The c-Jun NH 2 -terminal protein kinases (JNKs), as well as the extracellular signal-regulated protein kinases (ERKs) and p38 mitogen-activated protein kinase, are activated in renal cells in response to extracellular hypertonicity. To determine whether activation of JNKs by hypertonicity is isoform-specific, renal inner medullary collecting duct cells were stably transfected with cDNA's encoding hemagglutinin (HA)-tagged JNK1 and JNK2 isoforms, and the expressed kinases were immunoprecipitated with an anti-HA antibody. Whereas both recombinant kinases were equivalently expressed, only immunoprecipitates from the HA-JNK2 cells displayed hypertonicity-inducible JNK activity. Furthermore, expression of dominant-negative JNK2 (HA-JNK2-APF) in stable clones inhibited hypertonicity-induced JNK activation by 40 -70%, whereas expression of dominant-negative JNK1 (HA-JNK1-APF) had no significant inhibitory effect. Independent HA-JNK2-APF (but not HA-JNK1-APF) clones displayed greatly reduced viability relative to neomycin controls after 16 h of exposure to 600 mosM/kg hypertonic medium with percent survival of 20.5 ؎ 2.7 and 31.5 ؎ 7.3 for two independent HA-JNK2-APF clones compared with 80.1 ؎ 1.0 for neomycin controls (p < 0.001, n ؍ 5, mean ؎ S.E.). However, neither JNK mutant blocked either regulatory volume increase or hypertonicity-induced enhancement of uptake of inositol, an organic osmolyte putatively involved in long term adaptation to hypertonicity. These results define JNK2 as the primary hypertonicity-activated JNK isoform in IMCD-3 cells and demonstrate its central importance in cellular survival in a hypertonic environment by a mechanism independent of acute regulatory volume increase as well as regulation of organic osmolyte uptake.
In liver cells, the influx of Na؉ mediated by nonselective cation (NSC) channels in the plasma membrane contributes importantly to regulation of cell volume. Under basal conditions, channels are closed; but both physiologic (e.g. insulin) and pathologic (e.g. oxidative stress) stimuli that are known to stimulate tyrosine kinases are associated with large increases in membrane Na ؉ permeability to ϳ80 pA/pF or more. Consequently, the purpose of these studies was to evaluate whether volumesensitive tyrosine kinases mediate cell volume increases through effects on the activity or distribution of NSC channel proteins. In HTC hepatoma cells, decreases in cell volume evoked by hypertonic exposure increased total cellular tyrosine kinase activity ϳ20-fold. Moreover, hypertonic exposure (320 -400 mosM) was followed after a delay by NSC channel activation and partial recovery of cell volume toward basal values (regulatory volume increase (RVI)). The tyrosine kinase inhibitors genistein and erbstatin prevented both NSC channel activation and RVI. Similarly, hypertonic exposure resulted in an increase in p60 c-src activity, and intracellular dialysis with recombinant p60 c-src led to activation of NSC currents in the absence of an osmolar gradient. Utilizing FM1-43 fluorescence, exposure to hypertonic media caused a rapid increase in the rate of exocytosis of ϳ40% (p < 0.01), and genistein inhibited both exocytosis and channel activation. These findings indicate that volume-sensitive increases in p60 c-src and/or related tyrosine kinases play a key role in the regulation of membrane Na ؉ permeability, suggesting that increases in the NSC conductance may be mediated in part through rapid recruitment of a distinct pool of channelcontaining vesicles.
In cultured renal cells, hypertonicity activates multiple mitogen-activated protein kinases (MAPKs) and enhances the expression of heat shock proteins (HSPs). In rats, 24 h water restriction increased mean urinary osmolality ( U osm ) from 2,179 Ϯ 153 mOsm/kg to 2,944 Ϯ 294 mOsm/kg ( P Ͻ 0.001) and was associated with significant (
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