Cystine dimethyl ester reduces the forces driving sodium-dependent transport in LLC-PK1 cells

1992 ◽  
Vol 263 (2) ◽  
pp. C516-C520 ◽  
Author(s):  
A. Ben-Nun ◽  
N. Bashan ◽  
R. Potashnik ◽  
R. Cohen-Luria ◽  
A. Moran
Keyword(s):  
Potassium Concentration ◽  
Dimethyl Ester ◽  
Maximal Velocity ◽  
Intact Cells ◽  
Sodium Gradient ◽  
Sodium Dependent ◽  
Rubidium Influx ◽  
Significant Change ◽  
Dependent Transport ◽  
Intracellular Potassium Concentration

Cystinosis is an inherited metabolic disease characterized by accumulation of lysosomal cystine and renal impairment. In an attempt to better understand the link between cystine accumulation and renal functions, we studied the effects of cystine loading on the Na(+)-H+ antiporter and the sodium pump in renal epithelial cells (LLC-PK1) in culture. Incubation of LLC-PK1 with 1 mM cystine dimethyl ester (CDME) for 48 h caused lysosomal cystine loading and reduced by 22 +/- 2% the maximal velocity of sodium-hydrogen antiport with no significant change in the affinity of sodium for the transporter. Rubidium influx decreased to 46 +/- 5% of control. Ouabain binding experiments revealed a 10% reduction in the number of Na(+)-K(+)-ATPase units in the intact cells. Na(+)-K(+)-ATPase activity in the particulate fraction of the cells homogenate declined to 50 +/- 7.5% of controls. No significant change was observed in the activity of ouabain-insensitive phosphatases. The intracellular concentration of sodium increased from 20.6 +/- 3.7 to 64.8 +/- 10 mM, and potassium concentration decreased from 103 +/- 6 to 80 +/- 13 mM. In addition to the observed reduction in the sodium gradient and in agreement with the reduction in the intracellular potassium concentration, the membrane potential changed from -80.8 +/- 7.5 to -69.9 +/- 7.0 mV. The results suggest that intracellular accumulation of cystine is associated with reduction in the number and the activity of membrane transporters. The consequence of the changes in the activity of Na(+)-K(+)-ATPase is a reduction in the electrochemical forces that drive transport in the renal cells tested.(ABSTRACT TRUNCATED AT 250 WORDS)

Sodium-dependent transport of Pi by an established intestinal epithelial cell line (CaCo-2)

1986 ◽  
Vol 250 (3) ◽  
pp. G323-G330 ◽  
Author(s):  
I. Mohrmann ◽  
M. Mohrmann ◽  
J. Biber ◽  
H. Murer
Keyword(s):  
Cell Line ◽  
Transport System ◽  
Apical Membrane ◽  
Intestinal Cell ◽  
Epithelial Cell Line ◽  
Maximal Velocity ◽  
Intact Cells ◽  
High Sodium ◽  
Sodium Dependent ◽  
Dependent Transport

The uptake of inorganic phosphate (Pi) was analyzed in monolayers and in apical membrane vesicles (AMV) of the established intestinal cell line CaCo-2. AMV, prepared by a MgCl2 precipitation technique, were enriched approximately 10-fold in alkaline phosphatase activity. Pi uptake into intact cells as well as into AMV was specifically dependent on the presence of sodium. In the presence of high sodium concentrations, the apparent Km for Pi was 214 +/- 17 mumol/l in monolayers and 300 +/- 19.7 mumol/l in AMV. Increasing the sodium concentration increased the apparent affinity of the transport system for Pi but hardly affected the maximal velocity (Vmax). At 0.1 mmol/l Pi and pH 7.4, the apparent Km for sodium was approximately 70 mmol/l in intact cells as well as in AMV. The results obtained in both systems suggested the involvement of two sodium ions and one phosphate ion in the transport process. Advancing confluence--independently of the age of the monolayers--reduced sodium-dependent uptake of Pi significantly by a decrease in Vmax, whereas the apparent Km for Pi remained unchanged. It is concluded that the apical membrane of CaCo-2 cells contains a sodium-dependent transport system for Pi.

Effects of chlorpromazine on Na+-K+-ATPase pumping and solute transport in rat hepatocytes

1987 ◽  
Vol 253 (5) ◽  
pp. G613-G621 ◽  
Author(s):  
R. W. Van Dyke ◽  
B. F. Scharschmidt
Keyword(s):  
Steady State ◽  
Membrane Potential ◽  
Solute Transport ◽  
Primary Cultures ◽  
Rat Hepatocytes ◽  
Sodium Content ◽  
Intracellular Sodium ◽  
Intact Cells ◽  
Sodium Dependent ◽  
Dependent Transport

Inhibition of Na+-K+-ATPase and sodium-dependent bile acid transport has been suggested as a mechanism for the cholestasis produced by certain drugs such as chlorpromazine. We examined the effects of chlorpromazine (and in selected studies, two of its metabolites) on Na+-K+-ATPase cation pumping (ouabain-suppressible 86Rb uptake), exchangeable intracellular sodium content, membrane potential (assessed by 36Cl- distribution), and sodium-dependent transport of taurocholate and alanine in primary cultures of rat hepatocytes. Chlorpromazine (10-300 microM), 7,8-dihydroxychlorpromazine (10-300 microM), and ouabain (0.1-2 mM), but not chlorpromazine sulfoxide, produced a concentration-dependent decrease in Na+-K+-ATPase cation pumping and an increase in intracellular sodium content. Chlorpromazine (100 microM) and ouabain (0.75 mM) also modestly decreased hepatocyte membrane potential. In further studies, chlorpromazine (75 and 100 microM) and ouabain (0.1, 0.5, and 0.75 mM) decreased initial sodium-dependent uptake rates of taurocholate and alanine by 18-63%. Although the steady-state intracellular content of alanine was decreased 25-53% by both agents, chlorpromazine increased the steady-state content of taurocholate by 171% and decreased taurocholate efflux, apparently related to partitioning of taurocholate into a large, slowly turning over intracellular pool. These studies provide direct evidence that chlorpromazine inhibits Na+-K+-ATPase cation pumping in intact cells and that partial inhibition of Na+-K+-ATPase cation pumping is associated with a reduction of both the electrochemical sodium gradient and sodium-dependent solute transport. These effects of chlorpromazine may contribute to chlorpromazine-induced cholestasis in animals and humans.

Quantitative requirement for ATP for active transport in isolated renal cells

1986 ◽  
Vol 251 (1) ◽  
pp. C120-C127 ◽  
Author(s):  
N. Tessitore ◽  
L. M. Sakhrani ◽  
S. G. Massry
Keyword(s):  
Atpase Activity ◽  
Active Transport ◽  
Phosphate Uptake ◽  
Quantitative Relationship ◽  
Similar Degree ◽  
Renal Cells ◽  
Stepwise Manner ◽  
Sodium Gradient ◽  
Sodium Dependent ◽  
86Rb Influx

We investigated the quantitative relationship between cellular ATP concentration and Na+-K+-ATPase activity as measured by ouabain-sensitive 86Rb influx in rabbit proximal renal cells. Cellular ATP was reduced in a stepwise manner by rotenone (10(-7) to 10(-5) M) and was increased by 10 mM adenosine. During these maneuvers, ouabain-sensitive 86Rb influx was linearly related to cellular ATP and did not saturate up to 9.9 mM ATP. In contrast, Na+-K+-ATPase activity in membranes prepared from these cells saturated at 2.0 mM ATP at various sodium (10-100 mM) and potassium (4-100 mM) concentrations. Sodium-dependent phosphate uptake and alpha-methylglucoside (alpha-MG) uptake were both inhibited to a similar degree when cellular ATP was reduced. We conclude that 1) the ATP requirement for saturation of Na+-K+-ATPase is higher in intact renal cells than in the membranes, and 2) the uptake of phosphate and alpha-MG are similarly influenced by reduction in ATP. This effect of ATP on phosphate and AMG uptake is most likely an indirect one and is secondary to changes in the sodium gradient across the cell.

Altered norepinephrine uptake in neuronal cultures from spontaneously hypertensive rat brain

1985 ◽  
Vol 248 (5) ◽  
pp. C488-C497 ◽  
Author(s):  
C. Sumners ◽  
T. F. Muther ◽  
M. K. Raizada
Keyword(s):  
Spontaneously Hypertensive Rat ◽  
Neuronal Cell ◽  
Neuronal Cultures ◽  
Maximal Velocity ◽  
Spontaneously Hypertensive ◽  
Lower Affinity ◽  
Neuronal Cell Bodies ◽  
Sodium Dependent ◽  
Uptake Sites ◽  
Wistar Kyoto

Uptake of [3H]norepinephrine (NE) has been characterized and compared in neuronal cultures prepared from the brains of 1-day-old normotensive (Wistar-Kyoto, WKY) and spontaneously hypertensive (SH) rats. In cultures from both strains total [3H]NE uptake consisted of a sodium-dependent portion and a sodium-independent portion. The sodium-dependent [3H]NE uptake was inhibited by NE uptake blockers such as maprotiline or desmethylimipramine (both at 0.5-100 microM). This sodium-dependent, NE uptake blocker-sensitive portion of the uptake was also stereospecific, preferring the l-isomer of NE. In contrast, the sodium-independent uptake was not sensitive to maprotiline or desmethylimipramine. Autoradiograms of cultures incubated with [3H]NE showed label concentrated in certain, but not all, neurites and in a few neuronal cell bodies. Cultures incubated with label in sodium-free buffer did not show any such localization of grains but instead showed a diffuse pattern. Incubation of neuronal WKY or SH brain cultures with various concentrations of l-[3H]NE and unlabeled l-NE in the presence or absence of sodium enabled the construction of saturation curves for sodium-dependent uptake in each culture type. In WKY cultures, Km and maximal velocity of uptake (Vmax) values of 0.37-0.45 microM and 0.58-0.69 pmol X mg protein-1 X min-1, respectively, were obtained for sodium-dependent uptake. In contrast, the Km and Vmax values for [3H]NE uptake in SH neuronal cultures were 1.4 microM and 1.31 pmol X mg protein-1 X min-1, respectively. Kinetic analyses of the results show that in SH neuronal cultures the [3H]NE uptake sites are of lower affinity but higher capacity compared with those in WKY neuronal cultures.

Nature of taurodehydrocholic acid uptake in rat hepatocytes

1988 ◽  
Vol 254 (2) ◽  
pp. G269-G274 ◽  
Author(s):  
W. G. Hardison ◽  
P. J. Lowe ◽  
E. Gosink
Keyword(s):  
Membrane Potential ◽  
Rat Hepatocytes ◽  
Competitive Inhibitor ◽  
Acid Uptake ◽  
Isolated Rat Hepatocytes ◽  
Acid Analogue ◽  
Sodium Gradient ◽  
Sodium Dependent ◽  
Independent Process ◽  
Transport Site

We studied uptake into isolated rat hepatocytes of the bile acid analogue taurodehydrocholate (TDHC) over a concentration range of 2.5-4,000 microM. Uptake was mainly by a saturable sodium-dependent process with a Km of approximately 50 microM and a Vmax of 0.036 nmol.s-1.mg protein-1. A lesser sodium-independent process was evident but was linear in the range studied. Both processes were inhibited by incubation at 37 degrees C under nitrogen in the presence of 3 mM sodium cyanide or by incubation at 0 degrees C. A single transport site was suggested by the Eadie-Hofstee plot of TDHC uptake from 2.5 to 750 microM. TDHC was a weak competitive inhibitor of taurocholic acid (TCA) uptake (Ki = 236 microM) but was not itself taken up by the TCA transport site. TCA exhibited moderately potent mixed inhibition of TDHC uptake. Uptake of both compounds was strongly inhibited by bromosulfophthalein (BSP) and Rose Bengal, whereas 0.5 mM alanine uptake was not affected. BSP exhibited a complex pattern of inhibition of TDHC uptake: mixed partial inhibition. Degree of inhibition of both TDHC and TCA uptake did not increase as BSP concentrations were increased from 50 to 100 microM. BSP did not exert its inhibitory effects by alteration of membrane potential or sodium gradients; 50 microM BSP changed membrane potential less than 10% and sodium gradient not at all. The data indicate that despite close structural analogy between TDHC and TCA, the two compounds are taken up by different sodium-dependent mechanisms. Nonetheless, the similar qualitative and quantitative effects of BSP on their uptakes suggests the mechanisms are related.

Folic acid transport by mammalian small intestine.

1978 ◽  
Vol 235 (6) ◽  
pp. E678 ◽  
Author(s):  
R C Rose ◽  
M J Koch ◽  
D L Nahrwold
Keyword(s):  
Folic Acid ◽  
Acid Concentration ◽  
Bathing Solution ◽  
Acid Transport ◽  
Additional Information ◽  
Carrier Mechanism ◽  
Sodium Gradient ◽  
Saturation Kinetics ◽  
Sodium Dependent ◽  
Free Media

The unidirectional influx of folic acid across the mucosal border of hamster duodenum and rat jejunum was determined. Influx follows saturation kinetics, is sodium-dependent, and is inhibited by methotrexate and is sodium-dependent, and is inhbited by methotrexate and folinic acid in the mucosal bathing solution. In hamster duodenum, the maximal influx is 1.2 nmol/(cm2.h), and the folic acid concentration required to give a half maximal influx (Km) is 7.2 micron.. At mucosal folic acid concentration of 1.5 micron, influx is reduced at least 65% by removal of sodium from the bathing solution. The influx process is significantly inhibited by cyanide and 2,4-dinitrophenol. The possibility was evaluated that the acidic microclimate at the brush border regulates the rate of folic acid transport and that inhibition of transmural transport by sodium-free media, ouabain, and methotrexate is brought about indirectly by an increase in pH of the microclimate. The data favor the alternative concept of a sodium-dependent carrier mechanism for entry of folic acid into the cells. The information presented is consistent with active transport of folic acid by a sodium-gradient mechanism, but additional information will be necessary to substantiate such a model.

Osmotic regulation of Na-myo-inositol cotransporter mRNA level and activity in endothelial and neural cells

1996 ◽  
Vol 270 (4) ◽  
pp. C990-C997 ◽  
Author(s):  
T. J. Wiese ◽  
J. A. Dunlap ◽  
C. E. Conner ◽  
J. A. Grzybowski ◽  
W. L. Lowe ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Mrna Level ◽  
Cell Types ◽  
Neural Cells ◽  
Maximal Velocity ◽  
Mrna Levels ◽  
Osmotic Regulation ◽  
High Affinity ◽  
Sodium Dependent ◽  
Renal Origin

Myo-inositol (MI) is an important factor in the synthesis of phosphoinositides, and as an osmolyte, MI contributes to the regulation of cell volume. In cells of renal origin, hypertonicity causes an increase in sodium-dependent MI transporter (SMIT) mRNA levels and MI transport. However, it is unknown whether changes in osmolarity regulate transport of MI in neural or endxsothelial cells. IN these studies, neural and endothelial cells were exposed to hyperosmotic medium for up to 48 h, and the effect on MI transport was determined. Transport of MI was maximally increased by exposing the cells to hyperosmotic medium for 24 h. Kinetic analysis of high-affinity MI transport demonstrated an increase in the apparent maximal velocity with no significant change in the apparent Km. The hyperosmotic induction of MI transport was blocked by the addition of cycloheximide, indicating a requirement for protein synthesis, and was associated with increased levels of SMIT mRNA. In contrast to the effect of hypertonicity, exposure of neural and endothelial cells to hypotonic conditions caused a decrease in SMIT mRNA levels and MI transport in endothelial cells. These studies demonstrate that, in extrarenal cell types, changes in osmolarity also regulate SMIT activity and mRNA levels.

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