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F V Sepúlveda

Publications and source records attributed to F V Sepúlveda.

At least 73 records · Page 4Linked to original sources
PubMed1985

A rabbit jejunal isolated enterocyte preparation suitable for transport studies.

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.

Alanine↗
PubMed1985

Potassium movements associated with amino acid and sugar transport in enterocytes isolated from rabbit jejunum.

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.

Amino Acids↗
PubMed1984

The distribution of (Na+ + K+)-ATPase along the villus crypt-axis in the rabbit small intestine.

The migration of intestinal epithelial cells from the crypts to the tips of villi is associated with progressive cell differentiation. The changes in Na+-pump levels during migration have been measured in epithelial cells isolated from rabbit small intestine. A significant proportion of ouabain-sensitive (Na+ + K+)-ATPase in the cell homogenates was latent but could be unmasked by detergent treatment. Highest detergent activation was observed in villus cells. The distribution of pumping sites was also assessed by measuring ouabain binding to intact cells. The kinetics of specific binding was consistent with the interaction of the cardiac glycoside with a single population of binding sites with an apparent Kd of around 10(-7) M. Both enzyme assay and ouabain-binding measurements suggest that a 2-3-fold increase in the number of Na+-pumping sites accompanies cell differentiation in rabbit jejunal epithelium. This increase in pumping capacity might be an adaptation of the cells to their absorptive function.

Animals↗
PubMed1984

Localisation of alanine uptake by cultured renal epithelial cells (LLC-PK1) to the basolateral membrane.

Alanine uptake by LLC-PK1 cells has previously been demonstrated to be almost exclusively sodium dependent. We here confirm that when the cells are grown on an impermeable substratum there is a marked fall in uptake as confluence is reached. By applying an autoradiographic technique to visualize transported alanine, it is clear, however, that even in subconfluent cultures there is marked cellular inhomogeneity with regard to uptake, which takes place predominantly in those cells at the periphery of growing islands and not those at the interior. In contrast, when cells are grown on permeable substrata, a uniform distribution of silver grains is found. In two other types of experiment, we found that when confluent cell monolayers on an impermeable support were treated briefly with a chelating agent or suspended by mechanical treatment, there was a marked increase per cell in sodium-dependent alanine uptake and in ouabain-sensitive potassium uptake. We conclude that the apparent fall in alanine uptake as cells reach confluence on an impermeable support is due to masking of transport sites, which are predominantly, if not exclusively, located at the basolateral membrane.

Alanine↗
PubMed1984

Absence of gap junctional complexes in two established renal epithelial cell lines (LLC-PK1 and MDCK).

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.

Animals↗
PubMed1984

Deficiency in intercellular communication in two established renal epithelial cell lines (LLC-PK1 and MDCK).

We have studied the cell-to-cell passage of uridine nucleotides in two renal epithelial cell lines (LLC-PK1 and MDCK) and in porcine aortic endothelial cells (PAE). All three cell types incorporated tritiated uridine. After a 3 h incubation the radioactivity was predominantly in the form of acid-soluble compounds, mainly UTP. Prelabelled LLC-PK1 or MDCK cells were unable to transfer radioactivity to added adjacent, non-labelled cells, whereas PAE cells readily formed communicating intercellular junctions, as judged by autoradiographic analysis after a 3 h co-culture period. Cell-to-cell communication in either of the renal cell lines was not promoted by treatment with dibutyryl cyclic AMP and methylisobutylxanthine. Radioactivity incorporated into the acid-insoluble pool was not available for intercellular transfer, as assessed in experiments in which cells were prelabelled 24 h before co-culture.

Animals↗
PubMed1983

Transport of sugars or amino acids increases potassium efflux from isolated enterocytes.

A technique to isolate epithelial cells from rabbit jejunum using hyaluronidase is described. The cells obtained retained their abilities to accumulate sugars and potassium (86Rb) against concentration gradients. Potassium efflux was monitored using cells preloaded with 86Rb and the rate constant of efflux was seen to increase when actively transported sugars or amino acids are added to the bathing medium. The increase is related to the transport of the non-electrolyte, but not to volume regulatory events.

Amino Acids↗
PubMed1983

Cellular adaptation of amino acid transport following intestinal resection in the rat.

1. The ability of rat ileal enterocytes to take up alanine and lysine before and after proximal resection of the small intestine has been assessed using both autoradiographic and dual-isotope methods of analysis. 2. The length of individual villi was approximately doubled after resection. Alanine uptake measured in the presence or absence of Na, represented per cm2 mucosal surface area, decreased following intestinal resection. 3. Alanine and lysine uptake were confined to villus tip enterocytes in both control and ileal remnants. The net effect of intestinal resection was to increase amino acid uptake calculated per individual villus or per unit length of intestine. 4. Adaptational changes occurring as a result of resection included a shortening of the time needed for enterocytes to reach the stage where they first began to absorb amino acids and a doubling of the rate at which absorption increased during the later stages of enterocyte differentiation. 5. It is suggested that the physiological response to intestinal resection can best be appreciated by studying events taking place within individual enterocytes. The way in which adaptational changes become organized within the mucosa remains to be elucidated.

Adaptation, Physiological↗
PubMed1982

The development of gamma-glutamyltransferase in a pig renal-epithelial-cell line in vitro. Relationship to amino acid transport.

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.

Amino Acids↗
PubMed1982

Cell differentiation and L-ornithine decarboxylase activity in the small intestine of rats fed low and high protein diets.

L-Ornithine decarboxylase activity is higher in enterocytes from rats fed low protein rather than high protein diets. Intestinal cell proliferation rate is 50% higher in rats fed high protein than low protein diets. This is not consistent with a direct role of ornithine decarboxylase in intestinal proliferation. It is shown that ornithine decarboxylase is preferentially associated with differentiating villus cells in intestine from rats fed low protein diets.

Acyltransferases↗
PubMed1982

Relation between sodium-coupled amino acid and sugar transport and sodium/potassium pump activity in isolated intestinal epithelial cells.

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.

Alanine↗
PubMed1982

Characterization of neutral amino acid uptake by cultured epithelial cells from pig kidney.

Two transport systems for neutral amino acids have been characterised in LLC-PK, cells. The first, which transports alanine in a sodium-dependent manner, also mediates alanine exchange and is perferentially inhibited by serine, cysteine, and alpha-amino-n-butyric acid. This system resembles the ASC system in Ehrlich ascites and some other cell types. There is only a small contribution of other systems to alanine uptake. The second, which transports leucine with no requirement for sodium and mediates leucine exchange, is blocked by 2-aminonorbornane-2-carboxylic acid and hydrophobic amino acids. This system is similar to the L system described in other cell types. LLC-PK1 cells retain several other features implying renal proximal tubule origin; our results thus suggest that these transport systems may be involved in the reabsorption of neutral amino acids by the nephron in vivo.

Alanine↗
PubMed1982

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 Acids↗
PubMed1982

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.

Amino Acids↗
PubMed1981

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.

Alanine↗
PubMed1981

Cellular distribution of neutral and basic amino acid transport systems in rabbit ileal mucosa.

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.

Alanine↗