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Biomedical subjects

H Murer

Publications and source records attributed to H Murer.

At least 271 records · Page 15Linked to original sources
PubMed1988

Ion channels activated by osmotic and mechanical stress in membranes of opossum kidney cells.

For patch-clamp measurements cultured kidney (OK) cells were exposed to osmotic and mechanical stress. Superfusion of a cell in whole cell configuration with hypotonic media (190 mOsm) evokes strong depolarization, which is reversible by returning to the isotonic bath medium. In the cell-attached configuration the exposure to hypotonic media evokes up to six ion channels of homogeneous single-channel properties in the membrane patch. Subsequently, the channels became activated after a time lag of a few seconds. At an applied membrane potential of 0 mV, the corresponding membrane current is directed inward and shows a transient behavior in the time range of minutes. In the same membrane patch these ion channels can be activated by application of negative hydrostatic pressure. The channel has a single-channel conductance of about 22 pS and is permeable to Na+ and K+ as well as to C1-. It is suggested that volume regulation involves mechanoreceptor-operated ion channels.

Animals↗
PubMed1988

Transport of sulfate and phosphate in small intestine and renal proximal tubule: methods and basic properties.

1. Isolated brush border membrane vesicles, basolateral membrane vesicles, and cultured renal epithelial cells provide good material for studying transport systems. 2. The vesicle systems have been used to study the transport of labeled phosphate, sodium/phosphate cotransport, sodium/sulfate cotransport, basolateral transport of sulfate and basolateral transport of phosphate via anion exchange. 3. Cultured renal cells show sodium/phosphate cotransport and parathyroid dependent inhibition of phosphate transport.

Animals↗
PubMed1988

Separate control of regulatory volume increase and Na+-H+ exchange by cultured renal cells.

Suspensions of OK cells (a continuous epithelioid cell line from opossum kidney) are examined by electronic cell sizing, measurements of intracellular pH, and measurements of cellular Na+ and K+. The response of the cells to hypertonic solutions is evaluated in most detail. When shrunken by exposure to hyperosmotic medium (430 mosmol/kg), the cells do not demonstrate a regulatory volume increase (RVI) independent of the solute that is used to increase osmolality [NaCl, N-methyl-D-glucamine-HCl (NMGCl), or sucrose]. In contrast, when cells are preexposed to 190 mosmol/kg medium and then shrunken by exposure to 310 mosmol/kg medium, a volume increase is observed after the addition of 120 mosmol/kg NaCl or NMGCl, but not sucrose. This RVI is sensitive to 1 mM furosemide and removal of Na+ or K+ from the medium, but it is not inhibited by 1 mM amiloride. In the presence of a propionate-induced cellular acidification, a Na+-H+ exchanger in the cells is shown to have a large capacity for net solute uptake and to be inhibited by 1 mM amiloride. Net solute uptake by the Na+-H+ exchanger is sensitive to addition of parathyroid hormone or 8-bromoadenosine 3',5'-cyclic monophosphate but is not stimulated in response to cell shrinkage.

8-Bromo Cyclic Adenosine Monophosphate↗
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↗
PubMed1988

Regulation of the rabbit ileal brush-border Na+/H+ exchanger by an ATP-requiring Ca++/calmodulin-mediated process.

Brush-border vesicles purified from rabbit ileal villus cells were used to evaluate how Ca++/calmodulin (CaM) regulates the neutral linked NaCl absorptive process, part of which is a Na+/H+ exchanger. After freezing and thawing to allow incorporation of macromolecules into the vesicles, the effect of Ca++/CaM on brush-border Na+ uptake with an acid inside pH gradient, and on Na+/H+ exchange was determined. Freezing and thawing vesicles with 0.85 microM free Ca++ plus 5 microM exogenous CaM failed to alter Na+/H+ exchange as did the addition of exogenous ATP plus an ATP regenerating system, which was sufficient to elevate intravesicular ATP to 47 microM from a basal level of 0.4 microM. However, the combination of Ca++/CaM plus ATP inhibited Na+ uptake in the presence of an acid inside pH gradient and inhibited Na+/H+ exchange, while Na+ uptake in the absence of a pH gradient was not altered. This effect required a hydrolyzable form of ATP, and did not occur when the nonhydrolyzable ATP analogue, AMP-PNP, replaced ATP. Under the identical intravesicular conditions used for the transport studies, Ca++ (0.85 microM) plus exogenous CaM (5 microM), in the presence of magnesium plus ATP, increased phosphorylation of five brush-border peptides. These data are consistent with Ca++/CaM acting via phosphorylation to regulate the ileal brush-border Na+/H+ exchanger.

Adenosine Triphosphate↗
PubMed1988

Calcium transport mechanisms in epithelial cell membranes.

The data which we have reviewed suggest that, at steady state, the cytosolic [Ca2+] is determined by the balance between Ca2+ influx across both the brush border and the basolateral membrane, and Ca2+ extrusion through the basolateral membrane catalyzed predominantly by the calmodulin-activated Ca2+ ATPase. Na+-Ca2+ exchange does not appear to play an important quantitative role in calcium extrusion but may participate in the regulation of cytosolic calcium as a function of luminal sodium entry. Ca2+ transport systems in endomembranes are probably activated only during Ca2+ transients in the cell. Thus, the reticular high-affinity Ca2+ ATPase allows for accumulation of limited amounts of Ca2+ into the reticulum at normal cytosolic Ca2+ concentrations of 0.1-0.2 M; Ca2+ can be rapidly released from this pool by IP3 produced in response to hormonal stimulation, which results in a transient increase in cytosolic Ca2+ and stimulation of calmodulin-dependent enzymes. When Ca2+ entry is stimulated to levels which exceed the capacity of Ca2+ ATPase in the basolateral membrane, cytosolic [Ca2+] may rise above the mitochondrial set point and mitochondrial Ca2+ accumulation may occur. It remains to be investigated if the latter reaction occurs under physiological or only under pathological conditions.

Animals↗
PubMed1988

Calcium transport in plasma membrane subfractions of rat liver.

The selective demonstration of ATP dependent calcium uptake and calcium dependent phosphorylation of a 135 kD intermediate in isolated basolateral liver plasma membrane vesicles suggest that the basolateral plasma membrane of rat hepatocytes contains an ATP dependent calcium transport system with an apparent molecular weight of 135,000. The failure to demonstrate ATP dependent calcium uptake in simultaneously isolated canalicular plasma membrane vesicles suggests the absence of a similar ATP dependent calcium transport system on the canalicular domain. Additional phosphoproteins of 200 and 110 kD could be assigned to microsomal contamination of the isolated plasma membrane subfractions. A canalicular specific 70 kD phosphorylated intermediate presumably does not represent a Ca++ ATPase. Calcium dependent phosphorylation of the basolateral 135 kD intermediate is maximal at physiologic pH (7.0, 7.5), when the calcium pump is active but not at pH 8.0. The 135 kD phosphoprotein has also been shown for erythrocyte Ca++ ATPase and for kidney basolateral Ca++ ATPase (De Smedt et al., 1984; Niggli et al., 1979).

Adenosine Triphosphate↗
PubMed1987

Sodium-bicarbonate cotransport occurs in rat kidney cortical membranes but not in rat small intestinal basolateral membranes.

Basolateral membrane vesicles were isolated from rat kidney cortex and small intestinal enterocytes. Both membrane preparations show ATP-dependent calcium uptake and cytochalasin B-sensitive D-glucose transport. In renal membranes, sodium influx is stimulated by bicarbonate; bicarbonate-dependent sodium flux is membrane-potential-dependent and inhibited by 4,4'-di-isothiocyanato-2, 2'-stilbenedisulphanic acid ('DIDS'). Small intestinal basolateral membranes do not show bicarbonate-dependent sodium fluxes.

Adenosine Triphosphate↗
PubMed1987

Identification of proximal tubular transport functions in the established kidney cell line, OK.

OK cells, derived from an American opossum kidney, were analyzed for proximal tubular transport functions. In monolayers, L-glutamate, L-proline, L-alanine, and alpha-methyl-glucopyranoside (alpha-methyl D-glucoside) were accumulated through Na+-dependent and Na+-independent transport pathways. D-Glucose and inorganic sulfate were accumulated equally well in the presence or absence of Na+. Influx of inorganic phosphate was only observed in the presence of Na+. Na+/alpha-methyl D-glucoside uptake was preferentially inhibited by phlorizin and D-glucose uptake by cytochalasin B. An amiloride-sensitive Na+-transport was also identified. In isolated apical vesicles (enriched 8-fold in gamma-glutamyltransferase), L-glutamate, L-proline, L-alanine, alpha-methyl D-glucoside and inorganic phosphate transport were stimulated by an inwardly directed Na+-gradient as compared to an inwardly directed K+-gradient. L-Glutamate transport required additionally intravesicular K+. D-Glucose transport was similar in the presence of a Na+- and a K+-gradient. Na+/alpha-methyl D-glucoside uptake was inhibited by phlorizin whereas cytochalasin B had no effect on Na+/D-glucose transport. An amiloride-sensitive Na+/H+ exchange mechanism was also found in the apical vesicle preparation. It is concluded that the apical membrane of OK cells contains Na+-coupled transport systems for amino acids, hexoses, protons and inorganic phosphate. D-Glucose appears a poor substrate for the Na+/hexose transport system.

Amino Acids↗
PubMed1987

Parathyroid hormone regulates phosphate transport in OK cells via an irreversible inactivation of a membrane protein.

In a cultured, renal epithelial cell line, OK, parathyroid hormone (PTH) reduces Na/phosphate cotransport and upon removal of the hormone the activity is regained. Cycloheximide, an inhibitor of protein synthesis, did not interfere with the PTH action, but prevented the reduced phosphate transport from regaining its original activity. Drugs, such as colchicine, that disrupt the microtubular network, lessened both the action of PTH and prevented the recovery of activity. The results are consistent with irreversible inactivation of a plasma membrane protein necessary for full activity of the Na/phosphate cotransport.

Animals↗
PubMed1987

Adaptation of phosphate transport to low phosphate diet in renal and intestinal brush border membrane vesicles: influence of sodium and pH.

The possible role of changes in the sodium (Na) affinity of the carrier for inorganic phosphate (Pi) in the adaptation of Pi transport to low Pi diet was examined in both renal and intestinal brush border membranes vesicles (BBMV) obtained from the same animal. This role was assessed by measuring the Na concentration resulting in half maximal activation of Pi transport (K0.5 Na) in renal and intestinal BBMV prepared from animals adapted to either low (LPD) or high (HPD) phosphorus diet for 7 days. The K0.5 Na was not modified by dietary Pi, in both renal and intestinal BBMV. LPD increased maximal Pi transport from 1794.8 +/- 198.0 to 2964.0 +/- 362.0 in renal and from 28.2 +/- 3.4 to 80.5 +/- 7.2 pmol/mg 10 s in intestinal BBMV. For both LPD and HPD lowering pH from 7.4 to 6 dramatically increased K0.5 Na in renal and intestinal BBMV. As compared to pH 7.4, it was enhanced by approximately 200% in both renal and intestinal membranes. This change of Na affinity with acidic pH prevented the expression of Pi transport adaptation at 100 mM Na concentration. However, at saturating Na concentrations (500 mM for renal, 300 mM for intestinal membranes), Pi transport adaptation was equally expressed at pH 6 and 7.4 in both types of membranes. Hill coefficient analysis indicates a 2:1 stoichiometry of Na to Pi in renal and intestinal membranes isolated from high or low Pi diet animals. This ratio was not modified by changes of the medium pH.

Adaptation, Physiological↗
PubMed1987

Calcium-binding proteins in carcinoma, neuroblastoma and glioma cell lines.

Antisera against the Ca2+-binding proteins parvalbumin, calbindin D-28K, and the S-100 proteins were used to study the distribution of their target proteins in selected human carcinoma (LICR-HN6;Caco-2), mouse neuroblastoma (clone NB-2a), and rat glioma cell lines (clone C-6). Pronounced staining with anti-parvalbumin was observed in the cytosol of all cells as well as in some nuclei, in particular, mitotic nuclei were highly immuno-reactive. Applying light and immune-electron microscopy (colloidal gold labelling) the parvalbumin-fluorescence was associated with filaments in the LICR-HN6 cells. However, this immunoreactivity was not a result of the presence of parvalbumin itself--as shown by biochemical analyses (HPLC, 2D-PAGE)--but was due to the presence of a Ca2+-binding and tumour-associated protein with similar biochemical and immunological properties. S-100 proteins were present in all tumour cell lines but their intracellular distribution was different from calbindin D-28K. Calbindin-immunoreactivity was found on the membranes of the carcinoma cell lines whereas neuroblastoma and glioma cells remained unlabelled. It is suggested that these proteins might be involved in the modulation of the enhanced stimulation of Ca2+-dependent processes occurring in tumour cells.

Animals↗
PubMed1987

Measurements of intracellular pH in single LLC-PK1 cells: recovery from an acid load via basolateral Na+/H+ exchange.

LLC-PK1 cells (a continuous epithelioid cell line with renal characteristics) are examined by microspectrofluorometry as single cells, in order to determine the mechanism of intracellular pH (pHi) recovery from an acid load imposed by ammonium preincubation and removal (NH4 prepulse). Initial experiments evaluate the intracellular K+ levels through a null point analysis of total cellular K+ with flame photometry. The response of BCECF (a pH-sensitive fluorescent dye) is then calibrated, using saturating concentrations of nigericin to cause defined changes in pH. For experiments with the microspectrofluorometer, LLC-PK1 cells were grown on either glass coverslips or filters (the latter attached to plastic coverslips with a hole under the filter). The cells on glass coverslips demonstrate a Na+-dependent recovery from an (NH4 prepulse) acid load which is sensitive to 1 microM ethylisopropylamiloride. They also demonstrate a 'set point' of activation of Na+/H+ exchange. When examined for changes in pH, due to changes in membrane potential, plasma membrane proton conductance could not be detected at resting pHi. Cells grown on filters also demonstrate a pHi recovery from an acid load which is Na+ dependent and ethylisopropylamiloride sensitive, but in this configuration, the majority of cells (22/23 preparations) require Na+ at the basolateral membrane for rapid pHi recovery. The morphology and polarity of the cells grown on permeable supports appears normal at the electron-microscopic level. The results are not affected by changes in cell seeding density or collagen treatment of the filters.

Animals↗
PubMed1987

Calcium uptake into rat small intestinal brush border membrane vesicles: characterization of transmembrane calcium transport at short initial incubation times.

Calcium transport into brush border vesicles from rat small intestine was investigated by determining uptake rates at very short incubation periods. At incubation times up to 1 second a linear relationship between calcium uptake and time was observed at free calcium concentrations ranging from 1 microM to 5 mM. At time points above 1 second calcium uptake deviates progressively from linearity. Several lines of evidences (EGTA-wash, dependency on membrane potential, temperature sensitivity and effect of the calcium ionophore A23187) suggest transmembrane transport rather than extravesicular binding of calcium as being responsible for calcium uptake. Saturation experiments performed under initial linear and curvilinear uptake conditions show a saturable transport component in the mu molar and only a tendency to saturate in the molar concentration range. It is concluded that uptake values far from equilibrium are characteristic for transmembrane flux of calcium. Transmembrane flux of calcium is mediated by multiple and potential-sensitive mechanisms.

Animals↗
PubMed1987

Phosphate transport in brush-border membranes from control and rachitic pig kidney and small intestine.

1. Na-Pi co-transport was analysed using renal cortical and small intestinal brush-border membrane vesicles which were isolated from control (normal, heterozygotes) and rachitic piglets (homozygotes). 2. A kinetic analysis of Na-dependent initial linear uptake of Pi was performed using vesicles obtained from control animals. The results suggest similar kinetic properties for the renal and small intestinal co-transport system. (i) A sigmoidal dependence on Na concentration of Pi uptake suggests the involvement of more than one Na ion in the co-transport. (ii) Increasing Na concentration leads to an increase in the apparent affinity of the transport system for Pi and has minimal effect on the apparent Vmax (maximum velocity of uptake). (iii) Increasing pH leads to an increase in Pi transport rate. 3. The kinetic characteristics of the Na-Pi co-transport system in vesicles obtained from rachitic animals were similar to those in controls. The apparent Vmax, but not the apparent Km (Michaelis constant) for Na and Pi, is reduced in intestinal and renal brush-border membranes isolated from rachitic animals as compared to control animals. Injection of vitamin D3, three days prior to killing of rachitic litter-mates, increased the Na-Pi uptake rate in the brush-border membrane vesicles isolated from these piglets. 4. It is concluded that intestinal and renal brush-border membranes from piglets contain a similar Na-Pi co-transport system and that in vitamin-D-dependent rickets the number of operating transport units is reduced in both membranes.

Animals↗