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  • 1
    ISSN: 1432-2013
    Keywords: Platinum electrode ; Isolated perfused tubule ; Mouse ; Redox activity ; Uric acid ; Proximal tubule ; Probenecid ; Pyrazinamide
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Notes: Abstract The design and application of a micro-plantinum electrode for continuous monitoring of reducing activity in the isolated tubule preparation is described. The electrodes response to H2O2 up to 0.1 mmol/l, to uric acid up to 0.3 mmol/l, ascorbic acid up to 1.0 mmol/l and cysteine up to 2.0 mmol/l is almost linear. The electrode is insensitive to extracellular ions, to changes of pH (5.5–8.0), CO2 (1–10%) and O2 (1–100%). The reading of the electrodes is almost doubled when the temperature is increased from 20–40°C. When reducing substances are omitted from the perfusate for isolated perfused proximal tubules of the mouse, the reading is identical in perfusate and collected fluid, indicating that the tubular epithelium does not produce redox substances in sufficient amount to interfere with the electrode reading at flow rates ≈ 10 nl/min. When the tubule is perfused with solutions containing 0.3 mmol/l uric acid, the uric acid concentration in the collected fluid is 0.16±0.01 mmol/l after a contact time of 1.36±0.1 s, revealing net uric acid reabsorption. Adding probenecid to the luminal perfusion fluid, leads to a 37.5±1.0% increase of uric acid concentration in collected fluid, disclosing the inhibitory effect of probenecid on uric acid reabsorption. If 0.3 mmol/l uric acid is added to the bath, 0.017±0.002 mmol/l uric acid is detected in the luminal fluid. The entry of uric acid into the lumen is abolished by 10−4 mol/l pyrazinamide.
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  • 2
    ISSN: 1432-2013
    Keywords: Renal tubule ; Cystine/cysteine reabsorption ; Dibasic amino acids ; Microperfusion ; Diamide
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Notes: Abstract Interactions ofl-cysteine (=cys) andl-cystine (=cys-cys), and dibasic amino acids were investigated during tubular reabsorption by microperfusion experiments in rat kidney. The following results were obtained: The dibasic amino acidsl-ornithine andl-canavanine were strong inhibitors of cys-cys reabsorption. The arginine analogue agmatine and the lysine analogue 2,6-diaminopimelic acid had no effect. The oxidizing agent azodicarboxylic acid bis-dimethylamide (=diamide) decreased the fractional reabsorption rate (=FRR) of cys-cys (0.08 mmol·l−1) from 84% to 60% when present in the perfusion fluid in a concentration of 10 mmol·l−1. Diamide did not affect the reabsorption of a dibasic amino acid (l-arginine) nor of a neutral amino acid (l-phenylalanine). The FRR ofl-arginine andl-ornithine could not be decreased by adding cys-cys to the perfusion fluid. Cys had just as little effect on the reabsorption ofl-arginine like agmatine. In the presence of α-aminoisobutyric acid a slight reduction of the FRR ofl-arginine could be observed. The dibasic amino acidsl-arginine andl-canavanine had no influence on the FRR of cys when dithioerythritol was added to the perfusion fluid. Conclusions: More than one site exists for tubular reabsorption of cys-cys. One of these may be shared by dibasic amino acids. Cys is reabsorbed by a separate and specific transport system. A reduction of cys-cys to cys takes place rather in the tubular cell than in the lumen.
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  • 3
    Electronic Resource
    Electronic Resource
    Springer
    Pflügers Archiv 395 (1982), S. 190-195 
    ISSN: 1432-2013
    Keywords: Microperfusion ; Renal tubule ; Cystine ; Cysteine ; Cystathionine ; Neutral amino acids
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Notes: Abstract In microperfusion experiments renal tubular reabsorption of35S- and14C-labelledl-cysteine (=cys),l-cystine (= cys-cys), andl-cystathionine was measured in vivo et situ at different initial concentrations. The interactions of cys and cys-cys with several neutral amino acids were investigated. The cys reabsorption mechanism was found to be saturable and has a high capacity and a low affinity for cys. AnJ max-value of 3.22±0.88 nmol · m−1 · s−1 and aK m-value of 7.5±0.7 mmol · l−1 were estimated. A saturation of cys-cys reabsorption could not be demonstrated. The fractional reabsorption rate (=FRR) of cys-cys was about 85% at initial concentrations of 0.01, 0.08, and 0.4 mmol · l−1 after a perfusion distance of 2 mm. The FRR ofl-cystathionine at an initial concentration of 0.115 mmol · l−1 was only 30% under the same conditions. After perfusion of tubule segments between late proximal and early distal loops the recovery of cys, cys-cys, and cystathionine was smaller than 10%. The FRR of cys was decreased only byl-methionine. Six other neutral amino acids had no effect. On the other hand the FRR of cys-cys was reduced significantly by any of the tested neutral amino acids. The inhibitory effect increased in the orderl-alanine 〈l-methionine 〈l-citrulline 〈 α-aminoisobutyric acid 〈l-phenylalanine 〈 cycloleucine. The FRR ofl-methionine andl-phenylalanine was slightly reduced in the presence of cys. It is concluded from these results that cys-cys shares a transport system with other neutral amino acids which is not identical with the reabsorption mechanism for cys. Reabsorption of cys, cys-cys, and cystathionine occurs also in a tubular section between late proximal and early distal sites.
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  • 4
    Electronic Resource
    Electronic Resource
    Springer
    Pflügers Archiv 400 (1984), S. 388-392 
    ISSN: 1432-2013
    Keywords: Microelectrode ; Galactose ; Raffinose ; Isolated perfused tubule ; Mouse ; Proximal tubule ; Volume reabsorption ; Acetazolamide
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Notes: Abstract Manufacture, properties and use of a micro enzyme electrode for continuous monitoring of volume fluxes in the isolated tubule preparation is described. The specific electrode is a galactose-oxidase enzyme electrode, which can be used to detect changes in raffinose concentrations. The electrode's response to raffinose is almost linear over concentrations from 0–12 mmol/l. The electrode equally responds to galactose as to raffinose but is insensitive to other sugars, to pH changes (from 6.0–8.0), CO2 (from 1–10%) and electrolytes tested. Reducing O2 from 100 to 10% and to 1%, leads to a reduction of the reading by 10% and 30%, respectively. The reading is almost doubled when the temperature is increased from 20–40° C. Furthermore, reducing agents such as uric acid and ascorbic acid interfere with the reading. If these substances and raffinose are omitted from the perfusate for isolated perfused proximal mouse tubules, the reading is identical in perfusate and collected fluid, indicating that the tubular epithelium does not produce substances in sufficient amounts to interfere with the electrode reading. After addition of 6 mmol/l raffinose to the perfusate the raffinose concentration in the collected fluid of 0.76±0.05 mm segments of straight proximal mouse tubules (perfusion rate = 3.4±0.45 nl/min) is 10.2±0.3 mmol/l, indicating a volume reabsorption of 1.5±0.3 nl/min. Peritubular application of acetazolamide reduces the volume reabsorption by 42±4%.
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  • 5
    Electronic Resource
    Electronic Resource
    Springer
    Pflügers Archiv 411 (1988), S. 514-519 
    ISSN: 1432-2013
    Keywords: Cell volume regulation ; Proximal tubule ; K+-conductance ; Bicarbonate conductance ; Chloride conductance ; Cell membrane potential ; Microelectrodes
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Notes: Abstract The present study has been designed to test for the influence of cell swelling on the potential difference and conductive properties of the basolateral cell membrane in isolated perfused proximal tubules. During control conditions the potential difference across the basolateral cell membrane (PDbl) is −65±1 mV (n=74). Decrease of peritubular osmolarity by 80 mosmol/l depolarizes the basolateral cell membrane by +7.8±0.5 mV (n=42). An increase of bath potassium concentration from 5 to 20 mmol/l depolarizes the basolateral cell membrane by +25±1 mV (n=11), an increase of bath bicarbonate concentration from 20 to 60 mmol/l hyperpolarizes the basolateral cell membrane by −3.2±0.5 mV (n=13). A decrease of bath chloride concentration from 79.6 to 27 mmol/l hyperpolarizes the basolateral cell membrane by −1.8±0.7 mV (n=6). During reduced bath osmolarity, the influence of altered bath potassium concentration on PDbl is decreased (Δ PDbl=+16±2 mV,n=11), the influence of altered bicarbonate concentration on PDbl is increased (Δ PDbl=−6.0±0.8 mV,n=13), and the influence of altered bath chloride concentration on PDbl is unaffected (Δ PDbl=−1.8±0.6 mV,n=6). Barium depolarizes the basolateral cell membrane to −28±2 mV (n=16). In the presence of 1 mmol/l barium, decrease of peritubular osmolarity by 80 mosmol/l leads to a transient hyperpolarization of the basolateral cell membrane by −5.9±0.5 mV (n=16). This transient hyperpolarization is blunted in the absence of extracellular bicarbonate. In conclusion, cell swelling depolarizes straight proximal tubule cells and increases bicarbonate selectivity of the basolateral cell membrane at the expense of potassium selectivity. The data reflect either incrases of bicarbonate conductance or decrease of potassium conductance during exposure of proximal tubule cells to hypotonic media.
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  • 6
    Electronic Resource
    Electronic Resource
    Springer
    Pflügers Archiv 387 (1980), S. 253-259 
    ISSN: 1432-2013
    Keywords: Microperfusion ; Renal tubule ; l-Proline reabsorption ; Molecular specificity
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Notes: Abstract In microperfusion experiments the reabsorption of3H and14C labelledl-proline by two recently defined transport systems (one with high capacity and low affinity, the other one having the opposite characteristics) was measured in vivo et situ on addition of several amino acids and some N-methylated derivatives. The high capacity system is apparently an unspecific system for neutral amino acids. The methylation of the amino group does not change the affinity to the system. The affinity decreases in the order phenylalanine 〉glutamine〉alanine〉proline, hydroxyproline 〉glycine. The low capacity system seems to be a specific reabsorption mechanism for imino acids like proline, hydroxyproline, sarcosine and N-methylalanine. Common neutral amino acids are not accepted. The different characteristics of both transport systems are also demonstrated by the finding that the affinity of phenylalanine for the high capacity system is about 5 times higher but its affinity for the low capacity system is about 50 times lower than the affinity for proline.
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  • 7
    ISSN: 1432-2013
    Keywords: Kidney ; Tubular resorption ; Microperfusion ; Specificity ; Glutamate ; Aspartate ; Cysteate ; γ-Carboxyglutamate ; Pyroglutamate ; 5-oxo-proline
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Notes: Abstract Single sections of superficial proximal convolutions of rat kidney were microperfused in vivo and in situ. The perfusion fluids contained radioactively labelledl- ord-aspartate,l-glutamate,l-pyroglutamate, or N-methyl-d-aspartate.l-γ-Carboxyglutamate as well as the other amino acids were added in the unlabelled from. Results.l- andd-Aspartate (0.073 mmol·1−1) are quickly resorbed at about the same rate.d-Aspartate resorption was blocked byl-aspartate (5 mmol·1−1) but not by β-alanine (5 mmol·1−1).l-Aspartate resorption was inhibited byl-glutamate (2 mmol·1−1) but not byd-glutamate,l-asparagine,l-phenylalanine or by succinate (2 mmol·1−1, each). The fast resorption ofl-glutamate (0.073 mmol·1−1) was blocked byd-aspartate,l-cysteate (2 mmol·1−1), but not by 3-mercaptopicolinic acid (0.15 mmol·1−1),l-glutamine, 2-oxoglutarate, taurine, N-methyl-l-glutamate or kainic acid (2 mmol·1−1, each).l-γ-Carboxyglutamate (0.66 mmol·1−1) and N-methyl-d-aspartate (2μmol·1−1) were found to be resorbed only at an extremely small rate.l-pyroglutamate (0.076 mmol·1−1) resorption was not influenced byl-glutamate (1 mmol·1−1). Fractional excretion of γ-carboxyglutamate was 7–25% (l-from) or 45–70% (d-form) at an artificially elevated plasma level of 12μmol·1−1. It is concluded thatl- andd-aspartate,l-glutamate,l-cysteate and, to a much smaller extent,l-γ-carboxyglutamate, are accepted by the tubular resorption mechanism highly specific for “acidic” amino acids. N-Substitution, the amidation of the β- or γ-carboxyl group, or the removal of the α-amino moiety almost completely abolish the ability of such compounds to be resorbed via this carrier; N-methylated or γ-carboxylated derivatives of “acidic” amino acids are not resorbed at all from the proximal tubule. The resorption of glutamate, but not of aspartate, is highly stereospecific.
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  • 8
    Electronic Resource
    Electronic Resource
    Springer
    Pflügers Archiv 412 (1988), S. 1-6 
    ISSN: 1432-2013
    Keywords: Cell volume regulation ; Proximal tubule ; Bicarbonate ; Barium ; Acetazolamide ; Nitropropylphenylaminobenzoat
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Notes: Abstract The present study has been performed to test for the ionic requirement of regulatory cell volume decrease in isolated perfused straight proximal tubules of the mouse kidney. Reduction of peritubular osmolarity from 308 mosmol/l to 228 mosmol/l leads within 0.5 min to cell swelling by 16±1% (n=26) of original cell volume (V o). Within 2 min cell volume (V 2) approaches 105±1% ofV o (n=26) despite continued exposure to hypotonic bath perfusate. Reexposure of the tubules to isotonic bath perfusate shrinks the cells to 94±1% ofV o (n=25). Within 2 min from omission of extracellular bicarbonate and CO2 regulatory cell volume decrease is impaired (V 2=114±1% ofV o,n=14). Similarly, regulatory volume decrease is blunted upon prior removal of extracellular sodium (V o=115±2% ofV o,n=12). In constrast regulatory volume decrease is not affected by prior removal of extracellular chloride (V 2=104±2% ofV o,n=9). Regulatory volume decrease is impaired in the presence of 1 mmol/l potassium channel blocker barium (V 2=120±4% ofV o,n=7) and of 1 mmol/l carbonic anhydrase inhibitor acetazolamide (V 2=111±2% ofV o,n=16) but is preserved in the presence of 1 μmol/l chloride channel blocker NPPB, (V 2=105±2% ofV o,n=11). In conclusion, regulatory cell volume decrease apparently depends on potassium and bicarbonate, but does not depend on chloride.
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  • 9
    Electronic Resource
    Electronic Resource
    Springer
    The journal of membrane biology 117 (1990), S. 113-122 
    ISSN: 1432-1424
    Keywords: cell volume regulation ; potassium conductance ; intracellular potassium concentration ; proximal renal tubule ; cell membrane potential ; microelectrodes ; ouabain ; omcprazole ; barium
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Chemistry and Pharmacology
    Notes: Summary The present study was designed to assess for the influence of extracellular potassium and of inhibitors of potassium transport on cell volume regulatory decrease in isolated perfused straight proximal tubules of the mouse kidney. Volume regulatory decrease is virtually unaffected when bath potassium concentration is elevated from 5 to 20 mmol/liter, and still persists, albeit significantly retarded, in the presence of the potassium channel blocker barium on both sides of the epithelium and during virtually complete dissipation of the transmembrane potassium gradient by increasing extracellular potassium concentration to 40 mmol/liter. As evident from electrophysiologic observations, barium blocks the potassium conductance of the basolateral cell membrane. Reduction of bicarbonate concentration and increase of H+ concentration in the bath solution cannot compensate for enhanced potassium concentration and cell volume regulatory decrease is not affected in the presence of the K/H exchange inhibitor omeprazole. Similarly cell volume regulatory decrease is not affected by ouabain. In conclusion, potassium movements through potassium channels in the basolateral cell membrane are important determinants of cell volume and may participate in cell volume regulatory decrease. However, a powerful component of cell volume regulatory decrease in straight proximal tubules of the mouse kidney is apparently independent of potassium conductive pathways, K/H exchange and Na+/K+-ATPase.
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