Introduction
Two mitogen-activated protein (MAP) kinase family members have been identified to be activated by cellular stress and cytokines, and have therefore been termed stress-activated protein kinases or SAPKs (Cuenda et al., 1997; Tan et al.,1996). The mechanism by which hypo-osmotic stress activates the kinases is not fully understood. The present study focused on the role of cell volume in hypo-osmotic stress-induced activation of SAPKs, p38 kinase and JNKs, in renal epithelial A6 cells.
Methods
A6 cells were cultured onto Nunc filters for 9-13 days in NCTC-109 medium modified for amphibian cells supplemented with 10% fetal bovine serum (osmolality = 255 mOsm/kg H2O) at 27 °C with 2.0% CO2 in air (Niisato, Marunaka, 1999).
Results
Hypo-osmotic stress caused a rapid and transient activation in p38 MAP kinase which reached a peak around 5 min after exposure of A6 cells to a hypo-osmotic solution. The phosphorylation of p38 MAP kinase returned to basal levels within 30 min. Hypo-osmotic stress transiently and markedly activated JNKs, especially p46 JNK. p54 JNK was only slightly activated. Exposure to a hypo-osmotic solution elicits rapid cell swelling, followed by regulatory volume decrease (RVD) to recover to an original cell volume. NPPB (a Cl- channel blocker) inhibited RVD, resulting in continued cell swelling. When we blocked RVD by addition of NPPB, activation of SAPKs was maintained over 90 min. These results suggest that cell swelling activates SAPKs. To confirm the hypothesis that cell swelling but not hypo osmotic stress is essential for activation of SAPKs, we examined whether SAPKs are activated by cell swelling in iso-osmotic solution. A high K+ iso-osmotic solution containing nystatin, which causes cell swelling (Henson et al., 1997) mimicked the action of NPPB on SAPKs. These results strongly suggest that cell swelling but not hypo-osmotic stress is important for activation of SAPKs. Finally, we examined whether cell swelling induces activation of SAPKs via membrane deformation. In S. cerevisiae, PKC1 activation by low osmolality is mimicked by chlorpromazine (Kamada et al., 1995). Bilateral treatment with 100 µM chlorpromazine caused a gradual activation of SAPKs even under the iso-osmotic condition. Although maximal phosphorylation of SAPKs was approximately 5 min by hypo-osmotic stress or high K+ solution, chlorpromazine-induced activation of SAPKs reached maximal values at 30 min.
Conclusions
Cell swelling-induced change in membrane tension plays a crucial role in activation of SAPKs in renal epithelial A6 cells.
References
Cuenda, A., Cohen, P., Buee-Scherrer, V. & Goedert, M. (1997) EMBO J. 16, 295-305
Tan, Y., Rouse, J., Zhang, A., Cariati, S., Cohen, P. & Comb, M. J. (1996) EMBO J. 15, 4629-4642
Niisato, N.& Marunaka, Y. (1999) J. Physiol. (Lond) 518, 417-432
Henson, J. H., Rosener, C. D., Gaetano, C. J., Mendola, R. J., Forrest, J. N.,Jr., Holy, J. & Kleinzeller, A. (1997) J. Exp. Zool. 279, 415-424
Kamada, Y., Jung, U. S., Piotrowski, J. & Levin, D. E. (1995) Genes Dev. 9, 1559-1571