Arnost Kleinzeller

The effect of ATP and Ca2+ on the cell volume in isolated kidney tubules

The effect of external ATP on the steady-state levels of water and electrolytes in isolated renal tubules of healthy adult rabbits was studied: 1. 1. In the absence of external Ca2+, ATP produced an increase of tissue Na+, Cl−, and Ca2+, and a loss of K+, at all pH values tested (pH 6.2 to 8.2); only at pH 7.2 was a marked ATP-induced cellular swelling observed. No such effects of ATP were found in the presence of 2.5 mM [Ca2+]0. 2. 2. The swelling effect of ATP was not affected by 0.1 mM and 0.5 mM ouabain or the absence of Na+ (Li+ saline) and was in part reversed by raising [Ca2+]0 to 2.5 mM. Increased Mg2+ (5.5 mM final concn) could replace Ca2+ in preventing the effect of external ATP. 3. 3. ADP and GTP did not produce changes of cell volume under conditions where ATP was effective. 4. 4. Isolated kidney tubules were found to hydrolyse added ATP or other triphosphonucleotides. The rate of hydrolysis was increased by raising the medium pH from 6.2 (0.13 μmole P1/mg protein per 10 min) to 8.2 (0.24 μmole Pi/mg protein per 10 min) and was to some extent inhibited by Ca2+. 5. 5. A soluble protein with a nucleotidase specificity, activated by Mg2+ or Ca2+, was isolated from the tubules. At pH 7.2 this protein was precipitated from solution by the addition of ATP and Ca2+; this phenomenon was found to be specific for ATP. 6. 6. It is suggested that external ATP affects the water and ionic contents of the cells of kidney tubules by an interaction with the cell membrane and Ca2+, thus involving the physical (contractile?) properties of the membrane structure and/or membrane permeability.

The specificity of the active sugar transport in kidney cortex cells

Abstract The transport of some monosaccharides into renal tubular cells was investigated using slices of rabbit kidney cortex: 1. 1. An active, Na + -dependent and phlorrhizin-sensitive cellular accumulation of β-methyl- d -glucoside, d -glucosamine and d -galactosamine was demonstrated. d -Mannitol, d -glucose, N- acetyl- d -glucosamine , 3-deoxy- d -glucose, d -allose, d -ribose and 2,6-dideoxy- d -altrose were not actively accumulated against their concentration gradients. 2-Deoxy- d -allose, 2-deoxy- d -ribose and possibly 2-deoxy- d -xylose were transported against a small concentration gradient by a Na + -independent mechanism. A comparison of the transport properties of 26 monosaccharides indicates the following structural requirements for active transport: a hemiacetal group, C-3-OH (in a configuration identical with that of d -glucose) and C-6-OH. The Na + requirement for active transport is related to the presence of a hydrophilic (-OH or -NH 2 ) group on C-2. 2. 2. The apparent kinetic parameters, K m and v max , are given for the active transport of a α-methyl- d -glucoside, d -galactose, 2-deoxy- d -glucose and 2-deoxy- d -galactose into renal tubular cells. The values of the apparent transport K m for these sugars varied only slightly between 0.79 ± 0.07 for d -galactose to 3.0 ± 0.11 for 2-deoxy- d -galactose; rather wide variations of v max were found (12 μmoles per g cell water per h for 2-deoxy- d -glucose to 62 ± 4.7 for α-methyl- d -glucoside). 3. 3. The competition for transport between a variety of monosaccharides was investigated: the found K i was within the range of independently determined K m for the inhibition of galactose of 2-deoxy- d -galactose and 2-deoxy- d -glucose transport. However, the K i for the inhibition of d -galactose transport by 2-deoxy- d -galactose was one order of magnitude higher than the K m of the latter sugar. Similarly, no agreement between the respective K i and K m was found for the competitive inhibition between α-methyl- d -glucoside and galactose. d -Glucose, which competitively inhibited the transport of 2-deoxy- d -glucose, had practically no effect on the transport of 2-deoxy- d -galactose even at a molar ratio of 1:50. 4. 4. These kinetic data indicate the existence of several pathways for the active sugar transport into renal tubular cells. 5. 5. A correlation between the reabsorption of sugars in the kidney tubule ( in vivo ) and their active cellular accumulation ( in vitro ) is demonstrated.

The binding of phloridzin to the isolated luminal membrane of the renal proximal tubule

Abstract The binding of [3H]ploridzin by isolated luminal membranes of the rabbit proximal tubule and by slices of rabbit kidney cortex was studied. Kinetic analyses of the relationship between the concentration of phloridizin in the incubation medium and the binding of phloridzin to the membrane indicated two distinct classes of receptors sites. One class, comprising high affinity sites, reached saturation at 20–25 μM phloridzin, had a K(phloridzin) of 8 μM, and 8·10+2 nmoles interacted with 1 mg of brush border protein. The other class, comprising low affinity sites, had a K(phloridzin) of 2.5 mM, and the number of binding sites was 1.25 nmoles/mg Na+ was required for the binding of phloridzin at the high affinity sites. Na+ decreased the apparent Ki for phloridzin; the apparent V of binding was not altered. Binding at the low affinity sites was independent of Na+. Ca2+ was necessary for maximal binding at the high affinity sites. Binding of phloridzin at high affinity sites was more sensitive to N-ethylmalcimide and mersalyl than was binding at low affinity sites. Binding at high affinity sites, but not at low affinity sites, was temperature dependent. d -Glucose was a competitive inhibitor of the high affinity binding of phloridzin. The apparent K1 was 1 mM. D -Glucoe inhibited non-competitively at the low affinity sites. l -Glucose had no influence on phloridzin binding. Phloretin was a competitive inhibitor of high affinity phloridzin binding with an apparent Ki of 16 μM. Phloretin inhibited low affinity bindings of phloridizin non-competitively. Binding of phloridzin at high affinity sites was completely reversible. Binding at low affinity sites was only partially reversed. Phloridzin bound at high affinity sites on the brush border was displaced by phloridzin and phloretin but not by d -glucose. The mechanism of the high affinity binding of phloridzin was distinguished from that of the initial interaction of d -glucose with the membrane. Binding of phloridzin required Na+, whereas the interaction of d -glucose with the membranes had a prominent Na+-independent component. Intact renal cells in cortical slices accumulated phloridzin. The uptake did not saturate, was Na+ independent, and was not competitively inhibited by sugars. These characteristics resemble those for the low affinity binding of phloridzin by isolated membranes. It is suggested that low affinity binding may represent an initial binding followed by uptake of the glycoside into membrane vesicles.

Basal-lateral transport and transcellular flux of methyl α-d-glucoside across LLC-PK1 renal epithelial cells

The characteristics of methyl alpha-D-glucoside transport by the LLC-PK1 cell line are extended by a study of the interaction of this glucose analog with the basal-lateral membrane of these cells: 1 mM methyl alpha-D-glucoside enters LLC-PK1 cells across the basal-lateral membrane 10-times more slowly than when entering across the apical membrane; neither 10 mM glucose nor 10 mM methyl alpha-D-glucoside affect the rate of methyl glucoside uptake at the basal lateral membrane; 0.1 mM phlorizin in the apical hemichamber significantly decreases the rate at which methyl glucoside enter the cell when methyl glucoside is present in the basal-lateral hemichamber; the methyl glucoside transcellular flux ratio, Ja/Jb (apical to basal vs. basal to apical) is 15, whereas Ja/Jb for D-mannitol equals 1; and basal-lateral to apical fluxes (Jb) of mannitol consistently exceed those of methyl glucoside. These results demonstrate that methyl glucoside, unlike glucose, is accumulated within these cells to a high degree because of the limited ability of methyl glucoside to exit the cells by a carrier-mediated pathway. They also raise the important caveat for any studies with glucose (and other low-molecular-weight substrates) by showing that a monosaccharide presented to one surface of these cells will traverse the cell sheet (in part) by the intercellular route and will enter the cell at the unintended cell surface. The ability of the tight junctions of this intercellular route to discriminate between open-chain molecules, such as mannitol, vs. closed ring structures, like methyl glucoside, is also described.

Ca2+-activated ATPase from renal tubular cells.

Publisher Summary A Ca 2+ -activated ATPase (ATP hydrolase, EC 3.6.1.3) was isolated from erythrocyte membranes, and it has been suggested that this enzyme is involved in the active transport of Ca 2+ . On the other hand, ATP- and Ca 2+ -linked changes in the shape of the red blood cells have been described and related to physical changes of the cell membrane. Possibly, the Ca 2+ -transport as well as the Ca- and ATP-dependent physical changes of the membrane involve the same enzyme.

Confocal microscopic observation of cytoskeletal reorganizations in cultured shark rectal gland cells following treatment with hypotonic shock and high external K

The dogfish shark (Squalus acanthias) rectal gland (SRG) cell has served as a model experimental system for investigating the relationship between the actin cytoskeleton and cell volume regulation. Previous reports employing conventional fluorescence microscopy of tissue slices have shown that cells exposed to high external K+ and hypotonically-induced cell swelling displayed a fading of F-actin staining intensity, particularly at the basolateral cell borders. However, spectroscopic measurement of the F-actin present in similarly treated rectal gland slices failed to demonstrate a net change in F-actin amount. In an effort to resolve the structural reorganizations of F-actin which may be occurring during high K+ and hypotonic shock treatments, we have used cultured SRG cells in conjunction with confocal microscopic immunocytochemical localization techniques to examine actin filament, microtubule, and cytokeratin filament dynamics under these two experimental conditions. The results reveal that F-actin in control cells exists in an array of parallel linear bundles (which do not appear to be stress fiber-like given their lack of staining for myosin II or alpha-actinin) that is reorganized to a punctate pattern in hypotonic shock and a dense meshwork in high K+. The linear bundle pattern of F-actin returns in cells undergoing regulatory volume decrease. Quantitative western blotting of F-actin in SRG cell detergent extracted cytoskeletons indicates no significant difference in the relative amounts of F-actin present in control, hypotonic shocked, or high K+ cells. Anti-tubulin and anti-cytokeratin labeling of the treated SRG cells suggest that these other major cytoskeletal elements are not significantly altered by the treatments. Taken together, our results reinforce the concept that there is an association between the structural organization of the actin cytoskeleton and cell volume regulation in the SRG epithelial cells.

Active sugar transport in renal cortex cells: The electrolyte requirement

Abstract The electrolyte requirement for the active transport of α-methyl- d -glucoside, d -galactose, 2-deoxy- d -glucose and 2-deoxy-α-methyl- d -galactose was investigated using slices of rabbit kidney cortex: 1. 1. As compared with values obtained at [ Na + ] 0 = 0 , the presence of external Na+ (128 mM) increased the apparent v max of d -galactose transport without affecting the transport K m . 2. 2. The Na+ requirement for the transport of α-methyl- d -glucoside and d -galactose was not saturated at 128 mM Na+. Within the range of [Na+]0 25–128 mM, indications of a single Na+ entering a rate-limiting event in α-methyl- d -glucoside transport were found. 3. 3. A significant fraction of the d -galactose transport was found to be Na+ independent and insensitive to 2 mM ouabain. 4. 4. As compared with values obtained at [ K + ] 0 = 0 , the presence of 0.5–7 mM K+ activated the transport of α-methyl- d -glucoside and d -galactose; this K+ stimulation was saturated at [K+]0 1 mM and affected the v max , rather than the K m , of sugar transport. No such effect of [K+]0 on the transport of both 2-deoxyhexoses was observed. 5. 5. The absence of external Cl− had no effect on the transport of the four sugars tested. 6. 6. The absence of saline Ca2+ markedly depressed both the influx and the steady-state accumulation level of all four sugars tested; the efflux of d -galactose and 2-deoxy- d -galactose was accelerated. This Ca2+ stimulation of the sugar transport was saturated at [Ca2+]0 0.5 mM and affected the v max rather than the K m of sugar transport. The Ca2+ effect was found to be fairly specific for this ion: whereas Sr2+, Ba2+ and, to some extent, Mn2+ could replace Ca2+ in its effect on the sugar transport, Mg2+ and La3+ wer ineffective. 7. 7. The above results are discussed in relation to hypotheses put forward to explain the mechanism of the Na+-dependent nonelectrolyte transport in some animal cells. It is concluded that: (a) In kidney cortex cells, the presence of Na+ (external and intracellular) is not mandatory to bring about an active transport of 2-deoxyhexoses and also a fraction of galactose; (b) the K+ stimulation of sugar transport is related to the Na+ requirement for sugar transport; (c) Ca2+ stimulates a common step in the transport of all sugars tested.

Active renal hexose transport. Structural requirements

The active transport of methyl beta-D-galactoside and some other analogs of D-glucose and D-galactose was studied in slices of rabbit and renal cortex. 1. The non-metabolizable methyl beta-D-galactoside accumulates in renal cortical cells against its concentration gradient. At 1 mM substrate concentration (O2, 35 degrees C, 60 min incubation) the gradient was 2.36 +/- 0.11 S.E. (n = 33). The Kt was 1.50 +/- 0.02 mM. The active transport of the substrate was inhibited by dinitrophenol, phlorizin, absence of Na+ and by ouabain. This inhibition was incomplete, suggesting that the sugar may enter the cells by two separate pathways, only one of which was coupled to the down-hill electrochemical Na gradient. 2. The structural requirements for the interaction between substrate and the carrier were defined: (a) by testing the transport behavior of some analogs (1,5-anhydro-D-glucitol; methyl beta-thio-D-galadtoside; 3-deoxy-D-glucose; 4-deoxy-D-glucose; 5-thio-D-glucose; 6-deoxy-D-glucose and methyl-alpha-6-deoxy-D-glucoside); and (b) by inhibition analysis of methyl beta-D-galactoside transport. The role of each hydroxyl of the sugar molecule was tested by using a total of 41 analogs modified on each C by replacing -OH by -H, -O-CH3, -F and in some instances also by -SH. 3. The carrier is shared by D-glucose, D-galactose and their methyl glycosides. A pyranose ring is mandatory. The D-glucoconfiguration is preferred for the interaction with the carrier. 4. Replacement of -OH by -H or -S practically abolished (on C1, C2, C3) or greatly reduced (on C4) the affinity of the analog for the carrier. This was also confirmed by demonstrating that 1-deoxy-, and 3-deoxy-glucose and the thio-galactoside were not actively transported and their entry into the cells was not markedly affected by phlorizin, dinitrophenol, ouabain or absence of Na+. 4-Deoxy-D-glucose was taken up and its transport was inhibited by agents affecting the transport of methyl beta-D-galactoside. 5. Replacement of -OH by -F did not abolish the affinity of the analogs for the carrier, indicating hydrogen bonding between the carrier and the oxygens at C1, C2, C3, and C4. 6. 5-Thio-D-glucose was not transported against its concentration gradient and also poorly interacted with the carrier as shown by inhibition analysis. Hydrogen bonding between the carrier and the pyranose ring oxygen is suggested. 7. 6-Deoxyglucose is a potent inhibitor of methyl beta-D-galactoside transport although it is not actively taken up by the tissue. It is concluded that a hydroxyl at C6 is required for transport, but is not mandatory for an interaction with the carrier. However, 6-deoxy-D-galactose was ineffective as inhibitor. 8. The specificity of the carrier involved in the renal active transport of D-glucose, D-galactose and their methyl glycosides resembles qualitatively, and mostly also quantitatively that described for intestinal transport of these sugars.

Effect of pH on the water and electrolyte content of renal cells

Abstract The effect of external pH (varying from 6.2–8.2) on the water and electrolyte contents of preparations of rabbit kidney cortex (slices and isolated tubules) was studied: 1. 1. In sodium saline, increasing external pH produced primarily a decrease in cell K + . High pH increased both the passive membrane permeability for 42 K + , and also the compartmentation of K + within the cells. 2. 2. Inhibition of the Na + pump by ouabain (0.5 mM) or absence of external Na + (lithium, Tris, and choline salines) did not abolish the control of cell volume at pH 6.2 and 7.2, whereas a marked cellular swelling at pH higher than 7.5 was found. The ouabain-insensitive (and Na + -independent) volume control at pH 6.2 was dependent on metabolism, since it was inhibited by 0.1 mM dinitrophenol and anaerobiosis. 3. 3. Involvement of Ca 2+ in the ouabain-insensitive volume control was demonstrated by showing that the absence of Ca 2+ , ouabain, or absence of Na + , produced a marked swelling at pH 7.2. This phenomenon was accompanied by increased extra-cellular space, due to a swelling of the basal tubular membrane. 4. 4. A correlation between cellular swelling and a decrease in cell ATP was presented. 5. 5. The results are compatible with the mechanochemical hypothesis for the ouabain-insensitive (and Na + -independent) control of cell volume. It is suggested that in this mechanism, cell (membrane) ATP and Ca 2+ are determinants of the physical properties of the membrane.

The effect of pH on sugar transport and ion distribution in kidney cortex cells

Abstract The effect of pH (range: 6.2–8.2) on active sugar transport and the steady-state ionic distribution was studied using slices of rabbit kidney cortex. Sugars used: 2-deoxy- d -glucose, 2-deoxy- d -galactose, d -galactose and α-methyl- d -glucoside. 1. 1. An increase of pH from 6.2 to 8.2 affected sugar transport as follows: (a) A 3-fold increase in the rate and steady-state accumulation of 2-deoxy- d -glucose both in Na+- and Na+-free (Li+) salines was found; changes of pH affected the K m of 2-deoxy- d -glucose transport. (b) A marked decrease of the accumulation of 2-deoxy- d -galactose in both Na+- and Li+-salines was observed, the transport K m being affected. (c) A 4-fold stimulation of d -galactose transport in Na2+-saline took place, with only a minimial effect on the Na+-independent transport. This pH effect was reversible. An increase of pH from 7.2 to 8.2 markedly decreased the efflux of d -galactose from the cells. Evidence for complex influx kinetics was obtained. (d) No major changes of the steady-state accumulation of α-methyl- d -glucoside in Na+-salines could be detected; in Li+-saline, an accumulation of α-methyl- d -glucoside at pH 6.2 was found ( [S] 1 /[S] 0 : 1.725 ± 0.101 ( S.E. ) . This active accumulation at [ Na + ] 0 = 0 (and [ Na + ] i mM ) was inhibited to [S] 1 /[S] 0 values below 1.0 by anaerobiosis, and by 0.1 mM dinitrophenol or phlorrhizin, but was insensitive to 0.5 mM ouabain or acetazolamide. 2. 2. The increase of pH from 6.2 to 8.2 affected as follows the steady-state tissue water and the respective electrochemical ionic potential (referred to E 36 Cl − ): (a) In Na+-salines, tissue water, E 36 Cl − and E K + were minimally altered; E Na + and E H + decreased. (b) In Li+-salines, tissue water markedly increased with increasing pH; E 36 Cl − was of the same order as in Na+-salines; E K + , lower at pH 6.2 than in Na+-saline, significantly decreased; a small electrochemical gradient of Li+ at pH 6.2 (12 mV) decreased; E H + was of the same magnitude as in Na+-salines at all pH. 3. 3. The active transport of α-methyl- d -glucoside at pH 6.2 in the absence of Na+ was found to be independent of all ionic electrochemical gradients with the exception of Li+. It is concluded that the four sugars tested are transported into kidney cortex cells by a variety of pathways which do not appear to be related to the demonstrated active, independent mechanisms for H+ and K+ transport. A Li+ dependency for the active transport of α-methyl- d -glucoside in the absence of Na+ was observed.