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.