Search PubMed⌕ Search

Biomedical subjects

A Moran

Publications and source records attributed to A Moran.

At least 127 records · Page 7Linked to original sources
PubMed1988

Regulation of Na+-H+ exchange in cultured opossum kidney cells by parathyroid hormone, atrial natriuretic peptide and cyclic nucleotides.

The activity of Na+-H+, exchange was studied in a cultured cell line derived from opossum kidney (OK cells). The activity of the exchanger was measured either as the amiloride (2 mM) inhibitable 22Na flux in acid-loaded cells, or as the Na+-dependent and amiloride-sensitive recovery of intracellular pH (pHi) from an acid load. Initial rates of tracer flux were analyzed in confluent monolayers while changes in pHi were evaluated in suspensions of trypsinized cells which had been loaded with 2',7'-bis(2-carboxyethyl)-5,6-carboxyfluorescein. Both 8-bromo-cAMP and 8-bromo-cGMP inhibit the activity of the exchanger in a dose-dependent manner. Maximal inhibition due to 8-bromo-cAMP was about 50% and was attained with 0.75 mM of the cyclic nucleotide. Parathyroid hormone (10(-9)-10(-7) M) and atrial natriuretic peptide (10(-7) M) also inhibit the activity of the exchanger. By measuring the rate of Na+-dependent pHi recovery from different starting pHi values, evidence was obtained for a cyclic nucleotide-dependent decrease in the response of Na+-H+ exchange to intracellular acidification. We conclude that cAMP and cGMP are intracellular messengers in the hormone-dependent regulation of Na+-H+ exchange activity in renal epithelial cells.

Amiloride↗
PubMed1988

High affinity phlorizin binding to the LLC-PK1 cells exhibits a sodium:phlorizin stoichiometry of 2:1.

The phlorizin binding properties of luminal membrane vesicles isolated from the LLC-PK1 cells, a continuous epithelial cell line derived from pig kidney, are studied. Scatchard analysis of this binding indicates the existence of a single high affinity sodium-dependent site with KD = 0.4 microM at 266 mM sodium. The specificity properties of this site indicate that it represents the binding of phlorizin to the hexose binding site of the sodium-dependent D-glucose transporter previously identified in this cell line. Both phlorizin equilibrium binding and the rate of phlorizin binding were found to be sigmoidal functions of sodium concentration. A Hill analysis of these data was consistent with a sodium:phlorizin stoichiometry of 2:1 in good agreement with the sodium:glucose stoichiometry already established in these cells. Phlorizin dissociation was also found to be sodium-dependent. On the basis of the phlorizin binding data presented here, a number of models of the binding of phlorizin and sodium to the transporter can be excluded. An analysis of a random binding model consistent with the data is presented. The significance of the LLC-PK1 sodium-dependent D-glucose transporter as a model system for related renal and intestinal transporters is discussed.

Animals↗
PubMed1988

Sodium channels in membrane vesicles from cultured toad bladder cells.

Electrical potential-driven 22Na+ fluxes were measured in membrane vesicles prepared from TBM-18(c123) cells (a clone of the established cell line TB-M). Fifty to seventy percent of the tracer uptake in vesicles derived from cells that were cultivated on a porous support were blocked by the diuretic amiloride. The amiloride inhibition constant was less than 0.1 microM, indicating that this flux is mediated by the apical Na+-specific channels. Vesicles prepared from cells that were not grown on a porous support exhibited much smaller amiloride-sensitive fluxes. Two Ca2+-dependent processes that down-regulate the channel conductance and were previously identified in native epithelia were found in the cultured cells as well. Vesicles isolated from cells that were preincubated with 5 X 10(-7) M aldosterone for 16-20 h exhibited higher amiloride-sensitive conductance than vesicles derived from control, steroid-depleted cells. Thus membrane derived from TBM-18(c123) cells can be used to characterize the epithelial Na+ channel and its hormonal regulation.

8-Bromo Cyclic Adenosine Monophosphate↗
PubMed1987

Sodium-hydrogen exchange system in LLC-PK1 epithelium.

Sodium influx into LLC-PK1 cells has been characterized. The main amiloride-sensitive pathway for sodium entry through the apical membrane in this cell line is sodium-hydrogen exchange with an apparent Km for sodium of 19 mM. Influx is pH dependent and is amiloride sensitive with K1/2 of 30 microM. Inhibition of the sodium transport by protons (at 1 mM external sodium) is consistent with an interaction of H+ ions at a single site having an apparent pKH of 7.2. These data are similar to those reported previously for brush border membrane (BBM) isolated from kidney proximal tubule. However, in contrast to previously published reports, H+ does not compete with amiloride in blocking Na+ influx and exhibits a mixed inhibition with sodium ions. The latter, together with a direct and independent assessment of amiloride and sodium interaction, indicates that amiloride does not compete with sodium for the same site. Possible explanations for the discrepancy in the literature concerning the interaction of Na+, H+, and amiloride with the Na+-H+ exchanger and the characteristics of the Na+-H+ exchange system in LLC-PK1 are discussed.

Amiloride↗
PubMed1986

Effect of gamma radiation on sodium channels in different conformations in neuroblastoma cells.

We studied the dose-response relationship between gamma radiation and batrachotoxin-stimulated sodium influx in neuroblastoma cells in tissue culture. We also tested the hypothesis that changes in sodium channel conformation may alter the radiosensitivity of the channel. We found that gamma radiation inhibited toxin-stimulated 22Na uptake at doses beyond a threshold of 200-300 Gy. No effects were seen following doses below 100 Gy. This inhibition of sodium permeability was seen when the cells were irradiated with sodium channels in the closed or inactivated, nonconducting states. However, when the channels were in the toxin-opened, conducting state, gamma radiation had no effect at doses up to 2000 Gy. Our results support earlier electrophysiological studies that showed that high doses of ionizing radiation are required to produce a measurable decrease in sodium permeability. In addition, our data suggest that by changing the sodium channel conformation, batrachotoxin appears to alter radiosensitive chemical bonds in the gating or ion-conducting portion of the channel.

Batrachotoxins↗
PubMed1986

Role of cell replication in regulation of Na-coupled hexose transport in LLC-PK1 epithelial cells.

The glucose concentration in growth medium has been shown to regulate the number of sodium-coupled glucose transporters in LLC-PK1 epithelial cells. Epithelia grown in high concentrations of glucose express fewer transporters than epithelia grown in low concentrations of glucose. In the present work, the effect of a dose of ionizing radiation sufficient to block the incorporation of thymidine was examined in order to gauge the importance of cell replication in the hexose transport regulatory process. The low rate of thymidine incorporation in the plateau phase was completely eliminated by ionizing radiation. Under conditions of irradiation that completely blocked thymidine incorporation, down-regulation, namely the loss of alpha-methylglucoside-concentrating capacity, brought about by switching the epithelium from low to high glucose-containing medium, is independent of the irradiation and therefore most likely is also independent of cell replication. In contrast, the up-regulatory phenomenon is strongly impaired by radiation. This impairment may be due to specific radiation impairment of gene expression necessary for the up-regulatory process. It is apparent from the dose-response data that up-regulation is not inhibited by irradiation in a simple manner and is not inhibited at the same radiation dose as cell replication.

Animals↗
PubMed1986

A sodium-hydrogen exchange system in isolated apical membrane from LLC-PK1 epithelia.

We have monitored transmembrane pH gradients using acridine orange fluorescence quenching and traced Na+ flux to study the properties of Na+-H+ exchange in apical membrane vesicles isolated from LLC-PK1 epithelia. The membranes have low conductance for Na+, H+, and K+ ions. An outwardly directed K+ gradient in the presence of valinomycin and carbonyl cyanide p-trifluoromethoxyphenyl hydrazone produced intravesicular acidification. This pH gradient was collapsed by addition of extravesicular Na+ or Li+ ions but not by tetramethylammonium. Amiloride (10(-4) M) inhibited the effect of both Na+ and Li+. An outwardly directed Na+ gradient stimulated H+ influx, which was also inhibited by 10(-4) M amiloride. Membrane short-circuit conditions affected neither Na+ nor H+ flux, consistent with transport mediated by an electroneutral process. The interaction of amiloride and sodium is consistent with noncompetitive inhibition with Ki = 100 +/- 10 microM for amiloride and an apparent Km for Na+ of approximately 20 mM. This finding is in agreement with previous studies of intact LLC-PK1 epithelia but differs from observations in brush-border membrane vesicles isolated from kidney proximal tubule in which competitive and mixed inhibition have been reported. These observed differences can be reconciled if two types of Na+-H+ exchange systems exist along the nephron, one with competitive and the other with noncompetitive inhibition, and if only the latter is expressed in the homogeneous cultured cells.

Animals↗
PubMed1986

Effect of radiation on the regulation of sodium-dependent glucose transport in LLC-PK1 epithelial cell line: possible model for gene expression.

Low concentrations of glucose induce cultured kidney epithelial cells (LLC-PK1) to produce hexose transport proteins. We have investigated the effects of ionizing radiation on this induction process in plateau-phase cultures. The induced production of hexose transporters, requiring approximately 6 to 9 days for complete expression, can be inhibited by irradiation during the first 4 days. After the fourth postinduction day, radiation sensitivity decreases with almost no radiation effect on the induction of hexose transport apparent by the sixth day of the induction period. The D0 value associated with the induction block is approximately 25 Gy, a value which is considerably greater than that necessary to inhibit cell replication. Hexose transport, itself resistant to ionizing radiation at doses in excess of 100 Gy, is sensitive to cycloheximide throughout the induction period. The sensitivity to cycloheximide decreases during the last 2 days of the induction period, approximately 1 day after the reduction in radiosensitivity. Based on these properties hexose transport may be a convenient model for the study of radiation effects upon gene expression in this cell line.

Animals↗
PubMed1985

Regulation of expression of the sodium-coupled hexose transporter in cultured LLC-PK1 epithelia.

A variety of techniques have been used to study the sodium-coupled hexose transporter in epithelia formed by LLC-PK1 cells. The expression of the transporter is affected by the density and age of the culture and by the concentration of glucose in the growth medium. Sodium-coupled hexose transport appears as the epithelium becomes confluent and increases further as the epithelium matures. The increased transport is associated with increased transport in apical plasma membrane vesicles. Epithelia grown in medium containing 5 mM glucose express more transporters than epithelia grown in medium containing 25 mM glucose. The increase in transport is not the result of an extracellular signal that is generated as a consequence of the concentration of glucose. The response to different hexoses that are or are not transported on the carrier indicates that it is the metabolism of glucose that acts as the signal for expression of more or fewer transporters. The results are compared to similar studies of the effects of substrate concentration on expression of transporters in cultured fibroblasts and the intestines in situ.

Animals↗
PubMed1984

Hexose regulation of sodium-hexose transport in LLC-PK1 epithelia: the nature of the signal.

We have shown previously that the concentration of glucose in the growth medium regulates sodium-coupled hexose transport in epithelia formed by the porcine renal cell line LLC-PK1. Assayed in physiological salt solution, the ratio of the concentration of alpha-methyl glucoside (AMG) accumulated inside the cell at steady state to its concentration outside, and the number of glucose transporters, as measured by phlorizin binding, was inversely related to the glucose concentration in the growth medium. In this study, using a cloned line of LLC-PK1 cells, we provide evidence that the difference in AMG concentrating capacity is the result of a regulatory signal and not simply due to a selection process where the growth of cells with enhanced glucose transport is favored by low glucose medium or vice-versa. By adding glucose to conditioned medium (collected after 48 hr incubation with cells and therefore containing less than 0.1 mM glucose), we demonstrate that the signal in the growth medium is indeed the concentration of glucose rather than another factor secreted into or depleted from the medium. Fructose and mannose, two sugars not transported by the sodium-dependent glucose transporter, can substitute for glucose as a carbohydrate source in the growth medium and have a modest glucose-like effect on the transporter. Growth in medium containing AMG does not affect the transporter, indicating that the regulatory signal is not a direct effect of the hexose on its carrier but involves hexose metabolism.

Animals↗
PubMed1983

Regulation of sodium-coupled glucose transport by glucose in a cultured epithelium.

Cultured porcine kidney cells (LLC-PK1) form polarized epithelia that transport glucose from apical to basal surface as in the renal proximal tube. The ability of these cells to transport glucose is known to increase as the epithelium forms and matures in culture. We find that epithelia grown in medium containing 25 mM glucose have reduced hexose transport compared to epithelia grown in 5 mM glucose. This difference is not the result of differences in seeding efficiency and can be reversed by changing the concentration of glucose in the growth medium. Increased transport in epithelia grown in 5 mM glucose is the result of increased influx on the sodium-coupled apical membrane transporter rather than changes in efflux. This difference is apparently the result of more apical membrane transporters in epithelia grown in 5 mM glucose. The number of high affinity phlorizin-binding sites is greater in epithelia grown in 5 mM glucose (about 0.8 pmol/10(6) cells) than in 25 mM glucose (about 0.25 pmol/10(6) cells). The increase in the number of glucose transporters induced by the low glucose medium is specific in that there is not a comparable change in activity of marker enzymes (alkaline phosphatase, acid phosphatase, or glucose 6-phosphatase). The nature of the intracellular signal elicited by extracellular glucose remains to be determined.

Animals↗
PubMed1982

Stoichiometric studies of the renal outer cortical brush border membrane D-glucose transporter.

The stoichiometric properties of the renal outer cortical brush-border membrane D-glucose transporter are studied. Experiments which establish the glucose/sodium, glucose/phlorizin and phlorizin/sodium stoichiometries are reported. Three independent method of determining the substrate/activator (glucose/sodium) stoichiometry for coupled transport systems are presented and discussed. One of these, the "Static Head Method," is introduced here for the first time. This type of experiment appears to be more generally applicable than the usual procedure of directly measuring the coupled fluxes of substrate and activator to determine stoichiometric coupling ratios. The results presented in this paper demonstrate that the glucose/sodium/phlorizin stoichiometry of the renal outer cortical brush-border membrane D-glucose transport system is 1:1:1.

Animals↗
PubMed1982

Further studies of proximal tubular brush border membrane D-glucose transport heterogeneity.

The properties of two sodium-dependent D-glucose transporters previously identified in renal proximal tubule brush border membrane (BBM) vesicles are studied. The low-affinity system, found in BBM vesicles from the outer cortex (early proximal tubule), is shown to be associated with the high-affinity phlorizin binding site typically found in renal BBM preparations. The high-affinity system, found in BBM vesicles from the outer medulla (late proximal tubule), is almost two orders of magnitude less sensitive to inhibition by phlorizin and is apparently not associated with high-affinity phlorizin binding. The sodium/glucose stoichiometry of the outer medullary transporter is found to be 2:1 by two independent methods. Previous measurements have established that the stoichiometry of the outer cortical system is 1:1. It is suggested that this arrangement of transporters in series along the proximal tubule enables the kidney to reabsorb glucose from the urine in an energy-efficient fashion. The bulk of the glucose load is reabsorbed early in the proximal tubule at an energetic cost of one Na+ per glucose molecule. Then in the late proximal tubule a larger coupling ratio and hence a larger driving force is employed to reabsorb the last traces of glucose from the urine.

Animals↗