Figure 3.

Role of ETA and ETB in ET-1-induced PLC activation in myometrial and ELT3 cells. A, [³H]inositol-labeled myometrial cells were preincubated or not for 30 min with 1 μm FR 139317 (FR) or 100 nm BQ 788 (BQ) or for 14 h (during the labeling) with 100 ng/ml PTX before a 30 min-stimulation with 50 nm ET-1 or 50 nm S6c. Values, expressed as percentages of the ET-1 value, are the mean ± sem of three separate experiments performed in duplicate. *, P < 0.05 vs. ET1-treated cells. B, [³H]inositol-labeled ELT3 cells were preincubated or not with 1 μm FR 139317 (FR), 1 μm BQ 788 (BQ), or both 1 μm FR 139317 + 1 μm BQ 788 for 30 min or with 100 ng/ml PTX for 14 h (during the labeling) before a 30-min stimulation with 10 nm ET-1 or 10 nm S6c. Values, expressed as percentages of the ET-1 value, are the mean ± sem of four separate experiments performed in duplicate. *, P < 0.05 vs. ET1-treated cells; #, P < 0.05 vs. S6c-treated cells. C, Twenty-four hours after nucleofection of ELT3 cells with control or ETA- or ETB-targeted siRNA, the expression of ETR subtype was examined by PCR quantitative as described in Materials and Methods. Values, expressed as relative mRNA amounts, are the mean ± sem of three separate experiments performed in duplicate. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) transcript was used as a standard. *, P < 0.05 vs. corresponding mRNA in cells transfected with control siRNA. D, Twenty-four hours after nucleofection with control or ETA- or ETB-targeted siRNA, ELT3 cells were preincubated or not with 1 μm FR 139317 and treated at 4 C in the presence of 10 nm [¹²⁵I]ET-1 as described in Materials and Methods. ETA was determined as the FR139317-sensitive [¹²⁵I]ET-1 binding and ETB as the FR139317-insensitive [¹²⁵I]ET-1 binding. Values are the mean ± sem of three separate experiments performed in triplicate. *, P < 0.05 vs. control siRNA-nucleofected cells treated without FR 139317; #, P < 0.05 vs. control siRNA-nucleofected cells treated with FR 139317. E, Twenty-four hours after nucleofection with control or ETA- or ETB-targeted siRNA, ELT3 cells were labeled with [³H]inositol for 14 h before stimulation with 10 nm ET-1 or 10 nm S6c for 30 min. Values, expressed as percentages of the ET-1 value obtained with the control siRNA, are the mean ± sem of three separate experiments performed in duplicate. *, P < 0.05 vs. control siRNA-nucleofected cells treated with ET1; #, P < 0.05 vs. control siRNA-nucleofected cells treated with S6c.