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Trafficking and regulation of the epithelial brush border membrane (BBM) Na+/H+ exchanger 3 (NHE3) in the intestine involves interaction with four different members of the NHERF family in a signal-dependent and possibly segment-specific fashion. The aim of this research was to study the role of NHERF2 (E3KARP) in intestinal NHE3 BBM localization and second messenger-mediated and receptor-mediated inhibition of NHE3. Immunolocalization of NHE3 in WT mice revealed predominant microvillar localization in jejunum and colon, a mixed distribution in the proximal ileum but localization near the terminal web in the distal ileum. The terminal web localization of NHE3 in the distal ileum correlated with reduced acid-activated NHE3 activity (fluorometrically assessed). NHERF2 ablation resulted in a shift of NHE3 to the microvilli and higher basal fluid absorption rates in the ileum, but no change in overall NHE3 protein or mRNA expression. Forskolin-induced NHE3 inhibition was preserved in the absence of NHERF2, whereas Ca2+ ionophore- or carbachol-mediated inhibition was abolished. Likewise, Escherichia coli heat stable enterotoxin peptide (STp) lost its inhibitory effect on intestinal NHE3. It is concluded that in native murine intestine, the NHE3 adaptor protein NHERF2 plays important roles in tethering NHE3 to a position near the terminal web and in second messenger inhibition of NHE3 in a signal- and segment-specific fashion, and is therefore an important regulator of intestinal fluid transport.

Trafficking and regulation of the epithelial brush border membrane (BBM) Na+/H+ exchanger 3 (NHE3) in the intestine involves interaction with four different members of the NHERF family in a signal-dependent and possibly segment-specific fashion. The aim of this research was to study the role of NHERF2 (E3KARP) in intestinal NHE3 BBM localization and second messenger-mediated and receptor-mediated inhibition of NHE3. Immunolocalization of NHE3 in WT mice revealed predominant microvillar localization in jejunum and colon, a mixed distribution in the proximal ileum but localization near the terminal web in the distal ileum. The terminal web localization of NHE3 in the distal ileum correlated with reduced acid-activated NHE3 activity (fluorometrically assessed). NHERF2 ablation resulted in a shift of NHE3 to the microvilli and higher basal fluid absorption rates in the ileum, but no change in overall NHE3 protein or mRNA expression. Forskolin-induced NHE3 inhibition was preserved in the absence of NHERF2, whereas Ca2+ ionophore- or carbachol-mediated inhibition was abolished. Likewise, Escherichia coli heat stable enterotoxin peptide (STp) lost its inhibitory effect on intestinal NHE3. It is concluded that in native murine intestine, the NHE3 adaptor protein NHERF2 plays important roles in tethering NHE3 to a position near the terminal web and in second messenger inhibition of NHE3 in a signal- and segment-specific fashion, and is therefore an important regulator of intestinal fluid transport.

SLC26A7 (human)/Slc26a7 (mouse) is a recently identified chloride-base exchanger and/or chloride transporter that is expressed on the basolateral membrane of acid-secreting cells in the renal outer medullary collecting duct (OMCD) and in gastric parietal cells. Here, we show that mice with genetic deletion of Slc26a7 expression develop distal renal tubular acidosis, as manifested by metabolic acidosis and alkaline urine pH. In the kidney, basolateral Cl(-)/HCO3(-) exchange activity in acid-secreting intercalated cells in the OMCD was significantly decreased in hypertonic medium (a normal milieu for the medulla) but was reduced only mildly in isotonic medium. Changing from a hypertonic to isotonic medium (relative hypotonicity) decreased the membrane abundance of Slc26a7 in kidney cells in vivo and in vitro. In the stomach, stimulated acid secretion was significantly impaired in isolated gastric mucosa and in the intact organ. We propose that SLC26A7 dysfunction should be investigated as a potential cause of unexplained distal renal tubular acidosis or decreased gastric acid secretion in humans.

The epithelial anion channel CFTR interacts with multiple PDZ domain-containing proteins. Heterologous expression studies have demonstrated that the Na+/1H(+) exchanger regulatory factors, NHERF1, NHERF2, and PDZK1 (NHERF3), modulate CFTR membrane retention, conductivity, and interactions with other transporters. To study their biological roles in vivo, we investigated CFTR-dependent duodenal HCO3- secretion in mouse models of Nberf1, Nberf2, and Pdzk1 loss of function. We found that Nberf1 ablation strongly reduced basal as well as forskolin-stimulated (FSK-stimulated) HCO3- secretory rates and blocked beta(2)-adrenergic receptor (beta(2)-AR) stimulation. Conversely, Nberf2(-/-) mice displayed augmented FSK-stimulated HCO3- secretion. Furthermore, although lysophosphatidic acid (LPA) inhibited FSK-stimulated HCO3- secretion in WT mice, this effect was lost in Nberf2(-/-) mice. Pdzk1 ablation reduced basal, but not FSK-stimulated, HCO3- secretion. In addition, laser microdissection and quantitative PCR revealed that the beta(2)-AR and the type 2 LPA receptor were expressed together with CFTR in duodenal crypts and that colocalization of the beta(2)-AR and CFTR was reduced in the Nberf1(-/-) mice. These data suggest that the NHERF proteins differentially modulate duodenal HCO3- secretion: while NHERF1 is an obligatory linker for beta(2)-AR stimulation of CFTR, NHERF2 confers inhibitory signals by coupling the LPA receptor to CFTR.

G A A b st ra ct s of calcitonin on ion transport in IECs. Human colonic T84 cells grown on Transwell inserts were utilized as an in-vitro model of IECs. Chloride secretion was assessed by the measurement of short circuit current (Isc) across T84 monolayers mounted in Ussing chambers. We first examined the expression of CTR in IECs. Real time QRT-PCR and western blot analysis demonstrated the expression of CTR in T84 cells. Exposure of T84 cells to calcitonin from the basolateral but not apical side significantly increased Isc (a change of 287 ± 48 μA/cm2 from the baseline). Stimulation of Isc by calcitonin was dose-dependent (1-100 nM) and was completely blocked by the CTR antagonist, CT8-32. In addition, the increase in Isc was blocked by replacing chloride in the bath solutions with equal amount of gluconate (95 ± 6% reduction, P < 0.01) and was significantly inhibited by 10 μM of the specific CFTR inhibitor, CFTR127inh, (88 ± 9% reduction, P < 0.01) indicating the involvement of CFTR chloride channel in CT-induced effects. To further investigate the signaling pathways involved, we examined calcitonin-induced Isc in the presence of the chelator of [Cai], BAPTA-AM and PKA inhibitor, H89. Preincubation with 20 μMof BAPTAAM or 10μM H89 for 45 minutes significantly reduced calcitonin-induced Isc indicating the involvement of both Ca++and PKA-dependent pathways (58±13% reduction, P < 0.01 and 55±7%, P < 0.01, respectively). Simultaneous inhibition of both Ca++ and PKA pathways had an additive effect on the CT-induced stimulation of Isc (78 ± 8% reduction, P < 0.01). In conclusion, calcitonin-induced short circuit current appears to be mediated by CTR via direct activation of the CFTR channel and is Ca++ and PKA-dependent. This novel study showing the expression of calcitonin receptor in intestinal epithelial cells provides further insights into the molecular mechanisms underlying calcitonin-induced diarrhea. (Supported by NIDDK and Dept of Veteran Affairs).