Shrinkage-induced Activation of the Na+/H+ Exchanger in Ehrlich Ascites Tumor Cells: Mechanisms Involved in the Activation and a Role for the Exchanger in Cell Volume Regulation |
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Authors: | SF Pedersen B Kramhøft NK Jørgensen EK Hoffmann |
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Institution: | August Krogh Institute, Biochemical Department, 13, Universitetsparken, DK-2100 Copenhagen ?, Denmark, DK
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Abstract: | Amiloride-sensitive, Na+-dependent, DIDS-insensitive cytoplasmic alkalinization is observed after hypertonic challenge in Ehrlich ascites tumor cells.
This was assessed using the fluorescent pH-sensitive probe 2′,7′-bis-(2-carboxyethyl)-5,6-carboxyfluorescein (BCECF). A parallel
increase in the amiloride-sensitive unidirectional Na+ influx is also observed. This indicates that hypertonic challenge activates a Na+/H+ exchanger. Activation occurs after several types of hypertonic challenge, is a graded function of the osmotic challenge,
and is temperature-dependent. Observations on single cells reveal a considerable variation in the shrinkage-induced changes
in cellular pH
i
, but the overall picture confirms the results from cell suspensions.
Shrinkage-induced alkalinization and recovery of cellular pH after an acid load, is strongly reduced in ATP-depleted cells.
Furthermore, it is inhibited by chelerythrine and H-7, inhibitors of protein kinase C (PKC). In contrast, Calyculin A, an
inhibitor of protein phosphatases PP1 and PP2A, stimulates shrinkage-induced alkalinization.
Osmotic activation of the exchanger is unaffected by removal of calcium from the experimental medium, and by buffering of
intracellular free calcium with BAPTA.
At 25 mm HCO−
3, but not in nominally HCO−
3-free medium, Na+/H+ exchange contributes significantly to regulatory volume increase in Ehrlich cells.
Under isotonic conditions, the Na+/H+ exchanger is activated by ionomycin, an effect which may be secondary to ionomycin-induced cell shrinkage.
Received: 2 March 1995/Revised: 29 September 1995 |
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Keywords: | : Phosphorylation — Protein kinases — Protein phosphatases — Ca2+— cAMP — PKC inhibitors |
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