Regulation of apical membrane Na⁺/H⁺exchangers NHE2 and NHE3 in intestinal epithelial cell line C2/bbe

Abstract

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.

Ca2+-dependent Inhibition of Na+/H+ Exchanger 3 (NHE3) Requires an NHE3-E3KARP-α-Actinin-4 Complex for Oligomerization and Endocytosis

“…In small intestine, Na ϩ /H ϩ exchanger activity was reduced by elevation of [Ca 2ϩ ] i by treatment with carbachol and ionomycin (4). In addition, elevation of [Ca 2ϩ ] i by treatment with thapsigargin resulted in inhibition of NHE3 activity in C2bbe cells, a subclone of Caco-2 intestinal epithelial cells (8), which also have both NHERF and E3KARP (34). However, the inhibition of NHE3 activity by elevating Ca 2ϩ was not reproduced in PS120 fibroblasts (9), which lack E3KARP, but was reconstituted in these cells by stable expression of E3KARP (Fig.…”

“…However, the effect of elevation of [Ca 2ϩ ] i in cell culture models differs among cell lines. In human colon cancer Caco-2 epithelial cells (C2bbe) stably transfected with NHE3, elevation of [Ca 2ϩ ] i by treatment with thapsigargin inhibited NHE3 activity (8). In contrast, elevating [Ca 2ϩ ] i by treatment with ionomycin did not alter NHE3 activity in PS120 fibroblasts (9), although basal [Ca 2ϩ ] i is involved in the regulation of NHE3 activity in these cells in a calmodulin-or calmodulin kinase II-dependent manner (10).…”

Defective Fluid Secretion and NaCl Absorption in the Parotid Glands of Na+/H+ Exchanger-deficient Mice

“…However, the presence of NHE2 in mouse parotid acinar cells suggests a species-specific difference compared with rat, where immunohistochemistry using the same antibody used in this study combined with semi-quantitative reverse transcription-polymerase chain reaction failed to detect either NHE2 protein or NHE2 transcript in rat parotid (10) So what then is the function of the apical NHE2 in salivary acinar cells? In other epithelia this isoform is inhibited when the intracellular [Ca 2ϩ ] increases (43). Thus, muscarinic stimulation might be expected also to inhibit NHE2 activity in parotid acinar cells.…”

Inhibition of intestinal dipeptide transport by the neuropeptide VIP is an anti‐absorptive effect via the VPAC1 receptor in a human enterocyte‐like cell line (Caco‐2)

“…forskolin, cholera toxin or E. coli STa enterotoxin) that increase [cAMP] i or [cGMP] i (McEwan et al, 1988). NHE3 is highly regulated and is inhibited by factors that increase [cAMP] i and [cGMP] i (McSwine et al, 1998). There are relatively few studies of the short-term regulation of hPepT1 function, although dipeptide transport is modulated directly by some endogenous factors e.g.…”