Amylase transport across ileal epithelium in vitro

Amylase transport was measured across the rabbit ileum in vitro employing a modified Ussing chamber. Amylase was moved preferentially in the mucosal to serosal direction. Its rate of transfer was 2–3 orders of magnitude greater than that for inulin. Mucosal to serosal transport of exogenous amylase was completely inhibited in the absence of oxygen. There was also a constant release of endogenous amylase from intestinal tissue into both mucosal and serosal compartments in the absence of an exogenous source. An estimate of the rate of amylase absorption indicates that it may be of sufficient magnitude to account for the enteropancreatic circulation of amylase secreted by the pancreas during augmented secretion.     

Active phosphate transport across the urinary bladder of the toad, Bufo marinus.

Net transport of inorganic phosphate occurs in the absence of an electrochemical gradient from the mucosal to the serosal bathing solution in the isolated toad urinary bladder. This transport can be inhibited by metabolic inhibitors. The magnitude of this transport can be altered by changes in phosphate concentration or by the addition of parathyroid hormone.     

Active transport of D-xylose in the isolated small intestine of the bullfrog

Fluxes of D-xylose-1-C¹⁴ (xylose) across the wall of the isolated intestine of the bullfrog were studied. When sodium was the principal cation in the mucosal bathing fluid, the transport rate of xylose from the mucosa to the serosa was about 5 times greater than the transport rate from the serosa to the mucosa, indicating an active intestinal transport for this sugar. With potassium as the principal cation on the mucosal side, the transport rate of xylose from the mucosal to the serosal compartment is reduced about 5 to 6 times without appreciable change in the serosal to mucosal transport. The asymmetry was also considerably reduced when ouabain was added to the mucosal and serosal compartments. The data confirm the in vitro and in vivo observations indicating active transport of xylose and are also in accord with the earlier findings that active transport of sugars in the intestine is dependent upon the presence of sodium ions in the mucosal compartment and is inhibited by cardioactive steroids. Since the chemical constitution of xylose does not meet the requirements which were hitherto considered necessary for active transport of sugars in the intestine, this structural requirement has to be revised.     

Active phosphate transport across the urinary bladder of the toad,Bufo marinus

Summary Net transport of inorganic phosphate occurs in the absence of an electrochemical gradient from the mucosal to the serosal bathing solution in the isolated toad urinary bladder. This transport can be inhibited by metabolic inhibitors. The magnitude of this transport can be altered by changes in phosphate concentration or by the addition of parathyroid hormone.This work was presented in part at the Fall Meeting of the American Physiological Society Proceedings, October, 1975; Abstract inThe Physiologist, 1975,18:384.     

Alanine Efflux across the Serosal Border of Turtle Intestine

The exit of alanine across the serosal border of the epithelial cells of turtle intestine was measured by direct and indirect techniques. A decrease or an increase in cell Na did not affect the amino acid flux from cell to serosal solution. Cells loaded with Na and alanine did not exhibit any extrusion of alanine when their serosal membranes were exposed to an Na-free medium containing alanine. However, substantial amino acid extrusion was observed across the mucosal cell border under similar conditions. Although alanine flux across the serosal membrane appeared to be Na-independent, it showed a tendency toward saturation as cellular alanine concentration was elevated. The results are consistent with the postulate that the serosal and mucosal membranes of intestinal cells are asymmetrical with respect to amino acid transport mechanisms. The serosal membrane appears to have an Na-independent carrier-mediated mechanism responsible for alanine transport while transport across the mucosal border involves an Na-dependent process.     

The effects of bicarbonate and hydroxyl ions on chloride transport by toad bladders

The association between Cl-, HCO3- and H+ transported by toad bladders was investigated. Net mucosal to serosal Cl- transport by Colombian toad bladders was stimulated by incubation in HCO3- free solutions. In addition, when Colombian or Dominican toad bladders were exposed to low HCO3- concentrations on the mucosal side and 25 mM HCO3- on the serosal side, net mucosal leads to serosal Cl- transport was induced. Neither acetazolamide nor cyanide significantly inhibited Cl- transport under these conditions. The presence of a pH gradient, more acid on the mucosal side, also induced net mucosal leads to serosal Cl- transport. The results suggest that Cl- transport by toad bladders may occur by exchange with HCO3- or OH-; this process may not require carbonic anhydrase or oxidative metabolism. The Cl- transport by toad bladders is qualitatively different from the electrogenic Cl- transport of the thick limb of Henle's loop, but may be similar to a process which occurs in other portions of the nephron.     

The Role of Potassium in Active Transport of Sodium by the Toad Bladder

Studies were carried out on the isolated urinary bladder of the toad, Bufo marinus, in order to explain the dependence of active sodium transport on the presence of potassium, in the serosal medium. Attempts to obtain evidence for coupled sodium-potassium transport by the serosal pump were unsuccessful; no relation between sodium transport and uptake of K⁴² from the serosal medium was demonstrable. Rather, the predominant effect of serosal potassium appeared to be operative at the mucosal permeability barrier, influencing the permeability of this surface to sodium. The mucosal effects of serosal potassium were correlated with effects on cellular cation content. When sodium Ringer's solution was used as serosal medium, removal of potassium resulted in significant decrease in tissue potassium content, commensurate increase in tissue sodium content, and marked depression of mucosal permeability and sodium transport. When choline replaced sodium in the serosal medium, removal of potassium resulted in only slight alterations of tissue electrolyte content, and effects on mucosal permeability and sodium transport were minimal.     

Intestinal glucose and galactose transport in the cultured gilthead bream (Sparus aurata)

1. Electrical parameters and transepithelial glucose and galactose transport were determined in vitro across anterior and posterior intestine of the culture fish Sparus aurata. 2. Electrical potential difference (PD) and short-circuit current (Isc) were serosa-positive in anterior intestine, while they were serosa-negative or near zero in posterior intestine. 3. Tissue conductance (Gt) was higher in posterior than in anterior intestine. In both parts it was decreased when the Na ion was omitted in mucosal and serosal reservoirs. 4. Addition of glucose or galactose to the mucosal side of intestine caused an increase in PD and Isc in posterior intestine but did not significantly change PD and Isc in anterior intestine. 5. Isotopic flux of glucose and galactose measurements in short-circuit conditions showed a net active glucose and galactose absorption in posterior intestine, while in anterior intestine active transport of glucose or galactose was not observed. 6. The net transport of glucose and galactose in posterior intestine was decreased to zero in the absence of Na in mucosal and serosal reservoirs or in the presence of ouabain (1 mM) in serosal solution.     

Micropuncture analysis of the cellular mechanisms of electrolyte secretion by the in vitro rabbit pancreas

Micropuncture techniques have been used to study electrolyte secretion by the spontaneously secreting in vitro rabbit pancreas over a wide range of environmental conditions. Pancreatic secretion does not have a strong requirement for HCO3 and secretion continues at nearly normal rates when exogenous HCO3 is replaced by acetate. Acetate concentration in the juice averages 70 meq/liter, nearly three times the environmental concentration. The similar characteristics exhibited by HCO3 and acetate secretion indicate that they are secreted by a common mechanism involving active H transport. In vitro acid-base alterations demonstrate that the secretion rate is controlled by the environmental HCO3 concentration and to a much lesser extent by the pCO2. Secretion also requires active Na transport across the mucosal membrane. The effects of ouabain and a low Na environment strongly suggest coupling between the transport of Na and H and a cellular mechanism for electrolyte secretion is proposed involving Na-H exchange mechanisms at both the mucosal and serosal membranes.     

Stimulation of Na+ transport across the toad urinary bladder by p-chloromercuribenzene sulfonate.

The sulfhydryl reagent p-chloromercuribenzene sulfonate increased the ISC across substrate-replete toad urinary bladder when applied to the mucosal (apical) surface. This increase was accounted for by an increased mucosal to serosal net flux of Na+. In the absence of substrate, the rise in ISC was accompanied by an irreversible increase in tissue conductance which was not apparent in the replete preparation. These findings suggest that p-chloromercuribenzene sulfonate may be useful in marking mucosal functions associated with the Na+ transport apparatus.     

Calcium-dependence of sugar transport in rat small intestine

The involvement of Ca2+ in the theophylline action on sugar transport was investigated in isolated rat small intestinal mucosa. Theophylline significantly increased cell water free sugar accumulation and reduced mucosal to serosal sugar fluxes both in the presence and absence of calcium, but the effects of theophylline were significantly less in calcium free media. In theophylline untreated tissues, calcium-deprived bathing solutions decreased tissue galactose accumulation and increased mucosal to serosal sugar flux. The calcium-channel blocker verapamil produced similar effects on intestinal galactose transport to those induced by low extracellular calcium activity. RMI 12330A and the calmodulin antagonist trifluoperazine abolished the theophylline-effects on intestinal galactose transport. Both drugs also affected sugar transport in basal conditions. These studies suggest that calcium might modulate sugar permeability across the basolateral boundary of rat enterocytes, and that its effect may be mediated by calmodulin.     

Ouabain-dependent potassium-potassium exchange in the toad bladder

Summary Recent results from this laboratory have indicated the existence of two potassium compartments in the isolated toad bladder. Only one of these, containing less than 10% of total intracellular potassium, appears to be related to the sodium transport system, since potassium influx at the serosal border of this compartment is coupled to the sodium efflux which occurs there. Ouabain, which specifically inhibits serosal sodium exit, has no effect on potassium fluxes and compartment sizes in bladders mounted in normal (2.5mm K) Ringer's solution. However, in the presence of this inhibitor, removal of serosal potassium results in a significant decrease in the rate coefficient for potassium efflux into the serosal medium, while an increase in serosal potassium results in a significant rise in this parameter, which appears to saturate at approximately 5mm K. This sensitivity to serosal potassium is seen neither in the absence of ouabain nor when the sodium pump is inactivated by removal of sodium from the mucosal medium. Furosemide, which also inhibits the sodium transport system, both inhibits potassium transport parameters in normal Ringer's and abolishes the potassium-sensitive potassium efflux seen in the presence of ouabain. Thus, the Na–K pump appears to operate as a K–K exchanger when the sodium system is inhibited by ouabain; this K–K exchange mechanism is inhibited by furosemide. One explanation for these results is that ouabain effects an alteration in the affinities of the transport system for sodium and potassium.     

Intestinal SGLT1-mediated absorption and metabolism of benzyl beta-glucoside contained in Prunus mume: carrier-mediated transport increases intestinal availability

The intestinal absorption of benzyl beta-glucoside (BNZ beta glc) contained in the fruit of Prunus mume SIEB. et ZUCC. (Rosaceae), which is traditionally used as a medicinal food in Japan, was studied in rat intestines. BNZ beta glc was absorbed from the mucosal to serosal sides. Its metabolite, benzyl alcohol (BAL), was also detected on both the mucosal and serosal sides. In the presence of phloridzin (Na(+)/glucose cotransporter (SGLT1) inhibitor) or in the absence of Na+ (driving force), BNZ beta glc absorption was significantly decreased. Transport clearance of BNZ beta glc across the brush border membrane decreased as its concentration increased. These results indicate that BNZ beta glc is transported by SGLT1. Metabolic clearance of BNZ beta glc also decreased as its concentration increased. The amount ratio of BNZ beta glc to BAL on the serosal side increased with the increase of BNZ beta glc concentration. The intestinal availability of BNZ beta glc was lower in the absence of Na+ than in the presence of Na+, indicating that the SGLT1-mediated transport of BNZ beta glc increases intestinal availability by decreasing the intestinal extraction ratio. This neutraceutical study concluded that intestinal carrier-mediated transport across the brush border membrane improves the intestinal availability of nutritionally, pharmacologically or physiologically active compounds that undergo intestinal metabolism (first-pass effect).     

Glucose transport across the intestinal wall of the freshwater snail Biomphalaria glabrata

• 1.1. Isolated midguts of the freshwater snail Biomphalaria glabrata were mounted in an incubation chamber in saline containing 2 mM glucose and perfused with the same solution. External and internal media were continuously gassed with carbogen gas (95% O₂, 5% CO₂). In order to measure the flux rates of glucose [¹⁴C]glucose was applied in the perfusion medium or in the incubation medium. Net fluxes of glucose were calculated as the differences between unidirectional in- and effluxes.
• 2.2. A directed net flux from the mucosal to the serosal side of the intestine was demonstrated (mucosal to serosal = 50 ± 10 nmol cm⁻²hr⁻¹(N = 6) serosal to mucosal 7 ± 1 nmol cm⁻²hr⁻¹ (N = 6), net flux = 43 nmol cm⁻²hr⁻¹).r
• 3.3. The active transport of glucose was reduced by the presence of metabolic inhibitors, cyanide (1 mM) and dinitrophenol (1 mM) on the mucosal as well as on the serosal side. Ouabain (1 mM) inhibited the transport rate only when it was added on the serosal side. Amiloride (1 mM) had no effect on the transport rate whether added on the mucosal or on the serosal side.
• 4.4. Inhibition of glucose transport by oubain, a specific inhibitor of Na⁺/K⁺-ATPase, suggests that glucose transport is secondary active and coupled to Na⁺-transport.

     

Ion Transport in Isolated Rabbit Ileum : II. The interaction between active sodium and active sugar transport

The addition of actively transported sugars to the solution bathing the mucosal surface of an in vitro preparation of distal rabbit ileum results in a rapid increase in the transmural potential difference, the short-circuit current, and the rate of active Na transport from mucosa to serosa. These effects are dependent upon the active transport of the sugar per se and are independent of the metabolic fate of the transported sugar. Furthermore, they are inhibited both by low concentrations of phlorizin in the mucosal solution and by low concentrations of ouabain in the serosal solution. The increase in the short-circuit current, ΔI_sc, requires the presence of Na in the perfusion medium and its magnitude is a linear function of the Na concentration. On the other hand, ΔI_sc is a saturable function of the mucosal sugar concentration which is consistent with Michaelis-Menten kinetics suggesting that the increase in active Na transport is stoichiometrically related to the rate of active sugar transport. An interpretation of these findings in terms of a hypothetical model for intestinal Na and sugar transport is presented.     

Solute transport process in intestinal epithelial cells

In rat small intestine, the active transport of organic solutes results in significant depolarization of the membrane potential measured in an epithelial cell with respect to a grounded mucosal solution and in an increase in the transepithelial potential difference. According to the analysis with an equivalent circuit model for the epithelium, the changes in emf's of mucosal and serosal membranes induced by active solute transport were calculated using the measured conductive parameters. The result indicates that the mucosal cell membrane depolarizes while the serosal cell membrane remarkably hyperpolarizes on the active solute transport. Corresponding results are derived from the calculations of emf's in a variety of intestines, using the data that have hitherto been reported. The hyperpolarization of serosal membrane induced by the active solute transport might be ascribed to activation of the serosal electrogenic sodium pump. In an attempt to determine the causative factors in mucosal membrane depolarization during active solute transport, cell water contents and ion concentrations were measured. The cell water content remarkably increased and, at the same time, intracellular monovalent ion concentrations significantly decreased with glucose transport. Net gain of glucose within the cell was estimated from the restraint of osmotic balance between intracellular and extracellular fluids. In contrast to the apparent decreases in intracellular Na+ and K+ concentrations, significant gains of Na+ and K+ occurred with glucose transport. The quantitative relationships among net gains of Na+, K+ and glucose during active glucose transport suggest that the coupling ratio between glucose and Na+ entry by the carrier mechanism on the mucosal membrane is approximately 1:1 and the coupling ratio between Na+-efflux and K+-influx of the serosal electrogenic sodium pump is approximately 4:3 in rat small intestine. In addition to the electrogenic ternary complex inflow across the mucosal cell membrane, the decreases in intracellular monovalent ion concentrations, the temporary formation of an osmotic pressure gradient across the cell membrane and the streaming potential induced by water inflow through negatively charged pores of the cell membrane in the course of an active solute transport in intestinal epithelial cells are apparently all possible causes of mucosal membrane depolarization.     

Sodium-iodide symporter mediates iodide secretion in rat gastric mucosa in vitro

In vivo studies on rats have demonstrated that considerable amounts of iodide are transported from the bloodstream into the gastric lumen. The mechanisms for and functional significance of this transport are poorly understood. Active (driven by Na(+)/K(+)-ATPase) iodide transport into thyroid follicular cells is mediated by the sodium-iodide symporter (NIS), which is also abundantly expressed in gastric mucosa. We aimed to further investigate the iodide transport in gastric mucosa and the possible role of NIS in this transport process. Iodide transport in rat gastric mucosa was studied in vitro in an Ussing chamber system using (125)I as a marker. The system allows measurements in both directions over a mucosal specimen. A considerable transport of iodide (from the serosal to the mucosal side) was established across the gastric mucosa, whereas in the opposite direction (mucosa to serosa), iodide transport was negligible. Sodium perchlorate (NaClO(4)), a competitive inhibitor of NIS, and ouabain, an inhibitor of the Na(+)/K(+)-ATPase, both attenuated gastric iodide transport from the serosal to the mucosal side. To investigate a possible neuroendocrine regulation of the iodide transport identified to occur from the serosal to the mucosal side of the stomach, thyroid-stimulating hormone (TSH), thyrotropin-releasing hormone (TRH), vasoactive intestinal peptide (VIP), histamine, or nitric oxide donor S-nitroso-N-acetyl-D,L-penicillamine (SNAP) was added. None of these substances influenced the iodide transport. We conclude that iodide is actively transported into the gastric lumen and that this transport is at least partly mediated by NIS. Additional investigations are needed to understand the regulation and significance of this transport.     

Kinetics of bidirectional active sodium fluxes in the toad bladder.

The kinetics of isotopic Na+ flows was studied in urinary bladders of toads from the Dominican Republic. Initial studies of the potential dependence of passive serosal to mucosal 22Na+ efflux demonstrated the absence of isotope interaction and/or other coupling with passive Na+ flow. The electrical current I and mucosal to serosal 22Na+ influx were then measured with transmembrane potential clamped at deltapsi=0, 25, 50, 75 or 100 mV. Subsequent elimination of active Na+ transport with mucosal amiloride permitted calculation of the rates of active Na+ transport JaNa and active and passive influx leads to JaNa and leads to JpNa. The results indicate that for Dominican toad bladders mounted in chambers only Na+ contributes significantly to transepithelial active ion transport; hence JaNa=Ja. Ja was abolished at deltapsi=E=96.3+/-1.9 (S.E.) mV. As deltapsi approached E, active efflux comes from Ja became demonstrable. At deltapsi=100 mV, comes from Ja exceeded leads to Ja, so that Ja was negative. Experimental values of leads to Ja agreed well with theoretical values predicted by a thermodynamic formulation: leads to Jaexp=0.985 leads to Jatheor (r=0.993). The dependence of leads to Ja on deltapsi is curvilinear.     

Intestinal iodide secretion and its dependence upon mucosal I- permeability

Iodide secretion across different regions of rat small intestine has been investigated in vitro using the standard Wilson-Wiseman technique. Net I- secretion was observed along the entire small intestine, being significantly higher in the central region. Anaerobic conditions, ouabain (2 mM) and Na+ free Ringer solution prevented net I- secretion, whilst both theophylline (1 mM) and carbachol (0,1 mM) enhanced the observed basal intestinal I- secretion. Furthermore, Ca2+-deprived bathing solutions significantly reduced intestinal I- secretion. Epithelial I- uptake from both mucosal and serosal sides was measured by using a Ussing-type chamber technique. The initial rate of I- uptake across the mucosal membrane was significantly higher in the central region than in the proximal part of rat small intestine. No significant differences were observed in the rate of I- uptake from the serosal side. These studies suggest that mucosal I- permeability might determine the direction of net I- intestinal transport and that cytosolic Ca2+ may be a physiological regulator of intestinal I- transport.     

Intestinal SGLT1-mediated absorption and metabolism of benzyl β-glucoside contained in Prunus mume: carrier-mediated transport increases intestinal availability

The intestinal absorption of benzyl β-glucoside (BNZβglc) contained in the fruit of Prunus mume SIEB. et ZUCC. (Rosaceae), which is traditionally used as a medicinal food in Japan, was studied in rat intestines. BNZβglc was absorbed from the mucosal to serosal sides. Its metabolite, benzyl alcohol (BAL), was also detected on both the mucosal and serosal sides. In the presence of phloridzin (Na⁺/glucose cotransporter (SGLT1) inhibitor) or in the absence of Na⁺ (driving force), BNZβglc absorption was significantly decreased. Transport clearance of BNZβglc across the brush border membrane decreased as its concentration increased. These results indicate that BNZβglc is transported by SGLT1. Metabolic clearance of BNZβglc also decreased as its concentration increased. The amount ratio of BNZβglc to BAL on the serosal side increased with the increase of BNZβglc concentration. The intestinal availability of BNZβglc was lower in the absence of Na⁺ than in the presence of Na⁺, indicating that the SGLT1-mediated transport of BNZβglc increases intestinal availability by decreasing the intestinal extraction ratio. This neutraceutical study concluded that intestinal carrier-mediated transport across the brush border membrane improves the intestinal availability of nutritionally, pharmacologically or physiologically active compounds that undergo intestinal metabolism (first-pass effect).