Effect of dehydration on apical Na+-H+ exchange activity and Na+-dependent sugar transport in brush-border membrane vesicles isolated from chick intestine

 The current work examines the effect of 4 days of water deprivation on Na⁺-H⁺ exchange and Na⁺-sugar cotransport systems in brush-border membrane vesicles isolated from either the jejunum, ileum or the colon of the chick. Apical Na⁺-H⁺ exchange activity was evaluated by measuring the pH-gradient-dependent Na⁺ uptake. The contribution of the Na⁺-H⁺ exchangers NHE2 and NHE3 to total Na⁺-H⁺ exchange activity was evaluated from their sensitivity to the amiloride-related drug HOE694. Dehydration increased plasma aldosterone levels from 12 to 70 pg/ml and also the activities of both Na⁺-H⁺ exchange and Na⁺-dependent sugar transport in the three intestinal regions tested. Na⁺-independent sugar transport was not modified by 4 days of water deprivation. In the ileum and colon the increase in Na⁺-H⁺ exchange activity was due to an increase in NHE2 activity, whereas in the jejunum it was due to an increase in both NHE2 and NHE3. Since we have previously reported that chronic Na⁺ depletion increases plasma aldosterone levels and NHE2 activity in ileum and colon, decreased small and large intestine Na⁺-sugar cotransport activity and had no effect on jejunal apical Na⁺-H⁺ exchange activity, it can be concluded that: (1) aldosterone does not regulate intestinal Na⁺-dependent sugar transport, and (2) the regulation of jejunal Na⁺-H⁺ exchange activity differs from that of either the ileum or the colon. Received: 31 October 1997 / Received after revision: 17 December 1997 / Accepted: 8 January 1998     

Potassium transport in epithelial cells isolated from small intestine of the chicken

The transport of potassium has been studied in epithelial cells isolated from chicken small intestine using⁸⁶Rb as a tracer for K⁺. (i) The uptake studies revealed that about 60% of the total K⁺ net flux is inhibited by ouabain and therefore mediated by the Na⁺–K⁺-ATPase. About 20% of the ouabain-insensitive K⁺ net influx was inhibited by furosemide, bumetanide and by either Na⁺ or Cl^– removal from the incubation solution, suggesting that a Na⁺/Cl^–/K⁺ cotransport system might be present in chicken enterocytes. (ii) The efflux of K⁺ was measured from cells preloaded with⁸⁶Rb. K⁺ efflux was inhibited by Ba²⁺, quinine and verapamil; it was stimulated by A23187, and it was unaffected by 3,4-diaminopyridine. Apamin, that has no effect on basal rates of K⁺ efflux, abolished the effect of A23187. These findings suggest that K⁺ efflux appears to occur through Ca²⁺-activated K⁺ channels.     

Ionic basis of membrane potentials of epithelial cells in rat small intestine

1. Potentials across the mucosal and serosal membranes of the epithelial cells of rat jejunum together with transmural potentials were recorded using everted sac preparations.2. Ionic changes in either mucosal or serosal fluids affect mucosal or serosal membrane potentials respectively with comparable changes in the transmural potential. The contralateral membrane potential is relatively unaffected.3. Replacement of mucosal sodium chloride by potassium chloride or lithium chloride had little effect on potentials, but its replacement by mannitol or Tris chloride increased the negativity of the mucosal potential, giving linear relationships against log(10)[Na](m) with slopes of 41.4 and 30.7 mV respectively for tenfold change in [Na](m).4. At constant [Na](m), potassium or lithium increased the mucosal potential by 25.7 and 19.8 mV respectively for tenfold concentration changes.5. Qualitatively similar changes occurred in the serosal potential when the ionic composition of the serosal fluid was varied.6. Mucosal potential changes in response to modifications of the ionic composition of the mucosal fluid were the same in the presence and absence of galactose.7. Sodium and potassium diffusion potentials largely determine both the mucosal and serosal membrane potentials. For the mucosal membrane, P(K):P(Na) is 1.26:1, and is probably higher for the serosal membrane. Chloride makes no significant contribution to membrane potentials.8. Potentials generated by the electrogenic sodium pump are superimposed on diffusion potentials across the serosal membrane.     

Expression of GFP-tagged low affinity Na+-dependent glucose transporter in Xenopus oocytes and CHO cells

A low-affinity and high-capacity Na(+)-dependent glucose transporter (SGLT2) was inserted into the expression vector tagging of green fluorescent protein (EGFP). The protein expression and glucose transport activity were examined in Xenopus oocytes and Chinese hamster ovary (CHO) cells. In Western blotting analysis, EGFP-tagged SGLT2 protein expressed in both Xenopus oocytes and CHO cells. We also observed the EGFP fluorescence in both cells with a confocal laser microscope. To determine the function of EGFP-tagged SGLT2, we measured the uptake of [(14)C]-alpha-methyl glucopyranoside (AMG), a specific substrate for SGLT. The AMG uptake was time-dependently increased and inhibited by phloridzin in the EGFP-tagged SGLT2-expressing cells. The K(m) value of 1.7 mM for AMG and the IC(50) of 2 microM for phloridzin consist with the renal low affinity Na(+)-dependent glucose transporter. These results indicate that EGFP-tagged SGLT2 protein functionally expressed both in Xenopus oocytes and CHO cells, and these models are useful for studying the regulatory mechanisms of glucose reabsorption.     

Membrane potentials of epithelial cells in rat small intestine

1. Stripped sacs of rat jejunum in which the outer muscle layers had been removed were found to maintain substantial transport and electrical activities.2. Mucosal and serosal membrane potentials of epithelial cells of normal and stripped everted sacs of rat jejunum were recorded in vitro together with the transmural potential difference.3. The cell interior was negative relative to both serosal and mucosal fluids, the transmural potential being the sum of the two membrane potentials.4. Changes in the transmural potentials in the presence of actively transferred hexoses and amino acids were entirely due to variations in the serosal potential, the mucosal potential being unchanged.5. Serosal and transmural potential increases on the addition of galactose were consistent with Michaelis-Menten kinetics, giving apparent K(m) values of 14.9 and 14.1 mM respectively.6. Phlorrhizin, ouabain, 2,4-dinitrophenol and sodium fluoroacetate inhibited serosal potential changes in the presence of galactose.7. Osmotic potentials resulting from transmural osmotic gradients originated from the serosal layers of the tissue.8. The results are consistent with the concept of a serosally located, electrogenic sodium pump which is stimulated by actively transferred hexoses and amino acids. The sodium-dependent entry mechanism at the mucosal membrane is non-electrogenic.     

Electrophysiological studies in principal cells of rat cortical collecting tubules ADH increases the apical membrane Na+-conductance

The mechanism of ion transport across principal cells of rat cortical collecting tubules (CCT) and its regulation by vasopressin (ADH) has been studied in the isolated perfused tubule. To amplify the response to ADH rats were treated with 5 mg I. M. desoxycorticosterone 4–9 days prior to the experiments. Addition of 2·10^–10 mol·1^–1 ADH increased the transepithelial voltage from –5.1 ±0.7 mV to –16.1±1.4 mV (n=37) and decreased the transepithelial resistance from 51±4 cm² to 39±2 cm² (n=33). Optical and functional differentiation of impalements of principal and intercalated cells was made and only data of principal cells are presented. ADH depolarized the apical membrane from 79±1 mV to 66±2 mV (n=26) and decreased the fractional resistance of the apical membrane from 0.76±0.04 to 0.70±0.04 (n=13). These ADH effects were prevented by 10^–5 or 10^–4 mol·1^–1 luminal amiloride which hyperpolarized the apical membrane when added in the presence or absence of ADH. Apical and basolateral membranes were dominated by large K⁺ conductances and addition of 3 mmol·1^–1 barium to bath or lumen perfusates increased transepithelial resistance almost two-fold, whereas luminal amiloride increased the transepithelial resistance only by 26–35%. Ouabain (0.5 mmol·1^–1, bath) depolarized the basolateral membrane and decreased its K⁺ conductance. These effects were prevented by the simultaneous presence of apical amiloride suggesting that the only route of Na⁺ entry into the principal cells occurred via the amiloride sensitive Na⁺ conductance. We conclude that ADH stimulates Na⁺ reabsorption and K⁺ secretion in the rat CCT primarily by increasing the Na⁺ conductance in the apical cell membrane.Parts of this study have been presented at the 19th ASN meeting in Washington, DC, USA 1986     

Hexose transport across the apical and basolateral membrane of enterocytes from different regions of the chicken intestine

The properties of hexose transport across the apical and basolateral membranes of chicken enterocytes have been studied in the small and large intestine. Results show that (a) isolated epithelial cells from all segments except the coprodeum can accumulate 3-O-methylglucose (Glc3Me) against a concentration gradient, by a Na⁺-dependent and phloridzin-sensitive mechanism, (b) The cell cumulative capacity for Glc3Me (control/phloridzin-incubated cells) is lower in the small intestine than in the large intestine (rectum = proximal caecum = ileum > jejunum > duodenum). (c) Theophylline enhances the cell Glc3Me cumulative capacity 2.9-fold in the duodenum and 2.4-fold in the jejunum but has no effect in the other segments studied. (d) Analysis of sugar uptake indicates that net hexose influx rates decrease from proximal to distal regions: jejunum > duodenum > ileum = proximal caecum = rectum for the apical transport system (-methyl glucoside as substrate and phloridzin as inhibitor) and duodenum > jejunum > ileum = proximal caecum = rectum for the basolateral system (2-deoxyglucose; theophylline). (e) The duodenum and the jejunum show high apical and basolateral hexose transport rates, which confer a significant capacity for sugar absorption on the proximal intestine. More distal regions, including the ileum, the proximal caecum and the rectum, have transport systems analogous to those of the proximal intestine that keep a considerable potential capability to recover hexoses from the lumen.     

Role of epithelial basement membrane of the small intestine in providing immunologic homeostasis]

A morphometric method was used experimentally to prove the immune barrier function of the epithelial basal membrane of the small intestine mucous membrane. The epithelial basal membrane located on the border of the epithelium and connective tissue provides biochemical and immunological protection of the layer proper against penetrotion into it of antigens from the epithelium, being a barrier of immunological homeostasis. The damage of the epithelial membrane is accompanied by coming of the intestinal lumen antigens into the layer proper causing there a disturbance of immunological homeostasis. In response to this lymphoid cells migrate from the stroma to the epithelium towards the antigens, and lymphocytic reaction in the layer proper increases.     

Characterization of a Na+-dependent betaine transporter with Cl- channel properties in squid motor neurons

Most marine invertebrates, including squids, use transporters to accumulate organic osmolytes such as betaine, to prevent water loss when exposed to elevated salinity. Although a limited number of flux studies have shown the Na+ dependence of betaine transport, nothing is known about the electrogenic properties of osmolyte transporters. We used whole cell and perforated-patch voltage-clamp techniques to characterize the electrical properties of the betaine transporter in giant fiber lobe motor neurons of the squid Lolliguncula brevis. Betaine activated a large, Cl--selective current that was reversibly blocked by 100 microM niflumic acid (97 +/- 2% block after 40 s, SD; n = 7) and partially inhibited by 500 microM SITS (29 +/- 11%; n = 5). The Cl- current was Na+ dependent and was virtually eliminated by isotonic replacement of Na+ with Li+, NMDG+, or Tris+. Concentration-response data revealed an EC50 in a physiologically relevant range for these animals of 5.1 +/- 0.9 mM (n = 11). In vertebrates, the betaine transporter is structurally related to the GABA transporter, and although GABA did not directly activate the betaine-induced current, it reversibly reduced betaine responses by 34 +/- 14% (n = 8). Short-term changes in osmolality alone did not activate the Cl- current, but when combined with betaine, Cl- currents increased in hypertonic solutions and decreased in hypotonic solutions. Activation of the betaine transporter and Cl- current in hypertonic conditions may affect both volume regulation and excitability in L. brevis motor neurons. This study is the first report of a novel betaine transporter in neurons that can act as a Cl- channel.     

Fast Na+ current in circular smooth muscle cells of the large intestine

Whole-cell voltage clamp was carried out on freshly dispersed single smooth muscle cells from adult rat and human colons to investigate the regulation of the Ca²⁺ channels. In this study, we unexpectedly discovered the existence of a fast Na⁺ channel current. With normal physiological salt solution (PSS) plus 4-amino-pyridine (3 mM) in the bath and high-Cs⁺ solution in the pipette to inhibit outward K⁺ currents, an inward current possessing fast and slow components was observed when the cell membrane was depolarized to a value more positive than –20 mV from a holding potential of –100 mV. When Ca²⁺ ions were removed from the PSS, or when nifedipine (10 M) and Ni²⁺ (30 M) were simultaneously applied, the slow component disappeared and the fast component remained. The fast current component became almost completely inactivated within 10 ms. This fast component was dependent on extracellular Na⁺ concentration and was inhibited by tetrodotoxin (TTX) dose dependently (IC₅₀ of 130 nM in rat and 14 nM in human). These results suggest that the slow component of inward current was a Ca²⁺ channel current, whereas the fast component was a TTX-sensitive fast Na⁺ channel current. The threshold voltage, the voltage for peak current, and the reversal potential for the fast Na⁺ current were, respectively, about –50, –20, and + 50 mV in rats, and –40, 0, and + 60 mV in humans. The incidence of cells possessing fast Na⁺ currents depended on the region of the colon. In rat proximal colon, the incidence was 64% (14 out of 22 cells tested); in distal colon, it was 10% (2 out of 21 cells tested). In humans, the incidence in the ascending colon was 73% (16 out of 22 cells tested), and in the descending colon was 22% (7 out of 32 cells tested). The densities of fast Na⁺ and Ca²⁺ currents were 3.2 and 4.5 pA/pF in rats and 1.0 and 1.4 pA/pF in humans, respectively. The ratio of both current densities (Na⁺ vs Ca²⁺) was 0.71, in both rats and humans. We conclude that the major ion channels associated with the generation of inward currents in the circular smooth muscle cells of rat and human colon are voltage-dependent Ca²⁺ channels and TTX-sensitive Na⁺ channels. The fast Na⁺ current may facilitate propagation of excitation.     

Detergent-resistant membrane microdomains and apical sorting of GPI-anchored proteins in polarized epithelial cells

Detergent-insoluble microdomains or rafts play a crucial role in many cellular functions: membrane traffic, cell signalling and human diseases. In this work we investigate the role of rafts in the sorting of GPI-anchored proteins in polarized epithelial cells. In contrast to MDCK cells, the majority of endogenous GPI-anchored proteins are sorted to the basolateral surface of Fischer rat thyroid cells (Zurzolo et al., J. Cell Biol. 121, 1031-1039, 1993). We analyzed a set of transfected GPI proteins in order to understand the role of the GPI anchor and of association with rafts for apical sorting. We found that the GPI moiety is necessary but not sufficient for apical sorting of GPI proteins and that the ectodomain has a major role. We propose a new model in which the stabilization of proteins into rafts, probably mediated by interactions between protein ectodomains and a putative receptor, plays a crucial role in apical sorting.     

Structure of the musculature of the chicken small intestine

Summary The small intestine of the chicken was studied by light and electron microscopy. The musculature, measuring about 180 m in thickness in the distended intestine, consists of four layers (outer longitudinal, outer circular, inner circular and inner longitudinal) which are directly apposed to one another. There is no layer of connective tissue equivalent to the submucosa of mammalian intestine, and the intestinal glands lie close to the inner longitudinal muscle. Mucosal folds are not formed during isotonic contraction of the intestine. The muscle cells of the chicken small intestine are characterized by large, numerous and sharply outlined dense bodies, by the presence of an extremely thin basal lamina, by prominent dense bands at the cell surface but relatively few intermediate junctions. There are many areas of direct apposition between cell membranes of adjacent cells and little collagen between the muscle cells. The four muscle layers have each distinctive structural features. Gap junctions between muscle cells occur only in the outer circular layer. The outer circular and outer longitudinal layers are closely apposed and numerous junctions of the adherens type link cells of the two layers. Intramuscular blood capillaries are rare and are found virtually only in the outer circular layer; their endothelial cells are joined by tight junctions. In the outer circular layer (but not in the other layers) there are two further cell types, fibroblasts and interstitial cells, which can be clearly distinguished from one another. The latter cells are intimately related to nerve bundles and are connected by gap junctions to some muscle cells.     

Na+ and pH dependence of proline and beta-alanine absorption in rat small intestine

Aims: Early characterization of intestinal absorption of imino acids in mammals has demonstrated the existence of a Na⁺‐dependent, Cl^?‐independent transport system in rat small intestine, which is the only carrier for β‐alanine. Based on the substrate selectivity, it was proposed that the Proton Amino Acid Transporter 1 (PAT1) could be the same as this imino acid carrier. The present study characterizes the pH and Na⁺ dependence of proline and β‐alanine uptake in rat small intestine. Methods: Intestinal uptake of radiolabelled l ‐proline or β‐alanine was measured in brush border membrane vesicles and everted intestinal rings, in the presence and absence of Na⁺ and at different pH values. Results: The existence of an inwardly directed H⁺ gradient in the absence of Na⁺ enhanced the initial entry of proline and β‐alanine in brush border membrane vesicles, that reached a transient overshoot with maximal value around 30 s. In the absence of pH gradient, no overshoot was shown. In entire tissue, there was an increase of proline and β‐alanine uptake at acidic pH that was higher in the presence of Na⁺ than in its absence. This ion dependence and pH effect of the amino acids uptake also increased with the incubation period. Substrate inhibition studies confirmed that intestinal proline absorption in rat occurs mainly by system B and PAT1‐like transporter. Conclusions: There is a Na⁺‐independent, H⁺‐dependent transporter of amino acids at the apical membrane of the rat enterocytes.     

Effects of membrane potential on Na+ -dependent Mg2+ extrusion from rat ventricular myocytes

To study Mg2+ transport across the cell membrane, the cytoplasmic concentration of Mg2+ ([Mg2+](i)) in rat ventricular myocytes was measured with the fluorescent indicator furaptra (mag-fura-2) under Ca2+ -free conditions (0.1 mM EGTA) at 25 degrees C. The fluorescence ratio signal of furaptra was converted to [Mg2+](i) using calibration parameters previously estimated in myocytes (Watanabe and Konishi, Pflügers Arch 442: 35-40, 2001). After [Mg2+](i) was raised by loading the cells with Mg2+ in a solution containing 93 mM Mg(2+), the cells were voltage-clamped at a holding potential of -80 mV using the perforated patch-clamp technique with amphotericin B. At the holding potential of -80 mV, the reduction of extracellular Mg2+ to 1.0 mM caused a rapid decrease in [Mg2+](i) only in the presence of extracellular Na(+). The rate of the net Mg2+ efflux appeared to be dependent on the initial level of [Mg2+](i); the decrease in [Mg2+](i) was significantly faster in the myocytes markedly loaded with Mg2+. The rate of decrease in [Mg2+](i) was influenced little by membrane depolarization from -80 to -40 mV, but the [Mg2+](i) decrease accelerated significantly at 0 mV by, on average, approximately 40%. Hyperpolarization from -80 to -120 mV slightly but significantly slowed the decrease in [Mg2+](i) by approximately 20%. The results clearly demonstrate an extracellular Na(+)- and intracellular Mg2+ -dependent Mg2+ efflux activity, which is consistent with the Na(+)-Mg2+ exchange, in rat ventricular myocytes. We found that the apparent rate of Mg2+ transport depends slightly on the membrane potential: facilitation by depolarization and inhibition by hyperpolarization with no sign of reversal between -120 and 0 mV.     

Na+ selective channels in the apical membrane of rabbit late proximal tubules (pars recta)

Using the patch-clamp technique, Na⁺ selective channels were observed in the luminal membrane of rabbit straight proximal tubule segments. In the cell-attached configuration (NaCl-Ringers in pipette and bath) influx of Na⁺ ions from the pipette into the cell through fluctuating channels was observed was observed. The current-voltage curve of these Na⁺ channels yielded a zero-current potential of 84.3±30.9 mV (n=10), reflecting the electrochemical driving force for Na⁺ influx under resting conditions. The single channel conductance was 12.0±2.1 pS (n=13). In inside-out oriented cell-excised patches the single channel conductance was not significantly different with NaCl-Ringers on both sides. At clamp potentials ranging from +50 mV to –50 mV the single channel current was ohmic and channel kinetics were independent of the voltage. With KCl-Ringers on the bath side (corresponding to cell interior), the zero current potential was 62±19 mV (n=4), indicating a high selectivity of Na⁺ over K⁺ ions. Addition of 10^–5 mol/l amiloride to the bathing solution decreased the mean channel open time slightly. This effect was more pronounced with 10^–4 mol/l amiloride, whereas the single channel conductance was unaffected by the diuretic. 10^–3 mol/l amiloride caused a complete block of the channel. It is concluded that amiloride sensitive Na⁺ channels, with similar properties to those observed in tight epithelia, contribute to Na⁺ reabsorbtion in the straight portion of proximal tubules.