Volume-regulatory potassium release from isolated perfused rat kidney

The present study has been performed to test for cell volume regulatory potassium release from the isolated perfused rat kidney exposed to hypotonic perfusate and for its sensitivity to potassium channel blocker barium and calcium channel blocker verapamil. Replacement of 25 mmol/l NaCl with 50 mmol/l mannitol has little effect on effluent potassium activity, whereas subsequent omission of mannitol from the perfusate leads to a transient increase of effluent potassium activity, reflecting volume regulatory potassium release. Barium (1 mmol/l) leads to a marked transient decrease of effluent potassium activity, pointing to net cellular uptake of potassium. Verapamil (1 mumol/l) leads to a slight decrease of effluent potassium activity. Both barium and verapamil virtually abolish the rapid, transient increase of effluent potassium activity upon exposure to hypotonic perfusates. Thus, the substances either block or markedly retard volume regulatory potassium release. The apparent renal vascular resistance is transiently increased by exposure to hypotonic perfusates and by barium, but is reduced by verapamil. Cell volume regulation of isolated perfused mouse straight proximal tubules is retarded but not abolished by verapamil (0.1 mmol/l). In conclusion, cellular potassium release from rat kidney can be determined by continuous measurement of effluent potassium activity. The volume regulatory potassium release and cell volume regulation are impaired by both barium and verapamil. The persisting cell volume regulation could be due either to slow potassium release and/or some mechanism independent of potassium.     

Na+,K+ pump and Na+-coupled ion carriers in isolated mammalian kidney epithelial cells: regulation by protein kinase C.

This review updates our current knowledge on the regulation of Na+/H+ exchanger, Na+,K+,Cl- cotransporter, Na+,Pi cotransporter, and Na+,K+ pump in isolated epithelial cells from mammalian kidney by protein kinase C (PKC). In cells derived from different tubule segments, an activator of PKC, 4beta-phorbol 12-myristate 13-acetate (PMA), inhibits apical Na+/H+ exchanger (NHE3), Na+,Pi cotransport, and basolateral Na+,K+ cotransport (NKCCl) and augments Na+,K+ pump. In PMA-treated proximal tubules, activation of Na+,K+ pump probably plays a major role in increased reabsorption of salt and osmotically obliged water. In Madin-Darby canine kidney (MDCK) cells, which are highly abundant with intercalated cells from the collecting duct, PMA completely blocks Na+,K+,Cl- cotransport and decreases the activity of Na+,Pi cotransport by 30-40%. In these cells, agonists of P2 purinoceptors inhibit Na+,K+,Cl- and Na+,Pi cotransport by 50-70% via a PKC-independent pathway. In contrast with MDCK cells, in epithelial cells derived from proximal and distal tubules of the rabbit kidney, Na+,K+,Cl- cotransport is inhibited by PMA but is insensitive to P2 receptor activation. In proximal tubules, PKC-induced inhibition of NHE3 and Na+,Pi cotransporter can be triggered by parathyroid hormone. Both PKC and cAMP signaling contribute to dopaminergic inhibition of NHE3 and Na+,K+ pump. The receptors triggering PKC-mediated activation of Na+,K+ pump remain unknown. Recent data suggest that the PKC signaling system is involved in abnormalities of dopaminergic regulation of renal ion transport in hypertension and in the development of diabetic complications. The physiological and pathophysiological implications of PKC-independent regulation of renal ion transporters by P2 purinoceptors has not yet been examined.     

Effect of potassium on cell volume regulation in renal straight proximal tubules

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.     

Cell volume regulation in liver

The maintenance of liver cell volume in isotonic extracellular fluid requires the continuous supply of energy: sodium is extruded in exchange for potassium by the sodium/potassium ATPase, conductive potassium efflux creates a cell-negative membrane potential, which expelles chloride through conductive pathways. Thus, the various organic substances accumulated within the cell are osmotically counterbalanced in large part by the large difference of chloride concentration across the cell membrane. Impairment of energy supply leads to dissipation of ion gradients, depolarization and cell swelling. However, even in the presence of ouabain the liver cell can extrude ions by furosemide-sensitive transport in intracellular vesicles and subsequent exocytosis. In isotonic extracellular fluid cell swelling may follow an increase in extracellular potassium concentration, which impairs potassium efflux and depolarizes the cell membrane leading to chloride accumulation. Replacement of extracellular chloride with impermeable anions leads to cell shrinkage. During excessive sodium-coupled entry of amino acids and subsequent stimulation of sodium/potassium-ATPase by increase in intracellular sodium activity, an increase in cell volume is blunted by activation of potassium channels, which maintain cell membrane potential and allow for loss of cellular potassium. Cell swelling induced by exposure of liver cells to hypotonic extracellular fluid is followed by regulatory volume decrease (RVD), cell shrinkage induced by reexposure to isotonic perfusate is followed by regulatory volume increase (RVI). Available evidence suggests that RVD is accomplished by activation of potassium channels, hyperpolarization and subsequent extrusion of chloride along with potassium, and that RVI depends on the activation of sodium hydrogen ion exchange with subsequent activation of sodium/potassium-ATPase leading to the respective accumulation of potassium and bicarbonate. In addition, exposure of liver to anisotonic perfusates alters glycogen degradation, glycolysis and probably urea formation, which are enhanced by exposure to hypertonic perfusates and depressed by hypotonic perfusates.     

Evidence for chloride dependent potassium and water transport induced by hyposmotic stress in erythrocytes of the marine teleost,Opsanus tau

Summary Red blood cells of the marine teleost,Opsanus tau (oyster toadfish), were characterized as to their normal hemoglobin, ion and water contents. Cells were exposed to ouabain containing, hyposmotic salt solutions (osmolarity reduced to 2/3 of normal) in which the cation or anion composition was varied. It was found that the initial cell volume expansion due to water influx was independent of the anion present. However, a secondary volume reduction was dependent on the presence of chloride or bromide anions. During volume reduction, cellular potassium and chloride ion contents fell by about equal amounts. Potassium loss was commensurate to the total amount of potassium ions detected extracellularly about 1.5h after the initial osmotic shock. No major changes were seen in the cellular sodium ion contents. When chloride ions within the cells and in the suspending medium were replaced by nitrate, iodide or thiocyanate, the cells failed to return to volumes close to those of isosmotically suspended controls, and the cellular potassium content also remained constant. In hypotonic potassium chloride the cells failed to extrude potassium chloride and water, and hence retained their expanded volume. Neither potassium loss nor volume decrease occurred in cells swollen in hypotonic sodium chloride media containing furosemide or 4,4 diisothiocyano-2,2-stilbene-disulfonic acid (DIDS). These two compounds are known inhibitors of monovalent cation cotransport and anion self exchange, respectively, in mammalian red cells. Hence toadfish red cells respond to osmotic swelling primarily by activation of an ouabain-insensitive, chloride dependent potassium transport system which is sensitive to inhibition by furosemide and DIDS.     

Effect of amiloride on cell volume regulation in renal straight proximal tubules

Amiloride has been shown to impair cell volume regulatory decrease in amphiuma red cells. The present study has been performed to test for the influence of amiloride on volume regulatory decrease and electrical properties in isolated perfused mouse straight proximal tubules. Replacement of 40 mmol/l NaCl with 80 mmol/l mannitol in bath perfusate does not appreciably affect the cell volume or the potential difference across the basolateral cell membrane. Reduction of osmolarity by omission of mannitol leads to cell swelling by 16.7 +/- 0.7% (n = 7), followed by volume regulatory decrease to 107.2 +/- 1.2% (n = 7) of original cell volume within 2 min. 1 mmol/l amiloride (but not 0.1 mmol/l amiloride) in the bath depolarizes the basolateral cell membrane from -63 +/- 1 mV (n = 24) by +16 +/- 1 mV (n = 16), decreases the apparent potassium transference number from 0.69 +/- 0.02 (n = 5) to 0.36 +/- 0.05 (n = 5), and significantly impairs volume regulatory decrease without appreciably modifying cell volume in isotonic solutions. 1 mmol/l amiloride in the luminal perfusate leads to a slight hyperpolarization of the basolateral cell membrane but does not interfere with volume regulatory decrease. Reduction of bath osmolarity depolarizes the basolateral cell membrane within 30 s by +7.8 +/- 0.8 mV (n = 18) in the absence and by +18 +/- 2 mV (n = 8) in the presence of amiloride. In the presence of reduced bath osmolarity and amiloride the potassium transference number amounts to 0.36 +/- 0.04 (n = 8). The hyperpolarization following luminal application of amiloride is most likely due to inhibition of luminal sodium channels, whereas bath amiloride depolarizes the basolateral cell membrane by reduction of basolateral potassium selectivity. As in amphiuma red cells amiloride impairs volume regulatory decrease in proximal straight renal tubules.     

Localization of NBC-1 variants in human kidney and renal cell carcinoma

The Na(+)-HCO(3)(-) cotransporter (NBC-1) plays a major role in bicarbonate absorption from proximal tubules. However, which NBC-1 variant mediates proximal bicarbonate absorption has not been definitely determined. Moreover, the localization of this cotransporter in human kidney and renal cell carcinoma (RCC) tissues has not been clarified. To clarify these issues, immunohistochemical analysis was performed using the specific antibodies against kidney type (kNBC-1) and pancreatic type (pNBC-1) transporters. In Western blot analysis the expression of kNBC-1 but not of pNBC-1 was detected in both normal human kidney and RCC tissues. In immunofluorescence analysis on normal renal tissues the anti-kNBC-1 antibody strongly and exclusively labeled the basolateral membranes of proximal tubules, which was confirmed by electron microscopic observation. In RCC cells, the anti-kNBC-1 antibody labeled both plasma membranes and intracellular organelles. The labeling by anti-pNBC-1 antibody was not detected in both normal kidney and RCC tissues. These results indicate that kNBC-1 is the dominant variant that mediates bicarbonate absorption from human renal proximal tubules. They also suggest that NBC-1 may have distinct roles in cancer cells.     

ARF6-dependent interaction of the TWIK1 K+ channel with EFA6, a GDP/GTP exchange factor for ARF6

TWIK1 belongs to a family of K(+) channels involved in neuronal excitability and cell volume regulation. Its tissue distribution suggests a role in epithelial potassium transport. Here we show that TWIK1 is expressed in a subapical compartment in renal proximal tubules and in polarized MDCK cells. In nonpolarized cells, this compartment corresponds to pericentriolar recycling endosomes. We identified EFA6, an exchange factor for the small G protein ADP-ribosylation factor 6 (ARF6), as a protein binding to TWIK1. EFA6 interacts with TWIK1 only when it is bound to ARF6. Because ARF6 modulates endocytosis at the apical surface of epithelial cells, the ARF6/EFA6/TWIK1 association is probably important for channel internalization and recycling.     

Control of renal bicarbonate transport

Single rabbit renal tubules were perfused in vitro to elucidate the factors that control bicarbonate transport. One factor studied was the preexisting acid-base status of the rabbits. Cortical collecting ducts from acidotic rabbits (given ammonium chloride) transported bicarbonate from lumen to bath. Collecting ducts from alkalotic rabbits (given sodium bicarbonate) transported bicarbonate in the opposite direction. Thus, bicarbonate transport by collecting ducts in vitro was conditioned by the preexisting state of the rabbit in vivo. In contrast, bicarbonate transport by proximal straight tubules and cortical thick ascending limbs was not affected by ammonium chloride or sodium bicarbonate given to the rabbits. Parathyroid hormone, the second factor studied, strongly inhibited bicarbonate absorption by proximal straight tubules.     

Control of Renal Bicarbonate Transport

Single rabbit renal tubules were perfused in vitro to elucidate the factors that control bicarbonate transport. One factor studied was the preexisting acid-base status of the rabbits. Cortical collecting ducts from acidotic rabbits (given ammonium chloride) transported bicarbonate from lumen to bath. Collecting ducts from alkalotic rabbits (given sodium bicarbonate) transported bicarbonate in the opposite direction. Thus, bicarbonate transport by collecting ducts in vitro was conditioned by the preexisting state of the rabbit in vivo. In contrast, bicarbonate transport by proximal straight tubules and cortical thick ascending limbs was not affected by ammonium chloride or sodium bicarbonate given to the rabbits. Parathyroid hormone, the second factor studied, strongly inhibited bicarbonate absorption by proximal straight tubules.     

Ion channels in opossum kidney cells.

This review discusses the activation of ion transport pathways during regulatory volume decrease in opossum kidney (OK) cells. OK cells regulate their volume when exposed to a hypotonic medium. The changes in cell volume are caused by activation of ion transport pathways and the accompanying osmotically driven water movement so that the increased cell volume returns toward physiological levels. The reshrinking of hypotonically swollen cells is termed regulatory volume decrease. In OK cells separate K+ and Cl- conductances are activated. The Na+/H+ cotransport system seems not to be involved. The potassium pathway is mediated by a K+ channel with a slope conductance of about 12 pS. The occasionally observed widely distributed Ca2(+)- and voltage-dependent K+ channel of large unit conductance (120 pS) seems not to be involved. The volume regulatory decrease is accompanied by a cell depolarization from a resting potential of about -60 mV to about -20 mV followed by a repolarization. It will be discussed whether the depolarization is caused by the observed activation of stretch-sensitive ion channels of about 30 and 40 pS, respectively. The transient behavior of the cell volume parallels the time-dependent change of the total membrane current. For both recording techniques the volume regulatory decrease can be blocked by quinine. In addition an inward rectifying K+ channel of about 80 pS has been observed in high KCl solution.     

Evidence against bicarbonate reabsorption in the ascending limb, particularly as disclosed by free-water clearance studies.

Bicarbonate reabsorption in the thick ascending limb of Henle's loop was examined by studies of free-water clearance (CH2O) and free-water reabsorption (TcH2O). During maximal water diuresis in the dog, CH2O/GFR was taken as an indes of sodium reabsorption in, and urine flow (V/GFR) as an index of delivery of filtrate to, this scarbonate, infusion of a nonreabsorbable solute (hypotonic mannitol) and administration of an inhibitor of bicarbonate reabsorption (acetaent, but less than that achieved with hypotonic saline infusion. This suggests that sodium that sodium bicarbonate is not reabsorbed in the ascending limb. Rather, it is the sodium chloride, swept out of the proximal tubule by osmotic diuresis due to nonreabsorbed mannitol or sodium bicarbonate, that is reabsorbed in the ascending limb thereby increasing CH2O, whereas the nonreabsorption of mannitol and sodium bicarbonate results in a depressed CH20 per unit V when compared with hypotonic saline. V/GFR is not a satisfactory index of delivery to the ascending limb during osmotic diuresis, since it includes water obligated by nonreabsorbable solutes. When a better index of delivery, the sum of the clearances of chloride (CC1) and free-water (CH2O) is used, hypotonic bicarbonate infusion, hypotonic mannitol infusion and acetazolamide administration increase CH2O/GFR per unit delivery to the same extent as odes hypotonic saline infusion. Studies in dogs and rats on TcH2O also indicate that sodium bicarbonate is an impermeant solute in the ascending limb. Osmotic diuresis due to sodium bicarbonate diuresis, produced either by inhibition of sodium bicarbonate reabsorption (acetazolamide, L-lysine mono-hydrochloride) or infusion of sodium bicarbonate, or mannitol diuresis both produced marked chloruresis and increased TcH2O to the same extent as did hypertonic saline infusion. If chloride excretion was almost eliminated by hemodialysis against a chloride-free dialysate (dogs) or prolonged feeding of a salt-free diet (rats), TcH2O formation was unimpaired if hypertonic saline was infused but virtually obliterated during mannitol or sodium bicarbonate diuresis. Sodium reabsorption in the ascending limb, therefore, appears to be dependent upon chloride as the accompanying anion. At any given rate of bicarbonate excretion, more cloride is delivered out of the proximal tubule (as estimated from CC1 + CH2O) with hypotonic sodium bicarbonate infusion than with acetazolamide administration. This suggests that magnitude of the chlorutesis accompanying bicarbonate diuresis depends, not only on osmotic diuresis due to nonreabsorbed sodium bicarbonate, but also on the extent to which concomitant changes in effective extracellular volume influence overall sodium chloride reabsorption.     

Measurements of Electrical Potential Differences on Single Nephrons of the Perfused Necturus Kidney

Stable electrical potential differences can be measured by means of conventional glass microelectrodes across the cell membrane of renal tubule cells and across the epithelial wall of single tubules in the doubly perfused kidney of Necturus. These measurements have been carried out with amphibian Ringer's solution, and with solutions of altered ionic composition. The proximal tubule cell has been found to be electrically asymmetrical inasmuch as a smaller potential difference is maintained across the luminal cell membrane than across the peritubular cell boundary. The tubule lumen is always electrically negative with respect to the peritubular extracellular medium. Observations on the effectiveness of potassium ions in depolarizing single tubule cells indicate that the transmembrane potential is essentially an inverse function of the logarithm of the external potassium concentration. The behavior of the peritubular transmembrane potential resembles more closely an ideal potassium electrode than that of the luminal transmembrane potential. From these results, and the effects of various ionic substitutions on the electrical profile of the renal tubular epithelium, a thesis concerning the origin of the observed potential differences is presented. A sodium extrusion mechanism is considered to be located at the peritubular cell boundary, and reasons are given for the hypothesis that the electrical asymmetry across the proximal renal tubule cell could arise as a consequence of differences in the relative sodium and potassium permeability at the luminal and peritubular cell boundaries.     

Effect of lysophosphatidylcholine on salt permeability through the erythrocyte membrane under haemolytic conditions

Human erythrocytes were incubated in haemolytic salt or sucrose media and the amount of potassium and haemoglobin released were monitored. In hypotonic NaCl and KCl solutions potassium release and haemolysis increased with time showing that the cell membrane had been injured and became permeable to intra- and extracellular cations which, due to intracellular haemoglobin, causes water influx and continuous haemolysis. Both potassium release and haemolysis remained, however, at their 2-minute level in the presence of LPC. Thus, LPC could reseal the membrane and prevent continuous salt fluxes. It protected erythrocytes from hypotonic haemolysis and the protection was more efficient in NaCl than in sucrose media. This suggests that the increase in the critical volume of erythrocytes caused by LPC occurs both in electrolyte and sucrose media, and the additional protection observed in electrolyte media is due to the resealing of the injured cell membrane by LPC. The repairing mechanism was mediated via the membrane lipids or integral proteins, since the time-course of haemolysis of erythrocytes swollen in NaCl media at the spectrin-denaturing temperature of 49.5 degrees C was similar to that at room temperature with and without LPC. LPC did not protect erythrocytes from colloid osmotic haemolysis caused by ammonia influx in an isotonic NH4Cl medium, but protected the cells from colloid osmotic haemolysis caused by sodium influx through nystatin-channels in NaCl media without any area or volume increase. Hence, LPC could not prevent ammonia influx through the lipid bilayer, but suppressed sodium influx through nystatin-channels presumably via LPC interference with cholesterol.     

Characterization of low potassium-resistant mutants of the Madin-Darby canine kidney cell line with defects in NaCl/KCl symport

Three independent mutants of the Madin-Darby canine kidney cell line (MDCK) have been isolated which were capable of growth in media containing low concentrations of potassium. All three mutants were deficient to varying extents in furosemide- and bumetanide-sensitive 22Na+, 86+b+, and 36Cl- uptake. The two mutants most resistant to low K+ media had lost essentially all of the 22Na+, 86Rb+, and 36Cl- uptake activities of this system. The third mutant was partially resistant to low K+ media and had reduced levels of bumetanide-sensitive uptake for all three ions. Extrapolated initial uptake rates for 22Na+, 86Rb+, and 36Cl- revealed that the partial mutant exhibited approximately 50% of the parental uptake rates for all three ions. The stoichiometries of bumetanide-sensitive uptake in both the parental cell line and the partial mutant approximated 1 Rb+:1 Na+:2 Cl-. The results of this study provide genetic evidence for a single tightly-coupled NaCl/KCl symporter in MDCK cells. The correlation between the ability to grow in low K+ media and decreased activity of the bumetanide-sensitive co-transport system suggests that the bumetanide-sensitive transport system catalyzes net K+ efflux from cells in low K+ media. The results of 86Rb+ efflux studies conducted on ouabain-pretreated mutant and parental cells are consistent with this interpretation. Cell volume measurements made on cells at different densities in media containing normal K+ concentrations showed that none of the mutants differed significantly in volume from the parental strain at a similar cell density. Furthermore, all three mutants were able to readjust their volume after suspension in hypotonic media. These results suggest that in the MDCK cell line, the bumetanide-sensitive NaCl/KCl symport system does not function in the regulation of cell volume under the conditions employed.     

Activation of K+ and Cl− channels in MDCK cells during volume regulation in hypotonic media

Single-channel patch-clamp experiments were performed on MDCK cells in order to characterize the ionic channels participating in regulatory volume decrease (RVD). Subconfluent layers of cultured cells were exposed to a hypotonic medium (150 mOsm), and the membrane currents at the single-channel level were measured in cell-attached experiments. The results indicate that MDCK cells respond to a hypotonic swelling by activating several different ionic conductances. In particular, a potassium and a chloride channel appeared in the recordings more frequently than other channels, and this allowed a more detailed study of their properties in the inside-out configuration of the patch-clamp technique. The potassium channel had a linear I/V curve with a unitary conductance of 24 +/- 4 pS in symmetrical K+ concentrations (145 mM). It was highly selective for K+ ions vs. Na+ ions: PNa/PK less than 0.04. The time course of its open probability (P0) showed that the cells responded to the hypotonic shock with a rapid activation of this channel. This state of high activity was maintained during the first minute of hypotonicity. The chloride channel participating in RVD was an outward-rectifying channel: outward slope conductance of 63.3 +/- 4.7 pS and inward slope conductance of 26.1 +/- 4.9 pS. It was permeable to both Cl- and NO3- and its maximal activation after the hypotonic shock was reached after several seconds (between 30 and 100 sec). The activity of this anionic channel did not depend on cytoplasmic calcium concentration. Quinine acted as a rapid blocker of both channels when applied to the cytoplasmic side of the membrane. In both cases, 1 mM quinine reversibly reduced single-channel current amplitudes by 20 to 30%. These results indicate that MDCK cells responded to a hypotonic swelling by an early activation of highly selective potassium conductances and a delayed activation of anionic conductances. These data are in good agreement with the changes of membrane potential measured during RVD.     

Purinergic Modulation of Na+,K+,Cl− Cotransport and MAP Kinases is Limited to C11-MDCK Cells Resembling Intercalated Cells from Collecting Ducts

We demonstrated recently that in renal epithelial cells from collecting ducts of Madin-Darby canine kidneys (MDCK), Na⁺,K⁺,Cl⁻ cotransport is inhibited up to 50% by ATP via its interaction with P_2Y purinoceptors (Biochim. Biophys. Acta 1998. 1369:233–239). In the present study we examined which type of renal epithelial cells possesses the highest sensitivity of Na⁺,K⁺,Cl⁻ cotransport to purinergic regulation. We did not observe any effect of ATP on Na⁺,K⁺,Cl⁻ cotransport in renal epithelial cells from proximal and distal tubules, whereas in renal epithelial cells from rabbit and rat collecting ducts ATP decreased the carrier's activity by ∼30%. ATP did not affect Na⁺,K⁺,Cl⁻ cotransport in C7 subtype MDCK cells possessing the properties of principal cells but led to ∼85% inhibition of this carrier in C11-MDCK cells in which intercalated cells are highly abundant. Both C7- and C11-MDCK exhibited ATP-induced IP₃ and cAMP production and transient elevation of [Ca²⁺] _i . In contrast to the above-listed signaling systems, ATP-induced phosphorylation of ERK and JNK MAP kinases was observed in C11-MDCK only. Thus, our results reveal that regulation of renal Na⁺,K⁺,Cl⁻ cotransport by P_2Y receptors is limited to intercalated cells from collecting ducts and indicate the involvement of the MAP kinase cascade in purinergic control of this ion carrier's activity. Received: 10 June 1999/Revised: 23 August 1999     

Comparative cytotoxicity of 5-aminosalicylic acid (mesalazine) and related compounds in different cell lines

Nonsteroidal anti-inflammatory drugs can cause serious side-effects such as tubulo-interstitial nephritis. Mesalazine (5-ASA, 5-aminosalicylic acid) is used for the treatment of colitis ulcerosa, Crohn disease, and other diseases; it has been found to induce necrosis of both proximal convoluted tubules and renal papillaries. The comparative cytotoxicity of 3-, 4-, and 5- aminosalicylic acid, acetylsalicylic acid (AcSA), and the parent compound salicylic acid (SA) was investigated for the free acids and for their sodium salts. The interaction with endogenous glutathione (GSH) was also investigated. Four established cell lines were used: MDCK, LLC-PK1, NRK as renal cells, and HepG2 as hepatic cells. The free acid compounds were less toxic than their corresponding salts. Acidic 5-ASA was the most toxic of the three isomers in MDCK and LLC-PK1 cells, while NRK and HepG2 were more susceptible to acidic 3-ASA. Addition of NaOH modified the relative toxicity of 3-ASA and 5-ASA. The LLC-PK1 and HepG2 cells were more sensitive to the test chemicals as their salts than were the NRK and MDCK cells. SA and 5-ASA decreased the GSH content in renal cells and increased it in HepG2. GSH depletion with l-buthionine-(S,R)-sulfoximine enhanced the toxicity only for SA in NRK and for 5-ASA and AcSA in HepG2. No correlation between endogenous GSH and the susceptibility of MDCK and LLC-PK1 to the test compounds was observed. The results suggest that no typical nephrotoxic effect occurred. No explanation could be found for the tubulo-interstitial nephritis caused by 5-ASA therapy.     

Effect of osmolarity on potassium transport in isolated cerebral microvessels

Potassium transport in microvessels isolated from rat brain by a technique involving density gradient centrifugation was studied in HEPES buffer solutions of varying osmolarity from 200 to 420 mosmols, containing different concentration of sodium chloride, choline chloride, or sodium nitrate. The flux of 86Rb (as a tracer for K) into and out of the endothelial cells was estimated. Potassium influx was very sensitive to the osmolarity of the medium. Ouabain-insensitive K-component was reduced in hypotonic medium and was increased in medium made hypertonic with sodium chloride or mannitol. Choline chloride replacement caused a large reduction in K influx. Potassium influx was significant decrease when nitrate is substituted for chloride ion in isotonic and hypertonic media, whereas a slight decrease was found in hypotonic medium. The decrease of K influx in the ion-replacement medium is due to a decrement of the ouabain-insensitive component. Potassium efflux was unchanged in hypotonic medium but was somewhat reduced in hypertonic medium. The marked effect of medium osmolarity on K fluxes suggests that these fluxes may be responsible for the volume regulatory K movements. The possible mechanism of changes of K flux under anisotonic media is also discussed.     

Potassium transport in erythrocytes from patients with gentamicin intolerance

A study was made of rubidium (potassium analog) influxes via ouabain-sensitive Na/K pump, bumetanide-sensitive cotransport and resistant to ouabain bumetanide membrane ion pathways and of the intracellular potassium and sodium contents in red blood cells from patients with high gentamicin sensitivity (GSP) and healthy patients (HP). It is found that red blood cells from two groups of donors do not differ in both intracellular potassium and sodium contents and pump-mediated rubidium influxes, however, bumetanide-sensitive rubidium influxes were twice as low in GSP as compared to HP (0.28 against 0.46 mumole per gram of hemoglobin). In the presence of gentamicin (10(-6) M) bumetanide-sensitive rubidium influxes were shown to decrease in red blood cells of GSP, being unchanged in erythrocytes of HP. It is suggested that the increased rates of hemolysis in response to hypotonicity in red blood cells of GSP may be due to a decreased activity of bumetanide-sensitive cotransport in plasma membrane of these cells.