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C H van Os

Publications and source records attributed to C H van Os.

At least 73 records · Page 4Linked to original sources
PubMed1991

Quantification of Ca(2+)-ATPases in porcine duodenum. Effects of 1,25(OH)2D3 deficiency.

Previous studies have identified a calmodulin-stimulated ATP-dependent Ca2+ pump as the major Ca2+ efflux pathway in enterocytes. Here, we developed methods to quantify the number of Ca2+ pumps in basolateral and intracellular membranes from porcine duodenum. By the use of a pig strain with a genetic defect in renal 1 alpha-hydroxylase, we were able to investigate the influence of 1,25(OH)2D3-deficiency on the number of Ca(2+)-ATPases in porcine duodenum. The amount of Ca(2+)-ATPase in isolated basolateral membranes was 5.5 +/- 0.7 micrograms/mg protein, while the Vmax of ATP-dependent Ca2+ transport into inside-out resealed basolateral membrane vesicles was 2.6 +/- 0.4 nmol/mg protein per min. From these data we estimated roughly about 95 x 10(3) plasma membrane Ca2+ pump sites per enterocyte. In addition, the amount of intracellular Ca(2+)-ATPase in microsomal fractions was 0.41 +/- 0.02 microgram/mg protein. Comparison of these parameters between control and rachitic animals showed that Ca2+ pump capacities in both basolateral membranes and microsomal fractions of porcine duodenum are not influenced by 1,25(OH)2D3-deficiency. In conclusion, stimulatory effects of 1,25(OH)2D3 on intestinal Ca2+ transport most likely result from specific effects on apical influx and facilitation of cytosolic Ca2+ diffusion by Ca(2+)-binding proteins and not from an increase in Ca2+ pumping capacity in basolateral membranes.

Adenosine Triphosphate↗
PubMed1991

Glucose transporters do not serve as water channels in renal and intestinal epithelia.

Glucose carriers have been shown to serve as water channels in macrophages and in oocytes injected with messenger ribonucleic acid (mRNA) encoding the glucose carrier protein (Fischbarg et al. The contribution, therefore, of glucose carriers to osmotic water permeability (Pf) in renal and intestinal epithelial cells was investigated. Pf of brush border membrane vesicles (BBMVs) and of basolateral membrane vesicles (BLMVs) was studied using stopped-flow spectrophotometry. Osmotic shrinkage of renal vesicles exhibited fast and slow components at 4 degrees C and 37 degrees C. The fast component could be inhibited by HgCl2 or dimethylsulphoxide (DMSO) at these temperatures, whereas the slow component was inhibited only at 4 degrees C. Osmotic shrinkage of intestinal BBMVs and BLMVs was homogeneous at 4 degrees C and 37 degrees C and was slightly inhibitable by HgCl2 or DMSO at 4 degrees C but not 37 degrees C. In both tissues, vesicle uptake of glucose was sensitive to HgCl2, but not to DMSO. Phlorizin and phloretin inhibited D-glucose uptake in BBMVs and BLMVs respectively, but had no significant effect on Pf. In membrane vesicles of kidney origin, Pf was tenfold higher than in membranes from intestine. This difference was not reflected by the phlorizin- and phloretin-sensitive D-glucose uptakes. Our study concludes that glucose transporters do not serve as water channels in kidney or intestine. Although membrane proteins contribute slightly to Pf at 4 degrees C, this contribution is insignificant at 37 degrees C. A membrane protein serving specifically as a water channel could only be demonstrated in renal cortical membranes.

Alanine↗
PubMed1991

Immunocytochemical localization of calbindin-D28k, calbindin-D9k and parvalbumin in rat kidney.

Possible sites involved in active Ca2+ transport were traced by means of immunocytochemical detection of calcium-binding proteins (CaBP) in the mammalian kidney. Antisera were raised in rabbits against calbindin-D28k from chick kidney and calbindin-D9k from bovine intestine and parvalbumin from rabbit muscle. In the rat kidney, parvalbumin and calbindin-D9k were co-localized in the loops of Henle and distal convoluted tubule. In the collecting duct their presence was restricted to the intercalated cells. In all responsive cells parvalbumin and calbindin-D9k were present exclusively along the basolateral membrane. Calbindin-D28k was only present in the outer part of the cortex, where it was localized in the distal convoluted tubule and in the connecting tubule. In these cells calbindin-D28k was evenly distributed through the cytosol. Calbindin-D28k, unlike parvalbumin and calbindin-D9k, could not be demonstrated in the loops of Henle or collecting duct.

Animals↗
PubMed1991

Calbindin-D9k and parvalbumin are exclusively located along basolateral membranes in rat distal nephron.

There is strong evidence that vitamin D-dependent Ca(2+)-binding proteins, i.e., calbindin-D9k and calbindin-D28k, facilitate diffusion of Ca2+ through the cytosolic compartment of renal and intestinal cells, which transport Ca2+ transcellularly. In the study presented here, parvalbumin, calbindin-D9k, and calbindin-D28k were localized precisely by immunocytochemistry in rat kidney. Antisera recognizing specifically the thick ascending loop of Henle, the connecting tubules and collecting ducts, and the intercalated cells of the collecting ducts were used to identify different cell types. In rat kidney cortex, parvalbumin and calbindin-D9k colocalized in the thick ascending loop of Henle, the distal convoluted tubule, the connecting tubule, and the intercalated cells of the collecting duct. Strikingly, in all responsive cells, parvalbumin and calbindin-D9k were exclusively present in a thin layer along the basolateral membrane. In contrast, calbindin-D28k was only present in the distal convoluted and connecting tubule, where it was evenly distributed through the cytosol. In conclusion, the exclusive localization of parvalbumin and calbindin-D9k at the basolateral membrane of immunopositive renal cells implies their involvement in the regulation of transport processes located in these membranes rather than a role as intracellular Ca2+ buffer and Ca2+ shuttle between the two opposing membranes.

Animals↗
PubMed1990

Effects of dimethylsulfoxide and mercurial sulfhydryl reagents on water and solute permeability of rat kidney brush border membranes.

The effects of dimethylsulfoxide, DMSO, and mercurial sulfhydryl reagents have been studied on water and small solute permeability of rat renal brush border membrane vesicles. Water and solute permeability was measured by mixing membrane vesicles with hypertonic solutions in a stopped-flow apparatus and following osmotically-induced changes in vesicular volume via changes in scattered light intensity. The rate constant of the fast osmotic shrinkage is proportional to the osmotic water permeability, while the rate constant of the slow reswelling phase is proportional to the solute permeability. Using mannitol as the osmotic agent, the osmotic shrinkage of rat renal brush border membrane vesicles followed a biphasic time course. 80% of the vesicles shrunk with a rate constant of approx. 50 s-1 and 20% with a rate constant of approx. 2 s-1. DMSO decreased dose-dependently the amplitude of the fast osmotic shrinkage, without affecting its rate constant. In contrast to DMSO, HgCl2 decreased the rate constant but not the amplitude of the fast osmotic shrinkage of renal brush border vesicles. Between 40-50 microM HgCl2, the inhibition of the fast osmotic shrinkage was completed. DMSO and HgCl2 increase the activation energy of water permeation in renal membranes from 3 to 12-15 kcal/mol. DMSO and HgCl2 did not affect the rate constant of the slow osmotic shrinkage of renal membrane vesicles and were also without effect on osmotic shrinkage of small intestinal brush border and pure phospholipid vesicles. In renal brush border membranes, HgCl2 at low concentrations (less than 10 microM) increased by 15-fold the permeability to NaCl and urea but not to mannitol, an effect which precedes the inhibition of water permeability at higher HgCl2 concentrations. The increase in small solute permeability was irreversible while the inhibition of water permeability could be reversed with cysteine and dithiothreitol. We conclude that water and small solute pathways in rat renal brush border membranes are completely separate entities, which are effected differently by DMSO and HgCl2. These pathways for water and solutes must be membrane proteins since neither DMSO nor HgCl2 affect the permeability properties of pure phospholipid vesicles.

4-Chloromercuribenzenesulfonate↗
PubMed1990

Calcium absorption by fish intestine: the involvement of ATP- and sodium-dependent calcium extrusion mechanisms.

Measurements of unidirectional calcium fluxes in stripped intestinal epithelium of the tilapia, Oreochromis mossambicus, in the presence of ouabain or in the absence of sodium indicated that calcium absorption via the fish intestine is sodium dependent. Active Ca2+ transport mechanisms in the enterocyte plasma membrane were analyzed. The maximum capacity of the ATP-dependent Ca2+ pump (Vm: 0.63 nmol.min-1.mg-1, Km:27 nM Ca2+) is calculated to be 2.17 nmol.min-1.mg-1, correcting for 29% inside-out oriented vesicles in the membrane preparation. The maximum capacity of the Na+/Ca2+ exchanger with high affinity for Ca2+ (Vm:7.2 nmol.min-1.mg-1, Km:181 nM Ca2+) is calculated to be 13.6 nmol.min-1.mg-1, correcting for 53% resealed vesicles and assuming symmetrical behavior of the Na+/Ca2+ exchanger. The high affinity for Ca2+ and the sixfold higher capacity of the exchanger compared to the ATPase suggest strongly that the Na+/Ca2+ exchanger will contribute substantially to Ca2+ extrusion in the fish enterocyte. Further evidence for an important contribution of Na+/Ca2+ exchange to Ca2+ extrusion was obtained from studies in which the simultaneous operation of ATP- and Na(+)-gradient-driven Ca2+ pumps in inside-out vesicles was evaluated. The fish enterocyte appears to present a model for a Ca2+ transporting cell, in which Na+/Ca2+ exchange activity with high affinity for Ca2+ extrudes Ca2+ from the cell.

Adenosine Triphosphatases↗
PubMed1990

Active Ca2+ transport systems in basolateral membranes from rabbit distal colon.

Active Na+ absorption by tight epithelia such as frog skin and distal colon share common features like feedback inhibition of cellular [Na+] on Na+ influx through amiloride-sensitive Na+ channels. It is postulated that the negative feedback of increasing cell [Na+] is mediated via a rise in cell [Ca2+]. In this model, cell [Na+] is coupled to cell [Ca2+] via a Na+/Ca2+ exchange system in the basolateral membrane. In the present study, the Ca2+ transporting systems in rabbit distal colon basolateral membranes were characterized. ATP-dependent Ca2+ uptake could be demonstrated in membrane vesicles from surface cells with the following kinetic parameters: Km = 0.09 microM Ca2+ and Vm = 3.8 nmol Ca2+ mg-1 protein min-1. The ATP-dependent Ca2+ transport was not responsive to ruthenium red and oxalate, suggesting a plasmalemmal origin. The addition of 75 mM Na+ to the uptake medium, 10 min after addition of ATP, did not release Ca2+ from the vesicles in significant amounts. In the absence of ATP, outwardly directed Na+ gradients were incapable of stimulating Ca2+ uptake. This study demonstrates that rabbit distal colon epithelium lacks a well-defined Na+/Ca2+ exchange system, and (Ca2+, Mg2+)-ATPase appears to be the sole Ca2+ extrusion system. Alternatives for the coupling of cell [Na+] to cell [Ca2+] are discussed.

Animals↗
PubMed1989

Rabbit distal colon epithelium: I. Isolation and characterization of basolateral plasma membrane vesicles from surface and crypt cells.

A method has been developed for the simultaneous isolation of basolateral plasma membrane vesicles from surface and crypt cells of rabbit distal colon epithelium by sequential use of differential sedimentation, isopycnic centrifugation and Ficoll 400 barrier centrifugation. The protein yield was high (total 0.81 mg/g mucosa) and surface and crypt cell-derived basolateral membrane fractions have been purified 34- and 9-fold with respect to the homogenate. The pattern of marker enzyme enrichments revealed only minor contamination by subcellular organelles. Latency of ouabain-sensitive (Na+,K+)-ATPase activity prior and after trypsin treatment of membranes indicated a vesicle configuration of sealed right side-out: sealed inside-out: leaky of approximately 2:1:1. The presence of sealed vesicles was also evident from the osmotic sensitivity of the D-[1-14C] mannitol equilibrium space determined with either fraction. Although considerably different in protein profile, surface and crypt basolateral membranes were similar in cholesterol to phospholipid molar ratio and membrane fluidity as determined by steady-state fluorescence polarization. Stopped-flow light scattering experiments revealed a rather low water permeability of the membranes with a permeability coefficient of 6 microns/sec at 35 degrees C, which is one order of magnitude lower than reported for small intestinal plasma membranes. Both membrane fractions have been shown to effectively generate outward uphill potassium ion gradients, a process that is energized by ATP and inhibited by the membrane-permeant cardiac-glycoside digitoxin. These characteristics are consistent with the activity of a (Na+,K+) pump operating in inside-out vesicles.

Animals↗
PubMed1989

Rabbit distal colon epithelium: II. Characterization of (Na+,K+,Cl-)-cotransport and [3H]-bumetanide binding.

Loop diuretic-sensitive (Na+,K+,Cl-)-cotransport activity was found to be present in basolateral membrane vesicles of surface and crypt cells of rabbit distal colon epithelium. The presence of gradients of all three ions was essential for optimal transport activity. (Na+,K+) gradient-driven 36Cl fluxes were half-maximally inhibited by 0.14 microM bumetanide and 44 microM furosemide. While 86Rb uptake rates showed hyperbolic dependencies on Na+ and K+ concentrations with Hill coefficients of 0.8 and 0.9, respectively, uptakes were sigmoidally related to the Cl concentration, Hill coefficient 1.8, indicating a 1 Na+:1 K+:2 Cl stoichiometry of ion transport. The interaction of putative (Na+,K+,Cl-)-cotransport proteins with loop diuretics was studied from equilibrium-binding experiments using [3H]-bumetanide. The requirement for the simultaneous presence of Na+,K+, and Cl-, saturability, reversibility, and specificity for diuretics suggest specific binding to the (Na+,K+,Cl-)-cotransporter. [3H]-bumetanide recognizes a minimum of two classes of diuretic receptor sites, high-affinity (KD1 = 0.13 microM; Bmax1 = 6.4 pmol/mg of protein) and low-affinity (KD2 = 34 microM; Bmax2 = 153 pmol/mg of protein) sites. The specific binding to the high-affinity receptor was found to be linearly competitive with Cl- (Ki = 60 mM), whereas low-affinity sites seem to be unaffected by Cl-. We have shown that only high-affinity [3H]-bumetanide binding correlates with transport inhibition raising questions on the physiological significance of diuretic receptor site heterogeneity observed in rabbit distal colon epithelium.

Animals↗
PubMed1988

Na+ and H+ gradient-dependent transport of p-aminohippurate in membrane vesicles from dog kidney cortex.

The transport of p-aminohippurate (PAH) was studied in basolateral (BLMV) and brush border membrane vesicles (BBMV) isolated from dog kidney cortex. Imposition of an inwardly directed 100 mN Na+ gradient stimulated the uptake of 50 microM [3H]PAH into BLMV, whereas a pH gradient (pHout = 6.0, pHin = 7.4) only slightly enhanced uptake. The Na+ gradient-dependent uptake of PAH was electroneutral, saturable and sensitive to inhibition by probenecid and several anionic drugs, with (apparent) Km = 0.79 +/- 0.16 mM, Vmax = 0.80 +/- 0.05 nmol/mg protein, 15 sec and Ki for probenecid = 0.08 +/- 0.01 mM. Simultaneous imposition of the pH gradient (outward OH- gradient) and inward Na+ gradient stimulated PAH uptake significantly over that with an Na+ gradient alone. These results are consistent with an Na+ gradient-stimulated PAH/OH- exchange mechanism in the basolateral membrane. In BBMV, PAH uptake could be stimulated by an outwardly directed OH- gradient as well as an inward Na+ gradient. Both gradients could drive PAH transport via a mediated probenecid-sensitive pathway. Na+ gradient-stimulated uptake was electrogenic with a (apparent) Km = 4.93 +/- 0.57 mM, Vmax = 6.71 +/- 0.36 nmol/mg protein, 15 sec and Ki,prob = 0.13 +/- 0.01 mM. The kinetic parameters for PAH/OH- exchange were virtually the same, (apparent) Km = 5.72 +/- 0.49 mM, Vmax = 7.87 +/- 0.33 nmol/mg protein, 15 sec and Ki,prob = 0.16 +/- 0.02 mM. When both the Na+ and pH (outward OH-) gradient were simultaneously imposed an almost twofold stimulation in uptake was observed over that with either an Na+ or pH gradient alone. These results suggested that both gradients stimulate PAH transport in BBMV via the same pathway. However, inhibition experiments with various organic anions showed that the specificities of Na+ and pH gradient-stimulated PAH uptake do not entirely overlap. Thus, our results support a simple transport in BBMV, but it cannot be excluded that two separate pathways are involved.

Aminohippuric Acids↗
PubMed1988

Calcium homeostasis of epithelial cells.

1. Cytosolic free Ca2+ is an important regulator of ion transport processes in epithelial cells. 2. Free Ca2+ concentration is regulated by a concerted action of Ca2+ transport systems in plasma membranes and intracellular organelles. 3. These transport systems were studied in intestinal and renal cortical cells with emphasis on the transport capacities and Ca2+ affinities. 4. Ca2+ accumulation by permeabilized cells was compared to Ca2+ uptake by isolated organelles and membrane fractions. 5. Effects induced by cell or organelle isolation methods and the influence of temperature and pH on Ca2+ transport capacities were studied.

Adenosine Triphosphate↗
PubMed1987

Nanomolar concentrations of Cd2+ inhibit Ca2+ transport systems in plasma membranes and intracellular Ca2+ stores in intestinal epithelium.

The interactions of Cd2+ with active Ca2+ transport systems in rat intestinal epithelial cells have been investigated. ATP-driven Ca2+ transport in basolateral plasma membrane vesicles was inhibited by Cd2+ with an I50 value of 1.6 nM free Cd2+ at 1 microM free Ca2+, using EGTA and HEEDTA to buffer Ca2+ and Cd2+ concentrations, respectively. The inhibition was competitive in nature since the Km value of Ca2+ increased with increasing Cd2+ concentrations while the Vmax remained constant. Cd2+ had similar effects on ATP-dependent Ca2+ uptake by permeabilized enterocytes, indicating that non-mitochondrial and mitochondrial Ca2+ stores are also inhibited by nanomolar concentrations of Cd2+. We conclude that ATP-driven Ca2+ transport systems are the most sensitive elements so far reported in Cd2+ intoxication.

Animals↗