsaicin-induced calcium responses in cultured DRG neurons response, and their effect on bradykinin mediated sensitisation of this response. Capsaicin (80nm for 10 seconds) was applied twice with a 10 minutes interval between each application in absence and presence of BK (bar, 100nM for 3 minutes) with second application in presence of antagonist (500nM for; 10 minutes).Histogram showing the mean peak heights (F510 S-R) ± SEM for repeated capsaicin application in presence of BK (100nM, 3-minutes) and (EP4 antagonist, GW627368X) before second capsaicin application, C) Histogram showing mean ±SEM response ratio for 98 cells (6 coverslips) in control and 101 cells (6 coverslips) with BK. then in the presence of the BK along with each antagonist. The number in parentheses indicate number of cells, the dashed line represents the bradykinin response ratio for comparison, (*P < 0.05, one-way ANOVA followed by Bonferroni’s multiple comparison test).
Fig 5. Bradykinin enhances capsaicin responses and requires DAG lipase activity. A) 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). B)Histogram showing mean±SEM response ratio for control (98 cells; 6 coverslips), in the presence of bradykinin (101 cells; 6 coverslips), and in the presence of bradykinin + RHC-80267 (158 cells; 4 coverslips). The dashed line represents the bradykinin response ratio for comparison (**P < 0.01, ****P < 0.0001, one-way ANOVA followed by Bonferroni’s multiple comparison test).
Fig 6. Triple label of EP4, TRPV1 and B2 in (L4-L6) rat DRG neurons. A) Individual DRG neurons labelled with EP4 (green), B2 (red), and TRPV1 (green) antibodies. The panel on the right shows a composite image of triple labels clearly demonstrating neurons that are labelled with three antibodies. B) Cell size frequency distribution derived from 6 ganglia triple labelled with EP4, TRPV1 and B2. Triple labelling was observed mainly in small cells (<1,000 μm²) with a mean cell area of 651.9± 15.2 μm2.
Fig 7. Co-localisation of EP4 and COX-1 in lumbar (L4-L6) rat DRG neurons. A) Individual DRG neurons labelled with COX-1 (green) and EP4 (red) antibodies. The panel on the right shows a composite image of two labels clearly demonstrating neurons that are labelled with both antibodies.B) Cell size-frequency histogram derived from 6 ganglia shows that 36% of DRG neurons expressed both EP4 and COX-1 proteins and 77% of dual labelled cells have cell area less than 1,000μm2 with a mean cell area of 849.1± 22.4μm2.
Fig 8. Co-localisation of COX-1 and B2 in lumbar (L4-L6) rat DRG neurons. A) Individual DRG neurons labelled with COX-1 (green) and B2 (red) antibodies. The panel on the right shows a composite image of two labels clearly demonstrating neurons that are labelled with both antibodies.B) Cell size- frequency histogram derived from 6 ganglia shows that 39% of DRG neurons expressed both B2 and COX-1 proteins. The frequency histogram shows that 69% of dual labelled cells have cell area less than 1,000μm2 with a mean cell area of 955.7± 28.1μm2.
Fig 9 : A summary of the signalling pathways on the DRG neurones by bradykinin and PGE2.