Cytoskeletal control of alanine transport across the rat and turtle small intestine

Procaine inhibited significantly (P less than 0.01) alanine accumulation in the rat intestinal strips in a concentration-dependent pattern, whereas it showed no effect on alanine uptake by the turtle intestinal cells. Colchicine and Vinca alkaloids at 5 X 10(-4) and 1.5 X 10(-6) M respectively caused a significant inhibition (P less than 0.01) of intracellular alanine concentration in the rat with no effect noticed in the turtle. Unidirectional influx of alanine across the brush border membrane of the rat was significantly (P less than 0.01) reduced in the presence of procaine, colchicine and vincristine in the preincubation medium. The same drugs did not show any effect on alanine influx into the turtle small intestine. Electron microscopy showed major structural alterations in the cytoskeletal organization of the turtle intestine in response to procaine, colchicine or vincristine treatment. It is proposed that microtubular system may participate in the overall transport mechanism of alanine across the small intestine.     

Inhibition of epithelial chloride channels by a semi-purified fraction of Crotalus atrox venom

Rattlesnake venom has a strong effect on the ionic permeability of plasma membranes. Rattlesnake venom and one of its semipurified fractions (CAV-C2) has been implicated in affecting both sodium and chloride transfer across epithelia. The mechanism of CAV-C2 action on epithelia, however, is still open to question. Aplysia californica intestine has been shown to contain both chloride conductive channels and sodium dependent chloride uptake mechanisms in the mucosal membrane of its enterocytes which makes it an ideal model to differentiate the CAV-C2 action on either of these membrane transport processes. In the absence of sodium in the bathing medium, chloride is the only ion actively transported. CAV-C2 and piretanide have similar types of inhibitory effects on net chloride transport while furosemide had no effect on chloride movement across the intestine. It was concluded that CAV-C2 action is on chloride conductive channels located in the mucosal membrane of the enterocytes.     

Postnatal development of transport function in the pig intestine

1. In the newborn pig it appears that only prenatally produced enterocytes are capable of absorbing large amounts of protein. 2. The ability of the small intestine to transport sodium, lysine, lysine containing dipeptides and glucose declines markedly during the first week of post natal life. 3. Dexamethosone causes a doubling of the sodium dependent portion of alanine uptake. 4. EGF given between days three and six of postnatal life increases sucrase and maltase activity in the distal region of the small intestine. 5. Weaning induced problems are probably not due to direct inhibition of transport properties.     

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.     

Effect of Inhibitors on Alanine Transport in Isolated Rabbit Ileum

The effects of metabolic inhibitors and ouabain on alanine transport across rabbit ileum, in vitro, have been investigated. Net transport of alanine and Na across short-circuited segments of ileum is virtually abolished by cyanide, 2,4-dinitrophenol, iodoacetate, and ouabain. However, these inhibitors do not markedly depress alanine influx from the mucosal solution, across the brush border, into the intestinal epithelium, and they do not significantly affect the Na dependence of this entry process. The results of this investigation indicate that: (a) the Na dependence of alanine influx does not reflect a mechanism in which the sole function of Na is to link metabolic energy directly to the influx process; and (b) the inhibition of net alanine transport across intestine is, in part, the result of an increased rate coefficient for alanine efflux out of the cell across the brush border. Although these findings do not exclude a direct link between metabolic energy and alanine efflux, the increased efflux may be the result of the increased intracellular Na concentration in the presence of these inhibitors. The results of these studies are qualitatively consistent with a model for alanine transport across the brush border which does not include a direct link to metabolic energy.     

Sodium dependence of neutral amino acid uptake into rabbit ileum.

Alanine uses two mediated pathways to enter the rabbit ileal mucosa. Present results suggest that one of them (Km = 4.1 mM) is fully dependent on sodium in the mucosal medium, while the other (Km = 91 mM) is sodium-independent. Similar results are obtained for methionine and serine. Reinterpretation of previous alanine/sodium coupling coefficients suggests that two sodium ions per alanine molecule are transported via the high affinity system.     

Sodium-alanine cotransport in renal proximal tubule cells investigated by whole-cell current recording

Sodium-alanine cotransport was investigated in single isolated proximal tubule cells from rabbit kidney with the whole-cell current recording technique. Addition of L-alanine at the extracellular side induced an inward-directed sodium current and a cell depolarization. The sodium-alanine cotransport current was stereospecific and sodium dependent. Competition experiments suggested a common cotransport system for L-alanine and L-phenylalanine. Sodium-alanine cotransport current followed simple Michaelis-Menten kinetics, with an apparent Km of 6.6 mM alanine and 11.6 mM sodium and a maximal cotransport current of 0.98 pA/pF at -60 mV clamp potential. Hill plots of cotransport current suggested a potential-independent coupling ratio of one sodium and one alanine. The apparent Km for sodium and the maximal cotransport current were potential dependent, whereas the apparent Km for L-alanine was not affected by transmembrane potential. The increase in Km for alanine with decreasing inward-directed sodium gradients suggested a simultaneous transport mechanism. These results are consistent with a cotransport model with potential-dependent binding or unbinding of sodium (high-field access channel) and a potential-dependent translocation step.     

Sodium dependence of neutral amino acid uptake into rabbit ileum

Alanine uses two mediated pathways to enter the rabbit ileal mucosa. Present results suggest that one of them (K_m = 4.1 mM) is fully dependent on sodium in the mucosal medium, while the other (K_m = 91 mM) is sodium-independent. Similar results are obtained for methionine and serine. Reinterpretation of previous alanine/sodium coupling coefficients suggests that two sodium ions per alanine molecule are transported via the high affinity system.     

Vitamin D3 and calcium absorption in the chick

1. An attempt has been made to locate the site of action of vitamin D(3) as it affects the translocation of calcium across the intestine. 2. Calcium appears to be pumped out of cells by a process dependent on energy from metabolism. 3. The effects of cold, inhibitors and vitamin D(3) on the translocation of calcium by everted sacs of intestine were studied and compared with results obtained in vivo. 4. A model was proposed to explain the results which suggests that vitamin D(3) inhibits a metabolically operated pump that returns calcium from the mucosal cell to the lumen. 5. Some observations on the effect of sodium lauryl sulphate on the translocation of calcium in vivo and in vitro are reported.     

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.     

Reconstitution of Neutral Amino Acid Transport System from Ehrlich Ascites Tumor Cells

Amino acid transport systems for alanine and leucine have been reconstituted into artificial lipid vesicles. Purified plasma membrane vesicles from Ehrlich ascites cells were dissolved in 2% sodium cholate, 1 mM dithiothreitol, 0.5 mM EDTA, a mixture which solubilized approximately 50% of the membrane protein. This solubilized protein fraction was further purified by a combination of ammonium sulfate precipitations, gel filtration, and DEAE-cellulose chromatography. A fraction containing approximately 15 Coomassie blue staining bands on sodium dodecyl sulfate gels was obtained. This material was reconstituted into liposomes, and preliminary results demonstrated transport of alanine and leucine dependent on a sodium gradient. In addition, an electrogenic gradient mediated by valinomycin-induced potassium diffusion seemed to stimulate alanine uptake further.     

Reconstitution of neutral amino acid transport system from Ehrlich ascites tumor cells.

Amino acid transport systems for alanine and leucine have been reconstituted into artificial lipid vesicles. Purified plasma membrane vesicles from Ehrlich ascites cells were dissolved in 2% sodium cholate, 1 mM dithiothreitol, 0.5 mM EDTA, a mixture which solubilized approximately 50% of the membrane protein. This solubilized protein fraction was further purified by a combination of ammonium sulfate precipitations, gel filtration, and DEAE-cellulose chromatography. A fraction containing approximately 15 Coomassie blue staining bands on sodium dodecyl sulfate gels was obtained. This material was reconstituted into liposomes, and preliminary results demonstrated transport of alanine and leucine dependent on a sodium gradient. In addition, an electrogenic gradient mediated by valinomycin-induced potassium diffusion seemed to stimulate alanine uptake further.     

Reconstitution of Neutral Amino Acid Transport Systems from Ehrlich Ascites Tumor Cells

Amino acid transport systems for alanine and leucine were reconstituted into artificial lipid vesicles. Purified plasma membrane vesicles from Ehrlich ascites cells were dissolved in 2% sodium cholate, 1mM dithiothreitol, and 0.5 mM EDTA a mixture that solubilized approximately 50% of the membrane protein. This solubilized protein fraction was further purified by a combination of ammonium sulfate precipitations, gel filtration, and DEAE-cellulose chromatography. A fraction containing approximately 15 Coomassie blue-staining bands on sodium dodecyl sulfate gels was obtained. This material was reconstituted into liposomes, and preliminary results demonstrated transport of alanine and leucine dependent on a sodium gradient. In addition, an electrogenic gradient mediated by valino-mycin-induced potassium diffusion seemed to stimulate alanine uptake further.     

The maximal activity of phosphate-dependent glutaminase and glutamine metabolism in late-pregnant and peak-lactating rats.

The maximal activity of phosphate-dependent glutaminase was increased in the small intestine, decreased in the liver and unchanged in the kidney of late-pregnant rats. This was accompanied by increases in the size of both the small intestine and the liver. The maximal activity of phosphate-dependent glutaminase was increased in both the small intestine and liver but unchanged in the kidney of peak-lactating rats. Enterocytes isolated from late-pregnant or peak-lactating rats exhibited an enhanced rate of utilization of glutamine and production of glutamate, alanine and ammonia. Arteriovenous-difference measurements across the gut showed an increase in the net glutamine removed from the circulation in late-pregnant and peak-lactating rats, which was accompanied by enhanced rates of release of glutamate, alanine and ammonia. Arteriovenous-difference measurements for glutamine showed that both renal uptake and skeletal-muscle release of glutamine were not markedly changed during late pregnancy or peak lactation; but pregnant rats showed a hepatic release of the amino acid. It is concluded that, during late pregnancy and peak lactation, the adaptive changes in glutamine metabolism by the small intestine, kidneys and skeletal muscle of hindlimb are similar; however, the liver appears to release glutamine during late pregnancy, but to utilize glutamine during peak lactation.     

The effect of auxiliary conditions on intestinal unstirred layer diffusion modelled by numerical simulation.

Estimation of intestinal unstirred layer thickness usually involves inducing transmural potential difference changes by altering the content of the solution used to perfuse the small intestine. Osmotically active solutes, such as mannitol, when added to the luminal solution diffuse across the unstirred water layer (UWL) and induce osmotically dependent changes in potential difference. As an alternative procedure, the sodium ion in the luminal fluid can be replaced by another ion. As the sodium ion diffuses out of the UWL, the change in concentration next to the intestinal membrane alters the transmural potential difference. In both cases, UWL thickness is calculated from the time course of the potential difference changes, using a solution to the diffusion equation. The diffusion equation solution which allows the calculation of intestinal unstirred layer thickness was examined by simulation, using the method of numerical solutions. This process readily allows examination of the time course of diffusion under various imposed circumstances. The existing model for diffusion across the unstirred layer is based on auxiliary conditions which are unlikely to be fulfilled in the same intestine. The present simulation additionally incorporated the effects of membrane permeability, fluid absorption and less than instantaneous bulk phase concentration change. Simulation indicated that changes within the physiologically relevant range in the chosen auxiliary conditions (with the real unstirred layer length kept constant) can alter estimates of the apparent half-time. Consequently, changes in parameters unassociated with the unstirred layer would be misconstrued as alterations in unstirred layer thickness.     

Effects of Na-coupled alanine transport on intracellular K activities and the K conductance of the basolateral membranes ofNecturus small intestine

Intracellular electrical potentials and K activity, (K)c, were determined simultaneously in Necturus small intestine before and after the addition of alanine to the mucosal solution. As noted previously (Gunter-Smith, Grasset & Schultz, 1982), the addition of alanine to the mucosal solution resulted in a prompt depolarization of the electrical potential difference across the apical membrane (psi mc) and a decrease in the slope resistance of that barrier (rm). This initial response was followed by a slower repolarization of psi mc associated with a decrease in the slope resistance of the basolateral membrane (rs) so that when the steady state was achieved (rm/rs) did not differ significantly from control values in the absence of alanine. In the absence of alanine, psi mc averaged -32 mV and (K)c averaged 67 mM. When a steady state was achieved in the presence of alanine these values averaged -24 mV and 50 mM, respectively. The steady-state electrochemical potential differences for K across the basolateral membrane in the absence and presence of alanine did not differ significantly. Inasmuch as the rate of transcellular active Na transport or "pump activity" was increased two- to threefold in the presence of alanine, it follows that, if active Na extrusion across the basolateral membrane is coupled to active K uptake across that barrier with a fixed stoichiometry then, the decrease in rs must be due to an increase in the conductance of the basolateral membrane to K that parallels the increase in "pump activity". This "homocellular" regulatory mechanism serves to (i) prevent an increase in (K)c due to an increase in pump activity; and, (ii) repolarize psi mc and thus restore the electrical driving force for the rheogenic Na-coupled entry processes.     

Alanine and sodium fluxes across mucosal border of rabbit ileum

Unidirectional influxes of L-alanine and Na from the mucosal solution into the epithelium of in vitro rabbit ileum have been determined. In the presence of 140 mM Na, alanine influx is approximately 2.2 µmoles/hr cm², but is inhibited if the NaCl in the mucosal solution is replaced by choline Cl, Tris-Cl, mannitol, LiCl, or KCl. Although alanine influx is strongly dependent upon Na in the mucosal solution, it is uninfluenced by marked reduction of intracellular Na pools. In addition, alanine influx is unaffected by intracellular alanine concentration. Na influx is markedly inhibited by the presence of Li. Evidence is presented that Na transport across the mucosal border cannot be attributed to simple diffusion even though the net flux across this surface is in the direction of the electrochemical potential difference.     

Sodium-alanine cotransport in oocytes ofXenopus laevis: Correlation of alanine and sodium fluxes with potential and current changes

Summary The sodium-dependentl-alanine transport across the plasma membrane of oocytes ofXenopus laevis was studied by means of [¹⁴C]-l-alanine,²²Na⁺ and electrophysiological measurements. At fixed sodium concentrations, the dependence of alanine transport on alanine concentration follows Michaelis-Menten kinetics; at fixed alanine concentrations, the transport varies with sodium concentration with a Hill coefficient of 2. In the presence of sodium the uptake of alanine is accompanied by a depolarization of the membrane. Under voltage-clamp conditions this depolarization can be compensated by an inward-directed current. Assuming that this current is carried by sodium we arrive at a 21 stoichiometry for the sodium-alanine cotransport. The assumption was confirmed by direct measurements of both sodium and alanine fluxes at saturating concentrations of the two substrates, which also yielded a stoichiometry close to 21. The sodium-l-alanine cotransport is neither inhibited by furosemide (0.5 mmol/liter) nor by N-methyl amino isobutyric acid (5 mmol/liter). A 20-fold excess ofd-alanine overl-alanine caused about 60% inhibition.     

Sodium-dependent transport of neutral amino acids by whole cells and membrane vesicles of Streptococcus bovis, a ruminal bacterium.

Streptococcus bovis JB1 cells were able to transport serine, threonine, or alanine, but only when they were incubated in sodium buffers. If glucose-energized cells were washed in potassium phosphate and suspended in potassium phosphate buffer, there was no detectable uptake. Cells deenergized with 2-deoxyglucose and incubated in sodium phosphate buffer were still able to transport serine, and this result indicated that the chemical sodium gradient was capable of driving transport. However, when the deenergized cells were treated with valinomycin and diluted into sodium phosphate to create both an artificial membrane potential and a chemical sodium gradient, rates of serine uptake were fivefold greater than in cells having only a sodium gradient. If deenergized cells were preloaded with sodium (no membrane potential or sodium gradient), there was little serine transport. Nigericin and monensin, ionophores capable of reversing sodium gradients across membranes, strongly inhibited sodium-dependent uptake of the three amino acids. Membrane vesicles loaded with potassium and diluted into either lithium or choline chloride were unable to transport serine, but rapid uptake was evident if sodium chloride was added to the assay mixture. Serine transport had an extremely poor affinity for sodium, and more than 30 mM was needed for half-maximal rates of uptake. Serine transport was inhibited by an excess of threonine, but an excess of alanine had little effect. Results indicated that S. bovis had separate sodium symport systems for serine or threonine and alanine, and either the membrane potential or chemical sodium gradient could drive uptake.     

Amino acid-dependent sodium transport in plasma membrane vesicles from rat liver

Summary Thel-alanine-dependent transport of sodium ions across the plasma membrane of rat-liver parenchymal cells was studied using isolated plasma membrane vesicles. Sodium uptake is stimulated specifically by thel-isomer of alanine and other amino acids, whose transport is sodium-dependent in rat-liver plasma membrane vesicles. Thel-alanine-dependent sodium flux across the membrane is inhibited by an excess of Li⁺ ions, but not by K⁺ or choline ions. Sodium transport is sensitive to-SH reagents and ionophores, and is an electrogenic process: a membrane potential (negative inside) can enhancel-alanine-dependent sodium accumulation. The data presented provide further evidence for a sodium-alanine cotransport mechanism.