FIGURE 1.
The transport effects of 14,15-EET are amiloride-insensitive and, as with EGF, ERK1/2-mediated. A, changes in JNa+ induced by control, vehicle (black bars) or amiloride (10 μm) (white bars), or by 14,15-EET (5 μm) added in the presence of vehicle (black bars) or amiloride (10 μm) (white bars). Values (in pmol/h/cm2) are averages ± S.E. (error bars) calculated from five different cell samples. Differences from vehicle are indicated as follows: *, p < 0.0008; +, p < 0.0001; ≠, p < 0.002. The differences between amiloride and 14,15-EET or between amiloride added in the absence or presence of 14,15-EET were not significant (p > 0.05). The amiloride-sensitive components of the JNa+ responses to the agonists were 34, 26, and 37%, respectively, for amiloride, 14,15-EET, and a combination of both. B, cells incubated without (control) or with U0126 (apical; 10 μm) were exposed to vehicle, 14,15-EET (5 μm), or EGF (10 ng/ml) (black, gray, and white bars, respectively), and JNa+ was determined 2 h later. Values (in pmol/h/cm2) are averages ± S.E. (error bars) calculated from five different cell samples. Differences from controls are indicated as follows: *, p < 0.05; +, p < 0.0001; ≠, p < 0.01. The amiloride-sensitive components of the total cell JNa+ effects corresponded to 39, 30, and 25% for control untreated and to 46, 54, and 40% for U0126-treated cells for vehicle, 14,15-EET, and EGF, respectively. C and D, Western blots of cell lysates probed with anti-phospho-ERK1/2 (p-ERK) (upper panels) or -ERK1/2 (lower panels) antibodies. Shown are immunoreactive proteins with the mobilities of phospho-ERK1/2 and ERK1/2. C, lysates (50 μg of protein/lane) isolated from cells incubated for 10, 30, or 60 min with vehicle (lane 1) or 14,15-EET (5 μm) (lane 2). D, lysates from cells incubated without (control) or with U0126 (10 μm) for 1 h prior to a 30-min exposure to vehicle, 14,15-EET (5 μm), or EGF (10 ng/ml) (lanes 1, 2, and 3, respectively; 50–60 μg of protein/lane) probed with peptide antibodies.