Fig 1.
Duloxetine inhibits function of recombinant P2X4R.
(A) Effects of duloxetine (3–30 μM) and PPADS (10 μM) on ATP (10 μM)-induced [Ca2+]i responses in rP2X4R-1321N1 cells measured by the FDSS 7000EX system. Each column indicates the value as a percentage of control (Ctl: 10 μM ATP alone-induced [Ca2+]i responses) (n = 4, ***P < 0.001 vs. control). (B) Effect of duloxetine at various concentrations (1–10 μM) on ATP (10 μM)-induced [Ca2+]i respoznses in hP2X4R-1321N1 cells measured at a single cell level. Each column shows the effect of duloxetine evaluated by the S2 (2nd [Ca2+]i response) / S1 (1st [Ca2+]i response) (n = 4, ***P < 0.001 vs. control). Cells were pretreated with duloxetine and PPADS for 10 min prior to application of ATP. Data represent mean ± SEM for all groups.
Fig 2.
Comparison of the effect of duloxetine and other antidepressants on recombinant rP2X4R.
Effect of duloxetine and other antidepressants (30 μM) on ATP (10 μM)-induced [Ca2+]i in rP2X4R-1321N1 cells measured by the FDSS 7000EX system. Each column shows the value as a percentage of control (10 μM ATP alone-induced [Ca2+]i responses) (n = 6, ***P<0.001 vs. control). Cells were pretreated with duloxetine and other antidepressants 10 min prior to ATP application. Data represent mean ± SEM.
Fig 3.
Duloxetine inhibited microglial P2X4R function.
(A) Representative average traces of [Ca2+]i increases in response to ATP (100 μM for 30 s) with or without duloxetine (30 μM for 10 min) measured by a single cell analysis in C8-B4 cells. Each column shows the effect of duloxetine evaluated by the S2 (2nd [Ca2+]i response) / S1 (1st [Ca2+]i response) (n = 4, ***P < 0.001 vs. control). (B) Representative average traces of [Ca2+]i increases in response to ATP (50 μM for 30 s) with or without duloxetine (30 μM for 10 min) measured by single cell analysis in rat microglial cells. Each column shows the relative increase ratio (F340/F380) from the basal level prior to ATP application (n = 4, **P < 0.01 vs. control). (C) Representative average traces of [Ca2+]i increases in response to ATP (50 μM for 30 s) in combination with ivermectin (3 μM, 3 min) with or without duloxetine (30 μM for 10 min) measured by single cell analysis in rat microglial cells. Each column shows the relative increase ratio (F340/F380) from the basal level prior to ATP application (n = 4, **P < 0.01, ***P < 0.001 vs. control, #P < 0.05, ###P < 0.001). Data represent mean ± SEM for all groups.
Fig 4.
Effect of duloxetine on other P2 receptors.
(A) Effect of duloxetine at various concentrations (3–30 μM) and PPADS (10 μM) on BzATP (100 μM)-induced [Ca2+]i responses in rP2X7R-1321N1 cells measured by the FDSS 7000EX system. Each column shows the value as a percentage of control (100 μM BzATP alone-induced [Ca2+]i responses) (n = 4). (B, C) Effects of duloxetine (30 μM) on [Ca2+]i responses induced by BzATP (100 μM) (B) and ADP (10 μM) (C) measured by a single cell analysis in primary cultured rat microglial cells. Each column shows the relative increase ratio (F340/F380) from the basal level prior to BzATP or ADP application (n = 4, **P < 0.01). Cells were pretreated with duloxetine or PPADS for 10 min prior to application of BzATP or ADP. Data represent mean ± SEM for all groups.
Fig 5.
Intrathecal duloxetine attenuates allodynia after PNI: possible involvement of P2X4R.
(A) The paw withdrawal threshold (grams) of mechanical stimulation by von Frey filaments applied to the rat hindpaw after PNI. Duloxetine (20 or 50 μg/20 μl) or vehicle (Phosphate buffered saline, 20 μl) was intrathecally administered to rats on day 7 post-PNI. PCPA (300 mg/kg, i.p.) was administered once a day for 3 days from day 4 post-PNI, and DSP-4 (50 mg/kg, i.p.) was administered 3 days before PNI. The threshold was measured 300 min after duloxetine injection (n = 5–13, **P < 0.01, ***P < 0.001). Data represent mean ± SEM. (B) The antiallodynic effect (%) of duloxetine at 180 min after its intrathecal administration was calculated by the formula: antiallodynic effect (%) = 100 × (test value−control value)/(15 g−control value) [***P < 0.001 vs control group (Saline+PBS), §P < 0.05 and ##P < 0.01]. Data represent mean ± SEM.