We examined the regulation of the Na+/H+exchangers (NHEs) NHE2 and NHE3 by expressing them in human intestinal C2/bbe cells, which spontaneously differentiate and have little basal apical NHE activity. Unidirectional apical membrane22Na+influxes were measured in NHE2-transfected (C2N2) and NHE3-transfected (C2N3) cells under basal and stimulated conditions, and their activities were distinguished as the HOE-642-sensitive and -insensitive components of 5-( N, N-dimethyl)amiloride-inhibitable flux. Both C2N2 and C2N3 cells exhibited increased apical membrane NHE activity under non-acid-loaded conditions compared with nontransfected control cells. NHE2 was inhibited by 8-(4-chlorophenylthio)adenosine 3′,5′-cyclic monophosphate and thapsigargin, was stimulated by serum, and was unaffected by cGMP- and protein kinase C-dependent pathways. In contrast, NHE3 was inhibited by all regulatory pathways examined. Under acid-loaded conditions (which increase apical Na+ influx), NHE2 and NHE3 exhibited similar patterns of regulation, suggesting that the second messenger effects observed were not secondary to effects on cell pH. Thus, in contrast to their expression in nonepithelial cells, NHE2 and NHE3 expressed in an epithelial cell line behave similarly to endogenously expressed intestinal apical membrane NHEs. We conclude that physiological regulation and function of epithelium-specific NHEs are dependent on tissue-specific factors and/or conditional requirements.

Na+ retention by the colon in response to salt deprivation is mediated in part by the resulting secondary hyperaldosteronism. We show that experimental hyperaldosteronism, to levels seen with salt deprivation, causes an increase in the selective expression and activity of NHE3, an apically located isoform of the Na+/H+exchange family that functions in transepithelial Na+ absorption. The effect of aldosterone on NHE3 expression is tissue specific, occurring in intestine and not in kidney. Within the intestine, these effects are regional, being observed only in proximal colon, and different in distribution from that observed with glucocorticoids, where the predominant effect occurs in ileum. Although glucocorticoids are well known to exert many effects via regulation of transcript levels, the present study demonstrates that aldosterone stimulates intestinal Na+ absorption by increasing cellular NHE3 expression, a response that is tissue and region specific.

After massive small bowel resection (MSBR), the remnant small intestine adapts to restore Na absorptive function. The possibility that this occurs through increases in cellular Na absorptive capacity was examined by assessing the regional effects of 50% proximal MSBR on the function and expression of the apical membrane Na/H exchangers (NHEs) NHE2 and NHE3. Morphometric analysis confirmed adaptive changes consistent with villus hypertrophy, particularly distal to the anastomosis. Villus epithelium prepared by light mucosal scrapings from 2-wk-postresected and -posttransected control rats exhibited comparable brush-border hydrolase activities, total cell protein per DNA, and villin expression but increased basolateral Na-K-ATPase activity. Parallel increases of two- to threefold in protein and mRNA abundance of NHE2 and NHE3 were observed only in ileal regions distal to the anastomosis of resected rats. Basolateral NHE1 expression was unchanged. After 80% resection, increases in NHE2 and NHE3 became evident in proximal colon. We conclude that increased enterocyte expression and function of apical membrane NHEs in regions distal to the anastomosis play a role in the adaptive process after MSBR. The increased luminal Na load to distal bowel regions after proximal resection may stimulate increases in apical membrane NHE gene transcription and protein expression.

This report presents a study of the effects of the membrane fluidizer, benzyl alcohol, on NHE isoforms 1 and 3. Using transfectants of an NHE-deficient fibroblast, we analyzed each isoform separately. An increase in membrane fluidity resulted in a decrease of approximately 50% in the specific activities of both NHE1 and NHE3. Only Vmax was affected; KNa was unchanged. This effect was specific, as Na+, K+, ATPase activity was slightly stimulated. Inhibition of NHE1 and NHE3 was reversible and de novo protein synthesis was not required to restore NHE activity after washout of fluidizer. Inhibition kinetics of NHE1 by amiloride, 5-(N,N-dimethyl)amiloride (DMA), 5-(N-hexamethyl)amiloride (HMA) and 5-(N-ethyl-N-isopropyl)amiloride (EIPA) were largely unchanged. Half-maximal inhibition of NHE3 was also reached at approximately the same concentrations of amiloride and analogues in control and benzyl alcohol treated, suggesting that the amiloride binding site was unaffected. Inhibition of vesicular transport by incubation at 4 degrees C augmented the benzyl alcohol inhibition of NHE activity, suggesting that the fluidizer effect does not solely involve vesicle trafficking. In summary, our data demonstrate that the physical state of membrane lipids (fluidity) influences Na+/H+ exchange and may represent a physiological regulatory mechanism of NHE1 and NHE3 activity.

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.