Sodium ion transport in isolated intestinal epithelial cells. The effect of actively transported sugars on sodium ion efflux

A technique to measure Na⁺ efflux from isolated intestinal epithelial cells has permitted us to examine the mechanisms responsible for Na⁺ transport in absorptive cells without contamination by other cell types. We examined the effect of actively transported sugars on Na⁺ efflux from isolated rat jejunal epithelial cells to evaluate the mechanism by which actively transported non-electrolytes stimulate Na⁺ absorption. Glucose, galactose and 3-O-methylglucose, sugars known to be actively transported by the small intestine, stimulate total Na⁺ efflux from isolated epithelial cells. This stimulation results from an increase of active Na⁺ transport, since it is inhibited by ouabain. Glucose stimulation is significantly greater than that produced by galactose or 3-O-methylglucose, 2-Deoxyglucose, a sugar that is not actively transported, has no effect on total Na⁺ efflux from isolated cells. Phloridzin, which has no effect on Na⁺ efflux in a sugar-free medium, completely abolishes the effect of galactose. These findings (a) support the hypothesis that the increase in intestinal absorption of Na⁺ in the presence of actively transported non-electrolytes occurs by a transcellular route; and (b) are consistent with the ion-gradient model. The results are not compatible with the direct energy-coupling model.     

Biphasic action of growth hormone on intestinal amino acid absorption in striped bass hybrids

• 1.1. Weekly injections of bovine growth hormone (bGH) increased the maximal transport rate of both Na⁺-dependent and Na⁺ -independent l-leucine transport with little effect on the affinity constants in the intestine of striped bass hybrids.
• 2.2. The Na⁺-dependent and the Na⁺-independent transport of the non-metabolizable analog cycloleucine was also stimulated by bGH.
• 3.3. The Na⁺ -dependent active transport was stimulated 2 days after the hormone treatment, while the stimulation of the Na⁺-independent diffusional transport was not observed until after 2 weeks of treatment.
• 4.4. Studies of intestinal morphometry and l-leucine transport using brush border membrane vesicles suggested that bGH affects intestinal amino acid absorption initially by increasing the number of transporters per cell.
• 5.5. This phase is followed by a general increase of the intestinal mass after long-term treatment with the hormone.

     

(Na---K)-stimulated adenosinetriphosphatase in isolated intestinal villus tip and crypt cells

The migration of intestinal epithelial cells from the crypt area to the villus tip is associated with progressive differentiation of these cells. The distribution of (Na⁺---K⁺) stimulated adenosinetriphosphatase ((Na⁺---K⁺)-ATPase; EC 3.6.1.3) along the intestinal villus may have functional as well as developmental implications. To define this distribution, rat jejunal and ileal segments were incubated in vitro with a citrate solution that dissociates epithelial cells sequentially from villus tip to crypt area. ATPase activity in cell collections from villus tips and crypt areas were compared. The specific activity of (Na⁺---K⁺)-ATPase was higher in the villus tip than in the crypt cells of both jejunum and ileum. Crypt cell (Na⁺---K⁺)-ATPase activity in the jejunum and ileum were similar. Thus, (Na⁺---K⁺)-ATPase activity of villus tip cells in the jejunum was greater than in the ileum. There was no difference in villus tip and crypt cell Mg²⁺-ATPase activity in either jejunum or ileum. The steep gradient for (Na⁺---K⁺)-ATPase along the intestinal villus may signify an improtant difference in Na⁺ transport between the villus tip and crypt area. The higher level of (Na⁺---K⁺)-ATPase activity in the jejunal villi is consistent with the more important role of the jejunum in Na⁺ and substrate-linked Na⁺ transport.     

A novel sodium bicarbonate cotransporter-like gene in an ancient duplicated region: SLC4A9 at 5q31

Background  

Sodium bicarbonate cotransporter (NBC) genes encode proteins that execute coupled Na⁺ and HCO₃ ^- transport across epithelial cell membranes. We report the discovery, characterization, and genomic context of a novel human NBC-like gene, SLC4A9, on chromosome 5q31.     

Differences in epithelial morphology correlate to Na+‐transport: A study of the proximal,mid, and distal regions of the coprodeum from hens on high and low NaCl diet

A study was performed to correlate regional morphology and amiloride inhibitable Na⁺‐transport in the coprodeal epithelium in hens, Gallus domesticus, on low‐NaCl diet and in controls. Proximal (close to colon), mid and distal (close to urodeum) regions were examined using light microscopy, transmission‐ and scanning electron microscopy. Na⁺‐transport was measured electrophysiologically in Ussing‐chambers in the proximal and distal regions. The epithelium, simple and columnar, is composed of absorptive intestinal epithelial cells, goblet cells, brush cells, migrating lymphoid cells, and entero‐endocrine cells. Brush cells, identified in avians for the first time, occur in highest number in the proximal part of the coprodeum in low‐NaCl hens. Na⁺‐transport is high in the low‐NaCl hens, ranging from 347μA/cm² (proximal) to 187μA/cm² (distal). In control hens, which correspond to hens on high‐NaCl diet, it is low in all regions (0–4 μA/cm²). Absorptive intestinal epithelial cells as well as brush cells adapt to variations in transepithelial Na⁺‐transport by regulating height and packing density of their microvilli, number, size, and localization of apical vesicles, and the width of the intercellular space. Regional differences in the epithelial cell composition and ultrastructure are closely correlated to transepithelial Na⁺‐transport but only in low‐NaCl hens, as controls do not show these variations. J. Morphol. 239:75–86, 1999. © 1999 Wiley‐Liss, Inc.     

Effect of neutrophil elastase and its inhibitor EPI-hNE4 on transepithelial sodium transport across normal and cystic fibrosis human nasal epithelial cells

Background

Hyperactivity of the epithelial sodium (Na⁺) channel (ENaC) and increased Na⁺ absorption by airway epithelial cells leading to airway surface liquid dehydration and impaired mucociliary clearance are thought to play an important role in the pathogenesis of cystic fibrosis (CF) pulmonary disease. In airway epithelial cells, ENaC is constitutively activated by endogenous trypsin-like serine proteases such as Channel-Activating Proteases (CAPs). It was recently reported that ENaC activity could also be stimulated by apical treatment with human neutrophil elastase (hNE) in a human airway epithelial cell line, suggesting that hNE inhibition could represent a novel therapeutic approach for CF lung disease. However, whether hNE can also activate Na⁺ reabsorption in primary human nasal epithelial cells (HNEC) from control or CF patients is currently unknown.

Methods

We evaluated by short-circuit current (I_sc) measurements the effects of hNE and EPI-hNE4, a specific hNE inhibitor, on ENaC activity in primary cultures of HNEC obtained from control (9) and CF (4) patients.

Results

Neither hNE nor EPI-hNE4 treatments did modify I_sc in control and CF HNEC. Incubation with aprotinin, a Kunitz-type serine protease inhibitor that blocks the activity of endogenous CAPs, decreased I_sc by 27.6% and 54% in control and CF HNEC, respectively. In control and CF HNEC pretreated with aprotinin, hNE did significantly stimulate I_sc, an effect which was blocked by EPI-hNE4.

Conclusions

These results indicate that hNE does activate ENaC and transepithelial Na⁺ transport in both normal and CF HNEC, on condition that the activity of endogenous CAPs is first inhibited. The potent inhibitory effect of EPI-hNE4 on hNE-mediated ENaC activation observed in our experiments highlights that the use of EPI-hNE4 could be of interest to reduce ENaC hyperactivity in CF airways.     

Angiotensin II directly increases rabbit renal brush-border membrane sodium transport: Presence of local signal transduction system

Summary In the present study, we have examined the direct actions of angiotensin II (AII) in rabbit renal brush border membrane (BBM) where binding sites for AII exist. Addition of AII (10^–11–10^–7 m) was found to stimulate²²Na^– uptake by the isolated BBM vesicles directly. AII did not affect the Na⁺-dependent BBM glucose uptake, and the effect of AII on BBM²²Na⁺ uptake was inhibited by amiloride, suggesting the involvement of Na⁺/H⁺ exchange mechanism. BBM proton permeability as assessed by acridine orange quenching was not affected by AII, indicating the direct effect of AII on Na⁺/H⁺ antiport system.In search of the signal transduction mechanism, it was found that AII activated BBM phospholipase A₂ (PLA) and that BBM contains a 42-kDa guanine nucleotide-binding regulatory protein (G-protein) that underwent pertussis toxin (PTX)-catalyzed ADP-ribosylation. Addition of GTP potentiated, while GDP-ßS or PTX abolished, the effects of AII on BBM PLA and²²Na⁺ uptake, suggesting the involvement of G-protein in AII's actions. On the other hand, inhibition of PLA by mepacrine prevented AII's effect on BBM²²Na⁺ uptake, and activation of PLA by mellitin or addition of arachidonic acid similarly enhanced BBM²²Na⁺ uptake, suggesting the role of PLA activation in mediating AII's effect on BBM²²Na⁺ uptake.In summary, results of the present study show a direct stimulatory effect of AII on BBM Na⁺/H⁺ antiport system, and suggest the presence of a local signal transduction system involving G-protein mediated PLA activation.     

Ontogeny of d-Mannose Transport and Metabolism in Rat Small Intestine

Oral mannose therapy is used to treat congenital disorders of glycosylation caused by a deficiency in phosphomannose isomerase. The segmental distribution and ontogenic regulation of d-mannose transport, phosphomannose isomerase, and phosphomannose mutase is investigated in the small intestine of fetuses, newborn, suckling, 1-month-old, and adult rats. The small intestine transports d-mannose by both Na⁺-dependent and Na⁺-independent transport mechanisms. The activities of both systems normalized to intestinal weight peak at birth and thereafter they decreased. In all the ages tested, the activity of the Na⁺-independent mechanism was higher than that of the Na⁺/mannose transport system. At birth, the Na⁺-independent d-mannose transport in the ileum was significantly higher than that in jejunum. Phosphomannose isomerase activity and mRNA levels increased at 1 month, and the values in the ileum were lower than in jejunum. Phosphomannose mutase activity in jejunum increased during the early stages of life, and it decreased at 1 month old, as does the amount of mannose incorporated into glycoproteins, whereas in the ileum, they were not affected by age. The phosphomannose isomerase/phosphomannose mutase activity ratio decreased at birth and during the suckling period, and increased at 1 month old. In conclusion, intestinal d-mannose transport activity and metabolism were affected by ontogeny and intestinal segment.     

In vitro studies on intestinal calcium and phosphate transport in horses

Transepithelial transport mechanisms play a key role in regulating the absorption and secretion of calcium (Ca^2 +) and inorganic phosphate (P_i) in the gastrointestinal tract. Although intestinal disorders with imbalances in macromineral homeostasis are frequently observed in horses, available data on intestinal Ca^2 + and P_i transport are limited. The aim of the present study was to characterize the intestinal Ca^2 + and P_i transport functionally by using the in vitro radioisotope tracer technique with Ussing chambers and to identify components involved in Ca^2 + transport at both mRNA and protein level. Among the different intestinal segments, the duodenum showed significant and highest active Ca^2 + absorption. The findings from RT-PCR and Western blot analysis suggest that the epithelial Ca^2 + channel TRPV6, the cytosolic calcium binding protein calbindin-D_9K and the plasma membrane calcium ATPase PMCA may be involved in active transcellular Ca^2 + transport. Regarding the P_i transport, the results indicate significant active P_i secretion in the jejunum, but the contributing mechanisms remain unclear. A significant inhibiting effect of ouabain as an antagonist of the basolateral Na⁺/K⁺-ATPase on the serosal-to-mucosal P_i transport suggests a pivotal role of Na⁺ in jejunal P_i transport in the horse.     

Effect of K+ and K+ gradients on accumulation of sugars by isolated intestinal epithelial cells

Summary The role played by transmembrane K⁺ gradients in providing an energy input for Na⁺-dependent monosaccharide transport systems was evaluated with the use of isolated intestinal epithelial cells. Experimentally imposing a K⁺ gradient in a sense reversed from normal did not lead to extrusion of sugar from cells which had been pre-equilibrated with¹⁴C-3-OMG, even in situations where a reversed Na⁺ gradient was also imposed. Furthermore, cells preloaded with K⁺ have no better ability to accumulate 3-OMG than do cells depleted of K⁺, when the two populations are compared under identical incubation conditions. Fluxes of K⁺ associated with the sugar carrier could not be detected in terms of suspected sensitivity to agents which immobilize the sugar carrier. In addition, fluxes of sugar in response to imposed K⁺ gradients were not demonstrable in cells de-energized by preincubation with DNP, no matter in which direction the K⁺ gradient was imposed. Finally, the severe inhibitory effects of K⁺ on Na⁺-dependent sugar transport by the cells disappears in de-energized cells, despite the fact that Na⁺-dependent carrier-mediated sugar entry still occurs. All of these facts are difficult to reconcile with a significant role for cellular K⁺ gradients in supporting active sugar transport as envisioned by the ion gradient hypothesis. We have suggested instead a fundamental Na⁺-dependent energy transductive event which depends on ATP, and which can generate a membrane-bound energized intermediate which serves to support a variety of active transport events. An analogy is drawn between this concept for animal cell plasma membranes and the better documented phosphotransferase system for sugar transport described for certain microorganisms.     

1α,25-dihydroxy-vitamin D-3 regulates ATP-dependent calcium transport in basolateral plasma membranes of rat enterocytes

Basolateral plasma membrane vesicles of rat small intestinal epithelium accumulate calcium through an ATP-dependent pumping system. The activity of this system is highest in duodenum and decreases towards the ileum. This distribution along the intestinal tract is similar as the active calcium absorption capacity of intact intestinal epithelial segments. ATP-dependent calcium uptake in basolateral membrane vesicles from duodenum and ileum increased significantly after repletion of young vitamin D-3-deficient rats with 1α,25-dihydroxy-vitamin D-3. Ca²⁺-ATPase activity in duodenal basolateral membranes increased to the same extend as ATP-dependent calcium transport, but (Na⁺ + K⁺)-ATPase activity remained unaltered.     

Pyrophosphate-Fueled Na+ and H+ Transport in Prokaryotes

SUMMARY

In its early history, life appeared to depend on pyrophosphate rather than ATP as the source of energy. Ancient membrane pyrophosphatases that couple pyrophosphate hydrolysis to active H⁺ transport across biological membranes (H⁺-pyrophosphatases) have long been known in prokaryotes, plants, and protists. Recent studies have identified two evolutionarily related and widespread prokaryotic relics that can pump Na⁺ (Na⁺-pyrophosphatase) or both Na⁺ and H⁺ (Na⁺,H⁺-pyrophosphatase). Both these transporters require Na⁺ for pyrophosphate hydrolysis and are further activated by K⁺. The determination of the three-dimensional structures of H⁺- and Na⁺-pyrophosphatases has been another recent breakthrough in the studies of these cation pumps. Structural and functional studies have highlighted the major determinants of the cation specificities of membrane pyrophosphatases and their potential use in constructing transgenic stress-resistant organisms.     

Inhibition of Na+-coupled solute transport by calcium in brush border membrane vesicles

Intracellular Ca⁺⁺ is known to influence Na⁺ flux in luminal membranes. Abnormally elevated Ca⁺⁺ levels in some cells is believed to be the primary pathophysiologic defect in cystic fibrosis (CF). This in turn is thought to alter Na⁺ transport which accounts for certain clinical manifestations of this disease. Two Na⁺-dependent intestinal transport mechanisms have been reported to be suppressed or missing in CF. To examine whether alterations in cell Ca⁺⁺ may account for these findings, studies were performed to examine the influence of Ca⁺⁺ on Na⁺-solute co-transport across intestinal luminal membranes. Purified brush border membrane vesicles prepared from rat small bowel were preincubated in either Ca⁺⁺-free buffer or buffer containing 2.5 mM CaCl₂. Ca⁺⁺ loaded vesicles showed marked inhibition of Na⁺ co-transport of taurocholic acid, taurochenodeoxycholic acid, glucose and valine when compared to controls. The uptake of Na⁺ was also significantly reduced by intravesicular Ca⁺⁺. These data demonstrate that intravesicular Ca⁺⁺ inhibits Na⁺-coupled solute transport as well as Na⁺ influx across intestinal brush border membranes. These data suggest that intracellular Ca⁺⁺ may suppress Na⁺-dependent solute absorption in the intestine. Results presented here further support the theory that elevated intracellular Ca⁺⁺ may account for intestinal malabsorption and other altered transport phenomena reported in CF.     

2-Deoxyglucose transport by intestinal epithelial cells isolated from the chick

Summary Characteristics of 2-deoxyglucose uptake (2DG) by intestinal epithelial cells isolated from chickens were evaluated as a means of discriminating between the concentrative transport system for monosaccharides, associated with the mucosal brush border, and other possible routes of monosaccharide entry. 2DG was chosen as it is not a substrate for the mucosal transport system. The deoxysugar enters via a saturable pathway which is not Na⁺-dependent, is not inhibited by K⁺, does not accumulate solute against a concentration gradient; exhibits a high sensitivity to inhibition by phloretin; is relatively insensitive to phlorizin inhibition; and has low affinity [but high capacity relative to Na⁺-dependent mucosal transport of 3-O-methylglucose (3-OMG) and other monosaccharides]. These characteristics confirm those established in an earlier report for Na⁺-independent uptake of 3-OMG. Complications encountered in the use of 2DG as a test sugar include significant rates of metabolic conversion to an anionic form which presumably is a phosphorylated species. Methods for distinguishing between transport and subsequent metabolism are described. Inhibition of 2DG entry by several other sugars is described and inhibitory constants (K's) given for each.     

Cytochemical localization of adenylate cyclase in the sodium-transporting epithelium isolated from frog skin

Summary A modified cytochemical technique with 5-adenylylimidodiphosphate as substrate, was used to examine the distribution of adenylate cyclase in cells comprising the transepithelial Na⁺ transport pathway in isolated frog skin epithelium. Particular attention was paid to the effects of fixation on the activity and localization of adenylate cyclase. Fixation in glutaraldehyde alone or in combination with paraformaldehyde reduced the amount of reaction product, while better results were obtained using unfixed tissues. Optimum results were obtained following stimulation of adenylate cyclase with forskolin and in the presence of specific metabolic inhibitors. Adenylate cyclase was localized in the basolateral membranes of the principal cells which constitute a functional syncytium for Na⁺ transport and was absent from the apical membranes of the outermost granulosum cells. This distribution is consistent with the transepithelial Na⁺ transport model and defines the functional morphology of the cells involved in Na⁺ transport across frog skin. The results are compatible with the process of Na⁺ re-absorption across other epithelial cells, verifying that frog skin is a convenient model-tissue to study Na⁺ transport mechanisms. Adenylate cyclase was also found in membranes of the mitochondria-rich cells, a minor and parallel Na⁺ transporting pathway.