In vivo regulation of MAP kinases in Ratus norvegicus renal papilla by water loading and restriction.

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 (Uosm) from 2, 179+/-153 mOsm/kg to 2,944+/-294 mOsm/kg (P < 0.001) and was associated with significant (P < 0.05) increases in the papillary activity of c-Jun NH2-terminal protein kinase (JNK) by 22%, extracellular signal-regulated protein kinase (ERK) by 49%, and p38 MAPK by 15%. Conversely, 24 h of water-loading (Uosm 473+/-33 mOsm/kg) caused suppression of JNK activity by 43% (P < 0.001), ERK by 39% (P < 0.05), and p38 MAPK by 26% (P < 0.05). No such modulation was observed in the isotonic cortex. c-Jun phosphorylation was decreased in papilla from water-loaded rats by 45% versus controls. Expression of Hsp 110, inducible Hsp 70, and Hsp 25 was greater in the hyperosmotic papilla than the isosmotic cortex but was not affected by the animal's hydration state. In cultured inner medullary collecting duct cells, HSP expression was maximal at 500 mOsm/kg, while activation of JNK continued to increase. We conclude that under basal conditions of hydration, these HSPs are maximally expressed in the hypertonic inner medulla, while the activation of all three members of the MAPK family approaches but is not maximal.

p38 MAP kinase modulates liver cell volume through inhibition of membrane Na+ permeability.

In hepatocytes, Na+ influx through nonselective cation (NSC) channels represents a key point for regulation of cell volume. Under basal conditions, channels are closed, but both physiologic and pathologic stimuli lead to a large increase in Na+ and water influx. Since osmotic stimuli also activate mitogen-activated protein (MAP) kinase pathways, we have examined regulation of Na+ permeability and cell volume by MAP kinases in an HTC liver cell model. Under isotonic conditions, there was constitutive activity of p38 MAP kinase that was selectively inhibited by SB203580. Decreases in cell volume caused by hypertonic exposure had no effect on p38, but increases in cell volume caused by hypotonic exposure increased p38 activity tenfold. Na+ currents were small when cells were in isotonic media but could be increased by inhibiting constitutive p38 MAP kinase, thereby increasing cell volume. To evaluate the potential inhibitory role of p38 more directly, cells were dialyzed with recombinant p38alpha and its upstream activator, MEK-6, which substantially inhibited volume-sensitive currents. These findings indicate that constitutive p38 activity contributes to the low Na+ permeability necessary for maintenance of cell volume, and that recombinant p38 negatively modulates the set point for volume-sensitive channel opening. Thus, functional interactions between p38 MAP kinase and ion channels may represent an important target for modifying volume-sensitive liver functions.

Severely decreased MARCKS expression correlates with ras reversion but not with mitogenic responsiveness.

Phorbol ester-inducible phosphorylation of MARCKS, the '80-kDa' substrate of protein kinase C, was undetectable in several phenotypically dominant, non-transformed revertants independently derived from the ras-transformed cell line NIH3T3 DT-ras. Extremely low expression of MARCKS protein accounted for this apparent lack of phosphorylation. MARCKS-encoding mRNA levels were correspondingly decreased relative to normal and ras-transformed cells in all four ras revertant cell lines studied: C-11 and F-2, derived by 5-azacytidine treatment and selection with ouabain; CHP 9CJ, derived by ethylmethane sulfonate mutagenesis and selection with cis-hydroxy-L-proline; and 12-V3, derived by transfection with the human Krev-1 gene. However, re-expression of MARCKS after transfection of a cloned MARCKS cDNA into the C-11 ras revertant cells was not sufficient to induce retransformation. In fact, no significant difference in sensitivity to mitogenic stimulation by phorbol esters was observed among several cell lines expressing widely varying levels of MARCKS. This evidence argues against a direct role for MARCKS in mitogenic signaling. However, the strong correlation between attenuation of MARCIS expression and phenotypically dominant ras reversion suggests that a common negative regulatory mechanism might be responsible for both effects, presenting a potentially useful strategy for identifying factors involved in transducing the ras signal.

Activation of a novel form of phospholipase A2 during liver regeneration.

Activation of phospholipase A2 (PLA2) occurs following mitogenic stimulation of cells. This study examined PLA2 activation during liver regeneration. Increased activity was detected within 1 h after partial hepatectomy, was maximal by 6 h, and returned to control levels by 24 h. Fractionation of cell-free extracts revealed multiple peaks of PLA2 activity. One peak appeared identical to the previously described cPLA2, and was modestly stimulated during regeneration. A higher molecular weight form (hPLA2) was stimulated approximately 5-fold during regeneration. This enzyme was Ca(2+)-dependent and selective for arachidonoylphosphatidylethanolamine. The activation of this novel form of PLA2 represents an early event in liver regeneration, and is likely to contribute to the proliferative response.

Dominant-negative c-Jun NH2-terminal kinase 2 sensitizes renal inner medullary collecting duct cells to hypertonicity-induced lethality independent of organic osmolyte transport.

The c-Jun NH2-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.