Search PubMed⌕ Search

Biomedical subjects

H Murer

Publications and source records attributed to H Murer.

At least 307 records · Page 17Linked to original sources
PubMed1985

Defective jejunal brush-border Na+/H+ exchange: a cause of congenital secretory diarrhoea.

In a child with congenital secretory diarrhoea, jejunal perfusion studies showed net water and electrolyte secretion and suggested a defect in sodium/proton exchange. A technique was developed for investigation of such exchange in brush-border membrane vesicles from jejunal biopsy specimens. Vesicles from control specimens displayed proton-gradient-dependent sodium uptake, whereas those from the patient did not. These findings, as well as pointing to a cause of congenital secretory diarrhoea, confirm the existence and importance of jejunal sodium/proton exchange in man.

Cell Membrane↗
PubMed1985

Transport of sulphate in rat jejunal and rat proximal tubular basolateral membrane vesicles.

Basolateral membrane vesicles were isolated by a Percoll density gradient centrifugation method from small intestinal and renal proximal tubular epithelial cells. Transport of sulphate across the basolateral membrane was analyzed by measuring the uptake of tracer sulphate. In both membrane preparations, preloading the vesicles with sulphate- or hydroxyl-anions stimulated tracer sulphate uptake (trans-stimulation); an inwardly directed sodium gradient did not stimulate sulphate influx whether in the absence or in the presence of sulphate- or hydroxyl-ion-trans-stimulation. Under sulphate trans-stimulation conditions, DIDS (10(-4) mol/l) inhibited sulphate influx. In intestinal membranes, trans-stimulation of sulphate influx was obtained by preloading the vesicles with chloride, in renal membranes by preloading with bicarbonate. Under sulphate trans-stimulation conditions, in intestinal membranes, sulphate influx was strongly inhibited by chloride, in renal membranes, chloride inhibition was absent. Under bicarbonate trans-stimulation conditions, in renal membranes, sulphate transport was inhibited by lactate. It is concluded that small intestinal and renal proximal tubular basolateral membrane vesicles contain a transport mechanism for sulphate that cannot be energized by a sodium gradient. The transport system in small intestinal basolateral membranes seems to be different from that in renal membranes. It is suggested that the observed interaction between inorganic and organic anion transport in renal basolateral membranes is indirect.

Animals↗
PubMed1985

Identification of a voltage-dependent anion channel in the apical membrane of a Cl(-)-secretory epithelium (MDCK).

An anion-selective channel of large unit conductance is present in apical membranes of a secretory epithelial cell. For cell-attached patch-clamp configuration on cultured MDCK cells channels with a conductance of about 460 pS are observed at 37 degrees C. The channel becomes spontaneously activated only in rare cases. In inside-out membrane patches the probability of observing this type of channel increased significantly suggesting that the channel is controlled by an up to now unknown mechanism. The channel shows a voltage-dependent burst kinetic.

Anions↗
PubMed1985

Ca2+-dependent protein phosphorylation in brush border membranes of rat kidney proximal tubules.

Ca2+-dependent protein phosphorylation was studied in isolated brush border membrane vesicles from rat kidney cortex. Phosphorylation of about 20 target proteins with gamma-32P-ATP was observed after opening the vesicles transiently by detergent treatment or by hypotonic shock indicating that phosphorylation takes place from the cytoplasmic side. Five of these polypeptides (Mr 12, 19, 29, 97 and 138 kD) showed increased phosphorylation in the presence of micromolar calcium. Addition of exogenous calmodulin did not enhance the calcium induced phosphorylation significantly, nor did trifluoperazine depress it, suggesting that Ca2+-calmodulin-dependent protein kinase is not involved in the studied Ca2+-induced phosphorylation. 12-O-tetradecanoylphorbol-13-acetate (TPA) minimized the Ca2+ dependency for the 12 and 97 kD polypeptides. Cytotoxin I inhibited the incorporation of phosphate into the 12 and 97 kD polypeptides in a dose-dependent manner. Excess phosphatidylserine could reverse this inhibition but stimulated also the phosphorylation of the 19 kD polypeptide. These findings suggest that at least the 12 and the 97 kD polypeptides are substrates for an endogenous protein kinase C. When studied under conditions where Ca2+ increases membrane phosphorylation, no effects could be observed on the kinetic parameters (Km and Vmax) of the sodium-dependent phosphate uptake.

Animals↗
PubMed1985

Characterization of a Na : K : 2C1 cotransport system in the apical membrane of a renal epithelial cell line (LLC-PK1).

86Rb uptake into LLC-PK1 cells (an established renal epithelial cell line) was found to be comprised of an active ouabain-sensitive component, a loop diuretic-sensitive component which was passive and strictly dependent upon the presence of extracellular Na+ and Cl- for activity, and a "leak" component. The diuretic-sensitive component of influx was investigated further in apical membrane vesicles derived from these cells. A large fraction of 86Rb, 22Na and 36Cl flux into these vesicles was sensitive to inhibition by furosemide and dependent upon the presence of the other two co-ions, in keeping with the presence of a loop diuretic-sensitive Na+ : K+ : Cl- cotransport system. The kinetic parameters for Na+ and K+ interaction have been analyzed under initial linear zero trans conditions. The following values were obtained KmNa+ = 0.42 +/- 0.05 mmol/liter, Vmax = 303 +/- 24 pmol/mg/6 sec; KmK+ = 11.9 +/- 1.0 mmol/liter, VmaxK+ = 307 +/- 27 pmol/mg/6 sec. For Cl- interaction evidence for two cooperative binding sites with different affinities and different specificities were obtained. Thus, a stoichiometry of 1Na+ : 1K+ :2Cl- can be calculated. It is concluded that the apical membrane of LLC-PK1 cells contains a Na+ : K+ : 2Cl- co-transport system with properties similar to those described for the thick ascending limb of the loop of Henle.

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

Effect of pH on phosphate transport in rat renal brush border membrane vesicles.

The initial linear rate of phosphate uptake was analyzed in rat renal brush border membrane vesicles. An increase in medium pH from 6.0 to 8.0 increased the sodium gradient-dependent phosphate uptake about 20-fold. Sodium-independent phosphate uptake was not altered in this pH range. At pH 7.4 an intravesicular acid pH stimulated the initial linear uptake rate (20-25%). The apparent Km for sodium increased from about 100 to 200 mM when pH was decreased from 7.4 to 6.4. The Hill coefficient for sodium interaction was close to 2 and was unaffected by pH. Increasing external sodium reduced the apparent Km of the transport system for phosphate independent of pH. Variations of phosphate concentration had no influence on the apparent Km for sodium. At high sodium concentrations, small effects (20-30%) of pH on the apparent Vmax of the transport system were found; measured at saturating sodium concentrations, the apparent Km values calculated on the basis of total phosphate were increased (50-60%) when pH was decreased from 7.4 to 6.4. The data indicate that the major effect of pH is to modify the interaction of the transport system with sodium. At nonsaturating sodium concentrations, this resulted indirectly in a reduction in the affinity for phosphate related to a different occupancy of the sodium binding site. The differences of transport rate at low phosphate and high sodium concentrations could be explained by preferential transport of divalent phosphate as well as by pH effects on other carrier properties.

Animals↗
PubMed1985

Sodium-pyrazinoate cotransport in rabbit renal brush border membrane vesicles.

Pyrazinoate (PZA) is an organic anion actively reabsorbed and secreted in the mammalian kidney. In experiments with rabbit renal brush border membrane vesicles, we characterized a sodium-PZA cotransport mechanism that could be involved in reabsorption. An inwardly directed sodium gradient stimulated the influx of PZA. The sodium-dependent transport was electroneutral, suggesting a 1:1 stoichiometry. The kinetic constants for sodium-PZA cotransport were measured under initial linear flux and zero trans conditions for both sodium and PZA. The apparent Km for sodium was about 60 mM. At 90 mM sodium the apparent Km for PZA was about 1.1 mM; increasing the sodium concentration augmented the apparent affinity for PZA. Cis inhibition of sodium-dependent PZA uptake was observed by the addition of nicotinate, lactate, probenecid, succinate, beta-hydroxybutyrate, and salicylate. Urate had no effect. [14C]PZA uptake was trans stimulated by PZA itself, lactate, and nicotinate. PZA shares a transport system(s) involved in the proximal tubular reabsorption of these two anions.

Animals↗
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

Ca2+-stimulated, Mg2+-independent ATP hydrolysis and the high affinity Ca2+-pumping ATPase. Two different activities in rat kidney basolateral membranes.

The Mg2+-dependency of Ca2+-induced ATP hydrolysis is studied in basolateral plasma membrane vesicles from rat kidney cortex in the presence of CDTA and EGTA as Mg2+- and Ca2+-buffering ligands. ATP hydrolysis is strongly stimulated by Mg2+ with a Km of 13 microM in the absence or presence of 1 microM free Ca2+. At free Mg2+ concentrations of 1 microM and lower, ATP hydrolysis is Mg2+-independent, but is strongly stimulated by submicromolar Ca2+ concentrations (Km = 0.25 microM, Vmax = 24 mumol Pi/h per mg protein). The Ca2+-stimulated ATP hydrolysis strongly decreases at higher Mg2+ concentrations. The Ca2+-stimulated Mg2+-independent ATP hydrolysis is not affected by calmodulin or trifluoperazine and shows no specificity for ATP over ADP, ITP and GTP. In contrast, at high Mg2+ concentrations calmodulin and trifluoperazine affect the high affinity Ca2+-ATPase activity significantly and ATP is the preferred substrate. Control studies on ATP-dependent Ca2+-pumping in renal basolaterals and on Ca2+-ATPase in erythrocyte ghosts suggest that the Ca2+-pumping enzyme requires Mg2+. In contrast, a role of the Ca2+-stimulated Mg2+-independent ATP hydrolysis in active Ca2+ transport across basolateral membranes is rather unlikely.

Adenosine Triphosphate↗
PubMed1984

Comparison of Na+/H+ and Cl-/OH- exchange in rat jejunal brush border membrane vesicles: studies with acridine orange.

Vesicles can accumulate the fluorescent dye acridine orange if their intravesicular space is acidic compared with the external one. The quenching of the acridine orange fluorescence was used to monitor the formation and the dissipation of a delta pH in brush border vesicles isolated from rat kidney cortex or rat jejunum. Similar findings were obtained with both brush border membrane vesicles preparations. Under condition of potassium efflux, acridine orange fluorescence was quenched only in the presence of valinomycin showing a H+ greater than K+ conductance. Under condition of sodium efflux, an acidification of the intravesicular space occurred due to: a) indirect (electrical) coupling of sodium and proton fluxes; b) directly coupled Na+/H+ exchange. It is possible to preseta delta pH across the vesicles (pHin less than pHout) and to monitor the rate of the delta pH dissipation. The initial rate of dissipation of a preset delta pH was accelerated in a saturable manner by pulse-injections of sodium, this phenomenon is sensitive to the inhibition of Amiloride; Lithium can partially replace sodium. An inwardly directed chloride gradient did not induce an appreciable intravesicular acidification. On the other hand a Na-gluconate (in greater than out) gradient and a similar NaCl gradient produced the same intravesicular acidification. Furthermore the initial rate of dissipation of a preset delta pH was accelerated for the same extent by pulse injections of Na-gluconate and NaCl. These findings clearly confirm the existence of the Na+/H+ exchange. In contrast the Cl-/OH- exchange, if at all present, operates, in these brush border membrane preparation, at a much lower rate than Na+/H+ exchange.

Acridine Orange↗
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↗
PubMed1984

Na/H- and Cl/OH-exchange in rat jejunal and rat proximal tubular brush border membrane vesicles. Studies with acridine orange.

The quenching of the acridine orange fluorescence was used to monitor the formation and/or dissipation of a delta pH in brush border vesicles isolated from rat kidney cortex or rat jejunum. Similar findings were obtained with both brush border membrane vesicle preparations. Acridine orange fluorescence was quenched by a preset delta pH (intravesicular acid) or by the ionophore (valinomycin/CCCP) dependent development of a delta pH (intravesicular acid) under conditions of potassium efflux. Under sodium efflux conditions, an acidification of the intravesicular space occurred: a) due to indirect (electrical) coupling of sodium and proton fluxes; b) due to directly coupled sodium/proton exchange. The initial rate of the dissipation of a preset delta pH was accelerated by pulse injections of sodium in a saturable manner; lithium partially replaced sodium. The sodium dependent acceleration in the rate of dissipation of a preset delta pH was not altered by replacing gluconate with chloride. Amiloride was an inhibitor of directly coupled sodium/proton exchange. An inwardly directed chloride gradient did not induce intravesicular acidification. The initial rate of the dissipative proton fluxes (preset delta pH) was slightly accelerated by an outwardly directed chloride gradient. Sodium/proton exchange dependent acidification of the intravesicular space was not altered by replacing gluconate with chloride. These results clearly document the existence of sodium/proton exchange in both renal and intestinal brush border membrane vesicles. In contrast, Cl/OH exchange--under our experimental conditions--must have a much smaller rate than Na/H exchange.

Acridine Orange↗