p38 MAP kinase modulates liver cell volume through inhibition of membrane Na+ permeability
Andrew P. Feranchak, Tomas Berl, Juan Capasso, Paul A. Wojtaszek, Jiahuai Han, J. Gregory Fitz
Andrew P. Feranchak, Tomas Berl, Juan Capasso, Paul A. Wojtaszek, Jiahuai Han, J. Gregory Fitz
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p38 MAP kinase modulates liver cell volume through inhibition of membrane Na+ permeability

Abstract

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 p38α 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.

Authors

Andrew P. Feranchak, Tomas Berl, Juan Capasso, Paul A. Wojtaszek, Jiahuai Han, J. Gregory Fitz

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Intracellular dialysis with recombinant p38 MAP kinase protein inhibits ...
Intracellular dialysis with recombinant p38 MAP kinase protein inhibits volume-sensitive current activation. Under whole-cell patch-clamp conditions, the recombinant kinases p38α (5 μg/ml) and MEK-6 (5 μg/ml) were delivered, individually or together, to the cell interior by inclusion in the patch pipette, and cells were then exposed to hypertonic buffer (20 mM sucrose, ∼320 mOsm). (a) Whole-cell currents measured using the voltage-step protocol (as described in Figure 2a). Currents were small with intracellular dialysis of p38α and MEK-6 under isotonic conditions (top tracing). However, p38α and MEK-6 significantly inhibit the amplitude of hypertonic-induced currents (bottom tracing) as compared with control (middle tracing). (b) Cumulative data expressed as average current density at –80 mV. Control cells, dialyzed with heat-inactivated p38α and MEK-6, demonstrated characteristic current activation. Dialysis with either p38α or MEK-6 individually resulted in partial inhibition, which was not statistically significant compared with control. However, dialysis with p38α and MEK-6 together resulted in significant current inhibition. There was no effect of p38α and MEK-6 on basal (isotonic) currents. (c) Under control conditions, current density increased with increasing transmembrane osmolar gradients. In the presence of intracellular p38α + MEK-6, there was significant inhibition of current amplitude at lower transmembrane osmolar gradients (5–20 mM sucrose). However, this inhibitory effect was overcome at higher transmembrane osmolar gradients (50 mM sucrose). *P < 0.01.