Examination of the role for Ca2 in regulation and phosphorylation of the Na/H antiporter NHE1 via mitogen and hypertonic stimulation

Abstract

It has been suggested that Ca2+ 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 Ca2+ in the activation and phosphorylation of NHE1 induced by serum and hypertonicity. It is apparent that the microsomal Ca2+ 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 Ca2+ 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 Ca2+ plays the predominant role in the serum-induced activation of NHE1, while phosphorylation plays only a minor, if any, role in this process. However, Ca2+ does not appear to be involved in the osmotic regulation of NHE1.

Dynamics of Ca 2+ i and pH i in Ehrlich ascites tumor cells after Ca 2+ -mobilizing agonists or exposure to hypertonic solution

“…This is in agreement with a recent study in rabbit proximal tubule cells [30]. In contrast, thapsigargin activated the exchanger in human foreskin fibroblasts, although to a lesser degree than did mitogen stimulation [22]. Finally, cell shrinkage per se, induced by a hypertonic challenge, activated the exchanger in the absence of an increase in [Ca 2+ ] i (Fig.…”

“…The mechanism of osmotic activation of the exchanger is not known; however, in contrast to other types of stimuli (e.g. [22]; see also [43]), it is usually reported to be independent of changes in [Ca 2+ ] i ( [23,26,35] see [43]). …”

Activation of Na+/H+ and K+/H+ exchange by calyculin A in Amphiuma tridactylum red blood cells: implications for the control of volume-induced ion flux activity

“…Hence our data suggest that the phosphorylation-dependent event controlling the volume-dependent activation of NHE1 is not increased net phosphorylation of the NHE1 protein. This is supported by the observations of two other laboratories that net phosphorylation of NHE1 is not increased in response to osmotic cell shrinkage in human foreskin fibroblasts (35), Chinese hamster ovary cells (18), or human bladder carcinoma cells.…”

Angiostatin's molecular mechanism: Aspects of specificity and regulation elucidated

“…When extracellular pH is low, typically the sodium/proton antiporter, isoform 1 (NHE1) is activated to maintain intracellular pH in the viable range. This occurs as a function of attachment, phosphorylation, ATP binding, and the presence of cytokines [Schwartz et al, 1991;McSwine et al, 1996]. It has also been reported that NHE1 co-localizes with FAK [Schwartz et al, 1991] and clusters at the leading edge of lamellopodia in migrating cells [Akasaka et al, 1995].…”