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  • 1
    Electronic Resource
    Electronic Resource
    The mechanism of action of harmaline on renal solute transport (1977)
    Springer
    Pflügers Archiv 368 (1977), S. 83-88 
    Details
    ISSN: 1432-2013
    Keywords: Harmaline ; Active Na+ transport ; Na+−K+-ATPase ; HCO 3 − -ATPase ; Renal glucose transport
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Notes: Summary The effect of the hallucinogenic drug harmaline was tested on rat kidney proximal tubular solute and water transport, using in vivo micropuncture and electrophysiological techniques as well as in vitro biochemical techniques. During peritubular application harmaline (5 mmol/l) was found to block net tubular volume absorption reversibly (by 85%) through inhibition of active Na+ transport and possibly active HCO 3 − transport. The inhibition was accompanied by a rapid strong depolarization of the tubular cell membranes. As a biochemical equivalent harmaline inhibited the Na+−K+-ATPase and the Mg2+-ATPase of peritubular cell membrane fractions as well as the HCO 3 − -stimulated ATPase of a brush border membrane fraction with similar kinetics. By studying glucose tracer efflux and by measuring cell membrane potential and conductance changes in response to glucose perfusions, no evidence for a direct effect of harmaline on Na+-glucose (or amino acid) cotransport mechanisms in the brush border could be obtained. The data suggest that harmaline does not specifically compete with Na+ for transport sites. Neither are the cotransport systems in the brush border membrane specifically inhibited, nor could the inhibition of the Na+ pump in the peritubular cell membrane simply result from a competition between harmaline and Na+.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF01063458
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  • 2
    Electronic Resource
    Electronic Resource
    Electrophysiological analysis of rat renal sugar and amino acid transport (1982)
    Springer
    Pflügers Archiv 393 (1982), S. 210-214 
    Details
    ISSN: 1432-2013
    Keywords: Rat proximal tubule ; Transport of basic amino acids ; Electrophysiology
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Notes: Abstract Electrophysiological techniques were used to study the transport of the basic amino acidsl-arginine,l-lysine andl-ornithine in rat kidney proximal tubule in vivo. Tubular cells were punctured with microelectrodes and the response of the cell membrane potential to sudden applications of the amino acids was measured. In the presence of physiological Na+ concentrations luminal perfusions with millimolar concentrations of basic amino acids depolarized the tubular cells in a concentration dependent fashion by up to 15 mV, while in the absence of Na+ no significant potential changes were observed. These observations indicate that the basic amino acids are taken up into the cell across the brushborder in coupling with Na+ ions in a similar way as neutral and acidic amino acids, and that simple conductive pathways for uncoupled flow of the basic amino acids do either not exist or are quantitatively negligible in the brushborder. From kinetic measurements and competition experiments it was concluded that all basic amino acids are transported by the same transport system, which however does not accept acidic or neutral amino acids (with the possible exception ofl-cystine). Perfusion of the peritubular capillaries with millimolar concentrations of basic amino acids depolarized the cells only by approximately 1 mV, both in the presence and absence of Na+. This observation may indicate that a passive uncoupled transport pathway for basic amino acids is present in the peritubular cell membrane to allow exit from cell to interstitial space, if the intracellular concentration rises high enough to overcome the cell membrane potential.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF00584071
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  • 3
    Electronic Resource
    Electronic Resource
    Electrophysiological analysis of rat renal sugar and amino acid transport (1982)
    Springer
    Pflügers Archiv 393 (1982), S. 215-221 
    Details
    ISSN: 1432-2013
    Keywords: Renal transport of glutamate and aspartate ; Electrophysiology of rat proximal tubule ; Dicarboxylic aminoaciduria ; Metabolic acidosis and cellular transport of amino acids
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Notes: Abstract We have used electrophysiological techniques to study various aspects of the transport of glutamate and aspartate in proximal tubules of the rat kidney in vivo. Single tubular cells were punctured with microelectrodes and the response of the cell membrane potential to sudden luminal or peritubular applications of these amino acids was measured. The experiments indicated that a specific transport system exists forl-glutamate andl-aspartate in the brushborder membrane, which does not transport neutral or basic amino acids. The uptake of bothl-amino acids from the lumen into the cell was found to be rheogenic, probably reflecting the cotransport of two Na+ ions together with one amino acid molecule. The transport system has a slightly greater affinity forl-glutamate, but transports the smallerl-aspartate somewhat faster. Besides thel-isomers alsod-glutamate andd-aspartate were found to depolarize the tubular cells which suggests that also thed-isomers are absorbed in the tubule, however they do not seem to use the same transport system as thel-isomers. In addition to the transport system in the brushborder, a similar Na+-dependent, rheogenic transport system forl-glutamate andl-aspartate was also found in the peritubular cell membrane, as deduced from cell depolarizations in response to these substrates applied peritubularly. The simultaneous presence of Na-driven transport systems in the apical and basal cell membrane which is not found with other amino acids, may explain the high intracellular accumulation ofl-glutamate andl-aspartate in the kidney and provides a rational basis for explaining clinically observed cases of dicarboxylic aminoacidurias.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF00584072
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  • 4
    Electronic Resource
    Electronic Resource
    Electrophysiological analysis of rat renal sugar and amino acid transport (1982)
    Springer
    Pflügers Archiv 393 (1982), S. 190-197 
    Details
    ISSN: 1432-2013
    Keywords: Rat renal proximal tubule ; Electrophysiology ; Na+ glucose cotransport ; Sugar specificity ; Active Na+ pump
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Notes: Abstract Transepithelial and cellular electrical potential changes were measured in response to luminal perfusion ofd-glucose and related substrates in micropuncture experiments on rat kidney in vivo. By studying the dependence of the potential response on various experimental parameters, some insight was obtained into the mechanism of Na+ coupled glucose absorption. The experiments confirm the driving forces for glucose absorption in the living cell to be: a) the Na concentration gradient, b) the electrical potential gradient and c) the glucose concentration gradient across the brushborder membrane. Furthermore they describe the substrate specificity of the cotransport mechanism and the mechanism of inhibition ofd-glucose transport by various inhibitors, such as phlorizin, harmaline and oubain. The latter experiments suggest that the active Na+ pump in the peritubular cell membrane, which establishes the Na+ ion gradient and the electrical potential gradient across the brushborder, contributes a measurable partial conductance to the overall electrical conductance of the peritubular cell membrane.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF00582943
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  • 5
    Electronic Resource
    Electronic Resource
    Incubation in tissue culture media allows isolated rabbit proximal tubules to regain in-vivo-like transport function: response of HCO3 – absorption to norepinephrine (2000)
    Springer
    Pflügers Archiv 440 (2000), S. 908-917 
    Details
    ISSN: 1432-2013
    Keywords: Isolated proximal tubule Metabolic substrates Norepinephrine Tissue culture media Tubular bicarbonate absorption
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Notes: Abstract. Using a new stop-flow perfusion technique with microspectrofluorometric determination of luminal fluid pH, we have studied which substrates or incubation conditions allow isolated rabbit proximal tubules to attain in-vivo-like rates of HCO3 – absorption (J HCO3) and maximal responses of J HCO3 to norepinephrine (NE). Essentially three incubation media were tested: plasma-like HCO3 –-Ringer solution containing 5 mmol/l d-glucose (G-Ringer sol.), the same solution also containing 10 mmol/l lactate and 5 mmol/l l-alanine, (LAG-Ringer sol.), and two tissue culture media (DMEM and RPMI 1640). Compared to G-Ringer sol., application of LAG-Ringer sol. in the bath and/or lumen, or application of DMEM or RPMI 1640 in the bath either slightly increased or decreased J HCO3 with borderline significance. However, RPMI 1640 plus 1 mmol/l pyruvate stimulated J HCO3 by 55%. While NE (10–5 mol/l), if applied in G-Ringer sol., had no effect, in the presence of LAG-Ringer sol. it increased J HCO3 by ≅40%, and in the presence of DMEM or RPMI 1640 it increased J HCO3 by ≅100%. This stimulation by NE followed Michaelis–Menten kinetics with an EC50 value of 0.25 µmol/l and was probably mediated by α1-adrenergic receptors. Additional cell pH measurements suggest that NE stimulates the basolateral Na+-HCO3 – cotransporter which then becomes susceptible to inhibition by cAMP. We conclude that incubation in tissue culture media allows isolated proximal tubules to maintain a better functional state than the commonly used solutions with unphysiologically high substrate concentrations.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/s004240000361
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  • 6
    Electronic Resource
    Electronic Resource
    Partial recovery of in vivo function by improved incubation conditions of isolated renal proximal tubule. (1997)
    Springer
    Pflügers Archiv 434 (1997), S. 373-382 
    Details
    ISSN: 1432-2013
    Keywords: Key words Isolated renal proximal tubule ; Metabolic substrates ; Na/K pump ; Amiloride-inhibitable K+ conductance
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Notes: Abstract  Isolated microperfused rabbit renal proximal tubule S2 segments, if incubated in conventional substrate containing HCO3 –Ringer solution, exhibit lower cell membrane potentials (V b) and elevated intracellular Na+ concentrations ([Na]i) compared to rat tubules in vivo. Assuming that these and other differences reflect insufficient metabolic and/or hormonal stimulation of the cells, we have used microelectrode techniques to test whether improving substrate supply and applying norepinephrine (NE, to compensate for the missing nerve supply) reverts V b and [Na]i to values observed in vivo. Application of D-glucose (5.5 mmol/l) and additional application of pyruvate, lactate, or L-alanine (each 10 mmol/l), or bathing the tubules in Dulbecco’s modified Eagle’s tissue culture medium (DMEM) significantly increased V b and, whenever tested, reduced [Na]i as compared to substrate-free or D-glucose-containing control solution and these effects could be prevented – as tested in the case of pyruvate – by inhibition of the Na/K pump with ouabain. However, high concentrations of acetate, β-hydroxybutyrate, or L-glutamine had no significant effect. The largest effect was obtained with joint application of DMEM and NE (10 μmol/l) which increased V b from –42.8 ± 1.3 mV (SEM) to –55.3 ± 2.5 mV (n = 11). Interestingly we noticed that under the latter conditions the V b response to bath application of 1 mmol/l amiloride virtually disappeared, i.e. it changed from a depolarization of +14.6 ± 1.4 mV (in D-glucose Ringer solution) to +0.6 ± 0.7 mV (in DMEM plus NE) (n = 8), with some tubules showing even a small hyperpolarization. The latter implies partial restoration of the in vivo behaviour, since in experiments on rat proximal tubules in vivo amiloride regularly hyperpolarized the cells (by –3.4 ± 0.76 mV, n = 5). Obviously under conventional in vitro conditions an amiloride-inhibitable K+ conductance is activated which is inactive in vivo and also inactivates under improved conditions in vitro. In agreement with observations reported in the subsequent publication our results demonstrate that isolated proximal tubules undergo functional alterations which may be largely prevented by improved metabolic and stimulatory incubation conditions.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/s004240050410
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  • 7
    Electronic Resource
    Electronic Resource
    Electrophysiological analysis of rat renal sugar and amino acid transport (1982)
    Springer
    Pflügers Archiv 393 (1982), S. 199-209 
    Details
    ISSN: 1432-2013
    Keywords: Rat proximal tubule ; Neutral amino acid transport ; Electrophysiology ; Epithelial transport kinetics ; Competition experiments
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Notes: Abstract We have used electrophysiological techniques in rat kidney proximal tubule to study the transport of the followingl-α-amino acids: alanine, phenylalanine, glutamine, methionine, cysteine, cystine, proline, and hydroxyproline as well as the transport of glycine and β-alanine. When applied in millimolar concentrations in the tubular lumen in the presence of Na+, all amino acids tested were found to depolarize the tubular cell membranes partially. This depolarization arises from current flow that is associated with the cotransport of Na+ and amino acids across the brushborder membrane (Frömter 1982 [9]). Peritubular application did not alter the membrane potentials in a conclusive way. The magnitude of the depolarization in response to luminal perfusion increased with increasing amino acid concentration and obeyed simple Michaelis-Menten kinetics, except for proline, hydroxyproline, and glycine, which exhibited double-site saturation kinetics. By analyzing the time course of the potential changes and distinguishing between initial and steady state depolarizations, it was possible to separate kinetic properties of the brushborder transport mechanisms from the lumped kinetic properties of the overall epithelium. Knowing the concentration dependence of the depolarizations, the competition of different amino acids for the same transport site was investigated: Two amino acids were applied in saturating concentrations in the tubular lumen, either singly or jointly, to determine whether the depolarizations were additive and whether the additivity exceeded the predictions from the kinetic experiments. From such studies the presence of three separate rheogenic transport systems is postulated for neutral amino acids: System I transports all neutrall-α-amino acids with the exception of cystine, system II transports proline, hydroxyproline and glycine, and system III transports β-alanine or under physiological conditions taurine. Studies with the oxidant diamide suggest that cystine may be mostly reduced to cysteine and transported as cysteine, however it is likely that an extra transport system exists which transports cystine possibly in electroneutral fashion together with dibasic amino acids.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF00584070
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  • 8
    Electronic Resource
    Electronic Resource
    Determination of intracellular K+ activity in rat kidney proximal tubular cells (1978)
    Springer
    Pflügers Archiv 378 (1978), S. 37-45 
    Details
    ISSN: 1432-2013
    Keywords: Ion-selective microelectrode ; Renal tubular cell ; Cytoplasmic K+ activity ; Active K+ transport
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Notes: Abstract The intracellular K+ activity of rat kidney proximal tubular cells was determined in vivo, using intracellular microelectrodes. In order to minimize damage from the impaling electrodes, separate measurements on separate cells, were performed with single-barrelled KCl-filled non-selective electrodes and single-barrelled, K+-sensitive microelectrodes, which were filled with a liquid K+-exchanger resin that has also a small sensitivity to Na+. Both electrodes had tip diameters of 0.2 μm or below. The proper intracellular localization of the electrodes was ascertained by recording the cell potential response to intermittent luminal perfusions with glucose. The membrane potential measured with the non-selective microelectrodes was −76.3±8.1 mV (n=81) and the potential difference measured with the K+-sensitive microelectrode was −7.2±5.8 mV (n=32). Based on the activity of K+ in the extracellular fluid of ∼3 mmol/l the intracellular K+ activity was estimated to be ∼82 mmol/l. Assuming equal K+-activity coefficients to prevail inside and outside the cell, this figure suggests that the intracellular K+ concentration is ∼113 mmol/l which must be considered as a lower estimate, however. The data indicate that the K+-ion distribution between cytoplasm and extracellular fluid is not in equilibrium with the membrane potential, but that K+ is actively accumulated inside the cell. This result provides direct evidence for the presence of an active K+ pump in the tubular cell membranes, which in view of other observations, must be envisaged as a (not necessarily electroneutral) Na+/K+-exchange pump which operates in the peritubular cell membrane and is eventually responsible for the major part of the tubular solute and water absorption.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF00581956
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  • 9
    Electronic Resource
    Electronic Resource
    A stop-flow microperfusion technique for rapid determination of HCO3 – absorption/H+ secretion by isolated renal tubules (1999)
    Springer
    Pflügers Archiv 439 (1999), S. 208-215 
    Details
    ISSN: 1432-2013
    Keywords: BCECF H+ secretion HCO3– absorption Isolated renal proximal tubule Luminal pH
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Notes: Abstract. In the present experiments on microdissected tubules of rabbit kidney we present a refined stop-flow method for determining the rate of HCO3 – absorption (J HCO3) or H+ secretion (J H) that can be applied to isolated microperfused tubules. Using the pH-sensitive indicator dye BCECF (2',7'-bis [2-carboxyethyl]-5[6]-carboxyfluorescein) the luminal perfusate pH is continuously measured with a microspectrofluorometric set-up, and the pH change following a sudden stop of perfusion is analysed. Because the tubules partially collapse after stop-flow the calculation of fluxes requires a correction for volume loss. This is achieved by referring all fluxes to the remaining luminal volume, which can be estimated from the decay of the 440 nm reference fluorescence. During perfusion of the lumen with pure HCO3 – Ringer solution, and of the bath with the same solution but containing 5.5 mmol/l d-glucose as metabolic substrate, J HCO3 averaged 4.4±0.2 pmol cm–1·s–1 (n=40) and 13.4±0.8 pmol·cm–1·s–1 (n=5) in proximal straight tubules (PST) and in proximal convoluted tubules respectively. These values agree very well with data obtained in other laboratories with the picapnotherm technique. The present method has the advantage of requiring fewer micromanipulations and a shorter measuring time, thus allowing regulatory changes in J HCO3 to be analysed. Moreover it does not involve measurements of radioactivity, and it also allows J H to be measured in HCO3 –-free solutions which in PST averaged 0.9 pmol·cm–1·s–1 (n=8) in the present experiments.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/s004249900171
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