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Examination of the role for Ca 2+ in regulation and phosphorylation of the Na + /H + antiporter NHE1 via mitogen and hypertonic stimulation
Author(s) -
McSwine Rebecca L.,
Li Jing,
Villereal Mitchel L.
Publication year - 1996
Publication title -
journal of cellular physiology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.529
H-Index - 174
eISSN - 1097-4652
pISSN - 0021-9541
DOI - 10.1002/(sici)1097-4652(199607)168:1<8::aid-jcp2>3.0.co;2-t
Subject(s) - thapsigargin , antiporter , calmodulin , sodium–hydrogen antiporter , intracellular , intracellular ph , bapta , phosphorylation , microbiology and biotechnology , brefeldin a , chemistry , biology , biochemistry , endoplasmic reticulum , enzyme , sodium , organic chemistry , golgi apparatus , membrane
It has been suggested that Ca 2+ transients, acting through calmodulin‐binding proteins, play a role in the activation of the Na + /H + exchanger isoform NHE1 (Owen and Villereal, 1982a, Biochem. Biophys. Res. Commun., 109 :762–768; 1982b, Proc. Natl. Acad. Sci. U.S.A., 79 :3537–3541, Ober and Pardee, 1987, J. Cell. Physiol., 132 :311–317). This is supported by a recent report that NHE1 is a calmodulin‐binding protein and that loss of the high‐affinity calmodulin‐binding site results in alterations in antiporter function (Bertrand, et al., 1994, J. Biol. Chem., 269 :13703–13709). An additional mechanism by which NHE1 is activated by mitogens is thought to be phosphorylation (Sardet, et al., 1990, Science 247 :723–726). Although the calmodulin‐binding region appears vital to antiporter activation, the role of phosphorylation is unclear. The studies presented here examine a role for Ca 2+ in the activation and phosphorylation of NHE1 induced by serum and hypertonicity. It is apparent that the microsomal Ca 2+ ATPase inhibitor thapsigargin activates antiporter function in human foreskin fibroblasts (HSWP) as determined by increased intracellular alkalinization examined by image analysis. This effect is Ca 2+ ‐dependent as the alkalinization is blocked when cells are preincubated with BAPTA, an intracellular Ca 2+ chelator. Similarly, the effects of serum‐induced intracellular alkalinization are inhibited by BAPTA. In contrast, activation of NHE1 by increased osmolarity was not inhibited by BAPTA. This suggests that serum, and not hypertonicity, increases intracellular pH via a Ca 2+ ‐dependent process. It was also observed that both thapsigargin and hypertonicity activate NHE1 by a phosphorylation‐independent mechanism and that BAPTA did not block the serum‐induced increase in phosphorylation of NHE1. These results indicate that Ca 2+ plays the predominant role in the serum‐induced activation of NHE1, while phosphorylation plays only a minor, if any, role in this process. However, Ca 2+ does not appear to be involved in the osmotic regulation of NHE1. © 1996 Wiley‐Liss, Inc.