Heterogeneity of amino acid transport in horse erythrocytes: a detailed kinetic analysis of inherited transport variation.

1. Thoroughbred horses were divisible into five distinct amino acid transport subgroups on the basis of their erythrocyte permeability to L-alanine, measured uptake rates ranging from 5 to 625 mumol l cells-1 h-1 (0.2 mM-extracellular L-alanine, 37 degrees C). 2. Erythrocytes from animals belonging to the lowest L-alanine permeability subgroup (5-15 mumol l cells-1 h-1) (transport-deficient type) exhibited slow nonsaturable transport of this amino acid. In contrast, cells from horses of the four transport-positive subgroups possessed additional high-affinity (apparent L-alanine Km (Michaelis constant) congruent to 0.3 mM) and/or low-affinity (apparent L-alanine Km congruent to 13 mM) Na+-independent transport routes selective for L-neutral amino acids of intermediate size. The two transporters, designated systems asc1 and asc2, respectively, also possessed a significant affinity for dibasic amino acids. 3. Amino acid transport activity in horse erythrocytes behaved as if controlled by three co-dominant alleles (s, h and l), where s is a silent allele, and h and l code for the functional presence of systems asc1 and asc2, respectively. 4. At physiological temperature, system asc1 operated preferentially in an exchange mode. In contrast, system asc2 did not participate in exchange reactions at 37 degrees C, but did exhibit significant trans-acceleration at 25 degrees C. 5. Reduction of the incubation temperature also resulted in dramatic decreases in apparent Km and Vmax for L-alanine uptake by system asc2, whereas the effects of temperature on system asc1 were much less marked. At 5 degrees C the two transporters exhibited equivalent kinetic constants for L-alanine influx. L-Alanine uptake by transport-deficient cells was relatively insensitive to temperature. Influx by this route may represent the ground-state permeability of the lipid bilayer. 6. The effects of low temperature on system asc2 suggest a preferential impairment of the mobility of the unloaded carrier relative to that of the loaded transporter. Similarly, the different kinetic properties of systems asc1 and asc2 at physiological temperature are attributed to a difference in the mobilities of the empty carriers, this difference being minimized at 5 degrees C.

The measurement of liver blood flow in conscious calves.

Observations were made following single I.V. injections or during continuous I.V. infusions of sulphobromophthalein (BSP) in three Jersey calves (3-5 months of age) which had an indwelling hepatic vein catheter, surgically implanted under general anaesthesia. Simultaneous sampling of blood from a peripheral (jugular) vein and an hepatic vein enabled calculations of hepatic plasma flow (E.H.P.F.) based on the Fick principle. Estimates of E.H.P.F. in nine single injection experiments gave a mean flow of 38.6 ml X min-1 X kg-1 compared to 32.6 ml X min-1 X kg-1 estimated in seven continuous infusion experiments. The over-all mean haematocrit in the three calves was 30.0% and the E.H.P.F. values are equivalent to hepatic blood flows of 55 and 47 ml X min-1 X kg-1 respectively. In thirteen out of fourteen experiments the plasma clearance of BSP in jugular vein blood after a single I.V. injection of 5 mg BSP X kg-1 body weight was best fitted by a double exponential model of distribution of BSP. Parameters from these exponentials were used to calculate E.H.P.F. by the method of Clarkson, Hardy-Smith & Richards (1976) and gave values of 11.3 ml X min-1 X kg-1, clearly indicating that the method cannot be applied in conscious calves.

Effect of diet upon enterocyte differentiation in the rat jejunum.

Positional and temporal correlates for the development of microvillus membranes and for two of the hydrolytic enzymes they contain have been determined and compared with the ability of enterocytes to transport valine during migration from crypt base to villus tip in jejunal tissue taken from rats maintained on diets containing different amounts of protein. Microvillus elongation and the appearance of both aminopeptidase N (APN) and isomaltase (IM) activities reached maximal rates of expression in enterocytes located 16 +/- 5 micron from the crypt-villus junction. This close positional correlation was not found for the later development of the valine transport function. Feeding rats isoenergetic diets containing 20% instead of 5% protein caused significant increases in both villus height and crypt depth without changing the positional correlations described above. The maximal rates for microvillus elongation and APN and IM appearance were greater and occurred earlier in enterocytes taken from rats fed a high-protein diet. The time of onset and capacity to transport valine were found to be closely correlated for rats maintained on high- and low-protein diets. The ratio of APN to IM activity in fully differentiated enterocytes was either 0.7 or 1.2 depending on whether rats had been fed a low- or high-protein diet. The maximal length of microvillus membranes in fully differentiated enterocytes from rats on a low-protein diet was 1.4 times that found in rats maintained on a high-protein diet. Possible ways in which the microvillus membrane structure of enterocytes, enzyme activity and the ability to transport amino acids might be controlled are discussed. Relative estimates are also made of the probable effects that changes in diet will have on the capacity of the intestine to digest and absorb nutrients.

An improved model of the distribution and metabolism of progesterone and 20 alpha-dihydroprogesterone in sheep.

When [3H]progesterone is infused intravenously into ewes, blood 20 alpha-dihydroprogesterone (20 alpha-diHP) becomes labelled and the changes in [3H]20 alpha-diHP activity with time are clearly related to that of [3H]progesterone. Concentrations of 20 alpha-diHP in blood have now been estimated for these experiments. During the infusion, the specific radioactivity of 20 alpha-diHP at steady state was only 53% of the specific radioactivity of progesterone, indicating that 20 alpha-diHP was produced other than by C-20 reduction of secreted progesterone. The change in blood concentration of 20 alpha-diHP during pregnancy in ewes suggests that the placenta is its source. [3H]20 alpha-dihydroprogesterone was infused into pregnant ewes and the specific radioactivity of 20 alpha-diHP measured during and after infusion. Together with information from earlier experiments when [3H]progesterone was infused, there is now sufficient data to estimate, without constraint, the parameters of a four-pool model describing the distribution and metabolism of progesterone and 20 alpha-dyhydro-progesterone in sheep.

Dissipation of amino acid concentration gradients within rabbit ileal enterocytes.

The distribution of alanine, lysine and methionine within the cytoplasm of functionally mature enterocytes in rabbit ileum was measured by autoradiography after a short period of contact with tritiated substrate. Pronounced intracellular concentration gradients were noted for alanine and lysine, the concentration of these amino acids in the apical part of the enterocyte being 2- to 3-times that found near the base of the cell. No such concentration gradient was seen for methionine. Subsequent superfusion of the mucosal surface of the tissue with substrate-free medium caused intracellular concentration gradients for alanine and lysine to disappear. There was also a decrease in the enterocyte content of all three amino acids unassociated with backflux into the intestinal lumen. The ease with which intracellular concentration gradients for alanine and lysine can be manipulated is used as an argument against the possibility that their creation results from selective attachment to cytoplasmic structures in the apical part of the enterocyte.

Distribution of transported amino acid within rabbit ileal mucosa.

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.

Amino acid efflux from rabbit ileal enterocytes.

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.

Distinguishing transport systems having overlapping specificities for neutral and basic amino acids in the rabbit ileum.

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.

Stoichiometry versus coupling ratio in the cotransport of Na and different neutral amino acids.

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 acids (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 KA, the apparent dissociation constant of amino acid from the Na-dependent carrier in the absence of Na, and the ratio kappa 1/kappa 2, where kappa 1 and kappa 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 KA for methionine is 3.6 +/- 1.1 mM and the kappa 1/kappa 2 ratio is 1.01 +/- 0.07. The constant coupling ratio value for 1 for alanine indicates that the value for KA is extremely high or that the kappa 1 value is extremely low.

Delayed development of amiloride-sensitive sodium transport in lamb distal colon.

1. Na transport has been measured in vitro across distal colons taken from fetal and early post-natal lambs. 2. The mucosal to serosal Na flux fell by half during the first three weeks of post-natal life. Similar falls were recorded for measured short-circuit current and open-circuit voltage. The net transport of Na, measured isotopically at all stages of development, corresponded approximately to that calculated from measurements of short-circuit current. 3. The serosal to mucosal flux of Na was approximately half the mucosal to serosal flux at all stages in development. 4. Amiloride affected neither the short-circuit current nor the mucosal to serosal flux of Na measured during the first five days of post-natal life. The short-circuit current of an increasing proportion of distal colons (30-70%) began to show some sensitivity to amiloride during the second to third week of post-natal life. Sensitivity of the short-circuit to inhibition by amiloride appeared to be fully developed in the 8-week-old lamb colon. 5. The serum concentration of aldosterone was high at birth. It then fell rapidly to levels approaching those found in fetuses in the last week of pregnancy. The serum concentration of cortisol was two orders of magnitude higher than for aldosterone. Changes in cortisol concentration during development paralleled those for aldosterone. 6. There appears to be no aldosterone-induced change in Na transport across lamb distal colon during the first week of post-natal development. Comparisons are made between this situation and that found in the neonatal pig, where aldosterone acts to increase Na reabsorption immediately after birth. Possible reasons for this species difference are discussed.

Animal variation in alanine uptake by rabbit ileal mucosa.

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.

A sodium-indpendent low affinity transport system for neutral amino acids in rabbit ileal mucosa.

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.

Two-carrier influx of neutral amino acids into rabbit ileal mucosa.

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.

Aldosterone induced changes in colonic sodium transport occurring naturally during development in the neonatal pig.

1. Serum concentrations of aldosterone in later fetal, 3-6 week old and adult pigs are of the order of 300 pg ml.-1. This increases to about 2000 pg ml.-1 in the period immediately after birth. 2. Canrenoate injected into pigs from birth onwards stops the increase in colonic short-circuit current, seen to take place normally during early postnatal development. Amiloride has little or no effect on the short-circuit current of colons taken from canrenoate injected pigs. 3. Canrenoate stops the post-natal increase in colonic Na influx (and therefore net transport) seen to occur under normal conditions. 4. There is in the neonatal pig distal colon a portion of Na transport which appears to be resistant to inhibition by amiloride or canrenoate. 5. There is a second portion of Na transport, increasing in importance as the piglets become older, which is electrogenic and which is electrogenic and which is inhibited by prior injection of canrenoate. It is assumed that this fraction of Na transport is influenced by aldosterone. 6. There is a third part of Na transport, maximal in colons taken from one day old animals, which appears to be non-electrogenic. This is also blocked by prior injection of canrenoate. 7. The physiological relevance of these findings is discussed.

Progesterone and 20 alpha-dihydroprogesterone in sheep: a model of their distribution and metabolism.

The rates of metabolism of progesterone and 20alpha-dihydroprogesterone (20alpha-diHP) in sheep have been measured during and after the infusion of tracer amounts of [3H]progesterone. There were significant differences in the blood concentration of [3H]progesterone between experiments, but these were not attributable to the stage of gestation or to the difference between pregnant and non-pregnant animals. The mean (+/- S.E.M.) metabolic clearance rate of progesterone was 3-277 +/- 0-239 litres blood/min. The simplest model of the distribution of progesterone was one containing two pools, V1[P] and V2[P], where [p] is the blood concentration of progesterone, and in 23 experiments on 7 sheep the mean pool dimensions were 7-8[P] mug and 70[P] mug, respectively. This model was developed to include the formation of 20alpha-diHP from progesterone. Progesterone appeared to be the major source of 20alpha-diHP, though this did not seem to be an obligatory metabolite. The parameters obtained gave comparably low residual deviations for both labelled steroids and were consistent with other observations made on progesterone clearance.