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Biomedical subjects

C A Boyd

Publications and source records attributed to C A Boyd.

At least 19 recordsLinked to original sources
PubMed1992

Substrate-specific differences in the rate of bile acid carrier reorientation: studies on human placental basal vesicles.

The initial rate of transport of the bile acid glycocholic acid (GCA) has been measured in influx and efflux across placental basal membrane vesicles, and the mechanism of inhibition of its transport by the analogue taurochenodeoxycholic acid (TCDCA) analysed kinetically. This analogue, although trans-stimulating GCA efflux, inhibits influx in a way which does not depend upon substrate concentration; moreover, its potency as an inhibitor is markedly influenced by whether it is placed on one or on both sides of the vesicles membrane. These findings can be accounted for by postulating that both GCA and TCDCA are translocated through the carrier, but that the rate of loaded carrier reorientation is higher than that of the free carrier only when loaded with TCDCA and not with GCA.

Bile Acids and Salts↗
PubMed1992

Activation of cationic amino acid transport through system y+ correlates with expression of the T-cell early antigen gene in human lymphocytes.

Lysine influx (2 microM) into activated human B and T lymphocytes through transport systems y+ and y+L was measured. In activated T cells very substantial activation of system y+ was detected; system y+L was also activated in these cells but with a slower time course and to a smaller extent. No stimulation of either system was found in activated B cells. The time course of activation of system y+ precisely matched the expression of the T cell early antigen gene described by MacLeod et al, 1990. The functional significance of these observations with respect to L-arginine transport and nitric oxide synthesis in activated T lymphocytes is discussed.

Amino Acids↗
PubMed1992

Identification of a new transport system (y+L) in human erythrocytes that recognizes lysine and leucine with high affinity.

1. The effect of neutral amino acids on the transport of L-lysine across the human erythrocyte membrane was studied. 2. All neutral amino acids tested (range 0.3-5 mM) inhibit the influx of L-[14C]lysine (1 microM). The inhibition pattern is biphasic, and tends to reach a maximum at approximately 50% of the original flux. The concentrations that give 25% inhibition are (mM): L-cysteine (2.7), L-alanine (1.3), L-serine (0.9), L-isoleucine (0.6), L-phenylalanine (0.35), L-methionine (< 0.3), L-leucine (< 0.3). L-lysine and L-arginine completely inhibit the rate at the highest concentration. 3. These results can be explained by assuming that L-lysine transport occurs through two independent transporters that differ in their affinity for neutral amino acids. A detailed kinetic analysis of the effect of L-leucine on L-lysine entry is consistent with this hypothesis. 4. Using a new experimental strategy, the substrate and inhibitor transport parameters for the two systems were determined. The half-saturation constants for lysine (+/- S.E.M.) are found to be: KmA, 0.014 +/- 0.002 mM and KmB, 0.112 +/- 0.017 mM. The maximum rates differ by a factor of 8.2 (VmaxB/VmaxA). The leucine inhibition constants are: KiA, 0.022 +/- 0.003 mM and KiB, 30.36 +/- 7.9 mM. If the sodium in the incubation medium is replaced by potassium, the apparent affinity for leucine (1/KiA) is reduced approximately 30-fold. 5. The maximum inhibition caused by leucine decreases as the lysine concentration is raised, showing that leucine acts upon the higher affinity system. 6. When added to the trans side, L-leucine, L-phenylalanine and L-isoleucine do not cause inhibition, but stimulate the flux by approximately 30%. This indicates that these analogues are also transported. 7. In conclusion, in the concentration range 1-100 microM, lysine crosses the red cell membrane through two distinct transport systems, one of which recognizes both neutral and cationic amino acids with high affinity.

Amino Acids↗
PubMed1992

Pathways for glucose transport in type II pneumocytes freshly isolated from adult guinea pig lung.

Previous in vivo studies of sugar transport across the mature pulmonary epithelium have provided evidence for the existence of a specific phlorizin-inhibitable, sodium-dependent transport process for D-glucose, although no direct evidence for the cellular location of this transport system in fresh cells has been shown to date. With the use of elastase digestion and lectin agglutination, a pure preparation of type II alveolar epithelial cells was isolated from adult guinea pig lung. This preparation always contained >90% type II cells and typically showed approximately 85% cell viability 2-3 h after the isolation procedure had begun. At 37 degrees C, cells showed specific [3H]phlorizin binding that was attenuated by D-glucose and completely abolished by sodium replacement. Substantial accumulation of the hexose [14C]methyl alpha-D-glucopyranoside (14C-labeled AMG), a substrate specific for the sodium-dependent glucose cotransporter was found in the presence of extracellular sodium; this accumulation above equilibrium was abolished on removal of sodium, addition of phlorizin, or in the presence of a saturating concentration (69 mM) of D-glucose. The apparent inhibition constant (Ki) for glucose inhibition of AMG uptake was 0.4 mM and for phlorizin, 0.5 microM. The Hill plot of sodium activation of AMG uptake gave a coefficient of 2.8, suggesting cooperativeness between sodium and AMG transport. 3-O-[14C]methyl-D-glucose (3-OMG) transport was also blocked by phlorizin. Phloretin, in the presence of phlorizin, slowed the initial rate of entry but did not affect the equilibrium that was attained in the presence of phlorizin alone.(ABSTRACT TRUNCATED AT 250 WORDS)

3-O-Methylglucose↗
PubMed1990

Whole-cell K+ currents in type II pneumocytes freshly isolated from rat lung: pharmacological evidence for two subpopulations of cells.

The patch clamp technique was used to record whole cell K+ currents in type II pneumocytes freshly isolated from adult rats. Depolarizing voltage steps evoked outward K+ currents which were distinguished into low and high threshold types, only one type being apparent in any one cell. Low-threshold (LT) currents were activated at test potentials of -40 mV to -20 mV and were reduced in amplitude by 20 mM tetraethylammonium (TEA). High-threshold (HT) currents were activated only at test potentials positive to -20 mV and current noise was always greater than for LT currents. HT currents were also significantly more sensitive than were LT currents to block by TEA. Quinine (1 mM) blocked LT currents reversibly at all activating test potentials. HT currents were also reversibly blocked by 1 mM quinine, but in a voltage-dependent manner, the degree of block increasing with increasing test potential. 4-Aminopyridine (2 mM) further distinguished the two current types: it was virtually without effect on HT currents but caused large reductions in LT current amplitudes, apparently by acting on the open channels underlying this current. These data clearly distinguish type II pneumocytes into two subpopulations and suggest that they may play separate roles in the functioning of the intact alveolar epithelium.

4-Aminopyridine↗
PubMed1990

Characterization of amino acid transport systems in human placental basal membrane vesicles.

The amino acid transport systems have been characterized in basal membrane vesicles prepared from human full-term placental syncytiotrophoblasts. Transport of amino acids across basal membranes occurred via passive diffusion and Na(+)-independent and Na(+)-dependent carrier-mediated systems. Passive diffusion was responsible for a substantial fraction of transport. L-Glutamate and alpha-(methylamino)isobutyrate were transported only Na(+)-independently, while the transport of L-alanine was dependent solely on an Na+ gradient from the outside to the inside of the vesicles. L-Methionine, L-leucine, glycine and L-proline transport were supported by both Na(+)-independent and Na(+)-dependent systems. L-Lysine transport was decreased in the presence of cations, an inwardly directed Na+ gradient was much more effective than a K+ gradient at slowing L-lysine transport. A cross-inhibition analysis of these amino acids indicates that at least three Na(+)-independent and five Na(+)-dependent carrier-mediated systems exist in the human placental syncytiotrophoblast basal membranes. One Na(+)-independent system interacts with all substrates tested. Another Na(+)-independent system carries glycine, L-methionine, L-leucine and L-lysine; it is sensitive to L-glutamate, but not to L-proline or alpha-(methylamino)isobutyrate. The third system is selective for L-lysine, which is inhibited by L-methionine, glycine and L-leucine, but inaccessible to L-glutamate, L-proline and alpha-(methylamino)isobutyrate. One Na(+)-dependent system carries L-alanine, glycine, L-methionine and L-leucine, and it is sensitive to L-proline. The second system mediates transport of L-alanine, glycine, L-methionine and L-proline, but is not sensitive to L-leucine. The third system carries L-alanine, glycine and L-proline, and is inaccessible to L-methionine and L-leucine. The fourth system is responsible for L-methionine and L-leucine; it is sensitive to L-alanine and glycine, but not to L-proline. The fifth system is selective for L-proline.

Amino Acids↗
PubMed1990

Transport of amino acids by the human placenta: predicted effects thereon of maternal hyperphenylalaninaemia.

Brush border and basal plasma membrane vesicles prepared from normal term human placental syncytiotrophoblast have been used to study amino acid transport. Such studies are reviewed and novel results presented which confirm that saturation of placental transport by phenylalanine is unlikely to limit delivery of this amino acid to the fetus even with grossly raised maternal concentrations. Such raised maternal levels of phenylalanine are, however, likely to severely embarrass the delivery to the fetus across the placental brush border membrane of L-tyrosine and, to a lesser extent, of L-tryptophan. Reasons for thinking that this may be relevant to the fetal damage found in maternal PKU are discussed.

Female↗
PubMed1990

Bile acid transport by basal membrane vesicles of human term placental trophoblast.

The aim of this work was to investigate the first step in the vectorial translocation of bile acids from the fetus to the mother, which is the transfer across the basal (i.e., fetal-facing) plasma membrane of the trophoblast. Thus, the uptake of [14C]taurocholate by basal plasma membrane vesicles obtained from normal human term placentas was studied. Taurocholate retention into vesicles was studied using a rapid filtration technique that was modified to reduce the taurocholate binding to the filters and to the external surface of the vesicles. Using 100 mumol/L substrate, the membrane vesicles showed a temperature-dependent, Na(+)-independent transport of taurocholate into an osmotically reactive intravesicular space. The initial rate of taurocholate influx in the presence of 100 mmol/L KNO3 followed saturation kinetics (apparent Km for taurocholate = 670 +/- 128 mumol/L; Vmax = 1.86 +/- 0.28 nmol/mg protein.60 s at 37 degrees C). Over the 6.9-7.9 pH range neither internal nor external pH nor inward nor outward proton gradients affected the uptake of taurocholate. When the electrical potential difference across the basal membrane was manipulated by external anion replacement (Cl-, SCN-, SO4(2-), or NO3-) or by valinomycin-induced K(+)-diffusion potential (vesicle inside negative), taurocholate uptake was not significantly modified. Taurocholate uptake was cis-inhibited in the presence of 1 mmol/L glycocholate, 0.5 mmol/L 4,4'-diisothiocyanostilbene-2,2'-disulfonate and 0.5 mmol/L sulfobromophthalein. However, 1 mmol/L probenecid or 0.5 mmol/L p-aminohippurate had no effect. Moreover, preloading the vesicles with 100 mmol/L HCO3- (but not with 100 mmol/L Cl- or 50 mmol/L SO4(2-) induced a significant enhancement in the initial rate of taurocholate uptake. In summary, these findings provide strong evidence for the presence of an electroneutral transport system for taurocholate in the basal plasma membrane of human chorionic trophoblast. They also suggest that this is likely to be an anion-exchange system.

Basement Membrane↗
PubMed1990

Behavioral and neurochemical changes associated with chronic exposure to low-level concentration of pesticide mixtures.

In order to assess behavioral and neurochemical changes resulting from pesticide exposure, food-restricted male weanling rats were exposed for 90 d to low doses (1 ppb-10,000 ppb range) of individual pesticides (aldicarb, metribuzin, or methomyl) or mixtures of them. During exposure, rats were trained to run a T-maze and tested for spatial discrimination reversal learning. At sacrifice, three brain regions (cortex, hippocampus, and neostriatum) were assayed for the neurotransmitters dopamine, acetylcholine, and serotonin. Animals treated with a mixture of two insecticides and one herbicide were found to have slower speeds in maze-running (motor control) and also had altered levels of choline in their neostriatums. Rats treated with one herbicide compound (metribuzin) took longer to learn on two reversals; this group also had a significantly lower acetylcholine/choline ratio in their hippocampus.

Aldicarb↗
PubMed1990

Human placental L-tyrosine transport: a comparison of brush-border and basal membrane vesicles.

1. The mechanisms responsible for L-tyrosine transport at both the maternal-facing and fetal-facing surfaces of the human full-term placenta have been studied using isolated brush-border and basal membrane vesicles under conditions where a direct comparison of the transport properties of the two membranes can be made. 2. Brush-border vesicle uptake of L-tyrosine was substantially into an osmotically active space. Transport was Na(+)-independent, N-ethylmaleimide-sensitive (half-maximal inhibition, Ki = 1.1 mM), and insensitive to pH over the range 5.5-8.5. The initial rate of brush-border L-tyrosine uptake as a function of concentration showed saturation and obeyed Michaelis-Menten kinetics with Michaelis constant (Km) and maximum velocity (Vmax) values of 54.2 microM and 1.28 pmol (mg protein)-1 s-1, respectively. Influx of L-tyrosine was stereospecific and was virtually completely abolished by L-phenylalanine, L-tryptophan, L-leucine or by 2-aminobicycloheptane-2-carboxylic acid. These properties suggest that system L is responsible for brush-border L-tyrosine transport. 3. Basal membrane transport of L-tyrosine was more complex and uptake was slower than that found in the brush border. Although, as in the brush-border membranes, uptake was completely Na(+)-independent, N-ethylmaleimide was a less effective inhibitor, there was stimulation of transport at more alkaline pH and uptake did not show marked stereospecificity. An apparent Km of 168.9 microM and a Vmax of 0.31 pmol (mg protein)-1 s-1 were calculated for basal L-tyrosine transport. There was clear inhibition by L- and D-tyrosine, L-phenylalanine and L-tryptophan. 2-Aminobicycloheptane-2-carboxylic acid was not as effective. 4. These findings suggest the existence of non-identical carrier-mediated transport systems for L-tyrosine in brush-border and basal membranes. Brush-border transport resembles that by system L; L-tyrosine transport at the basal membrane may be via system t.

Amino Acids↗
PubMed1979

Movements of monosaccharides between blood and tissues of vascularly perfused small intestine.

1. A method involving the analysis of pulse transients of the vascular concentrations of test sugar and extracellular marker has been used to study the movements of non-metabolized sugars between the cells and vascular fluid of the vascularly perfused small intestine of R. ridibunda. Reasons are given for supposing that the method does properly measure the net entry of sugars into a cellular compartment that includes at least the epithelium. 2. It is found that while the glucose analogues, 3-O-methyl-D-glucose (3MG) and 2-deoxy-D-glucose (2)DG) are able to enter a cellular compartment when they are added to the vascular bed, alpha-methyl-D-glucoside (alpha MG) is able to enter the compartment only at a very slow rate from the vascular bed. In contrast, 3MG and alaph MG are well absorbed from the lumen whereas the inward permeability of 2DG across the lumen face of the epithelium is very low and, unlike 3MG and alpha MG, is not influenced by the presence in the lumen of other transported sugars. 3. The presence of phlorizin in the intestinal lumen increases the flux of alpha MG and of 3MG in the direction vascular bed--bulk phase of lumen. Reasons are given for supposing that the movement of sugars from the vascular bed into the lumen may involve a cellular route but occurs, at least in part, through a paracellular, extracellular route. 4. The exit of monosaccharides that have been loaded previously into the epithelium either from the lumen or from the vascular bed has been investigated. 3MG washes out rapidly into the vascular bed and the exit is stimulated by the addition of D-glucose to the intestinal lumen and by the addition of 2DG, but not alpha MG, to the arterial infusate. In contrast, alpha MG, loaded into the epithelium from the lumen, washes out of the cells only slowly into the vascular bed, so that even with high rates of vascular perfusion alpha MG accumulates within the tissue. The sustained accumulation of alpha MG implies that not only is the permeability for exit into the blood restricted for this sugar, but also the permeability is low across the brush border in an outward direction, cell to lumen ('lobster pot effect'). The wash-out of 3MG into the vascular effluent is sufficently rapid that only in the absence of vascular perfusion is 3MG accumulated within the tissue. 5. The contrasting properties of the monosaccharide transport systems accessible from the intestinal lumen and the vascular bed respectively are discussed in relation to the problems of epithelial transport of monosaccharide.

Animals↗
PubMed1979

Studies on amino acid inhibition of monosaccharide exit from anuran small intestinal epithelium.

1. The effect of the addition of amino acids to the intestinal lumen upon the movement of the monosaccharide alpha-methyl-D-glucopyranoside (alpha MG) from the preloaded epithelium into the blood and into the lumen of the vascularly perfused frog small intestine has been studied. 2. The neutral hydrophobic amino acids tryptophan, leucine, phenylalanine, tyrosine, isoleucine, valine, norleucine and cycloleucine all rapidly inhibit the exit of alpha MG out of the epithelium into the vascular bed. They stimulate backflux of the sugar from the epithelium into the lumen to a very much smaller extent. 3. L-Leucine is a more effective inhibitor of alpha MG exit into the blood than is D-leucine. Near-maximal inhibition of alpha MG exit is seen with 10 mM-L-leucine in the intestinal lumen. 4. The addition of leucine (10 mM) to the lumen of the intestine preloaded with alpha MG approximately halves the rate constant for alpha MG washout into the blood from 12.6 +/- 1.7 (4) x 10(-3) to 5.5 +/- 1.4 (4) x 10(-3) min-1, without appreciably altering the pool of monosaccharide in the tissue. The inhibitory effect of L-leucine upon alpha MG exit into the blood is not abolished by the presence of phlorizin (5 x 10(-5) M) in the intestinal lumen. 5. The complex pattern of inhibition of alpha MG transfer from the lumen to the blood observed upon the addition of L-leucine to the lumen is consistent with the finding that the amino acid inhibits the exit of the monosaccharide out of the epithelium into the blood in addition to any inhibitory effect upon sugar entry across the brush border. 6. It is suggested that alpha MG may be a substrate for a proposed transport system for neutral hydrophobic amino acids which, it is suggested, is present in the basolateral membrane of the epithelial cell.

Amino Acids↗
PubMed1979

Separation of the microvillous (maternal) from the basal (fetal) plasma membrane of human term placenta: methods and physiological significance of marker enzyme distribution.

Plasma membranes from normal, full-term human placental trophoblast have been isolated by a new procedure. The method depends upon isopycnic zonal centrifugation using linear sucrose/Ficoll density gradients. Enrichment of plasma membrane marker enzymes with respect to trophoblast homogenate is found in two distinct peaks (designated B and D) of the fractionated effluent recovered from the rotor. Fraction B is enriched with membrane-bound alkaline phosphatase and 5'-nucleotidase, but not with (Na+, K+)-ATPase of F(-)-stimulated adenylate cyclase. It is suggested that this material is derived from the maternal-facing microvillous plasma membrane. Fraction D, enriched with (Na+, K+)-ATPase, F(-)-stimulated adenylate cyclase and, to a smaller extent, with 5'-nucleotidase and alkaline phosphatase is, by exclusion, proposed to be derived from the fetal-facing basal plasma membrane. Both plasma membrane fractions are shown to be free of appreciable contamination, using specific markers for endoplasmic reticulum, mitochondria, nuclei and lysosomes. The separation of the two membrane fractions is shown to depend both upon these membranes forming closed vesicles during homogenization and upon the buoyant densities of such vesicles differing in such a way that microvillous plasma membranes band at a lower density than basal plasma membranes. No separation of the membranes is achieved in gradients in which the vesicles are collapsed.

Alkaline Phosphatase↗
PubMed1978

Effects of vascular perfusion on the accumulation, distribution and transfer of 3-O-methyl-D-glucose within and across the small intestine.

1. Factors affecting the transfer of the non-metabolized, ;actively transported' sugar, 3-O-methyl-D-glucose (3MG) across the small intestinal epithelium have been examined in vascularly perfused anuran intestine. Transfer has been studied during absorption in the steady state, and also during the period of transition from one steady state to another.2. During the steady state, the rate of absorption of 3MG from the intestinal lumen is equal to the rate of appearance in the portal venous effluent; this rate of transfer is to a small, but significant, extent directly related to the rate of arterial perfusion. With phlorizin in the intestinal lumen, transfer across the epithelium is reduced to very low rates which are independent of the rate of vascular perfusion.3. The apparent size of the tissue pool(s) of 3MG that have to be loaded to achieve the steady state rate of transfer are less than those that unload into the vascular bed after 3MG is removed from the intestinal lumen. This ;up-down asymmetry' is abolished when phlorizin is present in the intestinal lumen during the unloading phase.4. When 3MG is abruptly removed from the intestinal lumen after the tissue has been previously loaded with the sugar, the rate of washout into the vascular bed can be described by the sum of two exponential terms. The two terms differ in that the rate constant of the earlier ;fast' term is sensitive to the rate of vascular perfusion, while the later, ;slow', rate constant is insensitive to flow rate. The total quantity of 3MG that can be unloaded from the tissue into the portal venous effluent is decreased when phlorizin is present in the intestinal lumen during the unloading phase.5. Absorption from the lumen of the anuran intestine continues while the mesenteric circulation is interrupted. An estimate of the concentration of 3MG during the period of vascular stoppage can be made from the quantity recovered in the portal venous effluent when vascular perfusion is reinstituted (;vascular stop-flow'). The extent of the accumulation depends upon the duration of the vascular stoppage and the presence of Na ions in the intestinal lumen is essential for accumulation to occur.6. The findings are discussed in relation to the transfer of 3MG between various possible compartments in the tissue during absorption. Evidence is presented that a re-uptake of previously absorbed 3MG may occur across the brush border membrane. Such a recycling of 3MG across the epithelium implies that the apparent unidirectional fluxes measured across the epithelium between the bulk phase of the lumen and the blood may underestimate the size of fluxes across the epithelium at the cellular level.

Animals↗