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  • 1
    ISSN: 1432-1424
    Keywords: intracellular electrolytes ; epithelial transport ; Cl secretion ; cell volume regulation ; frog cornea ; isoproterenol ; ionophore A23187 ; ouabain ; bumetanide ; furosemide ; X-ray microanalysis
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Chemistry and Pharmacology
    Notes: Summary The intracellular electrolyte concentrations in the isolated cornea of the American bullfrog were determined in thin freeze-dried cryosections using energy-dispersive X-ray microanalysis. Stimulation of Cl secretion by isoproterenol resulted in a significant increase in the intracellular Na concentration but did not change the intracellular Cl concentration. Similar results were obtained when Cl secretion was stimulated by the Ca ionophore A23187. Inhibition of Cl secretion by ouabain produced a large increase in the intracellular Na concentration and an equivalent fall in the K concentration. Again, no increase or decrease in the intracellular Cl concentration was detectable. Clamping of the transepithelial potential to ±50 mV resulted in parallel changes in the transepithelial current and intracellular Na concentration, but, with the exception of the outermost cell layer, in no changes of the Cl concentration. Only when Cl secretion was inhibited by bumetanide or furosemide, together with a decrease in the Na concentration, was a large fall in the Cl concentration observed. Application of loop diuretics also produced significant increases in the P concentration and dry weight, consistent with some shrinkage of the epithelial cells. The results suggest the existence of a potent regulatory mechanism which maintains a constant intracellular Cl concentration and, thereby, a constant epithelial cell volume. Through the operation of this system any variation in the apical Cl efflux is compensated for by an equal change in the rate of Cl uptake across the basolateral membrane. Cl uptake is sensitive to loop diuretics, directly coupled to an uptake of Na, and dependent on the Na and K concentration gradients across the basolateral membrane. Isoproterenol and A23187 seem to increase the Cl permeability of the apical membrane and thus stimulate Cl efflux. Ouabain inhibits Cl secretion by abolishing the driving Na concentration gradient for Cl uptake across the basolateral membrane.
    Type of Medium: Electronic Resource
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  • 2
    ISSN: 1432-2013
    Keywords: X-ray microanalysis ; Electrolytes ; Picoliter fluid samples
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Notes: Summary A technique is reported for the application of the energy dispersive X-ray microanalysis for the simultaneous determination of electrolytes in picoliter samples of biological fluids. The preparation is characterized by the use of thin films as a specimen support. Using this arrangement, the X-rays generated in the support are kept to a minimum. At an acceleration voltage of 25 kV the specimen preparation can be regarded as ‘thin’, i.e. the intensity of an emitted characteristic radiation is almost uninfluenced by the gross elemental composition, and, therefore, is only dependent upon the content of elements. Quantification was achieved by comparing the intensities of the characteristic radiations with that of a standard. Electrolyte concentrations of 1 mM can be detected with an accuracy of 0.1 mM SD.
    Type of Medium: Electronic Resource
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  • 3
    ISSN: 1432-2013
    Keywords: X-ray microanalysis ; Freeze-dried cryosections ; Cellular electrolyte concentrations
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Notes: Summary A procedure is described which allows the evaluation of wet weight concentrations of diffusible substances in biological soft tissue on a cellular level by the use of energy dispersive X-ray microanalysis. Epithelia of frog skin and toad urinary bladder were used to prepare freeze-dried cryosections without the use of chemical fixatives, cryoprotectants, floating solutions or coating materials. The specimens were shock-frozen inl-propane (−180°C), cryosectioned (−80°C), sandwiched between collodion films and freeze-dried (−80°C). The analysis was performed in a scanning electron microscope at an acceleration voltage of 15 kV, probe current of 0.5 nA, using scanning areas of 1–2 μm2. The spatial resolution power using 1–2 μm thick sections was about 0.7 μm. In a superficial layer of about 30 μm the analysis was found not to be influenced by tissue damage due to ice crystal formation. The mass loss during electron bombardement was restricted to constituents of the organic matrix (30%). No changes of characteristic radiation were observed for Na, K and Cl. Albumin standards, containing electrolyte concentrations in the range of biological interest, revealed linear calibration curves. To obtain reliable wet weight concentrations, the characteristic X-rays of the tissue were compared to those of an internal standard which was applied to the specimen prior to freezing and analysed simultaneously.
    Type of Medium: Electronic Resource
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  • 4
    ISSN: 1432-2013
    Keywords: Novobiocin ; Amiloride ; Transepithelial Na transport ; Intracellular ion concentrations ; X-ray microanalysis ; Intracellular microelectrodes ; Na permeability
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Notes: Abstract Microelectrodes and electron microprobe analysis were employed to study the effect of novobiocin on membrane potential and intracellular electrolyte concentrations in the frog skin epithelium. In both species investigated (Rana esculenta andRana temporaria), novobiocin (1 mM, outer bath) caused a stimulation of transepithelial Na transport, a depolarization of apical membrane potential, a fall in the apical fractional resistance, and an increase in the intracellular Na concentration. The rise in the Na concentration was accompanied by an equivalent fall in the K concentration. All effects of novobiocin were fully reversible by subsequent application of amiloride. The depolarization as well as the Na increase suggests that the natriferic effect of novobiocin is due to a stimulation of the apical Na influx. Combining both measurements it was possible to calculate the effect of novobiocin on the Na permeability of the apical membrane directly. InRana esculenta novobiocin increased the permeability from 4.5 to 23.2 nm/s. InRana temporaria the increase was significantly smaller, from 8.7 to 16.9 nm/s. The transport rate as measured by the short-circuit current showed a non-linear dependence on the apical Na permeability. In the range of transport rates normally encountered, however, the current was a linear function of the Na permeability consistent with the view that the apical membrane is rate-limiting in transepithelial Na transport.
    Type of Medium: Electronic Resource
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  • 5
    ISSN: 1432-2013
    Keywords: Cortical collecting duct ; Principal cells ; Intercalated cells ; Cell electrolyte concentrations ; Ouabain ; Amiloride ; Na-H exchange ; Electron microprobe analysis
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Notes: Abstract Transmembrane sodium transport pathways were studied in principal and intercalated cells of the isolated perfused rabbit cortical collecting duct. Intracellular electrolyte concentrations in individual collecting duct cells were measured by electron microprobe analysis during blockage of basolateral Na-K-ATPase by ouabain and simultaneous inhibition of sodium entry across the apical and/or basolateral cell membrane. In principal cells the ouabain-induced rise in cell sodium concentration could only partially be blocked by amiloride (10−4mol/l) in the perfusion fluid. Amiloride (10−3mol/l) added to the bathing solution produced a further, significant reduction of sodium influx. In principal cells the ouabain-induced increase in sodium concentration was completely prevented by amiloride in the perfusion solution in combination with omission of sodium from the peritubular bathing solution. In intercalated cells ouabain caused a less pronounced increase in sodium concentration than in principal cells. Neither amiloride in the perfusate, nor amiloride in both bathing and perfusion solution, significantly reduced the ouabain-induced rise in intercalated cell sodium concentration. These results indicate that in principal cells amiloride-sensitive sodium channels constitute the predominant pathway for sodium entry across the apical cell membrane. In addition, substantial amounts of sodium enter principal cells across the basolateral cell membrane, probably via Na-H exchange. Finally, the data suggest that in intercalated cells sodium channels and the Na-H exchange are sparse or even absent.
    Type of Medium: Electronic Resource
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  • 6
    ISSN: 1432-2013
    Keywords: Cortical collecting duct ; Cell Na+ concentration ; Cell Rb+ uptake ; Na+/K+-ATPase activity ; Principal cell ; Intercalated cell
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Notes: Abstract The relation between transcellular Na+ absorption, intracellular Na+ concentration and Na+/K+-ATPase activity (the last estimated by the rubidium uptake across the basolateral cell membrane) was examined in the different cell types of the rabbit cortical collecting duct (CCD). Experiments were performed on isolated perfused CCD in which Na+ absorption was varied by perfusing the tubule with solutions containing different Na+ concentrations (nominally Na+-free, 30 mM and 144 mM). Experiments were terminated by shock-freezing the tubules during perfusion. Precisely 30 s before shock-freezing, the K+ in the bathing solution was exchanged for Rb+. Intracellular element concentrations, including Rb+, were determined in freeze-dried cryosections of the tubules using energy-dispersive X-ray analysis. Increasing Na+ concentration in the perfusion solution caused significant rises in intracellular Na+ concentration and Rb+ uptake of principal cells. Principal cell Na+ and Rb+ concentrations were 7.8±0.9 and 7.0±0.8 mmol/kg wet weight respectively, when the perfusion solution was Na+-free, 10.1±0.7 and 11.6±0.6 mmol/kg wet weight with 30 mM Na+ in the perfusion solution, and 14.5±1.5 and 14.9 ±0.9 mmol/kg wet weight with 144 mM Na+ in the perfusion solution. In contrast, a comparable relationship between lumen Na+ concentration, intracellular Na+ concentration and basolateral Rb+ uptake was not seen in intercalated cells. These results support the notion that principal, but not intercalated, cells are involved in transepithelial Na+ absorption. In addition, the data demonstrate that apical Na+ entry and basolateral Na+/K+-AT-Pase activity are closely coupled in principal cells of the rabbit CCD. A rise in lumen Na+ concentration leads to increased Na+ entry and augmented intracellular Na+ concentration, which then secondarily stimulates active basolateral Na+/K+(Rb+) exchange.
    Type of Medium: Electronic Resource
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  • 7
    ISSN: 1432-2013
    Keywords: Cortical collecting duct ; Isolated perfused tubules ; Principal cells ; Intercalated cells ; Cell electrolyte concentrations ; Ouabain ; Electron microprobe analysis
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Notes: Abstract Sodium, phosphorus, chloride and potassium concentrations were measured by a new method in individual principal and intercalated cells in the cortical collecting duct in vitro. Electron microprobe analysis was applied to freezedried cryosections of the isolated perfused rabbit cortical collecting duct. Cell analyses were performed under control conditions and after addition of ouabain to the bath. Under control conditions similar sodium, potassium, chloride, and phosphorus concentration (means±SEM) were observed in principal (10.0±0.6, 126.5±2.7, 24.6±1.0, and 121.5±3.5 mmol/kg wet weight, respectively) and intercalated cells (9.0±0.9, 127.1±4.2, 27.4±1.8, and 118.7±4.9 mmol/kg wet weight, respectively). In principal cells ouabain (10 min) caused an increase in sodium and chloride concentrations by 104 and 13 mmol/kg wet weight, and a decrease in potassium and phosphorus concentrations by 106 and 32 mmol/kg wet weight. These changes in cell element concentrations can be ascribed to an exchange of intracellular potassium against extracellular sodium and to cell swelling due to influx of extracellular fluid. The effects of ouabain on intercalated cells were far less pronounced than on principal cells. This different susceptibility to ouabain of principal and intercalated cells can be ascribed to differences in active and passive transmembrane ion transport pathways.
    Type of Medium: Electronic Resource
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