Abstract
In hypertonicity-stressed (i.e., 600 mOsm) SV40-immortalized rabbit and human corneal epithelial cell layers (RCEC and HCEC, respectively), we characterized the relationship between time-dependent changes in translayer resistance, relative cell volume and modulation of MAPK superfamily activities. Sulforhodamine B permeability initially increased by 1.4- and 2-fold in RCEC and HCEC, respectively. Subsequently, recovery to its isotonic level only occurred in RCEC. Light scattering revealed that in RCEC 1) regulatory volume increase (RVI) extent was 20% greater; 2) RVI half-time was 2.5-fold shorter. However, inhibition of Na-K-2Cl cotransporter and Na/K-ATPase activity suppressed the RVI response more in HCEC. MAPK activity changes were as follows: 1) p38 was wave-like and faster as well as larger in RCEC than in HCEC (90-and 18-fold, respectively); 2) increases in SAPK/JNK activity were negligible in comparison to those of p38; 3) Erk1/2 activity declined to 30–40% of their basal values. SB203580, a specific p38 inhibitor, dose de-pendently suppressed the RVI responses in both cell lines. However, neither U0126, which inhibits MEK, the kinase upstream of Erk, nor SP600125, inhibitor of SAPK/JNK, had any effect on this response. Taken together, sufficient activation of the p38 limb of the MAPK superfamily during a hypertonic challenge is essential for maintaining epithelial cell volume and translayer resistance. On the other hand, Erk1/2 activity restoration seems to be dependent on cell volume recovery.
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Araie, M., Maurice, D. 1987. The rate of diffusion of fluorophores through the corneal epithelium and stroma. Exp. Eye. Res. 44:73–87
Araki, K., Ohashi, Y., Sasabe, T., Kinoshita, S., Hayashi, K., Yang, X.Z., Hosaka, Y., Aizawa, S., Handa, H. 1993. Immortalization of rabbit corneal epithelial cells by a recombinant SV40-adenovirus vector. Invest. Ophthalmol. Vis. Sci. 34:2665–2671
Bildin, V.N., Yang, H., Crook, R.B., Fischbarg, J., Reinach, P.S. 2000. Adaptation by corneal epithelial cells to chronic hypertonic stress depends on upregulation of Na:K:2Cl cotransporter gene and protein expression and ion transport activity. J. Membrane Biol. 177:41–50
Bildin, V.N., Yang, H., Fischbarg, J., Reinach, P.S. 1998. Effects of chronic hypertonic stress on regulatory volume increase and Na-K-2C1 cotransporter expression in cultured corneal epithelial cells. Adv. Exp. Med. Biol. 438:637–642
Butterfield, L., Zentrich. E., Beekman, A., Heasley, L.E. 1999. Stress- and cell type-dependent regulation of transfected c-Jun N-terminal kinase and mitogen-activated protein kinase kinase isoforms. Biochem. J. 338:681–686
Candia, O.A., Patarca, R., Alvarez, L.J. 1998. Reduction of water permeability by anisotonic solutions in frog corneal epithelium. Invest. Ophthalmol. Vis. Sci. 39:378–384
Cohen, D.M. 1999. Signalling and gene regulation by urea and NaCl in the renal medulla. Clin. Exp. Pharmacol. Physiol. 26:69–73
Denkert, C., Warskulat, U., Hensel, F., Haussinger, D. 1998. Osmolyte strategy in human monocytes and macrophages: involvement of p38MAPK in hyperosmotic induction of betaine and myoinositol transporters. Arch. Biochem. Biophys. 354: 172–180
Duzgun, S.A., Rasque, H., Kito, H., Azuma, N., Li, W., Basson, M.D., Gahtan, V., Dudrick, S.J., Sumpio, B.E. 2000. Mitogen-activated protein phosphorylation in endothelial cells exposed to hyperosmolar conditions. J. Cell Biochem. 76: 567–571
Fischbarg, J., Li, J., Kuang, K., Echevarria, M., Iserovich, P. 1993. Determination of volume and water permeability of plated cells from measurements of light scattering. Am. J. Physiol. 265: C1412–1423
Gillis, D., Shrode, L.D., Krump, E., Howard, CM., Rubie, E.A., Tibbies, L.A., Woodgett, J., Grinstein, S. 2001. Osmotic stimulation of the Na+/H+ exchanger NHE1: relationship to the activation of three MAPK pathways. J. Membrane Biol. 181:205–214
Goss, G.G., Jiang, L., Vandorpe, D.H., Kieller, D., Chernova, M.N., Robertson, M., Alper, S.L. 2001. Role of JNK in hypertonic activation of Cl--dependent Na+/H+ exchange in Xenopus oocytes. Am. J. Physiol. 281:1978–1990
Graf, J., Haussinger, D. 1996. Ion transport in hepatocytes: mechanisms and correlations to cell volume, hormone actions and metabolism. J. Hepatol. 24:53–77
Haas, M., McBrayer, D., Lytle, C. 1995. [Cl- i-dependent phosphorylation of the Na-K-Cl cotransport protein of dog tracheal epithelial cells. J. Biol. Chem. 270:28955–28961
Hallows, K.R., Knauf, P.A. 1994. Regulatory volume decrease in HL-60 cells: importance of rapid changes in permeability of Cl- and organic solutes. Am. J. Physiol. 267:C1045–1056
Hoffert, J.D., Leitch, V., Agre, P., King, L.S. 2000. Hypertonic induction of aquaporin-5 expression through an ERK-dependent pathway. J. Biol. Chem. 275:9070–9077
Liedtke, CM., Cole, T.S. 2002. Activation of NKCC1 by hyperosmotic stress in human tracheal epithelial cells involves PKC-delta and ERK. Biochim. Biophys. Acta. 1589:77–88
Lu, L., Reinach, P.S., Kao, W.W. 2001. Corneal epithelial wound healing. Exp. Biol. Med. (Maywood) 226:653–664
Malek, A.M., Goss, G.G., Jiang, L., Izumo, S., Alper, S.L. 1998. Mannitol at clinical concentrations activates multiple signaling pathways and induces apoptosis in endothelial cells. Stroke 29:2631–2640
O’Neill, W.C. 1999. Physiological significance of volume-regulatory transporters. Am. J. Physiol. 276:C995-C1011
Palfrey, H.C., Pewitt, E.B. 1993. The ATP and Mg2+ dependence of Na+-K+-2Cl--cotransport reflects a requirement for protein phosphorylation: studies using calyculin A. Pfluegers Arch. 425:321–328
Pewitt, E.B., Hegde, R.S., Haas, M., Palfrey, H.C. 1990. The regulation of Na/K/2Cl cotransport and bumetanide binding in avian erythrocytes by protein phosphorylation and dephos-phorylation. Effects of kinase inhibitors and okadaic acid. J. Biol. Chem. 265:20747–20756
Reinach, P., Ganapathy, V., Torres-Zamorano, V. 1994. A Na:H exchanger subtype mediates volume regulation in bovine corneal epithelial cells. Adv. Exp. Med. Biol. 350:105–110
Roger, F., Martin, P.Y., Rousselot, M., Favre, H., Feraille, E. 1999. Cell shrinkage triggers the activation of mitogen-activated protein kinases by hypertonicity in the rat kidney medullary thick ascending limb of the Henle’ s loop. Requirement of p38 kinase for the regulatory volume increase response. J. Biol. Chem. 274:34103–34110
Romio, L., Zegarra, M., Varesio, L., Galietta, L.J.V. 2001. Regulation of taurine transport in murine macrophages. Amino Acids. 21:151–160
Russell, J.M. 2000. Sodium-potassium-chloride cotransport. Physiol. Rev. 80:211–276
Sheikh-Hamad, D., Di Mari, J., Suki, W.N., Safirstein, R., Watts, B.A., 3rd, Rouse, D. 1998. p38 kinase activity is essential for osmotic induction of mRNAs for HSP70 and transporter for organic solute betaine in Madin-Darby canine kidney cells. J. Biol. Chem. 273:1832–1837
Sinning, R., Schliess, F., Kubitz, R., Haussinger, D. 1997. Osmo-signalling in C6 glioma cells. FEBS Lett. 400:163–167
Sun, D., O’Donnell, M.E. 1996. Astroglial-mediated phosphorylation of the Na-K-Cl cotransporter in brain microvessel endothelial cells. Am. J. Physiol. 271:C620–627
Terada, Y., Tomita, K., Homma, M.K., Nonoguchi, H., Yang, T., Yamada, T., Yuasa, Y., Krebs, E.G., Sasaki, S., Marumo, F. 1994. Sequential activation of Raf-1 kinase, mitogen-activated protein (MAP) kinase kinase, MAP kinase, and S6 kinase by hyperosmolality in renal cells. J. Biol. Chem. 269:31296–31301
Watts, B.A., 3rd, Di Mari, J.F., Davis, R.J., Good, D.W. 1998. Hypertonicity activates MAP kinases and inhibits HCO-3 absorption via distinct pathways in thick ascending limb. Am. J. Physiol. 275:F478–486
Wojtaszek, P.A., Heasley, L.E., Siriwardana, G., Berl, T. 1998. Dominant-negative c-Jun NH2-terminal kinase 2 sensitizes renal inner medullary collecting duct cells to hypertonicity-induced lethality independent of organic osmolyte transport. J. Biol. Chem. 273:800–804
Zhuang, S., Hirai, S.I., Ohno, S. 2000. Hyperosmolality induces activation of cPKC and nPKC, a requirement for ERK1/2 activation in NIH/3T3 cells. Am. J. Physiol. 278:C102–109.
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Bildin, V.N., Wang, Z., Iserovich, P. et al. Hypertonicity-induced p38MAPK Activation Elicits Recovery of Corneal Epithelial Cell Volume and Layer Integrity. J. Membrain Biol. 193, 1–13 (2003). https://doi.org/10.1007/s00232-002-2002-8
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DOI: https://doi.org/10.1007/s00232-002-2002-8