Purchase this article with an account.
Z. Pan, J.E. Capo–Aponte, Z. Wang, F. Zhang, J.W. Du, V.N. Bildin, P.S. Reinach; Dependence of p38 MAPK Activation on Hypotonicity–Induced Regulatory Volume Decrease in Rabbit Corneal Epithelial Cells . Invest. Ophthalmol. Vis. Sci. 2006;47(13):2904.
Download citation file:
© ARVO (1962-2015); The Authors (2016-present)
To determine in rabbit corneal epithelial cells (RCEC) whether or not hypotonicity–induced RVD behavior is dependent on p38 MAPK activation.
RCEC were loaded for 60 min with the cytosolic volume marker, calcein–AM (10 µM). Regulatory volume responses were induced by a 25% hypotonic challenge and characterized using a microplate fluorescence reader. Cl– channel blockers were applied to cells for 30 min at 37°C prior to a challenge. Western blot analysis probed for p38 phosphorylation. Cl– channel blockers included 100 µM niflumic acid (NA), 100 µM 5–nitro–2–(3–phenylpropylamino) benzoic acid (NPPB), 1 mM disodium 4,4'–diisothiocyanatostilbene–2,2'–disulfonate (DIDS), 20 µM tamoxifen, and a Cl––free (D–gluconate) isotonic solution. Twenty µM SB203580 and 40 µM PD98059 were used to inhibit MAPK activity.
A RVD response was observed that caused nearly complete recovery to the isotonic baseline value with a time constant of 20 min. Such a challenge induced time dependent p38 MAPK phosphorylation, which reached a maximal value after 30 min followed by a gradual decline towards its baseline level after the next 30 min. SB203580 inhibited hypotonicity induced p38 MAPK phosphorylation. However, it failed to inhibit hypotonicity induced RVD. The rank order of the abovementioned Cl– channel blockers was: Cl––free isotonic solution (nearly complete blockade) >DIDS = tamoxifen >NPPB =NA. In parallel, each of these inhibitors also suppressed hypotonicity induced p38 MAPK phosphorylation.
P38 MAPK activation occurs as a consequence of RVD because following inhibition of p38 activity with SB203580 the RVD response was unaffected. On the other hand, inhibition of RVD with different Cl– channel blockers suppressed hypotonicity induced p38 MAPK activation. Therefore, hypotonicity–induced p38 phosphorylation is dependent on RVD response.
This PDF is available to Subscribers Only