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J Biber

Publications and source records attributed to J Biber.

At least 181 records · Page 10Linked to original sources
PubMed1990

Expression of rat liver canalicular sulfate carrier in Xenopus laevis oocytes.

Poly(A)+ RNA (mRNA)extracted from rat liver was injected into Xenopus laevis oocytes and the expression of sulfate transport was determined by measuring [35S] sulfate uptake. Compared to water-injected oocytes, which exhibited virtually no sulfate uptake, injection of rat liver mRNA resulted in a time- and dose-dependent increase in uptake of sulfate. Depending on the method used for the isolation of the mRNA, sulfate uptake was stimulated after injection (40 ng after 6 days) between 8- and 72-fold compared to water-injected oocytes. Sulfate uptake of oocytes injected with mRNA was found to be sensitive to 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (IC50 less than 20 microM) and could also be inhibited by thiosulfate. Sulfate uptake of injected oocytes showed Michaelis-Menten kinetics (apparent Km, 0.31 mM) which is similar to the Km of the sulfate/bicarbonate antiporter of rat liver canalicular plasma membranes. After fractionation by a sucrose density gradient, the mRNA encoding for the expressed rat liver sulfate carrier was found in fractions containing messages of 3.5-4.0 kilobases in length.

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗
PubMed1989

The Na+/Pi-cotransporter of OK cells: reaction and tentative identification with N-acetylimidazole.

Using an established renal epithelial cell line (OK cells) the effect of the amino-acid side-chain modifying reagent N-acetylimidazole (NAI) upon the sodium-dependent transport of phosphate (Pi) was investigated. After an incubation with 10 mM NAI for 20 min, cellular Na+/Pi uptake was inhibited by 70%. The presence of 5 mM Pi protected this transport function from being affected by NAI by 80 to 100%. Since the presence of sulfate was unable to protect the Na+/Pi transport inactivation by NAI and since the presence of Pi did not affect NAI inhibition of other transport systems, it is suggested that NAI interacts with the Pi transporter directly. The protective effect of Pi was used as a criterion to identify Pi-protectable [3H]NAI labelling of OK cell plasma membrane proteins. Pi protection was observed in four molecular mass regions: 31, 53, 104 and 176 kDa. Since the incorporation of [3H]NAI into these proteins was also affected by parathyroid hormone at 10(-10) M, it is concluded that the identified proteins represent possible candidates for the renal Na+/Pi cotransporter.

Animals↗
PubMed1989

Separation of hepatocyte plasma membrane domains by free flow electrophoresis.

We have applied free flow electrophoresis to separate the canalicular and basolateral (sinusoidal and lateral) domains of rat hepatocyte plasma membranes. Hepatocyte plasma membranes were prepurified by rat zonal and discontinous sucrose gradient centrifugation. In electrophoretic separation, the canalicular membranes were more deflected toward the anode than the basolateral membranes. Na+-dependent taurocholate uptake could be measured in both membrane fractions, transport activity being highest in fractions containing the highest specific activity in the basolateral marker enzyme Na+-K+-ATPase. Thus, differences in electrophoretic mobility permit the separation of functional intact plasma membrane vesicles derived from basolateral and canalicular plasma membrane domains of rat hepatocyte.

Absorption↗
PubMed1989

Cellular aspects of proximal tubular phosphate reabsorption.

In vivo manipulations to alter renal Pi reabsorption and the subsequent isolation of proximal tubular brush border membrane vesicles have greatly increased our knowledge about the regulation of renal Pi reabsorption via the Na+/Pi cotransport system. Only recently, direct biochemical and cell-biological access has become possible by the use of established and primary cell cultures. Based on the results obtained with isolated brush border membranes and cultured cells, a model has been presented, which might serve as a basis for future research of the regulatory control mechanisms of the renal Na+/Pi cotransport. At present, a major drawback is the fact that the molecular identity of the Na+/Pi cotransport system is still unknown. The identification of this transport system would certainly be a great step and would allow to verify or falsify one or the other hypotheses postulated in the past few years for the regulatory control mechanism(s) of the renal Na+/Pi cotransport.

Absorption↗
PubMed1989

Sodium-phosphate cotransport in OK cells: inhibition by PTH and "adaptation" to low phosphate.

Sodium-phosphate (Na-Pi) cotransport is principally regulated by parathyroid hormone (PTH) and by the intrinsic ability to "adapt" to ambient phosphate concentration. In the present study, these two control mechanisms were examined in a cloned opossum kidney (OK) cell line. PTH inhibited Na-Pi cotransport, half-maximal inhibition at 5 x 10(-12) M, by fractionally similar amounts irrespective of the initial transport rates predetermined by "adaptation" to media phosphate concentration. At maximal concentrations of PTH (10(-8) M), the residual Na-Pi cotransport activity was higher in cells exposed to low-phosphate media. Cells preexposed to PTH (10(-8) M) or dibutyryl adenosine 3'-5'-cyclic monophosphate (DBcAMP) (10(-5) M) and forskolin (10(-5) M) increase transport (adaptation) by fractionally similar amounts as control cells for any given external phosphate concentration. The protein kinase C inhibitor, staurosporine, prevented PTH action but did not alter the ability to adapt Na-Pi cotransport in response to low media phosphate concentration. These data support the notion that regulation of Na-Pi cotransport by PTH and the adaptive response to available media phosphate concentration are distinct regulatory control mechanisms.

Alkaloids↗
PubMed1988

Modulation of Na+-Pi cotransport in opossum kidney cells by extracellular phosphate.

The effect of the extracellular concentration of Pi on the Na+-dependent phosphate transport activity of OK cells was investigated. When incubated with extracellular Pi at concentrations of 200 microM or less, Na+-Pi cotransport increased approximately twofold in OK cells compared with control cells (kept in 0.85 mM Pi), whereas other Na+-dependent transport activities were not affected. After Pi deprivation, Na+-Pi cotransport could be inhibited to a similar extent (80%) by parathyroid hormone (PTH) as in control cells, suggesting that the PTH-sensitive Na+-Pi cotransport activity is also regulated by extracellular Pi. The increase of Na+-Pi cotransport was maximally expressed after 6 h and could be prevented by cycloheximide (70 microM) but not by actinomycin D (0.5-5 g/ml). However, the adaptive response was completely blocked by 3'-deoxyadenosine (cordycepin) at 100 microM. From these data, it is concluded that the upregulation of Na+-Pi cotransport in OK cells due to low extracellular Pi is controlled at a posttranscriptional level.

Animals↗
PubMed1986

Stimulation of Na+/phosphate cotransport in LLC-PK1 cells by 12-O-tetradecanoylphorbol 13-acetate (TPA).

We have tested for the effect of the phorbol ester 12-O-tetradecanoylphorbol 13-acetate (TPA) on Na+/phosphate cotransport in an established epithelial cell line of renal origin (LLC-PK1). Incubation of LLC-PK1 cells with TPA produced an increase in Na+/phosphate (Pi) cotransport. The maximal response was reached at a TPA concentration of 10 ng/ml. Other phorbol esters which have no potency or a smaller one to activate protein kinase C had no effect on Na+/Pi cotransport. Incubation of LLC-PK1 cells with 10 ng/ml TPA for 8 h led to a 300% increase in Na+/Pi cotransport; in the presence of cycloheximide the increase amounted only to a 100% and was reached within 2 h. Kinetic analysis of Na+/Pi cotransport indicated an increase in the apparent Vmax without an effect on the apparent Km. The increased Pi transport was retained in isolated apical vesicles. Na+-dependent alanine transport into LLC-PK1 monolayers was affected by TPA administration in a similar manner. TPA had under the chosen experimental conditions no effect on [3H]thymidine incorporation into DNA excluding a general proliferative effect. We conclude that TPA via activation of protein kinase C regulates the number of operating transport systems. As also other Na+-coupled transport systems are influenced, the TPA effect appears to be related to the expression of a general 'adaptive' alteration of membrane transport in LLC-PK1 cells.

Alanine↗
PubMed1986

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

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.

Adenocarcinoma↗
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↗
PubMed1985

Na-Pi cotransport in LLC-PK1 cells: fast adaptive response to Pi deprivation.

A short-term regulation of the Na-dependent transport of Pi in LLC-PK1 cells by the extracellular concentration of Pi is described. Pi deprivation of the cells for 10 min led to an increase (30%) of the Na-Pi cotransport activity, whereas the Na-dependent D-glucose transport system was not affected. This regulatory phenomenon was not affected by the presence of cycloheximide. The same adaptive response was observed in apical membrane vesicles isolated from rapidly adapted cells. In the isolated membranes, increased Na-Pi cotransport is characterized by an increase of the maximal rate of uptake (control: 193 +/- 15; adapted: 306 +/- 51 pmol X mg-1 X 15 s-1), whereas the apparent Km for Pi remained constant. The results suggest that LLC-PK1 cells possess a mechanism or mechanisms that allow a rapid "activation" and "inactivation" of Na-dependent Pi transport systems as a function of the concentration of the extracellular Pi.

Adaptation, Physiological↗
PubMed1985

Adaptation of phosphate transport in phosphate-deprived LLC-PK1 cells.

Sodium-dependent transport of phosphate was studied in LLC-PK1 cells that had been deprived of phosphate (Pi). Compared with control cells (fed with 2 mM Pi) a twofold increase in the rate of Na-Pi cotransport was observed in cells incubated for 15 h in a phosphate-free medium, whereas transport of L-alanine and the specific activity of alkaline phosphatase were not changed. The same adaptive response was observed with apical membrane vesicles isolated from Pi-deprived cells. In both experimental systems Pi deprivation caused a change in the Vmax but not in the apparent Km (for Pi) of the cotransport system. Adaptation of the Na-Pi cotransport was triggered by free phosphate concentrations between 0 and 100 microM. Over the first 20 h the adaptive response was found to be a linear process that could be prevented by 70 microM cycloheximide. Adapted cells that were re-treated with phosphate showed a rapid (less than 3 h) decrease in the Na-Pi transport. The data suggest that LLC-PK1 cells adapt to low Pi conditions by increasing the rate of the Na-Pi cotransport, which is located in the apical membrane. Two mechanisms may be involved in the adaptive response: a long-term process involving new protein synthesis, and a short-term response involving activation-inactivation of transport systems already existing.

Adaptation, Physiological↗
PubMed1985

In vitro phosphorylation of rat kidney proximal tubular brush border membranes.

The phosphorylation of rat renal brush border membrane protein was analyzed after incubation of cortical slices with 32P-orthophosphate and compared with the phosphorylation by gamma-32P-ATP of isolated brush border vesicles. Phosphate incorporation into brush border membranes isolated from slices was linearly related to the incubation time as well as to the specific activity of orthophosphate present during slice incubation. Incorporation of phosphate into proteins reached an equilibrium after about 60 min, whereas incorporation of phosphate into lipids increased continuously. In brush border membranes isolated from slices incubated with orthophosphate (32P), the addition of cAMP or theophylline produced a dephosphorylation of a 47,000-dalton protein; no increased phosphorylation was observed. In brush border membranes, phosphorylated with gamma-32P-ATP, cAMP and dibutyryl cAMP (dB-cAMP) produced an increase in phosphorylation but no dephosphorylation. Sodium-dependent phosphate transport in brush border membranes was not altered by an incubation of slices with cAMP or dB-cAMP. These observations suggest that the phosphorylation machinery of isolated rat renal brush border membranes does not correspond with the mechanisms leading to phosphate incorporation into brush border membrane proteins in the intact cell.

Animals↗
PubMed1984

Sodium-dependent phosphate transport by apical membrane vesicles from a cultured renal epithelial cell line (LLC-PK1).

Apical membrane vesicles were prepared from confluent monolayers of LLC-PK1 cells grown upon microcarrier beads. The final membrane preparation, obtained by a modified divalent cation precipitation technique, was enriched in alkaline phosphatase, leucine aminopeptidase and trehalase (8-fold compared to the initial homogenate). Analysis of phosphate uptake into the vesicles identified a specific sodium-dependent pathway. Lithium and other cations were unable to replace sodium. At 100 mmol/l sodium and pH 7.4, an apparent Km for phosphate of 99 +/- 19 mumol/l and an apparent Ki for arsenate of 1.9 mmol/l were found. Analysis of the sodium activation of phosphate uptake gave an apparent Km for sodium of 32 +/- 12 mmol/l and suggested the involvement of two sodium ions in the transport mechanism. Sodium modified the apparent Km of the transport system for phosphate. The rate of sodium-dependent phosphate uptake was higher at pH 6.4 than at pH 7.4. At both pH values, an inside negative membrane potential (potassium gradient plus valinomycin) had no stimulatory effect on the rate of the sodium-dependent component of phosphate uptake. It is concluded that the apical membrane of LLC-PK1 cells contains a sodium-phosphate cotransport system with a stoichiometry of 2 sodium ions: 1 phosphate anion.

Animals↗