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C H van Os

Publications and source records attributed to C H van Os.

At least 91 records · Page 5Linked to original sources
PubMed1987

Effect of pH on the kinetics of Na+-dependent phosphate transport in rat renal brush-border membranes.

The kinetics of Na+-dependent phosphate uptake in rat renal brush-border membrane vesicles were studied under zero-trans conditions at 37 degrees C and the effect of pH on the kinetic parameters was determined. When the pH was lowered it turned out to be increasingly difficult to estimate initial rates of phosphate uptake due to an increase in aspecific binding of phosphate to the brush border membrane. When EDTA or beta-glycerophosphate was added to the uptake medium this aspecific binding was markedly reduced. At pH 6.8, initial rates of phosphate uptake were measured between 0.01 and 3.0 mM phosphate in the presence of 100 mM Na+. Kinetic analysis resulted in a non-linear Eadie-Hofstee plot, compatible with two modes of transport: one major low-affinity system (Km approximately equal to 1.3 mM), high-capacity system (Vmax approximately equal to 1.1 nmol/s per mg protein) and one minor high-affinity (Km approximately equal to 0.03 mM), low-capacity system (Vmax approximately equal to 0.04 nmol/s per mg protein). Na+-dependent phosphate uptake studied far from initial rate conditions i.e. at 15 s, frequently observed in the literature, led to a dramatic decrease in the Vmax of the low-affinity system. When both the extra- and intravesicular pH were increased from 6.2 to 8.5, the Km value of the low-affinity system increased, but when divalent phosphate is considered to be the sole substrate for the low-affinity system then the Km value is no longer pH dependent. In contrast, the Km value of the high-affinity system was not influenced by pH but the Vmax decreased dramatically when the pH is lowered from 8.5 to 6.2. These results suggest that the low-affinity, high-capacity system transports divalent divalent phosphate only while the high-affinity, low-capacity system may transport univalent as well as divalent phosphate. Raising medium sodium concentration from 100 to 250 mM increased Na+-dependent phosphate uptake significantly but the pH dependence of the phosphate transport was not influenced. This observation makes it rather unlikely that pH changes only affect the Na+ site of the Na+-dependent phosphate transport system.

Animals↗
PubMed1987

Increased plasma calcitonin levels in young spontaneously hypertensive rats: role in disturbed phosphate homeostasis.

In young spontaneously hypertensive rats (SHR) and in normotensive Wistar-Kyoto controls (WKY) several parameters of phosphate and calcium homeostasis were determined. At 6 and 8 weeks, blood analysis revealed a significant hypophosphatemia (p less than 0.001) in SHR and twice as high plasma calcitonin levels in SHR than in WKY controls. At 8 weeks, 1,25-dihydroxycholecalciferol concentration was 20% higher in SHR (p less than 0.02) while 25-hydroxycholecalciferol was unaltered (p greater than 0.51). In addition total immunoreactive PTH, iPTH, was slightly increased (p less than 0.07) but intact PTH (1-84) (p greater than 0.90) was not significantly different from age matched WKY controls. Also at 8 weeks, a slightly reduced serum ionized Ca2+ concentration (p less than 0.001) with no change in total serum calcium was found in SHR (p greater than 0.39). Balance studies at 6 and 8 weeks of age revealed no significantly different balances for phosphate (F = 2.5, p greater than 0.10) and for calcium (F = 2.6, p greater than 0.09), although a tendency for slightly more positive balances existed in SHR when compared to WKY. However, SHR excreted significantly less phosphate in the urine than WKY control (F = 0.2, p less than 0.0009). Bone analysis was performed on femora of SHR and WKY of 6 weeks of age. Femora were significantly shorter in SHR (20.54 +/- 0.35 vs. 21.50 +/- 0.05 mm in WKY), whereas bone dry weight (127 +/- 6 vs. 107 +/- 2 mg).(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1987

Kinetics of ATP-dependent Ca2+ uptake by permeabilized rat enterocytes. Effects of inositol 1,4,5-trisphosphate.

Isolated rat enterocytes were permeabilized by saponin treatment. 45Ca2+ was accumulated by these cells when provided with ATP in a medium containing Ca2+ ligands. The use of oxalate, vanadate and mitochondrial inhibitors indicated that both non-mitochondrial and mitochondrial pools are involved. Kinetic analysis of non-mitochondrial Ca2+ uptake revealed a Km of 0.1 microM Ca2+ and a Vmax of 0.4 nmol Ca2+/mg protein X min for this Ca2+-pumping ATPase activity. Mitochondria started to take up Ca2+ between 0.2 and 0.3 microM free Ca2+ reaching maximal rates around 2 microM. At 1 microM free Ca2+ mitochondria accumulated 20 times more Ca2+ than the non-mitochondrial pool. Inositol 1,4,5-trisphosphate released 40% of the Ca2+ content of the non-mitochondrial pool. Half-maximal release was observed at 0.5 and 1.5 microM IP3 in duodenal and ileal cells respectively. These findings support the possibility that the phosphatidyl inositide metabolism plays a role in regulation of electrolyte transport in enterocytes.

Adenosine Triphosphate↗
PubMed1987

The adenosine triphosphate-dependent calcium pump in rat small intestine: effects of vitamin D deficiency and cell isolation methods.

Duodenal villus cells from vitamin D-deficient (-D) and replete rats (+D) were isolated either in citrate buffers according to the method of Stern or by vibration in EDTA-containing solutions according to the method of Harrison and Webster. Basolateral plasma membrane vesicles (BLMV) were purified, and active Ca2+ transport was studied. The rates of ATP-dependent Ca2+-transport in BLMV from -D and +D animals were 2.6 +/- 1.0 and 10.6 +/- 0.6 nmol Ca2+/min X mg protein when cells had been isolated in citrate buffers. These transport rates were 9.2 +/- 0.7 and 9.6 +/- 0.5, respectively, when cells were isolated by vibration. The specific activities of other enzyme markers and vesicle parameters, such as the degree of resealing or purification factors, were not influenced by the cell isolation procedure. Addition of lipase and protease inhibitors to the citrate buffer or fasting the animals before death increase the active Ca2+ transport rates in BLM from -D rats to control levels. These results indicate that the ATP-driven Ca2+ pump in duodenal plasma membranes from vitamin D-deficient rats is more prone to inactivation during enterocyte isolation procedures. With the vibration technique, six populations of cells could be obtained that are sequentially released from villus tip to crypt base. A similar villus-crypt gradient of active Ca2+ transport was present in duodenal BLMV from -D and +D animals. In ileal BLMV from vitamin D-deficient rats a significantly lower Ca2+ transport rate was found compared to that in +D animals, even when villus cells were isolated by vibration. It is thought that the mechanisms by which 1,25-dihydroxyvitamin D3 regulate transcellular Ca2+ fluxes in duodenum and ileum must be different.

Animals↗
PubMed1987

Effects of chlorothiazide, furosemide and PTH on Na+ and Ca2+ handling in isolated perfused kidneys of the spontaneously hypertensive rat.

The role of the kidney in a disturbed calcium metabolism in spontaneously hypertensive rats (SHR) was investigated. The hemodynamics of isolated perfused SHR kidneys were not basically altered compared to Wistar-Kyoto (WKY) control kidneys. Renal calcium handling by isolated perfused WKY and SHR kidneys at 12 weeks of age was not significantly different. In addition, the effects of chlorothiazide and furosemide on renal calcium handling were studied in isolated perfused kidneys from both rat strains. Both diuretics increased glomerular filtration rate, diuresis and excretion of sodium and calcium. However, both diuretics stimulated diuresis and calcium excretion significantly less in SHR than in WKY kidneys. The calciuric action of chlorothiazide was completely abolished by administration of human-parathyroidhormone (hPTH), while the natriuric effect was unchanged by hPTH. This observation suggests that a hypocalciuric action of chlorothiazide 'in vivo' is possibly mediated by PTH. Our study suggests that the kidney is not responsible for the disturbance in calcium metabolism in SHR. A surprising finding is that the SHR kidney was less responsive to the diuretics furosemide and chlorothiazide than the kidney of the WKY control.

Animals↗
PubMed1986

Subcellular distribution of ATP-dependent calcium transport in rat duodenal epithelium.

Subcellular fractionation studies were performed to delineate plasma membrane and intracellular membrane populations which might be involved in intracellular Ca2+-homeostasis of rat small intestinal epithelial cells. After a low-speed supernatant fraction had been suspended in 5% sorbitol and subjected to equilibrium centrifugation in a zonal rotor, the Golgi and endoplasmic reticulum markers, galactosyltransferase and NADPH-cytochrome -c reductase, were concentrated in a density region designated Window II. The basal-lateral membrane marker (Na+-K+)-ATPase was concentrated in a higher-density region designated Window III. ATP-dependent Ca2+ transport was equally distributed between the two windows. Several membrane populations could be resolved from each window with good recovery of Ca2+-transport activity by a second density gradient centrifugation step. Second density gradient fractions were subjected to counter-current partitioning in an aqueous polymer two-phase system. Basal-lateral membranes, characterized by an 11-fold enrichment of (Na+-K+)-ATPase, contained ATP-dependent Ca2+-transport activity with Vmax = 3.7 nmol/mg per min and Km = 0.5 microM. A major Golgi-derived population exhibited Ca2+-transport activity with Vmax and Km values similar to those of the basal-lateral membranes. One membrane population, presumed to have been derived from the endoplasmic reticulum, contained Ca2+-transport activity with Vmax = 4 nmol/mg per min and Km = 0.5 microM. In addition to demonstrating that ATP-dependent Ca2+-transport activity has a complex distribution within enterocytes, this study raises the possibility that the basolateral plasma membranes might account for a relatively minor portion of the cell's Ca2+-pumping ability.

Adenosine Triphosphate↗
PubMed1986

Membrane vesicles of cellular dimensions fit in two geometric series.

Preparations of basal-lateral plasma membranes from rat intestinal epithelial cells were analyzed with the analytical centrifuge. In these preparations a number of well-defined membrane fractions were observed. The particle weights of these fractions appear to fit in two geometric series. Until now only relatively small vesicles up to a diameter of about 1 micron were observed. In our preparation we have observed vesicles up to a diameter of 7.5 microns. Therefore, even vesicles with the same size as the plasma membranes of intact cells fit in the two geometric series.

Animals↗
PubMed1986

Osmotic water permeabilities of brush border and basolateral membrane vesicles from rat renal cortex and small intestine.

The osmotic water permeability Pf of brush border (BBM) and basolateral (BLM) membrane vesicles from rat small intestine and renal cortex was studied by means of stopped-flow spectrophotometry. Scattered light intensity was used to follow vesicular volume changes upon osmotic perturbation with hypertonic mannitol solutions. A theoretical analysis of the relationship of scattered light intensity and vesicular volume justified a simple exponential approximation of the change in scattered light intensity. The rate constants extracted from fits to an exponential function were proportional to the final medium osmolarity as predicted by theory. For intestinal membranes, computer analysis of optical responses fitted well with a single-exponential treatment. For renal membranes a double-exponential treatment was needed, implying two distinct vesicle populations. Pf values for BBM and BLM preparations of small intestine were equal and amount to 60 microns/sec. For renal preparations, Pf values amount to 600 microns/sec for the fast component, BBM as well as BLM, and to 50 (BBM) and 99 (BLM) microns/sec for the slow component. The apparent activation energy for water permeation in intestinal membranes was 13.3 +/- 0.6 and in renal membranes 1.0 +/- 0.3 kCal/mole, between 25 and 35 degrees C. The mercurial sulfhydryl reagent pCMBS inhibited completely and reversibly the high Pf value in renal brush border preparations. These observations suggest that in intestinal membranes water moves through the lipid matrix but that in renal plasma membranes water channels may be involved. From the high Pf values of renal membrane vesicles a transcellular water permeability for proximal tubules can be calculated which amounts to approximately 1 cm/sec. This value allows for an entirely transcellular route for water flow during volume reabsorption.

Animals↗
PubMed1986

Enterocyte isolation procedure specifically effects ATP-dependent Ca2+-transport in small intestinal plasma membranes.

Epithelial villus cells from rat small intestine were isolated either in citrate buffers or by vibration in EDTA containing solutions. Basolateral plasmamembrane vesicles (BLMV) were isolated and active Ca2+-transport studied. The rate of ATP-dependent Ca2+-transport in BLMV was 11.2, 1.2 and 0.8 nmol Ca2+/min.mg protein in duodenum, mid-jejunum and terminal ileum when enterocytes had been isolated in citrate buffers. These transport rates were 10.5, 4.8 and 5.8 respectively when cells were isolated by vibration. The specific activities of various marker enzymes were not influenced by the cell isolation procedure. The enrichment factors for (Na+-K+)-ATPase and the latency of this enzyme activity, the mannitol spaces and the half-times for mannitol equilibration among the three BLMV preparations were independent of the cell isolation method. It was demonstrated that active Ca2+-transport in BLMV from jejunum and ileum could be destroyed when cells isolated by vibration were incubated with low proteolytic activity (1 microgram/ml trypsin). Therefore, Ca2+-ATPase in jejunum and ileum is far more susceptible to extracellular proteolytic activity than Ca2+-ATPase more proximally located. Addition of various protease inhibitors to the citrate buffer only partly prevented the selective damage of Ca2+-transport in basolateral membranes.

Adenosine Triphosphate↗
PubMed1985

Distribution of Ca2+-ATPase, ATP-dependent Ca2+-transport, calmodulin and vitamin D-dependent Ca2+-binding protein along the villus-crypt axis in rat duodenum.

The migration of intestinal epithelial cells from the crypts to the tips of villi is associated with progressive cell differentiation. The changes in Ca2+-ATPase activity and ATP-dependent Ca2+-transport rates in basolateral membranes from rat duodenum were measured during migration along the crypt-villus axis. In addition, vitamin D-dependent calcium-binding protein and calmodulin content were measured in homogenates of six cell populations which were sequentially derived from villus tip to crypt base. Alkaline phosphatase activity was highest at the tip of the villus (fraction I) and decreased more than 20-fold towards the crypt base (fraction VI). (Na+ + K+)-ATPase activity also decreased along the villus-crypt axis but in a less pronounced manner than alkaline phosphatase. ATP-dependent Ca2+-transport in basolateral membranes was highest in fraction II (8.2 +/- 0.3 nmol Ca2+/min per mg protein) and decreased slightly towards the villus tip and base (fraction V). The youngest cells in the crypt had the lowest Ca2+-transport activity (0.9 +/- 0.1 nmol Ca2+/min per mg protein). The distribution of high-affinity Ca2+-ATPase activity in basolateral membranes correlated with the distribution of ATP-dependent Ca2+-transport. The activity of Na+/Ca2+ exchange was equal in villus and crypt basolateral membranes. Compared to the ATP-dependent Ca2+-transport system, the Na+/Ca2+ exchanger is of minor importance in villus cells but may play a more significant role in crypt cells. Calcium-binding protein decreased from mid-villus towards the villus base and was undetectable in crypt cells. Calmodulin levels were equal along the villus-crypt axis. It is concluded that vitamin D-dependent calcium absorption takes primarily place in villus cells of rat duodenum.

Alkaline Phosphatase↗
PubMed1984

Kinetic properties of the ATP-dependent Ca2+ pump and the Na+/Ca2+ exchange system in basolateral membranes from rat kidney cortex.

Basolateral plasma membranes from rat kidney cortex have been purified 40-fold by a combination of differential centrifugation, centrifugation in a discontinuous sucrose gradient followed by centrifugation in 8% percoll. The ratio of leaky membrane vesicles (L) versus right-side-out (RO) and inside-out (IO) resealed vesicles appeared to be L:RO:IO = 4:3:1. High-affinity Ca2+-ATPase, ATP-dependent Ca2+ transport and Na+/Ca2+ exchange have been studied with special emphasis on the relative transport capacities of the two Ca2+ transport systems. The kinetic parameters of Ca2+-ATPase activity in digitonin-treated membranes are: Km = 0.11 microM Ca2+ and Vmax = 81 +/- 4 nmol Pi/min X mg protein at 37 degrees C. ATP-dependent Ca2+ transport amounts to 4.3 +/- 0.2 and 7.4 +/- 0.3 nmol Ca2+/min X mg protein at 25 and 37 degrees C, respectively, with an affinity for Ca2+ of 0.13 and 0.07 microM at 25 and 37 degrees C. After correction for the percentage of IO-resealed vesicles involved in ATP-dependent Ca2+ transport, a stoichiometry of 0.7 mol Ca2+ transported per mol ATP is found for the Ca2+-ATPase. In the presence of 75 mM Na+ in the incubation medium ATP-dependent Ca2+ uptake is inhibited 22%. When Na+ is present at 5 mM an extra Ca2+ accumulation is observed which amounts to 15% of the ATP-dependent Ca2+ transport rate. This extra Ca2+ accumulation induced by low Na+ is fully inhibited by preincubation of the vesicles with 1 mM ouabain, which indicates that (Na+-K+)-ATPase generates a Na+ gradient favorable for Ca2+ accumulation via the Na+/Ca2+ exchanger. In the absence of ATP, a Na+ gradient-dependent Ca2+ uptake is measured which rate amounts to 5% of the ATP-dependent Ca2+ transport capacity. The Na+ gradient-dependent Ca2+ uptake is abolished by the ionophore monensin but not influenced by the presence of valinomycin. The affinity of the Na+/Ca2+ exchange system for Ca2+ is between 0.1 and 0.2 microM Ca2+, in the presence as well as in the absence of ATP. This affinity is surprisingly close to the affinity measured for the ATP-dependent Ca2+ pump. Based on these observations it is concluded that in isolated basolateral membranes from rat kidney cortex the Ca2+-ATPase system exceeds the capacity of the Na+/Ca2+ exchanger four- to fivefold and it is therefore unlikely that the latter system plays a primary role in the Ca2+ homeostasis of rat kidney cortex cells.

Adenosine Triphosphate↗
PubMed1983

Effect of amiloride on electrolyte transport parameters of the main duct of the rabbit mandibular salivary gland.

We have studied the response of the rabbit mandibular main duct perfused in vitro to luminally administered amiloride. The half-maximal inhibitory concentrations (KI) when the duct was bathed in Cl solutions were: for net Na+ transport, 3 X 10(-6) mol l-1; for transepithelial potential difference, 6 X 10(-6) mol l-1; and for transepithelial conductance, 3 X 10(-7) mol l-1. Substitution of the impermeant SO2-(4) anion for Cl- changed the KI for conductance to 3 X 10(-6) mol l-1. Within Cl- -containing media, the time course of the amiloride effect on potential difference showed an early rapid fall of 10 mV with a half-time 2 s, followed by a slower depolarization of 9 mV, and the conductance change followed the slower component of the potential change. In SO2-(4)-containing media, the potential difference and conductance changes followed time courses similar to one another. Finally, experiments on the effect of serosal applications of ouabain revealed that, although, in general, ouabain reduced resistance, it caused an increase in resistance in those ducts where the initial resistance was low. We conclude that: i) luminal Na+ transport occurs via amiloride-sensitive, conductive Na+ channels; ii) the Cl- conductance is the major determinant of transepithelial conductance; iii) the first phase of the potential response is due to blocking of the Na+ conductive channels, whilst the slow phase reflects secondary inhibition of an electrogenic Na+ pump; and iv) duct resistance changes are secondary to alterations in intracellular Cl- concentration.

Amiloride↗