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1. The whole-cell configuration of the patch-clamp technique has been used to investigate the conductance properties of villus enterocytes isolated from guinea-pig small intestinal epithelium. 2. With near physiological ionic gradients inward and outward rectification was observed in the hyperpolarizing and depolarizing voltage domains respectively. 3. Replacement of intra- and extracellular K+ with N-methyl-D-glucamine (NMG) eliminated inward rectification but did not alter outward currents. In symmetrical low Cl- solutions outward currents were reduced but inward rectification was not affected. Under these conditions increases in extracellular K+ shifted both the current-voltage relation and the extrapolated reversal potential as expected for a K(+)-selective current. 4. The inwardly rectifying nature of the K+ current observed here remained unaltered after chelation of internal Mg2+ with ATP or EDTA. 5. Extracellular application of 5 mM-Ba2+ or 50 micrograms ml-1 of the venom of the scorpion Leiurus quinquestriatus abolished the inward K+ current, while 5 mM-extracellular tetraethylammonium (TEA) had little effect. 6. The current remaining in the presence of symmetrical Cl- solutions and in the complete absence of K+ rectified outwardly and reversed at 0 mV. The anionic nature of this current was confirmed by replacing Cl- with different anions. SCN- and Br- carried more current than Cl-, while F- and gluconate were less permeant. 7. Anionic currents of villus guinea-pig enterocytes were not stimulated by cyclic AMP and were strongly and reversibly inhibited by the Cl- channel blocker 5-nitro-2-(3-phenylpropylamino) benzoic acid (NPPB, 10(-5) M). 8. The inwardly rectifying K+ current described here shares some, but not all, characteristics with others previously described. It is postulated that this conductance might function to couple K+ permeability and the Na(+)-K+ pump rate in enterocytes. Absorption of chloride may be mediated by the Cl- channels.

The whole-cell mode of the patch-clamp technique has been used to monitor ionic currents in T84 colonic carcinoma cells. The cells were stimulated by either a cAMP cocktail, ionomycin or hypotonicity. Sizeable currents with distinct kinetics were observed after the stimulation with the different agonists. These kinetically distinct Cl- currents also presented a differential sensitivity to the anti-oestrogen Tamoxifen and to the halide I-. Tamoxifen only inhibits the volume activated Cl- current without affecting the other two. Substitution of extracellular Cl- by I- shifted the reversal potential towards more negative values both in the hypotonicity and ionomycin activated Cl- currents. The cAMP activated current responded to the Cl- substitution by I- with a blockade of both outward and inward currents, in addition to the displacement of the zero current level towards positive values. Thus, the use of these two simple tools, I- and tamoxifen, allows the distinction of Cl- channels in epithelial cells.

Background & Aims: K + secretion is believed to require the presence of a basolateral Na + /K + /2Cl − cotransporter. The aim of this study was to identify this transport system in epithelial cells from guinea pig colon and to study its possible regulation by phosphorylation. Methods: Cells were selectively isolated from crypt or surface epithelium of proximal or distal colon. Radioisotopes were used to measure K + , Na + , or Cl − influx. Bumetanide was used to discriminate for influx mediated by Na + /K + /2Cl − cotransport. Results: Under basal conditions, no bumetanide-sensitive K + influx was observed. Pretreatment with the protein-phosphatase inhibitor calyculin A (50% effective concentration, 23 nmol/L) or ionomycin showed a bumetanide-sensitive K + influx specifically in distal colon crypt cells. Okadaic acid and protein kinases C or A activators did not have effect. Bumetanide-sensitive K + uptake was abolished by the removal of external Na + or Cl − and occurred by cotransport in a 1Na + /1K + /2Cl − stoichiometry. Conclusions: Evidence is presented for the presence of an Na + /K + /2Cl − cotransporter in crypt cells from distal colon epithelium. The activity of this transporter is proposed to be regulated by a phosphorylation/dephosphorylation cycle, controlled by a type I protein phosphatase. It is possible that this phosphatase(s) is modulated by intracellular Ca 2+ .

The contribution of possible ionic conductances to the membrane potential (Em) of cells in guinea-pig small intestinal crypts has been studied using the nystatin “perforated-patch” approach in current-clamp experiments. Changes in extracellular K+ produced shifts in Em, with a 37 mV change in potential per ten-fold increase in extracellular K+ concentration. Reduction of extracellular Cl− by 130 mM led to a 7 mV hyperpolarisation while Na+ replacement was without effect on Em. The muscarinic agonist carbachol produced a hyperpolarisation which could be ascribed to an increase in basolateral K+ conductance. This effect was sustained in the presence of extracellular Ca2+ but was transient in its absence. We conclude that the conductance of the basolateral membrane of small intestinal crypts is mainly K+ selective and can be reversibly increased by muscarinic activation.

Certain cell lines derived from epithelial tissues have been demonstrated to retain in vitro some of the characteristics of their tissue of origin. Polarity of membrane proteins involved in transepithelial transport has been one of the most interesting characteristics that are preserved in these lines, thus allowing the study of ion and non-electrolyte transport in epithelia of simplified cellular composition. In addition they have become the model of choice in studies of the cell biology of membrane polarity generation.

The possible regulation of guinea pig enterocyte ion channels by GTP-binding proteins has been investigated by using the whole-cell recording mode of the patch-clamp technique and non-hydrolysable analogues of GTP. The main K+ currents in these cells are mediated by inwardly-rectifying K+ channels. Intracellular dialysis with hydrolysis-resistant GTP analogues leads to the deactivation of the inward K+ currents. Non-hydrolysable analogues of ATP or GDP were without effect.The effect occurs after a lag phase of 2 to 7 min, suggesting a multistep mechanism. Cl− currents were not affected by any of the nucleotide analogues. It is suggested that inwardly-rectifying K+ currents are deactivated by a G-protein-dependent process.

Intestinal epithelial cells isolated from rabbit small intestine and whose plasma membrane had been rendered highly permeable were used to study the role of intracellular structures in Ca2+ buffering. Monitoring free Ca2+ with a selective electrode revealed that the cells could reduce Ca2+ concentration in the medium to a level of 3.6 X 10(-7) M independently, within a certain range, of the initial Ca2+ concentration or amount of cells used. Ca2+ buffering by permeabilised enterocytes was Mg2+- and ATP-dependent and was abolished in the presence of the Ca2+ ionophore A23187. The rapidity of Ca2+ buffering, but not the final Ca2+ level attained, was reduced in the combined presence of the mitochondrial inhibitors azide and oligomycin or in the presence of ruthenium red. Buffering of Ca2+ was abolished in the presence of vanadate, although some uptake was still observed. Complete blocking occurred in the presence of both vanadate and mitochondrial inhibitors. Measurement of initial rates of uptake with radioactive calcium revealed that mitochondrial uptake plays a role at relatively high Ca concentrations but that at the presumably physiological levels most of the uptake is into a non-mitochondrial compartment. Non-differentiated crypt cells seemed to handle intracellular Ca2+ in a similar way as mature villus cells, although they appeared to buffer at a level about 2 X 10(-7) M lower.

1. Influx of neutral and basic amino acids into the mucosal epithelium of the rabbit ileum was measured in the presence of high and low concentrations of Na. Uptake measured at an amino acid concentration of 1 mM varied from 10 to 16 nmole . cm-2 . min-1. Removal of Na inhibited the uptake of short-chain amino acids more than long-chain amino acids. 2. Inhibition of uptake of glycine, alanine, leucine and phenylalanine by a selection of non-radioactive amino acids was found to follow a particular pattern. Long-chain amino acids inhibited the uptake of all test amino acids; short-chain amino acids inhibited preferentially the uptake of glycine. 3. The maximum inhibition serine could cause to the uptake of other amino acids was found to vary. Serine inhibited completely the uptake of glycine but a portion of uptake of long-chain amino acids was found to persist, even in the presence of high concentrations of serine. This is taken as evidence for the presence of an amino acid uptake mechanism having no affinity for serine. 4. The apparent affinities of neutral amino acids for the serino-inhibitable system (system 1) varied from about 0.5 mM (for long-chain amino acids) to about 3 mM (for short-chain amino acids). The total uptake of individual amino acids by system 1 was essentially similar when compared at an amino acid concentration of 1 mM. 5. The serine-resistant uptake of neutral amino acids (system 2) constituted up to two fifths of total uptake for long-chain amino acids, measured at amino acid concentrations of 1mM. The affinities of long-chain amino acids for system 2 is thought to be less than for system 1. Serine appears not to interact with system 2. 6. A second component to serine uptake was found to be related linearly to the concentration of serine in the medium. A similar component may contribute to the total uptake of phenylalanine. The possibility that such a component could arise as a space marker artifact is discussed. 7. An independent kinetic analysis of phenylalanine uptake by rabbit ileal mucosa showed that it could not be accounted for on the basis of a single entry system. However uptake could be described kinetically, assuming two systems of mediated entry to be present. The possible presence of a third non-saturable component to uptake does not affect these conclusions. 8. It is concluded that least two systems exist for the mediated entry of neutral amino acids into rabbit ileal mucosa. This fact should be taken into account in any future mechanistic interpretation of carrier-mediated amino acid transport in the small intestine.

Harmaline strongly inhibits the uptake of phenylalanine by slices of guinea-pig intestine in vitro. The lowest concentration having a significant effect is 0.1 mM. The drug also inhibits the unidirectional flux of phenylalanine from the mucosal to serosal face of the tissue provided it is added to the solution bathing the mucosal surface. The unidirectional flux of sodium from the mucosa to the serosa was similarly reduced. Ion and water absorption in the perfused dog intestine in vivo is also diminished in the presence of harmaline. These results support the hypothesis, previously proposed in view of the rapid onset of harmaline inhibition of sodium-dependent uptake mechanisms in a variety of tissues, that harmaline interacts with the sodium-site of non-electrolyte carrier complexes. The effect of harmaline on phenylalanine uptake by the intesting is duplicated by other psychotropic indole analogues. The actions of harmine and harnalol are similar to that of harmaline, despite great differences in the liposolubility of the different compounds. N:N-dimethyl-tryptamine is equally inhibitory, but serotonin is inactive. Mescaline and lysergic acid diethylamide also inhibit phenylalanine transport, but to a much lesser extent than harmaline.

Slices of dog gall bladder are capable of accumulating amino-acids and sugars against considerable concentration gradients across the luminal membrane of the cell. The epithelium of the common bile duct also absorbs these substrates. The transport systems are sodium-dependent, saturable and inhibited by ouabain and metabolic poisons. The specificity of the mechanisms is more reminiscent of kidney than of intestinal transport. Glycine is preferentially transported across dog gall bladder from the mucosa to the serosa, but the net flux is small. It is concluded that the contraluminal membrane of the epithelial cell might be relatively impermeable to the amino-acid. concentrative uptake also occurs in guinea-pig gall bladder; however it appears to be practically non-existent in rabbit tissue. Human gall bladders, obtained at random from the operating room, also displayed active accumulation. The gall bladder epithelium is remarkably resistant to anoxia. Furthermore, following obstruction of the common bile duct for 2 weeks in dogs, the gall bladder is still able to concentrate amino-acids and sugars, but the extent of the uptake is significantly reduced.

The pig kidney-cell line, LLC-PK1, possesses gamma-glutamyltransferase with properties similar to those of the purified renal enzyme. gamma-Glutamyltransferase activity increases after cells are seeded at low density to reach values comparable with those found in kidney cortex when the cells are fully confluent. This increase is paralleled by an increase in alpha-methyl D-glucoside uptake. In contrast, the uptakes of L-leucine and L-alanine decrease during this time. These results suggest that gamma-glutamyltransferase is not important as a mediator of the renal transport of neutral amino acids.

1. Pieces of rabbit distal ileum, incubated for short periods of time in solutions containing tritiated amino acids, have been processed for autoradiography and the profiles of amino acid concentration across the villi determined by microdensitometry. 2. The concentration profiles of a series of amino acids could be described in terms of two descending exponentials, one extending from the brush border to the basal membrane of the enterocyte and the other from the base of the epithelial layer to the centre of the villus. 3. Exponential coefficients describing the steepness of these gradients were highest for basic amino acids. Coefficients for short-chain amino acids were greater than for long-chain neutral amino acids. None of these values changed for times of incubation varying from 5 to 180 sec. 4. Enterocytes accumulated amino acids in the apical cytoplasm, against a concentration gradient, within the first few seconds of incubation. This step-up in concentration decreased as the external concentration was increased, in a manner dependent on the amino acid used. 5. It is suggested that amino acid concentration gradients within enterocytes arise by diffusion and that the amino acid specificity of this process originates from an ability of the more lipophilic amino acids to permeate structures acting as barriers to the more hydrophilic molecules.

1. The cell-attached and excised inside-out configurations of the patch-clamp technique were employed to probe isolated enterocytes of Necturus maculosus for the presence of Cl(-)-selective channels. 2. Chloride-selective channels were rarely observed unless cells were previously stimulated by agonists that raise cyclic AMP. In cell-attached patches forskolin (20 microM) or dibutyryl cyclic AMP 2 mM) evoked single-channel activity that reversed, depending on the cell, between 9 and 27 mV positive to the spontaneous membrane potential. This is close to the Cl- equilibrium potential in those cells; the single-channel current-voltage relationship was linear with a unitary slope conductance between 17 and 25 pS (pipettes filled with 100 mM-NaCl). 3. Large depolarizing voltage steps also activated Cl- channels in excised inside-out membrane patches that were previously quiescent. This mode of activation produced a distinctive single-channel current-voltage relationship with strong outward rectification at depolarizing membrane potentials. Single-channel cord conductance at negative potentials was 15-18 pS and increased to 45 pS at + 100 mV. 4. Altering the Cl- concentration in the bathing solution of excised inside-out patches displaced the observed reversal potential (Erev) to values predicted for Cl- equilibrium potential. Replacement of K+ for Na+ was without effect. 5. The effect of different anions upon Erev was used to determine the channel anion selectivity in excised inside-out patches. The permeability sequence was SCN- greater than I- greater than Br- greater than Cl- greater than F- greater than HCO3- greater than gluconate which corresponds to Eisenman's sequence 1. Neither ionic size nor diffusion rates determine the permeation of ions through the channel. 6. In channels activated by depolarization the open probability (Po) was insensitive to changes in the Ca2+ concentration (less than 10(-8)-10(-3) M) bathing the cytoplasmic face of excised inside-out patches. Depolarization was also without marked effect on Po. 7. Chloride channels in excised inside-out patches were inhibited by stilbene and diphenylamine-2-carboxylate derivatives. 4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulphonic acid (SITS, 5 x 10(-5) M) and 5-nitro-2-(3-phenylpropylamino) benzoic acid (NPPB, 1 x 10(-5) M) caused an irreversible 'flickery' blockade without altering single-channel current. 3'5-Dichlorodiphenylamine-2-carboxylic acid (DDPC, 5 x 10(-5) M) reduced the currents at every voltage without apparent effects on gating properties of the channel.(ABSTRACT TRUNCATED AT 400 WORDS)

The dog jejunum is a much denser tissue than the ileum, with a greater weight per unit length and higher proportion of mucosal tissue. Morphometric analysis reveals longer and wider villi, deeper crypts and larger enterocytes in the jejunal mucosa. Uptake of phenylalanine or beta-methyl-glucoside by tissue slices in vitro is slightly greater in jejunal than in ileal tissue. The levels of acid phosphatase and alkaline phosphatase in the individual enterocytes are significantly greater in the jejunum, according to quantitative histochemical analysis. The absorption of water, sodium, potassium, chloride and glucose in vivo is significantly smaller in the jejunal than in ileal loops, particularly when expressed in terms of unit mucosal weight. Sodium and water absorptions are stimulated by glucose at both sites, but the stimulation is significantly greater in the ileum. Opposite results have been obtained in rats where the transport of phenylalannie in vitro is greater in the ileum, but water, electrolyte and glucose absorption in vivo is greater in the jejunum.

1. A method is described for converting tissue concentrations of amino acid, determined autoradiographically using sections of rabbit distal ileum, into measurements of total uptake. 2. Using this method the quantity of amino acid recovered from the villus core following short-term incubation with tritiated amino acid was shown to be directly related to the intra-enterocyte concentration of amino acid determined at a site immediately adjacent to the basal membrane. No evidence was obtained for saturation of efflux across the basal membrane of the enterocyte. 3. Amino acid efflux from the mucosa to the villus core, calculated for a constant intra-enterocyte concentration of substrate, was found to be greater for methionine and leucine than for alanine, serine, lysine or arginine. 4. The distribution of amino acids within the core of the villus following efflux from the enterocyte could not be explained by diffusion alone. 5. It is suggested that quantitative autoradiography can be used as an alternative method to study mechanisms responsible for amino acid movement across the basal membranes of enterocytes. Advantages and limitations of the technique are discussed.

The effects of ouabain, ethacrynic acid andn-butyl-biguanide, alone or in combination, on the dog kidney have been studied by infusion of the drugs into the renal artery in vivo, with simultaneous determinations of the clearances of creatinine and PAH, renal blood flow, oxygen consumption, and reabsorptions of sodium, potassium, chloride and glucose; after removal of the organ, the tissue water and intracellular ion contents, the oxygen consumption and transport capacity for glycine, β-methylglucoside and PAH in cortical slices, and the activities of Na+−K+-ATPase in microsomal fractions from cortical homogenates were determined.

Active transport of amino acids and the sugar, alpha-methyl-D-glucoside (alpha-MG) caused an increase in the rate of K efflux from isolated rabbit enterocytes. These effects were inhibited by apamin (5 X 10(-7) M), quinidine (10(-3) M), Ba (5 X 10(-3) M) and trifluoperazine (5 X 10(-5) M) but not by the loop diuretic furosemide (10(-4) M). None of these drugs affected the basal rate of K efflux. The stimulatory effects of amino acids or alpha-MG on K efflux are too great to be explained in terms of an increase in the electrical driving force across the plasma membrane of these cells and a change in membrane permeability is envisaged. An apparent Ca-dependent K permeability in isolated enterocytes can be demonstrated using the Ca ionophore A23187. The effect of the ionophore on K efflux is abolished by apamin or Ba. It is proposed that Ca-dependent K channels mediate the sugar and amino acid induced increases of K efflux. Under control conditions there is a decrease in intracellular K concentration during accumulation of alanine or alpha-MG. Ba by itself does not alter K concentration but it did produce a marked increase when used in conjunction with alanine or alpha-MG. The accumulation of alpha-MG was inhibited in the presence of Ba. This is consistent with an interference with the driving force for sugar accumulation. It is suggested that the increase in K permeability described has a role in both maintaining ion homoeostasis during Na-coupled transport and contributing to the driving force for sugar and amino acid absorption.

1. The kinetic parameters for serine, alanine and methionine uptake by rabbit ileal mucosa have been determined in the absence of Na. 2. Uptake of all three amino acids took place through a single mediated system. The apparent Km values of serine, alanine and methionine for this system were equal to their respective apparent K1 values (approximately 89, 75 and 23 mM respectively). 3. Autoradiography was used to measure the cellular location of alanine uptake by rabbit ileum. Approximately 80% of the total uptake took place in the upper third of each villus. This uptake was reduced by 75% either by removal of Na or addition of serine. The proportional distribution of Na-dependent and Na-independent alanine uptakes along the villus was found to be equal. 4. The kinetic properties of the low affinity uptake mechanism for neutral amino acids, seen in the absence of Na, were virtually identical with those of one of the uptake mechanisms seen previously in the presence of Na. 5. The low affinity uptake mechanism appears to be Na-independent. It is suggested that the Na-coupled uptake of amino acid takes place through the high affinity system.

Alanine uptake by rabbit ileal mucosa has been measured in the presence and absence of Na+ to establish the characteristics of biological variation. Common Km values, calculated for two systems of alanine entry, were used for estimation of two Jmax values for individual rabbits. The distribution of Jmax1 and Jmax2 within the population was normal and there was minimal interaction between these two parameters, judged by a cross-correlation analysis. Ways of processing data to account for this type of animal variation are discussed.

The inhibitory aciton of harmaline on L-phenylalanine uptake by guinea-pig intestinal rings is fully reversible provided only low concentrations of the inhibitor are used; if the concentration is raised to a sufficient extent to enable the drug to interfere with sodium pumping or cellular metabolic reactions, as witnessed by its effect on tissue oxygen consumption, then the inhibition of L-phenylalanine uptake is only reversible if short contact times are employed.

The ability of dog renal cortex slices to accumulate beta-methyl-glucoside or glycine is enhanced by the flavonoid (+)-catechin at a concentration of 3.5 mM. This stimulatory effect is apparently due to a decreased rate of efflux of either substrate. On the other hand, the uptake of p-amino-hippuric acid and N1-methyl-nicotinamide is inhibited by (+)-catechin. The drug at the same concentration is without action on amino-acid transport by guinea-pig intestine in vitro.

Cells isolated from the epithelium of the small intestine are used to study the relationship between amino acid or sugar-coupled sodium transport and potassium uptake through the sodium/potassium pump. Potassium influx is a saturable function of the external potassium concentration. Uptake in the presence of ouabain, a specific pump inhibitor, is greatly reduced. This remaining influx is linearly related to the concentration up to 6 mM potassium. Sugars and amino acids are actively accumulated by the intestinal cells. Their transport is accompanied by an initial extra influx of sodium. Although cells seem to regulate their internal sodium concentrations, this is not accompanied with a concomitant increase in potassium uptake through the pump. Thus L-alanine, 3-0-methyl-D-glucoside, and alpha-methyl-D-glucoside all fail to increase the rate of ouabain-sensitive potassium uptake. A very high coupling ratio of sodium efflux to potassium influx through the pump would be a likely explanation of the present results though they cannot be regarded as conclusive.

The object of the present review is to describe the characteristics of amino acid transport studied at the cellular level. Such studies have been carried out using enterocytes isolated from villi; they have also involved transport studies carried out on enterocytes cultured from the intestine of germ-free animals. More recently, studies have also been carried out using whole tissue, enterocytes involved in amino acid transport being identified and analysed subsequently using quantitative autoradiography.

LLC-PK1 and MDCK cells take up cationic amino acids (lysine and arginine) by a specific sodium independent transport system. Uptake is inhibited by ornithine in LLC-PK1 and MDCK cells either in the presence or absence of sodium and by glutamine or homoserine in MDCK cells in the presence of sodium. Trans-stimulation of uptake occurs in the presence of intracellular cationic amino acids. Experiments with valinomycin or with different extracellular potassium concentrations suggest that uptake is dependent on the membrane potential of these cells. These transport features are similar to those previously ascribed to a transport system denominated y+ in other cells. Further experiments suggested that this carrier system is localised to the basolateral membrane in each cell type.

Harmaline is a psychotomimetic alkaloid extracted from Banisteriopsis caapi, a Colombian liana. A recent study1 has disclosed its action as an inhibitor of various transport systems in epithelial tissues, where its mode of action differs from that of other known transport inhibitors. Thus the drug reduces both sugar and amino acid uptake by the guinea-pig intestine in vitro;furthermore1 this inhibition can be extrapolated to zero time, revealing that the effect of the drug, at least in short incubations, is on the first step of the transport process, namely the translocation of the substrate across the brush-border membrane of the enterocyte.

The kinetics of phenylalanine influx into guinea-pig small intestinal rings at different external sodium-ion concentrations have been examined. A change in the sodium concentration alters the value of Kt for phenylalanine influx without affecting the Jmax for the process. By studying the relationship between the value of Kt and the sodium-ion concentration, the applicability of various models to the overall process of sodium-coupled phenylalanine influx has been considered. The results are most consistent with a general non-compulsory model for the formation of a ternary complex between carrier, phenylalanine molecule and a sodium ion, which can be formed from either binary complex, i.e., either species can combine first to the carrier. The consistency of the model was tested by determining phenylalanine influx as a function of the sodium-ion concentraion at different amino-acid concentrations, and a good fit was obtained with the predictions of the non-compulsory model.

Amino acid transport at the luminal side of brain capillary endothelium and the basal side of salivary epithelium were compared using a nondestructive, first-circulation, paired-tracer dilution method. In the brain, the reference molecule was an intravascular marker (Crone’s method) whereas in the salivary gland the reference was an extracellular marker of similar size to the test molecule. The unidirectional flux was related to a maximal uptake, U: $${\text{U = 1}} - {\text{test concentration/reference concentration}}$$ Uptake and cross-inhibition experiments in brain suggest the presence of only a long-chain neutral L-amino acid transport system (leucine, phenylalanine, tryptophan, tyrosine, valine, methionine). In contrast, results in the salivary gland suggest 4 transport systems: 1. large neutral, 2. small neutral (alanine, serine), 3. basic (lysine) and 4. acidic (aspartic, glutamic). The same method was applied to localize binding sites. Ouabain bound very significantly to the salivary epithelium but not at all to the brain endothelium. The method described is of interest since it can be extended to any organ and possibly to man.

1. The kinetic parameters of lysine and alanine uptake by rabbit ileal mucosa have been measured in the presence and absence of Na. 2. Using lysine as an inhibitor of part of alanine uptake in the absence of Na it has been possible to define two mediated systems for alanine entry. One of these systems (Km 14.2 mM; Jmax 41.3 n-mole cm-2 min-1) is inhibited by lysine (Ki 0.96 mM) while the other (Km 115 mM; Jmax 323 n-mole cm-2 min-1) is not. 3. Methionine is an effective inhibitor of both Na-independent uptake mechanisms for alanine. 4. Na-independent lysine uptake also takes place through two mediated systems. One of these systems (Km 1.0 mM; Jmax 12.8 n-mole cm-2 min-1) is inhibited by alanine (Ki 4.3 mM) while the other (Km 108 mM; Jmax 194 n-mole cm-2 min-1) is not. 5. The use of a naturally occurring basic amino acid to inhibit a portion of neutral amino acid uptake extends considerably the ability to distinguish multiple acid transport systems present in the same cell membrane. The physiological implications of these findings are discussed.

Amino acid tmransport at the luminal side of brain capillary endothelium and the basal side of salivary epithelium were compared using a nondestructive, first-circulation, paired-tracer dilution method. In the brain, the reference molecule was an intravascular marker (Crone's method) whereas inthe salivary gland the reference was an extracellular marker of similar size to the test molecule. The unidirectional flux was related to a maximal uptake, U: U = 1 --test concentration/reference concentration Uptake and cross-inhibition experiments in brain suggest the presence of only a long-chain neutral L-amino acid transport system (leucine, phenyl-alanine, tryptophan, tyrosine, valine, methionine). In contrast, results in the salivary gland suggest 4 transport systems: 1. large neutral, 2. small neutral (alanine, serine), 3. basic (lysine) and 4. acidic (aspartic, glutamic). The same method was applied to localize binding sites. Ouabain bound very significantly to the salivary epithelium but not at all to the brain endothelium. The method described is of interest since it can be extended to any organ and possibly to man.

1. Mucosal amino acid uptake by pig proximal colon, measured independently for fourteen different amino acids each used at a concentration of 1 mM, ranged from 0.6 to 8.6 n-mole. cm(-2). min(-1) in the new-born to 0 to 0.3 n-mole. cm(-2). min(-1) in the 2-day-old animal. Long chain amino acids entered the mucosa of new-born pig proximal colon much more readily than did short chain amino acids.2. Glycine was used extensively to inhibit the uptake of other neutral amino acids. The degree of maximal inhibition produced depended on the amino acid used. The relative inability of glycine to inhibit the uptake of long chain amino acids suggested that these compounds could cross the brush border on a carrier inaccessible to glycine. The glycine-sensitive uptake remained more or less constant for all amino acids tested (1-2 n-mole.cm(-2).min(-1)); the glycine-insensitive uptake varied from 0 to 7 n-mole.cm(-2).min(-1) (glycine and methionine respectively).3. It is suggested that at least two mechanisms exist for the entry of neutral amino acids into pig proximal colon, one showing specificity for hydrophobic amino acids and the other having broad specificity. The mechanism responsible for the uptake of long chain essential amino acids predominates over the less specific mechanism.4. These results are discussed in relation to previous work carried out on the rabbit ileum where two similar systems for neutral amino acid entry have been shown to be present. Both tissues transport hydrophobic amino acids on their own specific carrier at approximately the same rate; the ability of the pig colon to transport amino acids on the broad specificity carrier is eight times less than in the rabbit ileum. The possibility is raised that this system is subject to regulation.

1. The influx of serine, alanine and methionine across the brush border membrane of the rabbit ileal mucosa has been measured during short periods of incubation. 2. A kinetic analysis of the uptake data, assuming one mediated entry mechanism or one mediated entry mechanism plus a diffusion component to be present, does not provide an adequate explanation for the results obtained. Methionine inhibition of serine uptake provided direct evidence that the diffusive entry of serine into the rabbit ileum was small or non-existent. 3. Data taken from amino acid inhibition and substrate-uptake experiments, fitted simultaneously to a double hyperbolic model of amino acid uptake, give good agreement between predicted and experimental results. There is also good quantitative agreement between computer-derived kinetic constants in the present work and similar constants obtained previously using a different method of analysis. 4. Present work supports the general hypothesis that neutral amino acids use two mediated pathways to enter the rabbit ileal mucosa. The possible physiological significance of these results and their probable effect on currently held concepts of how amino acids cross the brush border membrane of the rabbit intestinal mucosa is discussed.

1. An autoradiographic technique is described whereby the cellular location of tritiated amino acids can be determined following uptake by rabbit ileal mucosa. 2. Stirring solutions in contact with the intestinal mucosa during measurement of rapid influx changes the quantity, but not the distribution, of alanine taken up by the tissue. 3. Conditions predicted to favour either a high affinity system (Ly1) or a low affinity system (Ly2) were used to measure lysine distribution following uptake. Maximal uptake for both transport systems occurred in fully differentiated enterocytes at the tips of villi. Initial maturation of the Ly1 system, which was slow, was followed by a rapid phase of development. The Ly2 system lacked this rapid phase of late development. 4. The cellular distribution of alanine entering on a low affinity Na-independent neutral amino acid carrier closely resembles that determine for Ly1 system for lysine entry. 5. Arginine is a potent inhibitor of lysine uptake through the Ly2 system. Little or no diffusion of lysine appears to take place into rabbit ileal enterocytes. 6. The different distribution of the high and low affinity systems for lysine transport provides further support for their independent existence. It also suggests that more than one message exists fo the switching on of amino acid transport function in differentiating enterocytes.

1. Intracellular potentials, Cl- activity and membrane resistances were measured in Necturus small intestinal epithelium during Cl- replacement experiments using conventional or Cl- -selective double-barrelled microelectrodes. A Cl- conductance, located in the apical membrane and activated by cyclic nucleotides is demonstrated by ion-substitution experiments. 2. The mean mucosal membrane potential (Em) was -35.5 mV. Removal of Cl- from the mucosal medium by replacement with gluconate, evoked a sudden depolarization of Em and an immediate increase in the fractional resistance of the mucosal membrane (f(Rm)). The size of the change in Em varied between 3 and 65 mV, corresponding to Cl- to K+ permeability ratios between 0.2 and 20. It was inversely related to the initial f(Rm), which ranged from 0.04 to 0.50. 3. Prolonged incubation in low-Cl- solutions led to a reversal of the initial depolarization and to a sustained hyperpolarization accompanied by a marked increase in f(Rm). The new value of Em was close to the K+ equilibrium potential, consistent with a depletion of cellular Cl- and the preponderance of a K+ membrane permeability in the absence of Cl-. This emphasizes the role of Cl- in establishing Em. 4. Removal of mucosal Cl- produced a fast decrease in intracellular Cl-, as measured with Cl- -selective microelectrodes. The efflux was consistent with electrodiffusion across the mucosal membrane. Changes in Em paralleled changes in intracellular Cl- activity, indicating the presence of a large Cl- conductance. 5. Dibutyryl cyclic AMP or forskolin produced a slow depolarization, a decrease in f(Rm) and an increased change in intracellular potential in low mucosal Cl- which on average corresponds to an approximately 15-fold increase in the relative Cl- permeability. These results are consistent with an activation of apical Cl- conductance. 6. The selectivity of Cl- channels of Necturus enterocytes to different anions was obtained from potential measurements. The sequence of permeabilities was SCN- greater than I- greater than or equal to Br- greater than NO3- greater than Cl- much greater than HCO3- greater than gluconate. This is consistent with a model involving a weak interaction of the anions with the selectivity filter. 7. The selectivity of the anion conductance was maintained after activation with cyclic nucleotides, suggesting a single channel for the permeation of the different anions tested, rather than parallel channels. 8. Derivatives of 9-anthracene which are potent inhibitors of Cl- channels in other systems failed to block the apical Cl- conductance of Necturus enterocytes. Chloride conductance was also insensitive to furosemide.(ABSTRACT TRUNCATED AT 400 WORDS)

A method is described for isolating viable enterocytes from rabbit jejunum. Estimates of sucrase and gamma-glutamyl transferase activities in cells isolated by this method suggest that they originate from the upper villus only. Isolated cells accumulate both alpha-methyl-D-glucoside and alanine, maintaining high intracellular concentrations for at least 60 and 40 min respectively. Accumulation of alpha-methyl-D-glucoside is inhibited by the presence of phloridzin. The cells accumulate 42K and 86Rb in an identical manner. This uptake, which is maintained for at least 60 min, is inhibited in the presence of ouabain. Passive efflux of 42K and 86Rb occurs with rate constants which are virtually identical. The efflux follows a single exponential suggesting that it originates from only one intracellular compartment. It is suggested that the preparation can be used to study the effect of sugars and amino acids on K efflux. The advantages of using such a preparation are discussed.

The type of junctions present in the membranes of the two renal epithelial cell lines, LLC-PK1 and MDCK, and of subcultured porcine aortic endothelial (PAE) cells have been studied by freeze-fracture. No gap junctions were observed in the two renal cell lines, while they were numerous in the endothelial cells. Tight junctions were abundant in LLC-PK1 and MDCK cells and varied in numbers of ridges from one to ten. ONly a few simple tight junctions unconnected with gap junctions were observed in PAE cells. The occurrence of gap junctions in these cells correlates with their ability to form intercellular communicating channels.

Harmaline inhibits both the Na+ -K+ -ATPase activity and the uptake of L-phenylalanine in guinea-pig intestinal mucosa. The latter effect is not a direct consequence of the former, since higher concentrations are needed to inhibit the enzyme than the influx into the mucosa; Furthermore the uptake is still sensitive to harmaline when the Na+ -K+ -ATPase has been fully inhibited by ouabain. Harmaline can inhibit L-phenylalanine influx at a concentration at which it does not affect intracellular ion concentrations. Ouabain, however, inhibits the uptake of L-phenylalanine only after a 30 min preincubation period, when the intracellular sodium concentration reached the extracellular level. Harmaline also interferes with the influx of beta-methyl-D-glucoside in the mucosa of the dog colon. Addition of harmaline at the mucosal face of the tissue suppresses all net transport of sodium and chloride ions and L-phenylalanine across the mucosa. Thus the same mode of action appears to apply in both the guinea-pig ileum and the dog colon.

The validity of a general non-compulsory model for the description of the co-transport of sodium and phenylalanine in the small intestinal mucosa of the guinea-pig has been examined by measuring the influx of sodium and the flux ratios. The simultaneous influxes of sodium and phenylalanine have been determined by incubating intestinal tissues at a fixed sodium and variable phenylalanine concentrations. The experiment was repeated at a number of sodium concentrations, and a straight-line relationship between the fluxes was always obtained. The slope of this line, the flux ratio, is dependent on the sodium concentration; the function is biphasic inasmuch as it rises with sodium concentration at low external sodium, but decreases when the sodium concentration is raised above a level of about 70 mM. This response is compatible with the model under examination. No saturable component of sodium influx could be detected in the absence of phenylalanine, but the values of this influx corresponded with those predicted from the flux ratio experiment. In the presence of phenylalanine, sodium influx could be resolved into saturable and non-saturable components, and the Kt for sodium influx via the saturable mechanism agreed with that predicted from the constants derived from measurements of phenylalanine influx. The maximal velocity for sodium influx was similar to the maximal velocity for phenylalaline influx. Equations were derived to examine the behaviour of sodium influx as a function of the external phenylalanine concentration, and a Kt for phenylalanine was deduced which agrees closely with that obtained by studying phenylalanine fluxes directly. These results provide evidence in favour the applicability, in this species, of a model involving no compulsory pathway for the formation of a ternary complex between the carrier, a sodium ion and the phenylalanine molecule. Examination of the literature suggests that in other species different models may describe more accurately this co-transport mechanism.

A characteristic secretory response in the dog jejunumis observed following three hours' perfusion with cholera toxin. When this mucosa in the secretory phase is examined in vitro, the steady-state accumulation of phenylalanine and beta-methyl-glucoside is considerably enhanced. Since the initial rates of influx of these substrates are unchanged and the tissue ion contents are the same as normal, it is concluded that the increase in steady-state uptake may be attributed to a change in the rate of efflux of the substrates across the baso-lateral membrane of the cell. This change might be related to the reported collapse of the intercellular channels when the intestine is in a secretory state.

The stoichiometry of Na coupling to amino acid movement across the brush border membrane of the rabbit distal ileum has been determined under initial rate conditions. The coupling ratio, defined as the amino acid-dependent Na influx/the Na-dependent amino acid influx, was equal to unity for alanine, measured over a 10-fold range of Na and alanine concentrations. Coupling ratio values determined under a single set of conditions for a number of amino acids varied from 1 for serine to 4.6 for methionine. Reducing the methionine concentration from 12.5 to 1.5 mM caused the coupling ratio value to fall from 4.6 to 1.2. These results are explained by assuming a fixed stoichiometry of 1 : 1 under all conditions, with initial binding of the amino acid (A) to the Na-dependent carrier (E) but with some amino acids being able to cross on the Na-dependent carrier in the absence of Na. The variation in coupling ratio values can be used to calculate K A , the apparent dissociation constant of amino acid from the Na-dependent carrier in the absence of Na, and the ratio k 1 k 2 , where k 1 and k 2 are first-order rate constants for translocation of the complexes EA and EANa, respectively. This method of processing results has been defined as delta analysis. The value of K A for methionine is 3.6 ± 1.1 mM and the k 1 k 2 ratio is 1.01 ± 0.07. The constant coupling ratio value of 1 for alanine indicates that the value for K A is extremely high or that the k 1 value is extremely low.