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R Kinne

Publications and source records attributed to R Kinne.

At least 37 records · Page 2Linked to original sources
PubMed1986

The anion specificity of the sodium-potassium-chloride cotransporter in rabbit kidney outer medulla: studies on medullary plasma membranes.

Plasma membrane vesicles were isolated from rabbit kidney outer medulla and employed in sodium, rubidium, and chloride flux studies. Chloride dependence and bumetanide sensitivity of (part of) the sodium and rubidium flux indicate that this plasma membrane fraction can be used to study the properties of Na-K-2Cl cotransport system present in the luminal membrane of the medullary thick ascending limb. The anion specificity of the cotransporter was investigated by determining the effect of anion replacement on sodium fluxes. When chloride was completely replaced by bromide, iodide, nitrate, or thiocyanate only bromide could effectively substitute for chloride (90% activity), whereas sodium uptake in the presence of iodide, nitrate, and thiocyanate amounted to only 25% of the sodium uptake observed in the presence of chloride. When similar replacement experiments were performed in the presence of 10 mmol/l chloride, bromide could substitute for chloride by 110%, iodide and nitrate by 60%, and thiocyanate by 70%. In the presence of 10 mmol/l bromide iodide, nitrate, and thiocyanate were similarly effective. The effect of nitrate and chloride on sodium flux was additive. Bumetanide-sensitive chloride uptake was inhibited by nitrate, the inhibition was however only partly, amounting to 60%. The results obtained are compatible with the view that the two anion binding sites of the Na-K-2Cl cotransporter can exhibit a different substrate specificity and that the transporter in addition to a 2Cl mode can also operate in a 2Br, Cl-, A- and Br-, A- mode, A- representing iodide, nitrate, or thiocyanate.

Animals↗
PubMed1986

Proton pump activity and Mg-ATPase activity in rat kidney cortex brushborder membranes: effect of 'proton ATPase' inhibitors.

In order to further characterize the ATP driven proton pump present in the luminal membrane of the renal proximal tubule, brushborder membranes were isolated from rat kidney cortex and the effect of various proton ATPase inhibitors on intravesicular ATP hydrolysis in sealed brushborder membrane vesicles and on Mg-ATPase activity in permeabilized brushborder membranes was investigated. The protonophor induced intravesicular ATP hydrolysis (ATP driven proton pump) was inhibited by DCCD and filipin but not by diethylstilbestrol and duramycin. All four compounds decreased Mg-ATPase activity, the two former inhibited the ATPase activity with a lower potency than the proton pump. NEM--up to 10 mM--and orthovanadate did not affect intravesicular ATP hydrolysis nor Mg-ATPase activity. From the relative sensitivity of the proton pump and the Mg-ATPase activity to the inhibitors it is concluded that about 35% of the Mg-ATPase activity found in the brushborder membrane can be attributed to the ATP-driven proton pump. Furthermore, the results obtained with NEM and duramycin suggest that the brushborder membrane proton pump has different properties than the proton pump in clathrin-coated vesicles or endosomes. The results presented above raise the possibility that the brushborder membrane proton pump is predominantly involved in acid secretion by the proximal convoluted tubule whereas the proton pump in clathrin-coated vesicles may be predominantly involved in the endocytosis of larger peptides and proteins.

Animals↗
PubMed1986

Ammonium transport in medullary thick ascending limb of rabbit kidney: involvement of the Na+,K+,Cl(-)-cotransporter.

In order to investigate the question whether ammonium reabsorption in the thick ascending limb of Henle's loop (TALH) proceeds via the Na+,K+,Cl(-)-cotransporter, plasma membrane vesicles were prepared from TALH cells isolated from rabbit kidney outer medulla and the effect of NH+4 on their transport properties was investigated. It was found that, in the presence of a 78-mmol/liter NaCl gradient, 5 mmol/liter NH+4 inhibited bumetanide-sensitive rubidium flux by 86%; a similar decrease was observed for 5 mmol/liter, K+. Inhibition of bumetanide-sensitive rubidium uptake by NH+4 was competitive and an apparent Ki of 1.9 mmol/liter was found. Bumetanide-sensitive sodium uptake measured in the presence of a 83 mmol/liter KCl gradient was not inhibited by 5 mmol/liter NH+4. A 100-mmol/liter NH4Cl gradient was, however, capable of stimulating bumetanide-sensitive sodium uptake to the same extent as a KCl gradient. These data suggest that NH+4 is accepted by the K+ site of the Na+,K+,Cl-cotransport system and that the transporter can function in a Na+,NH+4,2Cl mode. Since the affinity of the transporter for NH+4 lies in the concentration range found in the TALH lumen in vivo, it is concluded that Na+,NH+4,2Cl-cotransport can contribute to the NH+4 reabsorption in this tubular segment.

Ammonia↗
PubMed1986

Development of differentiated characteristics in cultured kidney (thick ascending loop of Henle) cells.

This study describes the characterization of epithelial cells in culture following their isolation from the thick ascending limb of Henle's loop of rabbit kidney, by enzymatic digestion and subsequent purification using density gradient centrifugation. In culture, these cells expressed a variety of morphological, enzymatic and functional parameters expected of such cells in vivo. These cells were polarised, formed tight junctions and exhibited considerable lateral interdigitation between adjacent cells. They also developed characteristically high levels of activity of Na,K-ATPase, comparable to those seen in freshly isolated cells, and also expressed the functionally important Na,K,Cl-co transport system. The development of these systems in culture, however, was not coincident and their activities were reduced upon extended culture. The ability of these cells to develop and express differentiated characteristics in culture indicates that cells derived from defined kidney cell populations should provide valuable models for the study of the factors involved in the development and regulation of kidney cell type-specific characteristics.

Alkaline Phosphatase↗
PubMed1986

Maintenance of expression of differentiated function of kidney cells following transformation by SV40 early region DNA.

This study describes the isolation and characterization of epithelial cell lines that maintain their differentiated phenotype following the stable integration of SV40 genes. Epithelial cells were derived from a defined location of rabbit kidney, the thick ascending limb of Henle's loop, and were co-transfected with genes from the early region of SV40 together with pSV2-neo DNA (which confers resistance to the antibiotic G418). These cells were shown to be resistant to G418, express SV40 large T-antigen and continued to express differentiated characteristics typical of cells of their origin. Such characteristics include the expression of high levels of activity of both Na,K-ATPase and the functionally important Na,K,Cl-co-transport system, the synthesis of Tamm-Horsfall glycoprotein and the presence of a barium-sensitive K+ channel on the apical membrane surface.

Animals↗
PubMed1986

Amphotericin B and amphotericin B methylester: effect on brush border membrane permeability.

In order to explain the nephrotoxicity of polyene antibiotics such as Amphotericin B (AM), an effect on the tubule membrane permeability has been postulated. However, studies on the action of AM have been complicated by the use of sodium deoxycholate (DOC), a membrane dissociating detergent as a solvent. Recently, a derivative, the methylester aspartate salt of Amphotericin B (AME) has been synthesized, which is highly water soluble in the absence of organic solvents. We have tested the action of AM, DOC, and AME on the sodium permeability of brush border (BBM) vesicles isolated from rat kidney cortex. It was found that both AM and AME increased the 22Na uptake as measured by a rapid filtration technique. However, a large fraction of the AM action was due to the effect of DOC on the BBM sodium permeability. We also investigated the time- and dose-dependent action of AME on 22Na and 3H-D-mannitol efflux from BBM vesicles. After 15 sec of exposure, efflux from 22Na-preloaded vesicles was unchanged in the presence of 1 microgram AME/mg protein compared to control vesicles. With 10, 50, and 100 micrograms AME/mg protein, the efflux increased 16, 25, and 35% respectively; 260 micrograms AME/mg protein did not elicit a further increment in the 22Na efflux. In the same membrane vesicles 3H-D-mannitol efflux did not change. After preincubation of the membranes for 60 min with different concentrations of AME, the 15 sec 22Na efflux increased 26% in the presence of 1 microgram/mg protein AME with no change in the 3H-D-mannitol efflux.(ABSTRACT TRUNCATED AT 250 WORDS)

Amphotericin B↗
PubMed1986

Photoinactivation of sodium-potassium-chloride cotransport in LLC-PK1/Cl 4 cells by bumetanide.

Rb+ uptake into LLC-PK1/Cl 4 cells can be subdivided into three components: 1) ouabain-sensitive uptake, 2) bumetanide-sensitive uptake, and 3) ouabain- and bumetanide-insensitive uptake. Exposure of cells to near-UV light in the presence of low concentrations of bumetanide produces a specific, irreversible inhibition of the bumetanide-sensitive uptake component, while not affecting the other two uptake components. Irreversible inhibition of bumetanide-sensitive transport is observed when measuring either cellular uptake or efflux and also when measuring 86Rb+ uptake into membrane vesicles. The irreversible inhibition is both concentration and time dependent and is blocked under conditions where the interaction of bumetanide with the Na+-K+-Cl- cotransporter is disturbed. We conclude that bumetanide, at low concentrations, can specifically and irreversibly inhibit the Na+-K+-Cl- cotransporter of LLC-PK1/Cl 4 cells. We suggest that this irreversible inhibition is the result of the photoactivation of an ether linkage in the bumetanide molecule, leading to a covalent binding of bumetanide to the Na+-K+-Cl- cotransporter.

Animals↗
PubMed1986

Renal bicarbonate reabsorption in the rat. I. Effects of hypokalemia and carbonic anhydrase.

Free-flow micropuncture studies were carried out on superficial rat proximal and distal tubules to assess the participation of different nephron segments in bicarbonate transport. Particular emphasis was placed on the role of the distal tubule, and micro-calorimetric methods used to quantitate bicarbonate reabsorption. Experiments were carried out in control conditions, during dietary potassium withdrawal, and after acute intravenous infusions of carbonic anhydrase. We observed highly significant net bicarbonate reabsorption in normal acid-base conditions as evidenced by the maintenance of significant bicarbonate concentration gradients in the presence of vigorous fluid absorption. Distal bicarbonate reabsorption persisted in hypokalemic alkalosis and even steeper transepithelial concentration gradients of bicarbonate were maintained. Enhancement of net bicarbonate reabsorption followed the acute intravenous administration of carbonic anhydrase but was limited to the nephron segments between the late proximal and early distal tubule. The latter observation is consistent with a disequilibrium pH along the proximal straight tubule (S3 segment), the thick ascending limb of Henle, and/or the early distal tubule.

Absorption↗
PubMed1985

The use of membrane vesicles to study the NaCl/KCl cotransporter involved in active transepithelial chloride transport.

Properties of the NaCl/KCl cotransport system were investigated in isolated membranes by flux measurements and binding studies. Chloride competes with "furosemide-like loop diuretics" for its two binding sites at the cotransporter as evidenced by the decrease in piretanide sensitivity of sodium flux and inhibition of high affinity N-methylfurosemide binding by chloride in rectal gland plasma membranes. In the rectal gland lithium inhibits sodium flux but is not translocated whereas in the renal thick ascending limb (TALH) it is also transported. Ammonium is a substrate for the sodium and potassium site in the rectal gland but only for the potassium site in the TALH. The latter finding raises the possibility that part of the ammonium reabsorption in the TALH is mediated by the cotransport system as NaCl/NH4Cl cotransport.

Animals↗
PubMed1985

Short term effect of low doses of tri-iodothyronine on proximal tubular membrane Na-K-ATPase and potassium permeability in thyroidectomized rats.

Tri-iodothyronine (T3), even when administered for short time and at low doses, induces a large increase in the isotonic fluid reabsorption (Jv) in proximal tubules of thyroidectomized rats (TX). In order to investigate the role of the Na-K-ATPase in this process, we measured the Na-K-ATPase activity in early proximal convoluted tubules (S1) and proximal straight tubules (S2) microdissected from TX rats and rats treated with low doses of T3 (10 micrograms/kg body wt), either for 3 days (TX + 3T3) or for 7 days (TX + 7T3). In both segments no changes in Na-K-ATPase activity were found in TX + 3T3 rats versus TX rats, while an increase was registered in TX + 7T3 rats. Using micropuncture techniques, Jv measured on the same tubular segments increased by 68% in TX + 3T3 rats versus TX. Thus, no correlation between Jv and Na-K-ATPase activity measured in vitro could be detected after short term treatment of TX rats with T3. Na-K-ATPase activity in vivo is also regulated by the potassium permeability of the membrane, which might be altered by tri-iodothyronine. This hypothesis was tested by perfusing intraluminally and peritubularly proximal tubules of TX rats with the K ionophore, valinomycin (1 microgram/ml). In the dual perfusion experiments valinomycin elicited 40% of the action induced on Jv by 3 days treatment with T3. On the other hand, no further increase in Jv was recorded when valinomycin was applied in TX rats pretreated with T3.(ABSTRACT TRUNCATED AT 250 WORDS)

Absorption↗
PubMed1985

Regulation of volume reabsorption by thyroid hormones in the proximal tubule of rat: minor role of luminal sodium permeability.

In order to investigate whether changes in luminal membrane sodium permeability can explain the increase in isotonic fluid reabsorption (Jv) found in proximal tubules of thyroidectomized rats (TX) treated with tri-iodothyronine (T3), experiments were carried out on TX rats and TX rats treated for 3 days (TX + T3) with physiological doses (10 micrograms/kg body wt) of T3. Two sets of experiments were performed: 1) in vivo, using the micropuncture technique for the measurements of Jv; 2) in vitro, using isolated brush border membrane vesicles for the direct measurement of Na+ permeability. In micropuncture studies a 65% increase in Jv of TX rats was observed after treatment with T3. Luminal perfusion of proximal tubules of TX rats with Amphotericin B (10 micrograms/ml), to increase luminal sodium permeability, enhanced Jv only by 15%. Brush border membrane vesicles isolated from TX and TX + T3 rats showed the same sodium permeability in uptake or efflux experiments. These results were confirmed by the fact that sodium gradient dependent histidine transport into brush border membrane vesicles did not change after T3 treatment. Finally, measuring the amiloride sensitive sodium uptake, it was also found that Na+-H+ exchange was also only slightly affected by T3. These micropuncture and vesicle data indicate that the large effect of T3 on the trans-cellular sodium transport and volume reabsorption in the proximal tubule, cannot be explained by an action of T3 on the sodium entry step across the brush border membrane.

Absorption↗
PubMed1985

Active chloride transport in rabbit thick ascending limb of Henle's loop and elasmobranch rectal gland: chloride fluxes in isolated plasma membranes.

To investigate directly whether a sodium-potassium-chloride cotransport system is operating in the mammalian thick ascending limb of Henle's loop (TALH) and in the elasmobranch rectal gland, plasma membrane vesicles were prepared from TALH cells isolated from rabbit kidney outer medulla and from rectal glands of Squalus acanthias, and chloride uptake was measured by a rapid filtration technique. Chloride uptake into TALH vesicles in the presence of a 25 mM Na2SO4, 25 mM K2SO4 gradient reached 70% of equilibrium at 2.5 min. In the presence of both sodium and potassium, the 15 s chloride uptake was inhibited 35% by 1 mM bumetanide. When either sodium or potassium was removed from the incubation medium, chloride uptake decreased to the level observed in the presence of 1 mM bumetanide, 0.5 mM SITS had no effect on chloride uptake by the plasma membrane vesicles. This sodium and potassium dependent, bumetanide sensitive chloride uptake was also observed under tracer exchange conditions. Chloride uptake into rectal gland plasma membrane vesicles in the presence of a 50 mM Na2SO4, 50 mM K2SO4 gradient reached 80% of equilibrium at 2.5 min. 1 mM bumetanide inhibited the 15 s uptake of chloride by 34% and removal of either sodium or potassium from the incubation medium reduced chloride uptake to the level observed in the presence of bumetanide under both gradient and tracer exchange conditions. These studies provide additional support for the hypothesis that a sodium-potassium-chloride cotransport system is operating in these epithelia.

4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfo↗
PubMed1984

Structural state of the Na+/D-glucose cotransporter in calf kidney brush-border membranes. Target size analysis of Na+-dependent phlorizin binding and Na+-dependent D-glucose transport.

Target sizes of the renal sodium-D-glucose cotransport system in brush-border membranes of calf kidney cortex were estimated by radiation inactivation. In brush-border vesicles irradiated at -50 degrees C with 1.5 MeV electron beams, sodium-dependent phlorizin binding, and Na+-dependent D-glucose tracer exchange decreased exponentially with increasing doses of radiation (0.4-4.4 Mrad). Inactivation of phlorizin binding was due to a reduction in the number of high-affinity phlorizin binding sites but not in their affinity. The molecular weight of the Na+-dependent phlorizin binding unit was estimated to be 230 000 +/- 38 000. From the tracer exchange experiments a molecular weight of 345 000 +/- 24 500 was calculated for the D-glucose transport unit. The validity of these target size measurements was established by concomitant measurements of two brush-border enzymes, alkaline phosphatase and gamma-glutamyltransferase, whose target sizes were found to be 68 570 +/- 2670 and 73 500 +/- 2270, respectively. These findings provide further evidence for the assumption that the sodium-D-glucose cotransport system is a multimeric structure, in which distinct complexes are responsible for phlorizin binding and D-glucose translocation.

Alkaline Phosphatase↗
PubMed1984

Biliary transport of glutathione disulfide studied with isolated rat-liver canalicular-membrane vesicles.

Canalicular plasma membrane vesicles isolated from rat liver (right side out) were used to study glutathione disulfide (GSSG) transport. GSSG is transported into an osmotically sensitive compartment. Extrapolation to an external osmolarity of infinity indicates 35% binding after 10 min of incubation, e.g. as mixed disulfides between glutathione and SH-groups in membrane proteins. Glutathione disulfide uptake occurred with equal rates in the presence of KCl or NaCl. After equilibration, no significant GSSG concentration gradient across the canalicular membrane was found. The temperature dependence of uptake is consistent with a carrier-mediated transport for GSSG (Q10 X 1.6) and seems to exclude simple diffusion. Concentration dependence of GSSG uptake shows Michaelis-Menten kinetics at concentrations up to 1 mM; a Km of 0.4 mM and a V of 1.1 nmol X min-1 X mg protein-1 is calculated. At higher concentrations a linear dependence is observed without saturation kinetics. It is concluded that transport of glutathione disulfide is mediated by a carrier present in the canalicular membrane of the hepatocyte.

Animals↗
PubMed1984

Biliary transport of glutathione S-conjugate by rat liver canalicular membrane vesicles.

Transport of S-dinitrophenyl glutathione, a model compound of glutathione S-conjugates, was studied in isolated rat liver canalicular membrane vesicles by a rapid filtration technique. The membrane vesicles exhibited time-dependent uptake of [2-3H]glycine-glutathione conjugate into an osmotically sensitive intravesicular space. Inactivation of vesicle-associated gamma-glutamyltransferase by affinity labeling with L-(alpha-S,5S)-alpha-amino-3-chloro-4,5-dihydro-5-isoxazole-acetic acid had no effect on the initial rate of transport. Chemical analysis revealed that the intact glutathione conjugate accounted for most vesicle-associated radioactivity, reflecting the low transferase activity in the liver and membrane vesicles. The initial rate of transport followed saturation kinetics with respect to conjugate concentrations; an apparent Km of 1.0 mM and Vmax of 1.7 nmol/mg of protein X 20 s were calculated. These results indicate that transport of the glutathione S-conjugate across the canalicular membranes is a carrier-mediated process. Sodium chloride in the transport medium could be replaced by KCl, LiCl, or choline chloride without any changes in transport activity. The rate of conjugate transport was enhanced by a valinomycin-induced K+ diffusion potential (vesicle-inside-positive). The rate of conjugate uptake was enhanced by replacing KCl in the transport medium with K gluconate, providing a less permeant anion, and was reduced by replacing KCl with KSCN, providing a more permeant anion. These data indicate that conjugate transport is electrogenic and involves the transfer of negative charge. Transport of S-dinitrophenyl glutathione was inhibited by S-benzyl glutathione, oxidized glutathione, or reduced glutathione. This transport system in canalicular membranes may function in biliary secretion of glutathione S-conjugates of xenobiotics whose synthesis in hepatocytes requires glutathione S-transferases.

Animals↗
PubMed1984

Glutathione transport across hepatocyte plasma membranes. Analysis using isolated rat-liver sinusoidal-membrane vesicles.

Transport of reduced glutathione (GSH) and its derivatives was studied in rat-liver sinusoidal plasma-membrane vesicles by a rapid filtration technique. The membrane vesicles exhibited transport of GSH into an osmotically active intravesicular space. Equilibrium uptake of vesicle-associated GSH was similar to that of free ligands which can be trapped by the intravesicular space of sinusoidal membrane samples. Kinetic analysis of the transport process revealed that the sinusoidal membrane vesicles have high-affinity and low-affinity GSH transport systems; the former has an apparent Km of 0.34 mM for GSH and V of 1.4 nmol X mg protein-1 X 20 s-1, and the latter has an apparent Km of 3.3 mM and V of 3.9 nmol X mg protein-1 X 20 s-1. Both Km values are lower than intrahepatic GSH levels, indicating that GSH transport across the sinusoidal membrane occurs via carrier-mediated mechanism and argues simple diffusion mechanism. The presence of oxidized glutathione (GSSG) or S-benzylglutathione inhibited GSH transport by the vesicles; the low-affinity transport system was inhibited more markedly than the high-affinity transport system. This suggests that these glutathione derivatives interacted preferentially with the low-affinity transport system for GSH. S-Dinitrophenylglutathione was also transported by the vesicles by a process which was inhibited by GSH and GSSG. The vesicles also transported GSSG, and this transport was markedly inhibited by S-benzylglutathione or GSH. The transport systems in sinusoidal plasma membranes may function in vivo in translocating GSH and its derivatives from hepatocytes into plasma and play an important role in inter-organ metabolism of these compounds.

Animals↗