Mechanism of regulation of rabbit intestinal villus cell brush border membrane Na/H exchange by nitric oxide

In the mammalian small intestine, coupled NaCl absorption occurs via the dual operation of Na/H and Cl/HCO(3) exchange on the villus cell brush border membrane (BBM). Although constitutive nitric oxide (cNO) has been demonstrated to alter gastrointestinal tract functions, how cNO may specifically alter these two transporters to regulate coupled NaCl absorption is unknown. In villus cells, inhibition of cNO synthase (cNOS) with l-N(G)-nitroarginine methylester (l-NAME) stimulated Na/H exchange whereas Cl/HCO(3) exchange was unaffected. In villus cell BBM vesicles (BBMV) prepared from rabbits treated with l-NAME, Na/H exchange was also stimulated. d-NAME, an inactive analog of l-NAME, and N(6)-(1-imonoethyl)-l-lysine dihydrochloride, a more selective inhibitor of inducible NO synthase, did not affect Na/H exchange. Kinetic studies demonstrated that the mechanism of stimulation is secondary to an increase in the maximal rate of uptake of Na, without an alteration in the affinity of the transporter for Na. Northern blot studies demonstrated an increase in the message for the BBM Na/H exchanger NHE3, and Western blot studies showed that the immunoreactive protein levels of NHE3 was increased when cNOS was inhibited. Thus these results indicate that cNO under nominal physiological states most likely maintains an inhibitory tone on small intestinal coupled NaCl absorption by specifically inhibiting BBM Na/H expression.     

Regulation of sodium glucose co-transporter SGLT1 through altered glycosylation in the intestinal epithelial cells

Inhibition of constitutive nitric oxide (cNO) production inhibits SGLT1 activity by a reduction in the affinity for glucose without a change in Vmax in intestinal epithelial cells (IEC-18). Thus, we studied the intracellular pathway responsible for the posttranslational modification/s of SGLT1. NO is known to mediate its effects via cGMP which is diminished tenfold in L-NAME treated cells. Inhibition of cGMP production at the level of guanylyl cyclase or inhibition of protein kinase G also showed reduced SGLT1 activity demonstrating the involvement of PKG pathway in the regulation of SGLT1 activity. Metabolic labeling and immunoprecipitation with anti-SGLT1 specific antibodies did not show any significant changes in phosphorylation of SGLT1 protein. Tunicamycin to inhibit glycosylation reduced SGLT1 activity comparable to that seen with L-NAME treatment. The mechanism of inhibition was secondary to decreased affinity without a change in Vmax. Immunoblots of luminal membranes from tunicamycin treated or L-NAME treated IEC-18 cells showed a decrease in the apparent molecular size of SGLT1 protein to 62 and 67 kD, respectively suggesting an alteration in protein glycosylation. The deglycosylation assay with PNGase-F treatment reduced the apparent molecular size of the specific immunoreactive band of SGLT1 from control and L-NAME treated IEC-18 cells to approximately 62 kD from their original molecular size of 75 kD and 67 kD, respectively. Thus, the posttranslational mechanism responsible for the altered affinity of SGLT1 when cNO is diminished is secondary to altered glycosylation of SGLT1 protein. The intracellular pathway responsible for this alteration is cGMP and its dependent kinase.     

NHE1, NHE2, and NHE3 contribute to regulation of intracellular pH in murine duodenal epithelial cells

Na(+)/H(+)-exchangers (NHE) mediate acid extrusion from duodenal epithelial cells, but the isoforms involved have not previously been determined. Thus we investigated 1) the contribution of Na(+)-dependent processes to acid extrusion, 2) sensitivity to Na(+)/H(+) exchange inhibitors, and 3) molecular expression of NHE isoforms. By fluorescence spectroscopy the recovery of intracellular pH (pH(i)) was measured on suspensions of isolated acidified murine duodenal epithelial cells loaded with 2', 7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein. Expression of NHE isoforms was studied by RT-PCR and Western blot analysis. Reduction of extracellular Na(+) concentration ([Na(+)](o)) during pH(i) recovery decreased H(+) efflux to minimally 12.5% of control with a relatively high apparent Michaelis constant for extracellular Na(+). The Na(+)/H(+) exchange inhibitors ethylisopropylamiloride and amiloride inhibited H(+) efflux maximally by 57 and 80%, respectively. NHE1, NHE2, and NHE3 were expressed at the mRNA level (RT-PCR) as well as at the protein level (Western blot analysis). On the basis of the effects of low [Na(+)](o) and inhibitors we propose that acid extrusion in duodenal epithelial cells involves Na(+)/H(+) exchange by isoforms NHE1, NHE2, and NHE3.     

Involvement of G protein-coupled receptor kinase 4 and 6 in rapid desensitization of dopamine D1 receptor in rat IEC-6 intestinal epithelial cells

Dopamine-induced inhibition of Na(+)-K(+)-ATPase has been suggested to play a role in the regulation of Na(+) absorption at the intestinal level, and these effects were mediated by dopamine D(1)-like receptors. The aim of this work was to evaluate the effect of the activation of the D(1)-like receptors on the activity of the Na(+)/H(+) exchanger (NHE) in the rat intestinal epithelial cell line IEC-6. The presence of D(1) receptors was confirmed by immunoblotting. The dopamine D(1)-like receptor agonist SKF-38393 produced a concentration-dependent inhibition of NHE activity and stimulation of adenylyl cyclase (AC), this being antagonized by the D(1) selective antagonist SKF-83566. Effects of SKF-38393 on NHE and AC activities were maximal at 5 min of exposure to the agonist and rapidly diminished with no effect at 25 min. Exposure of cells for 25 min to dibutyryl-cAMP (0.5 mM) or to the AC activator forskolin (3 microM) effectively inhibited NHE activity. Pretreatment of cells with heparin (1 microM), a nonselective G protein-coupled receptor kinase (GRK) inhibitor, prevented the loss of effects on NHE activity after 25 min exposure to SKF-38393. The presence of GRK4, GRK6A, and GRK6B was confirmed by immunoblotting. Overnight treatment with the anti-GRK4-6 antibody complexed with Lipofectin was also effective in preventing loss of the effects of SKF-38393 on NHE and AC activities. It is concluded that dopamine D(1) receptors in IEC-6 rapidly desensitize to D(1)-like agonist stimulation and GRK4 and 6 appear to be involved in agonist-mediated responsiveness and desensitization.     

Na-glucose and Na-neutral amino acid cotransport are uniquely regulated by constitutive nitric oxide in rabbit small intestinal villus cells

Na-nutrient cotransport processes are not only important for the assimilation of essential nutrients but also for the absorption of Na in the mammalian small intestine. The effect of constitutive nitric oxide (cNO) on Na-glucose (SGLT-1) and Na-amino acid cotransport (NAcT) in the mammalian small intestine is unknown. Inhibition of cNO synthase with N(G)-nitro-l-arginine methyl ester (L-NAME) resulted in the inhibition of Na-stimulated (3)H-O-methyl-D-glucose uptake in villus cells. However, Na-stimulated alanine uptake was not affected in these cells. The L-NAME-induced reduction in SGLT-1 in villus cells was not secondary to an alteration in basolateral membrane Na-K-ATPase activity, which provides the favorable Na gradient for this cotransport process. In fact, SGLT-1 was inhibited in villus cell brush-border membrane (BBM) vesicles prepared from animals treated with L-NAME. Kinetic studies demonstrated that the mechanism of inhibition of SGLT-1 was secondary to a decrease in the affinity for glucose without a change in the maximal rate of uptake of glucose. Northern blot studies demonstrated no change in the mRNA levels of SGLT-1. Western blot studies demonstrated no significant change in the immunoreactive protein levels of SGLT-1 in ileal villus cell BBM from L-NAME-treated rabbits. These studies indicate that inhibition of cNO production inhibits SGLT-1 but not NAcT in the rabbit small intestine. Therefore, whereas cNO promotes Na-glucose cotransport, it does not affect NAcT in the mammalian small intestine.     

Mechanism of leukotriene D4 inhibition of Na-alanine cotransport in intestinal epithelial cells

In a rabbit model of chronic intestinal inflammation, we previously demonstrated inhibition of neutral Na-amino acid cotransport. The mechanism of the inhibition was secondary to a decrease in the affinity for amino acid rather than the number of cotransporters. Since leukotriene (LT)D4 is known to be elevated in enterocytes during chronic intestinal inflammation, we used rat intestinal epithelial cell (IEC-18) monolayers to determine the mechanism of regulation of Na-alanine cotransport (alanine, serine, cysteine transporter 1: ASCT1) by LTD4. Na-alanine cotransport was inhibited by LTD4 in IEC-18 cells. The mechanism of inhibition of ASCT1 (solute carrier, SLC1A4) by LTD4 is secondary to a decrease in the affinity of the cotransporter for alanine without a significant change in cotransporter numbers and is not secondary to an alteration in the Na+ extruding capacity of the cells. Real-time quantitative PCR and Western blot analysis results indicate that ASCT1 message and protein levels are also unchanged in LTD4-treated IEC-18 cells. These results indicate that LTD4 inhibits Na-dependent neutral amino acid cotransport in IEC. The mechanism of inhibition is secondary to a decrease in the affinity for alanine, which is identical to that seen in villus cells from the chronically inflamed rabbit small intestine, where LTD4 levels are significantly increased.     

NHERF1 and NHERF2 are necessary for multiple but usually separate aspects of basal and acute regulation of NHE3 activity

Na(+)/H(+) exchanger 3 (NHE3) is expressed in the brush border (BB) of intestinal epithelial cells and accounts for the majority of neutral NaCl absorption. It has been shown that the Na(+)/H(+) exchanger regulatory factor (NHERF) family members of multi-PDZ domain-containing scaffold proteins bind to the NHE3 COOH terminus and play necessary roles in NHE3 regulation in intestinal epithelial cells. Most studies of NHE3 regulation have been in cell models in which NHERF1 and/or NHERF2 were overexpressed. We have now developed an intestinal Na(+) absorptive cell model in Caco-2/bbe cells by expressing hemagglutinin (HA)-tagged NHE3 with an adenoviral infection system. Roles of NHERF1 and NHERF2 in NHE3 regulation were determined, including inhibition by cAMP, cGMP, and Ca(2+) and stimulation by EGF, with knockdown (KD) approaches with lentivirus (Lenti)-short hairpin RNA (shRNA) and/or adenovirus (Adeno)-small interfering RNA (siRNA). Stable infection of Caco-2/bbe cells by NHERF1 or NHERF2 Lenti-shRNA significantly and specifically reduced NHERF protein expression by >80%. NHERF1 KD reduced basal NHE3 activity, while NHERF2 KD stimulated NHE3 activity. siRNA-mediated (transient) and Lenti-shRNA-mediated (stable) gene silencing of NHERF2 (but not of NHERF1) abolished cGMP- and Ca(2+)-dependent inhibition of NHE3. KD of NHERF1 or NHERF2 alone had no effect on cAMP inhibition of NHE3, but KD of both simultaneously abolished the effect of cAMP. The stimulatory effect of EGF on NHE3 was eliminated in NHERF1-KD but occurred normally in NHERF2-KD cells. These findings show that both NHERF2 and NHERF1 are involved in setting NHE3 activity. NHERF2 is necessary for cGMP-dependent protein kinase (cGK) II- and Ca(2+)-dependent inhibition of NHE3. cAMP-dependent inhibition of NHE3 activity requires either NHERF1 or NHERF2. Stimulation of NHE3 activity by EGF is NHERF1 dependent.     

Rapid protein kinase D1 signaling promotes migration of intestinal epithelial cells

We have examined the role of protein kinase D1 (PKD1) signaling in intestinal epithelial cell migration. Wounding monolayer cultures of intestinal epithelial cell line IEC-18 or IEC-6 induced rapid PKD1 activation in the cells immediately adjacent to the wound edge, as judged by immunofluorescence microscopy with an antibody that detects the phosphorylated state of PKD1 at Ser(916), an autophosphorylation site. An increase in PKD1 phosphorylation at Ser(916) was evident as early as 45 s after wounding, reached a maximum after 3 min, and persisted for ≥15 min. PKD1 autophosphorylation at Ser(916) was prevented by the PKD family inhibitors kb NB 142-70 and CRT0066101. A kb NB 142-70-sensitive increase in PKD autophosphorylation was also elicited by wounding IEC-6 cells. Using in vitro kinase assays after PKD1 immunoprecipitation, we corroborated that wounding IEC-18 cells induced rapid PKD1 catalytic activation. Further results indicate that PKD1 signaling is required to promote migration of intestinal epithelial cells into the denuded area of the wound. Specifically, treatment with kb NB 142-70 or small interfering RNAs targeting PKD1 markedly reduced wound-induced migration in IEC-18 cells. To test whether PKD1 promotes migration of intestinal epithelial cells in vivo, we used transgenic mice that express elevated PKD1 protein in the small intestinal epithelium. Enterocyte migration was markedly increased in the PKD1 transgenic mice. These results demonstrate that PKD1 activation is one of the early events initiated by wounding a monolayer of intestinal epithelial cells and indicate that PKD1 signaling promotes the migration of these cells in vitro and in vivo.     

IFN-gamma downregulates expression of Na(+)/H(+) exchangers NHE2 and NHE3 in rat intestine and human Caco-2/bbe cells

Diarrhea associated with inflammatory bowel diseases has traditionally been attributed to stimulated secretion. The purpose of this study was to determine whether chronic stimulation of intestinal mucosa by interferon-gamma (IFN-gamma) affects expression and function of the apical membrane Na(+)/H(+) exchangers NHE2 and NHE3 in rat intestine and Caco-2/bbe (C2) cells. Confluent C2 cells expressing NHE2 and NHE3 were treated with IFN-gamma for 2, 24, and 48 h. Adult rats were injected with IFN-gamma intraperitoneally for 12 and 48 h. NHE2 and NHE3 activities were measured by unidirectional (22)Na influx across C2 cells and in rat brush-border membrane vesicles. NHE protein and mRNA were assessed by Western and Northern blotting. IFN-gamma treatment of C2 monolayers caused a >50% reduction in NHE2 and NHE3 activities and protein expression. In rats, region-specific, time- and dose-dependent reductions of NHE2 and NHE3 activities, protein expression, and mRNA were observed after exposure to IFN-gamma. Chronic exposure of intestinal epithelial cells to IFN-gamma results in selective downregulation of NHE2 and NHE3 expression and activity, a potential cause of inflammation-associated diarrhea.     

Activated H-ras transforms rat intestinal epithelial cells with expression of α-TGF

We describe malignant transformation of cultured rat intestinal epithelial cells (IEC-18) by transfection with the activated human H-ras gene cloned from the EJ bladder carcinoma. Transformed cells showed a marked morphological change, expressed high levels of the transfected H-ras gene, were able to grow in agar and expressed antigenic markers identical with parental IEC-18 cells. When injected into syngeneic rats these cells formed rapidly growing tumors expressing the same intestinal-specific antigenic markers as the injected cells. Parallel to the high expression of H-ras mRNA in the transformants we document overexpression of rat α-TGF mRNA.     

RNA interference reveals a differential role of FAK and Pyk2 in cell migration, leading edge formation and increase in focal adhesions induced by LPA in intestinal epithelial cells

In the gastrointestinal mucosa, cell migration plays a crucial role in the organization and maintenance of tissue integrity but the mechanisms involved remain incompletely understood. Here, we used small-interfering RNA (siRNA)-mediated depletion of focal adhesion kinase (FAK) protein to determine the role of FAK in wound-induced migration and cytoskeletal organization in the non-transformed intestinal epithelial cells IEC-6 and IEC-18 stimulated with the G protein-coupled receptors (GPCR) agonist lysophosphatidic acid (LPA). Treatment of these cells with FAK siRNA substantially reduced FAK expression, but did not affect the expression of proline-rich tyrosine kinase 2 (Pyk2). Knockdown of FAK protein significantly inhibited LPA-induced migration of both IEC-18 and IEC-6 cells. LPA induced reorganization of actin and microtubule cytoskeleton in the leading edge was largely inhibited in FAK siRNA-transfected IEC-18 cells. Interestingly, in contrast to the FAK-/- cells, which exhibit an increased number of prominent focal adhesions when plated on fibronectin, FAK knockdown IEC-18 cells exhibited dramatically decreased number of focal adhesions in response to both LPA and fibronectin as compared with the control cells. We also used siRNAs to knockdown Pyk2 expression without reducing FAK expression. Depletion of Pyk2 did not prevent LPA-induced migration or cytoskeletal reorganization in IEC-18 cells. In conclusion, our study shows that FAK plays a critical role in LPA-induced migration, cytoskeletal reorganization, and assembly of focal adhesions in intestinal epithelial cells whereas depletion of Pyk2 did not interfere with any of these responses elicited by LPA.     

Evidence for NHE3-mediated Na transport in sheep and bovine forestomach

Na absorption across the cornified, multilayered, and squamous rumen epithelium is mediated by electrogenic amiloride-insensitive transport and by electroneutral Na transport. High concentrations of amiloride (>100 μM) inhibit Na transport, indicating Na(+)/H(+) exchange (NHE) activity. The underlying NHE isoform for transepithelial Na absorption was characterized by mucosal application of the specific inhibitor HOE642 for NHE1 and S3226 for NHE3 in Ussing chamber studies with isolated epithelia from bovine and sheep forestomach. S3226 (1 μM; NHE3 inhibitor) abolished electroneutral Na transport under control conditions and also the short-chain fatty acid-induced increase of Na transport via NHE. However, HOE642 (30 μM; NHE1 inhibitor) did not change Na transport rates. NHE3 was immunohistochemically localized in membranes of the upper layers toward the lumen. Expression of NHE1 and NHE3 has been previously demonstrated by RT-PCR, and earlier experiments with isolated rumen epithelial cells have shown the activity of both NHE1 and NHE3. Obviously, both isoforms are involved in the regulation of intracellular pH, pH(i). However, transepithelial Na transport is only mediated by apical uptake via NHE3 in connection with extrusion of Na by the basolaterally located Na-K-ATPase. The missing involvement of NHE1 in transepithelial Na transport suggests that the proposed "job sharing" in epithelia between these two isoforms probably also applies to forestomach epithelia: NHE3 for transepithelial transport and NHE1 for, among others, pH(i) and volume regulation.