The Na+/H+ exchanger
isoforms NHE1, NHE2, and NHE3 were all found to be expressed in
Ehrlich ascites tumor cells, as evaluated by Western blotting and confocal microscopy. Under unstimulated conditions, NHE1 was found predominantly in the plasma membrane, NHE3 intracellularly, and NHE2 in both compartments. Osmotic cell shrinkage elicited a rapid intracellular alkalinization, the sensitivity of which to
EIPA (IC50 0.19 microM) and
HOE 642 (IC50 0.85 microM) indicated that it predominantly reflected activation of NHE1. NHE activation by osmotic shrinkage was inhibited by the
protein kinase C inhibitors
chelerythrine (IC50 12.5 microM), Gö 6850 (5 microM), and Gö 6976 (1 microM), and by the
p38 MAPK inhibitor
SB 203580 (10 microM). Furthermore, hypertonic cell shrinkage elicited a biphasic increase in
p38 MAPK phosphorylation, with the first significant increase detectable 2 minutes after the hypertonic challenge. Neither
myosin light chain kinase-specific concentrations of
ML-7 (IC50 40 microM) nor ERK1/2 inhibition by
PD 98059 (50 microM) had any effect on NHE activation. Under isotonic conditions, the
serine/
threonine protein phosphatase inhibitor
calyculin A elicited an
EIPA- and HOE 642-inhibitable intracellular alkalinization, indicating NHE1 activation. Similarly, shrinkage-induced NHE activation was potentiated by
calyculin A. The
calyculin A-induced alkalinization was not associated with an increase in the free, intracellular
calcium concentration, but was abolished by
chelerythrine. It is concluded that shrinkage-induced NHE activation is dependent on PKC and
p38 MAPK, but not on MLCK or ERK1/2. NHE activity under both iso- and hypertonic conditions is increased by inhibition of
serine/
threonine phosphatases, and this effect appears to be PKC-dependent.