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  • 1
    ISSN: 1432-2013
    Keywords: Ouabain ; Cell membrane potential ; Cell membrane resistance ; Potassium conductance ; Bicarbonate conductance ; Proximal tubule ; Amphibian kidney
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Notes: Abstract According to a previous study from this laboratory, the electrochemical gradient for potassium across the peritubular cell membrane of proximal tubules in the isolated perfused frog kidney increases following the application of ouabain. In order to test, if this phenomenon were due to a decrease of potassium conductance, the effects of ouabain on cell membrane resistances and the sensitivity of the peritubular cell membrane potential difference (PDpt) to step changes of peritubular potassium and bicarbonate concentration were studied. In the absence of ouabain, PDpt averaged −60±3 mV (n=25). A step increase of peritubular potassium concentration from 3 to 18 mmol/l (pH 8.07) depolarises PDpt (ΔPDk) by +24±2 mV (n=8). An increase of bicarbonate from 20 to 40 mmol/l (pH 8.07) hyperpolarises PDpt (ΔPDb) by −2.8±0.4 mV (n=9). The resistance of the luminal and peritubular cell membranes in parallel (R m) amounts to 45±9 kΩ cm (tubule length) (n=4) and the voltage divider ratio (VDR) to 1.4±0.2 (n=7). The resistance of the cellular cable (cellular core,R c) approaches 131±37 MΩ/cm (n=4). Peritubular application of 0.1 mmol/l ouabain leads to a gradual decline of PDpt (t 1/2 approx. 30 min), to an increase ofR m, a decrease of ΔPDk and an increase of ΔPDb. VDR andR c are not changed significantly. The data point to a functional link between the sodium/potassium ATPase and the potassium conductance of the peritubular cell membrane.
    Type of Medium: Electronic Resource
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  • 2
    ISSN: 1432-2013
    Keywords: Frog kidney ; Proximal tubule ; Glucose transport ; Ouabain ; Cell membrane potential ; Intracellular sodium ; Microelectrodes
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Notes: Abstract Inhibition of basolateral Na+/K+ ATPase by ouabain eventually abolishes transport of glucose. The present study was performed to test, if this effect is due to a dissipation of the electrochemical gradient for sodium or due to a regulatory inhibition of sodium-coupled glucose entry across the luminal membrane at increasing intracellular sodium activity. To this end, proximal convoluted tubules of the doubly perfused isolated frog kidney were perfused alternatively with solutions containing either 5 mmol/l glucose or raffinose. The potential difference across the peritubular cell membrane (PDpt) and across the epithelium (PDpt) has been recorded with conventional and across the peritubular cell membrane with ion selective microelectrodes (PDpt). In the absence of luminal glucose PDpt is (±SEM) −54.0±2.4 mV, PDte=−1.2±2.0 mV and PD pt Na =−96±5 mV. The electrochemical gradient for sodium (μNa+) amounts to 95 mV and intracellular sodium activity to 14 mmol/l (extracellular sodium activity is 74 mmol/l). Luminal application of glucose leads to a rapid depolarisation of PDpt (ΔPDpt=8.6±0.9 mV and PD pt Na (ΔPD pt Na =11.1±3.0 mV) and to hyperpolarisation of PDte (ΔPDte=−0.8±0.2 mV). The peritubular application of ouabain leads to a gradual, reversible and proportional decline of PDpt, PD pt Na and μNa+. Glucose induced ΔPDpt and ΔPD pt Na decrease in parallel to PDpt and PD pt Na , resp. In a separate series, the lumped conductance (G m) of the luminal and basolateral cell membrane has been determined, which amounts to 2.4±0.3 μS/mm (tubule length).G m decreases 23±4%, when PDpt is decreased to half. ΔPDpt andG m allow the calculation of an apparent transport rate (T Glu). Following the application of ouabain,T Glu decreases in linear proportion to PDpt and PD pt Na . There is no evidence for a significant regulatory inhibition ofT Glu. Rather, glucose transport operates in linear proportion to the potential difference across the luminal membrane.
    Type of Medium: Electronic Resource
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  • 3
    ISSN: 1432-2013
    Keywords: cAMP ; Frog kidney ; Proximal tubule ; Ionsensitive microelectrodes ; Sodium transport
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Notes: Abstract Experiments were performed in proximal tubule of the isolated perfused frog kidney to evaluate peritubular cell membrane potentials (PDpt), and the intracellular ion activities of sodium (Nai ü), chloride (Cli −) and potassium (Ki ü) under control conditions and following peritubular application of dibutyryl-cyclic AMP (cAMP, 2·10−4mol·l−1). Conventional and ion-sensitive microelectrodes were applied to record continuously cAMP-induced changes of these parameters in individual proximal tubule cells. Within a few minutes a significant hyperpolarisation of PDpt (Δ=2.0±0.2 mV) occurs simultaneously with a decrease of Nai ü (Δ=2.5±0.5 mmol·l−1). Ki ü increases (Δ=3.6±0.9 mmol·l−1) and Cli − decreases (0.4±0.07 mmol·l−1) slightly, but significantly. With both ions the alterations of the chemical gradient is significantly smaller than the potential shift. PDte is not significantly altered by cAMP. The cAMP-induced hyperpolarisation of PDpt can be observed in presenceand absence of luminal glucose. However, omission of Naü from the luminal perfusate abolishes the hyperpolarising effect of cAMP on PDpt. The results suggest that cAMP reduces sodium entry from the lumen into the cell, thus hyperpolarising the cell membrane and decreasing Nai ü. Persistance of sensitivity of PDpt to cAMP after omission of glucose indicates that other Naü coupled transport processes and/or passive Naü conductance are affected by cAMP. the changes of Ki ü and Cli − are secondary, following the change of PDpt.
    Type of Medium: Electronic Resource
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  • 4
    ISSN: 1432-2013
    Keywords: Ouabain ; Frog kidney ; Proximal tubule ; Ion sensitive microelectrodes ; Na+/K+ pump ; Intracellular K+, Na+, Cl−, pH, Ca2+
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Notes: Abstract Using conventional and ion selective microelectrodes, the effect of ouabain (10−4 mol/l) on peritubular cell membrane potential (PDpt), on intracellular pH (pHi) as well as on the intracellular ion activities of Cl− (Cl i − ), K+ (K i + ), Na+ (Na i + ) and Ca2+ (Ca i 2+ ) was studied in proximal tubules of the isolated perfused frog kidney. In the absence of ouabain (PDpt=−57.0±1.9 mV), the electrochemical potential difference of chloride (apparent {ie6-1} and of potassium {ie6-2} is directed from cell to bath, of H+ {ie6-3}, of Na+ {ie6-4} and of Ca2+ {ie6-5} from bath to cell. Ouabain leads to a gradual decline of PDpt, which is reduced to half (PDpt, 1/2) within 31±4.6 min (in presence of luminal glucose and phenylalanine), and to a decline of the absolute values of apparent {ie6-6}, of {ie6-7}, {ie6-8} and {ie6-9}. In contrast, an increase of {ei6-10} is observed. At PDpt, 1/2 apparent Cl i − increases by 6.2±1.0 mmol/l, pHi by 0.13±0.03, Ca i 2+ by 185±21 nmol/l, and Na i + by 34.2±4.6 mmol/l, whereas K i + decreases by 37.7±2.2 mmol/l. The results suggest that the application of ouabain is followed by a decrease of peritubular cell membrane permeability to K+, by an accumulation of Ca2+, Na+ and HCO 3 - in the cell and by a dissipation of the electrochemical Cl− gradient.
    Type of Medium: Electronic Resource
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