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  • 1
    Electronic Resource
    Electronic Resource
    Culturing induced expression of basolateral Na+-K+-2Cl− cotransporter BSC2 in proximal tubule, aortic endothelium, and vascular smooth muscle (1996)
    Springer
    Pflügers Archiv 431 (1996), S. 458-460 
    Details
    ISSN: 1432-2013
    Keywords: bumetanide ; rubidium uptake
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Notes: Abstract So far, two isoforms of the neutral Na+-K+-2Cl− cotransporter have been cloned in mammals. One isoform, BSC1, mediates apical ion entry in the renal thick ascending limb of Henle and a second, BSC2, appears to be an ubiquitously expressed Na+-K+-2Cl− cotransporter. In primary cultures of rabbit proximal tubule, porcine aortic endothelial cells, and rat vascular smooth muscle cells, expression of the second isoform BSC2 was demonstrated by Northern blot analysis and bumetanide-sensitive86Rb+ uptake studies. A surprising finding was the absence of BSC2 in fully differentiated freshly-isolated proximal tubule, porcine aortic endothelial cells, and rat vascular smooth muscle cells. Several studies have reported modulation of the cotransport activity by vasoactive substances and suggested a role for disturbed cotransport in, for example, the pathogenesis of essential hypertension. All these observations, however, were made in cultured cells which, in view of our findings, makes the physiological relevance questionable.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF02207286
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  • 2
    Electronic Resource
    Electronic Resource
    A modified confocal laser scanning microscope allows fast ultraviolet ratio imaging of intracellular Ca2+ activity using Fura-2 (1997)
    Springer
    Pflügers Archiv 433 (1997), S. 653-663 
    Details
    ISSN: 1432-2013
    Keywords: Key words Confocal microscopy ; Acousto-optic tunable filter ; Fura-2 ; Ratio imaging ; HT29 cells
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Notes: Abstract  A confocal, ultraviolet laser scanning microscope (LSM) for reliable ratio measurements of localized intracellular Ca2+ gradients using the Ca2+-sensitive dye Fura-2 was developed. In a commercial LSM, the filter wheels for the excitation band-pass filters and the grey filters were replaced by acousto-optic tunable filters (AOTF) for rapid switching (≤1.5 μs) of the ultraviolet (351 and 364 nm) and the visible (457, 476, 488, 514 nm) excitation light. This enabled dual wavelength excitation of Fura-2, or 2’7’-biscarboxyethyl-5(6)-carboxyfluorescein (BCECF) for pH measurements. Changing to a transmitted-light detector of high sensitivity allowed for simultaneous recording of differential interference contrast images of the preparation with the excitation light. The AOTF fine control of the intensity of the excitation light and improvements in the emission detector sensitivity enabled the acquisition of up to 120 ratio pairs of high-quality images from a single cell. The optical capabilities and limitations of the instrument were evaluated with fluorescent beads and dye-loaded cultured cells. Agonist-induced intracellular Ca2+ transients in HT29 cells were recorded to test for the instrument’s ability to measure changes in [Ca2+]i. Ratio z-sections from Fura-2-loaded cells showed an inhomogeneity of the Fura-2 loading with an accumulation of the dye mostly in the mitochondria. We show, as an example of the microscope’s achievable resolution, the spatial and temporal heterogeneity of [Ca2+]i signals in mitochondria and the cytosol in response to agonist-evoked stimulation of HT29 cells. In addition, we show that the lipophilic, membrane-bound Fura-2 derivative Fura-C18, for measurements of near-membrane Ca2+ changes, can be used with this confocal microscope. This new LSM is expected to deepen our understanding of localized [Ca2+]i signals; for example, the nuclear Ca2+ signalling or the [Ca2+]i changes that occur during stimulation of ion secretion in polarized epithelial cells.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/s004240050327
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  • 3
    Electronic Resource
    Electronic Resource
    Cellular acidification occurs during anoxia in cultured, but not in freshly isolated, rabbit proximal tubular cells (1995)
    Springer
    Pflügers Archiv 429 (1995), S. 722-728 
    Details
    ISSN: 1432-2013
    Keywords: Ischaemia ; Intracellular pH ; Proximal tubule ; Primary culture
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Notes: Abstract In a variety of cells it has been shown that acidosis is protective against anoxic injury. We have demonstrated previously that proximal tubule (PT) cells in primary culture were more resistant to anoxiainduced cell injury than were freshly isolated cells. Therefore, we asked the question of whether a difference in cellular acidification during anoxia could explain this difference in susceptibility to anoxia. To answer this question, intracellular pH (pHi) was measured during anoxic incubation of PT cells in culture and those that were freshly isolated. PT cells were incubated in an anoxic chamber at 37°C after loading with 2′,7′-bis-(2-carboxyethyl)-5,6-carboxyfluorescein acetoxymethyl ester (BCECF-AM) or fura-2 acetoxymethyl ester (fura-2-AM). pHi and cytosolic free Ca2+([Ca2+]i) were measured by digital imaging fluorescence microscopy. During anoxia, pHi in cultured PT cells decreased from 7.3±0.1 to 6.8±0.1, whereas pHi in freshly isolated cells did not decrease significantly. In addition, the intrinsic buffering capacities (β i) in cultured and freshly isolated PT cells were determined and turned out to be the same at a pHi greater than or equal to 7.3. Below pHi 7.3, β i increased several fold in freshly isolated PT cells, and rose to significantly higher levels than in cultured PT cells. During 1 h of anoxia, cell viability of freshly isolated PT cells decreased significantly to 54%±2% (P〈0.05), while no loss in viability was observed in cultured PT cells. Clamping the pHi during anoxia at 6.7 and 6.1 significantly increased cell viability in freshly isolated PT cells to 76%±5% and 72%±4%, respectively (P〈0.05). In contrast, prevention of acidification in cultured PT cells during anoxia did not lead to increased cell death. Therefore, the differences in susceptibility to anoxic injury between cultured and freshly isolated PT cells cannot be explained by cellular acidification in cultured cells, but must be sought elsewhere.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF00373995
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  • 4
    Electronic Resource
    Electronic Resource
    Effects of substrate-free anoxia and veratridine on intracellular calcium concentration in isolated rat ventricular cardiomyocytes (1994)
    Springer
    Pflügers Archiv 428 (1994), S. 142-149 
    Details
    ISSN: 1432-2013
    Keywords: Ischaemia ; Ca2+ overload ; Cell injury ; Cell volume ; R 56865
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Notes: Abstract Cytosolic free Ca2+ concentration ([Ca2+]i) was measured in freshly isolated rat ventricular cardiomyocytes during substrate-free anoxia. Cardiomyocytes were loaded with fura-2 and incubated in an anoxic chamber in which a pO2 equal to 0 mmHg was realized by inclusion of Oxyrase. [Ca2+]i was measured in individual cells using digital imaging fluorescence microscopy. During anoxia, the shape of cardiomyocytes changed from a relaxed-elongated form into a rigor configuration within 15 min after the onset of anoxia. After the cells had developed the rigor state, a delayed rise in [Ca2+]i reached a stable maximal level within 45 min. The mean values for the pre-anoxic and maximal anoxic [Ca2+ i were 52±3 nM (N=42) and 2115±59 nM (N=45), respectively. The purported Na+ overload blocker R 56865, significantly reduced maximal anoxic [Ca2+]i to 553±56 nM (P〈0.05), implicating a role of elevated intracellular Na+ in the anoxia-induced increase in [Ca2+]i. Veratridine (30 μM), which induces Na+ overload, increased [Ca2+]i to 787±39 nM. The compound R 56865 reduced veratridine-induced increases in [Ca2+]i to 152±38 nM. Upon reperfusion, after 45 min of anoxia, two distinct responses were observed. Most often, [Ca2+]i decreased upon reperfusion without a change in morphology or viability, while in the minority of cases, [Ca2+]i increased further followed by hypercontraction and loss of cell viability. The mean value for [Ca2+]i 10 min after reperfusion of the former group, was 752±46 nM (N=38). The cardiomyocyte cell shape could be followed by monitoring changes in the total fura-2 fluorescence (340+380 nm signal). Within 15 min after the onset of anoxia, the total fluorescence signal increased suddenly, before [Ca2+]i started to rise, coinciding with the onset of rigor contraction induced by ATP depletion.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF00374851
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  • 5
    Electronic Resource
    Electronic Resource
    The colon carcinoma cell line Caco-2 contains an H+/K+-ATPase that contributes to intracellular pH regulation (1992)
    Springer
    Pflügers Archiv 421 (1992), S. 591-597 
    Details
    ISSN: 1432-2013
    Keywords: Caco-2 ; H+/K+-ATPase ; SCH 28080 ; Intracellular pH ; Na+/H+ exchange ; N-Ethylmaleimide ; Bafilomycin A1 ; Rabbit distal colon
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Notes: Abstract The presence of an H+/K+-ATPase and its contribution to the regulation of intracellular pH (pHi) was investigated in Caco-2 cells. The H+/K+-ATPase was detected immunologically using the monoclonal antibody 5-B6, which was raised against hog gastric H+/K+-ATPase. Cell pH was determined using the pH-sensitive dye 2′,7′-bis(carboxyethyl)-carboxyfruorescein. Control pHi, measured in HCO 3 − -free medium, was 7.62±0.03 (n=27) when cells were cultured for 14 days and decreased to 7.40±0.03 (n=18) after 35 days in culture. Recovery of pHi following a NH 4 + /NH3 pulse could be reduced by either 100 μM SCH 28080 or 1 mM amiloride, or by removing extracellular Na+. The inhibitory effects of SCH 28080 and amiloride were additive, demonstrating the involvement of a gastric-like H+/K+-ATPase and a Na+/H+ exchanger in regulating pHi. Recovery rates at pHi 6.8 were not significantly different in cells cultured for up to 21 days, but were significantly lower in cells cultured for 28 and 35 days. This decrease in recovery rate was due to a decrease in the SCH-28080-insensitive recovery, indicating a reduction of the relative importance of Na+/H+ exchange to the recovery. Recovery of pHi was also inhibited by 1 mM N-ethylmaleimide. However, it is unlikely that N-ethylmaleimide inhibited a vacuolar type of H+-ATPase, since bafilomycin A1 had no effect on pHi recovery. In conclusion, Caco-2 cells contain a SCH-28080-sensitive mechanism for regulating pHi, which is most conveniently studied after 28 days in culture, when the relative contribution of a Na+/H+ exchanger to pHi regulation is decreased.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF00375056
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  • 6
    Electronic Resource
    Electronic Resource
    Protection against hypoxic injury of rat proximal tubules by felodipine via a calcium-independent mechanism (1995)
    Springer
    Pflügers Archiv 431 (1995), S. 20-27 
    Details
    ISSN: 1432-2013
    Keywords: Ischaemia ; Calcium channel blocker ; potassium channels
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Notes: Abstract Most evidence for a key role of calcium entry in hypoxia-induced renal damage stems from studies with calcium channel blockers. In proximal tubules, a primary site of renal ischaemic injury, only phenylalkylamines, especially verapamil, have been studied. In the present study the effect of the dihydropyridine felodipine on hypoxic injury in isolated rat proximal tubules was investigated. To discriminate between the block of calcium entry and other effects, the enantiomers and a non-calcium blocking derivative of felodipine (H186/86) were included. Cell membrane injury was assessed by measuring the release of lactate dehydrogenase (LDH). At high concentrations (100 μM) felodipine, H186/86 and the two enantiomers all protected rat proximal tubules against hypoxiainduced injury to the same extent. Absence of extracellular calcium did not offer protection, but rather enhanced hypoxic injury. All dihydropyridines used increased the intracellular potassium concentration during normoxia. Felodipine attenuated the hypoxiainduced loss of cellular potassium. We have tried to mimic the effects of felodipine by using potassium channel blockers. The potassium channel blockers quinidine and glibenclamide afforded some protection against hypoxic injury, although their effects on cellular potassium were equivocal. We conclude that the dihydropyridine calcium channel blocker felodipine protects rat proximal tubules against hypoxic injury via a calcium-independent mechanism. We propose that high levels of intracellular potassium and attenuation of potassium loss during hypoxia are important in this protection.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF00374373
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  • 7
    Electronic Resource
    Electronic Resource
    Role of Na+/Ca2+ exchange in transcellular Ca2+ transport across primary cultures of rabbit kidney collecting system (1992)
    Springer
    Pflügers Archiv 420 (1992), S. 566-572 
    Details
    ISSN: 1432-2013
    Keywords: Na+/Ca2+ exchange ; Rabbit kidney ; Connecting tubule ; Cortical collecting duct ; Ca2+ reabsorption ; Bepridil ; Ca2+ entry blockers ; Ca2+ transport
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Notes: Abstract Cells from connecting tubule and cortical collecting duct of rabbit kidney were isolated by immunodissection with mAb R2G9 and cultured on permeable filters. Confluent monolayers developed an amiloride-sensitive transepithelial potential difference of −50±1 mV (lumen negative) and a transepithelial resistance of 507±18 Ω cm2. Transepithelial Ca2+ transport increased dose-dependently with apical [Ca2+] and, in solutions containing 1 mM Ca2+, the active transcellular Ca2+ transport rate was 92±2 nmol h−1 cm−2. Transcellular Ca2+ transport was dependent on basolateral Na+ (Na b + ). Isoosmotic substitution of Na b + for N-methylglucamine resulted in a concentration-dependent decrease in Ca2+ absorption, with maximal inhibition of 67±5%. A Hill plot of the Na+-dependence yielded a coefficient of 1.9±0.4, indicating more than one Na+ site on a Na+-dependent Ca2+ transport system. In addition, the absence of Ca b 2+ resulted in a significant increase in Ca2+ transport both in the presence and absence of Na b + . Added basolaterally, ouabain (0.1 mM) inhibited Ca2+ transport to the same extent as did Na+-free solutions, while bepridil (0.1 mM), an inhibitor of Na+/Ca2+ exchange, reduced Ca2+ transport by 32±6%. Methoxyverapamil, felodipine, flunarizine and diltiazem (10 μM) were without effect. Depolarisation of the basolateral membrane, by raising [K+]b to 60 mM, significantly decreased transcellular Ca2+ transport, which is indicative of electrogenic Na+/Ca2+ exchange. In conclusion, active Ca2+ transport in the collecting system of rabbit kidney is largely driven by basolateral Na+/Ca2+ exchange. However, a residual Ca2+ absorption of about 30% was always observed, suggesting that other Ca2+ transport mechanisms, presumably a Ca2+-ATPase, participate as well.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF00374634
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  • 8
    Electronic Resource
    Electronic Resource
    The effect of L-type Ca2+ channel blockers on anoxia-induced increases in intracellular Ca2+ concentration in rabbit proximal tubule cells in primary culture (1993)
    Springer
    Pflügers Archiv 423 (1993), S. 378-386 
    Details
    ISSN: 1432-2013
    Keywords: Proximal tubule ; Kidney ; Ca2+ channel blockers ; Phenylalkylamine ; Dihydropyridine ; Anoxia ; Intracellular Ca2+
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Notes: Abstract Ca2+ channel blockers (CCB) have been shown to be protective against ischaemic damage of the kidney, suggesting an important role for intracellular Ca2+ ([Ca2+]i) in generating cell damage. To delineate the mechanism behind this protective effect, we studied [Ca2+]i in cultured proximal tubule (PT) cells during anoxia in the absence of glycolysis and the effect of methoxyverapamil (D600) and felodipine on [Ca2+]i during anoxia. A method was developed whereby [Ca2+]i in cultured PT cells could be measured continuously with a fura-2 imaging technique during anoxic periods up to 60 min. Complete absence of O2 was realised by inclusion of a mixture of oxygenases in an anoxic chamber. [Ca2+]i in PT cells started to rise after 10 min of anoxia and reached maximal levels at 30 min, which remained stable up to 60 min. The onset of this increase and the maximal levels reached varied markedly among individual cells. The mean values for normoxic and anoxic [Ca2+]i were 118±2 (n=98) and 662±22 (n=160) nM, respectively. D600 (1 μM), but not felodipine (10 μM), significantly reduced basal [Ca2+]i in normoxic incubations. During anoxia 1 μM and 100 μM D 600 significantly decreased anoxic [Ca2+]i levels by 22 and 63% respectively. Felodipine at 10 μM was as effective as 1 μM D600. Removal of extracellular Ca2+ and addition of 0.1 mM La3+ completely abolished anoxia-induced increases in [Ca2+]i. We conclude that anoxia induces increases in [Ca2+]i in rabbit PT cells in primary culture, which results from Ca2+ influx. Since this Ca2+ influx is partially inhibited by low doses of CCBs, Ltype Ca2+ channels may be involved.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF00374931
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  • 9
    Electronic Resource
    Electronic Resource
    Increased plasma calcitonin levels in young spontaneously hypertensive rats: role in disturbed phosphate homeostasis (1987)
    Springer
    Pflügers Archiv 408 (1987), S. 395-400 
    Details
    ISSN: 1432-2013
    Keywords: Spontaneously hypertensive rat ; Calcitonin ; 1,25-dihydroxycholecalciferol ; Parathyroid hormone ; Phosphate metabolism ; Bone
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Notes: Abstract 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〈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〈0.02) while 25-hydroxycholecalciferol was unaltered (p〈0.51). In addition total immunoreactive PTH, iPTH, was slightly increased (p〈0.07) but intact PTH (1–84) (p〉0.90) was not significantly different from age matched WKY controls. Also at 8 weeks, a slightly reduced serum ionized Ca2+ concentration (p〈0.001) with no change in total serum calcium was found in SHR (p〉0.39). Balance studies at 6 and 8 weeks of age revealed no significantly different balances for phosphate (F=2.5,p〉0.10) and for calcium (F=2.6,p〉0.09), although a tendency for slightly more positive balaces existed in SHR when compared to WKY. However, SHR excreted significantly less phosphate in the urine than WKY control (F=0.2,p〈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±2mg), bone ash weight (79±4 vs. 66±1 mg) and bone volume (0.196±0.007 vs. 0.165±0.004 cm3) were significantly greater in SHR. Calcium content per femur (717±35 vs. 617±11 μmol Ca/femur) and phosphate content per femur (512±23 vs. 447±8 μmol P/femur) were also significantly higher in SHR. It is discussed that the disturbances in phosphate homeostasis may be secondary to the strikingly increased plasma calcitonin levels present in young SHR.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF00581135
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  • 10
    Electronic Resource
    Electronic Resource
    Interaction of ethidium bromide with yeast cells investigated by electron probe X-ray microanalysis (1983)
    Springer
    The journal of membrane biology 73 (1983), S. 131-136 
    Details
    ISSN: 1432-1424
    Keywords: electron probe X-ray microanalysis ; Saccharomyces cerevisiae ; ethidium ; brontophenol blue ; cationic dye ; cytolysis
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Chemistry and Pharmacology
    Notes: Summary K+ efflux provoked by ethidium proceeds partially as an all-or-none effect by which the diffusion barrier for K+ is disrupted and partially from still intact cells, presumably by exchange against ethidium. This is shown by the application of an electron probe microanalysis X-ray technique by which the K+ content of a number of individual cells is analyzed.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF01870436
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