Cell Swelling Activates Stress-Activated Protein Kinases, p38 MAP Kinase and JNK, in Renal Epithelial A6 Cells Naomi Niisato,* Martin Post,* Willy Van Driessche,† and Yoshinori Marunaka* ,1 *Lung and Cell Biology Programmes, Hospital for Sick Children, University of Toronto, Toronto, Ontario M5G 1X8, Canada; and †Laboratorium voor Fysiologie, K. U. Leuven, Campus Gasthuisberg, B-3000 Leuven, Belgium Received November 9, 1999 Osmotic shock is well recognized as one of the factors activating stress-activated protein kinases (SAPKs), p38 MAP kinase and c-Jun N-terminal ki- nases (JNKs). In renal epithelial A6 cells, hypo-osmotic shock transiently activated SAPKs with maximal acti- vation at 5 min. A6 cells showed a regulatory volume decrease (RVD) after swelling when the cells were exposed to a hypo-osmotic solution. In contrast, acti- vation of SAPKs was maintained over 90 min after hypo-osmotic shock in the presence of 5-nitro-2-(3- phenylpropylamino)benzoic acid (NPPB, a Cl  chan- nel blocker), which completely blocked the RVD and kept the cells continuously swelling. Exposure of the cells to a high K  iso-osmotic solution containing nystatin, which induces continuous cell swelling, also continuously activated SAPKs. Furthermore, mem- brane deformation induced by chlorpromazine acti- vated SAPKs. These results suggest that changes in membrane tension by cell swelling or chlorpromazine, but not osmolality, are important steps for activation of SAPKs in A6 cells. © 1999 Academic Press Two mitogen-activated protein (MAP) kinase family members have been identified to be activated by cellular stress (chemical, heat and osmotic shock, UV radiation) and cytokines, and have therefore been termed stress-activated protein kinases or SAPKs (1, 2). SAPK1 (also termed c-Jun N-terminal kinases (JNKs)) isoforms phosphorylate and activate transcrip- tion factors such as c-Jun, ATF2 and Elk1. SAPK2 (also termed p38 MAP kinase) activates two protein kinases, termed MAP kinase-activated protein kinase 2 (MAPKAP2) and MAPKAP3. Although many reports have shown that hyper- osmotic shock activates JNK and p38 MAP kinase (3– 5), little is known about the effects of hypo-osmotic shock on activation of JNKs and p38 kinase. In intes- tine 407 cell (6) and cardiac myocytes (7), hypo-osmotic shock activates p38 kinase and JNKs, respectively. However, the mechanism by which hypo-osmotic shock activates these kinases is not fully understood. The present study focused on the role of cell volume in activation of SAPKs, p38 kinase and JNKs, in renal epithelial A6 cells by hypo-osmotic shock. We report here that cell swelling-induced changes in membrane tension are important for activation of p38 kinase and JNKs in renal epithelial cells under iso-osmotic and hypo-osmotic conditions. MATERIAL AND METHODS Solutions. The iso-osmotic solution (255 mOsm/kg H 2 O) con- tained the following ion concentrations (in mM); 120 NaCl, 3.5 KCl, 1 CaCl 2 , 1 MgCl 2 , 5 glucose, 10 N-2-hydroxyethyl-piperazine-N-2- ethanesulfonic acid (HEPES). The hypo-osmotic solution (135 mOsm/kg H 2 O) contained the following ion concentrations (in mM); 55 NaCl, 3.5 KCl, 1 CaCl 2 , 1 MgCl 2 , 5 glucose, 10 HEPES. The iso-osmotic high K solution contained the following ion concentra- tions (in mM); 123.5 KCl, 1 CaCl 2 , 1 MgCl 2 , 5 glucose, 10 HEPES. The pH of solutions used in the present study was adjusted to 7.4 by NaOH. Bathing solutions were stirred with air. Cell culture. A6 cells were derived from Xenopus laevis distal nephron and purchased from American Type Culture Collection (ATCC). Briefly, A6 cells (passage 72– 84) were grown on plastic culture flasks in NCTC-109 medium modified for amphibian cells supplemented with 10% fetal bovine serum (osmolality = 255 mOsm/kg H 2 O) (8 –10). The flasks were kept in a humidified incuba- tor at 27°C with 2.0% CO 2 in air. Cells were seeded onto Nunc filters at density of 5  10 4 cells/well and were cultured for 9 –13 days. Western blotting. A6 cells were lysed by lysis buffer (50 mM HEPES, 150 mM NaCl, 1.5 mM MgCl 2 , 1 mM EGTA, 10% glycerol, 1% Triton X-100, 100 mM NaF, 10 mM pyrophosphate, 200 M Na-orthovanadate, 250 g/ml leupeptin, 0.1 mM phenylmethylsulfo- nyl fluoride, 100 kallikrein inactivator units/ml aprotinin, pH 7.4) after various treatments. Cells were homogenized by sonication and centrifuged at 12,000  g for 10 min at 4°C to remove insoluble debris. The cell lysates containing 20 g of protein were boiled in SDS sample buffer (60 mM Tris-HCl, 2% (w/v) SDS, 5% (v/v) glycerol, pH 6.8) and then subjected to 10% SDS-polyacrylamide gel electro- phoresis (SDS-PAGE). After electrophoresis, proteins were trans- ferred to nitrocellulose membranes. Nonspecific binding was blocked 1 To whom correspondence should be addressed at Lung Biology, Hospital for Sick Children, University of Toronto, 555 University Avenue, Toronto, Ontario M5G 1X8, Canada. Fax: + 1 (Country code) 416-813-5771. E-mail: marunaka@sickkids.on.ca. Biochemical and Biophysical Research Communications 266, 547–550 (1999) Article ID bbrc.1999.1843, available online at http://www.idealibrary.com on 547 0006-291X/99 $30.00 Copyright © 1999 by Academic Press All rights of reproduction in any form reserved.