Coupling of a loop diuretic-sensitive Na+ influx with the net loop diuretic-sensitive K+ efflux in mouse NIH 3T3 cells

Mouse 3T3 fibroblasts have a loop diuretic sensitive Na+ transport system, responsible for more than 50% of the total Na+ influx. This transport system is dependent on the simultaneous presence of all three ions; Na+, K+, (Rb+) and Cl- in the extracellular medium. The same requirement for these three ions was also found for the loop diuretic-sensitive K+ efflux. In addition, the sensitivities of Na+ influx and Rb+ efflux for the two loop diuretics, furosemide and bumetanide were found to be similar. The similar ionic requirement and sensitivity towards loop diuretics of the two fluxes, support the hypothesis, that this loop diuretic-sensitive Na+ influx in mouse 3T3 cells, is accompanied by the net loop diuretic-sensitive K+ efflux.     

Na+, Cl− cotransport in Ehrlich ascites tumor cells activated during volume regulation (regulatory volume increase)

Ehrlich ascites cells were preincubated in hypotonic medium with subsequent restoration of tonicity. After the initial osmotic shrinkage the cells recovered their volume within 5 min with an associated KCl uptake. The volume recovery was inhibited when NO-3 was substituted for Cl-, and when Na+ was replaced by K+, or by choline (at 5 mM external K+). The volume recovery was strongly inhibited by furosemide and bumetanide, but essentially unaffected by DIDS. The net uptake of Cl- was much larger than the value predicted from the conductive Cl- permeability. The undirectional 36Cl flux, which was insensitive to bumetanide under steady-state conditions, was substantially increased during regulatory volume increase, and showed a large bumetanide-sensitive component. During volume recovery the Cl- flux ratio (influx/efflux) for the bumetanide-sensitive component was estimated at 1.85, compatible with a coupled uptake of Na+ and Cl-, or with an uptake via a K+,Na+,2Cl- cotransport system. The latter possibility is unlikely, however, because a net uptake of KCl was found even at low external K+, and because no K+ uptake was found in ouabain-poisoned cells. In the presence of ouabain a bumetanide-sensitive uptake during volume recovery of Na+ and Cl- in nearly equimolar amounts was demonstrated. It is proposed that the primary process during the regulatory volume increase is an activation of an otherwise quiescent, bumetanide-sensitive Na+,Cl- cotransport system with subsequent replacement of Na+ by K+ via the Na+/K+ pump, stimulated by the Na+ influx through the Na+,Cl- cotransport system.     

Na+ participates in loop diuretic-sensitive Cl(-)-cation co-transport in the pancreatic beta-cells

In order to investigate whether Na+ participates in loop diuretic-sensitive Cl(-)-cation co-transport in the beta-cells, we tested the interaction between the effects of Na+ deficiency, furosemide and D-glucose on 86Rb+ fluxes in beta-cell-rich mouse pancreatic islets. Removal of extracellular Na+ slightly reduced the ouabain-resistant 86Rb+ influx and the specific effect of 1 mM furosemide on this influx was significantly smaller in Na(+)-deficient medium. The capacity of 20 mM D-glucose to reduce the ouabain-resistant 86Rb+ influx was not changed by removal of extracellular Na+. The 86Rb+ efflux from preloaded islets was rapidly and reversibly reduced by Na+ deficiency. Furosemide (1 mM) reduced the 86Rb+ efflux and the effect of the combination of Na+ deficiency and 1 mM furosemide was not stronger than the effect of furosemide alone. 22Na+ efflux was reduced by both ouabain and furosemide and the effects appeared to be additive. The data suggest that Na+ participates in loop diuretic-sensitive Cl(-)-cation co-transport in the pancreatic beta-cells. This adds further support to the idea that beta-cells exhibit a Na+, K+, Cl- co-transport system. Since some of the furosemide effect on 86Rb+ efflux persisted in the Na(+)-deficient medium, it is likely that also loop diuretic-sensitive K+, Cl- co-transport exists in this cell type.     

Na+-independent Mg2+ efflux from Mg2+-loaded human erythrocytes

Net Mg2+ efflux from Mg2+-loaded human erythrocytes was maximal after reincubation in sucrose. Net Mg2+ efflux was not inhibited by furosemide or bumetanide and, therefore, was not performed by the (Na,K,Cl)- or (K,Cl)-cotransport system. A component of net Mg2+ efflux was inhibited by extracellular NaC1, KCl, LiCl, choline Cl and SITS, in analogy to the inhibition of net Cl- and SITS. Therefore, it was concluded that net Mg2+ efflux is dependent on net Cl- efflux for charge compensation. Cl- -dependent net Mg2+ efflux was inhibited by amiloride. Only 10% of the maximal net Mg2+ efflux may depend on extracellular Na+.     

Effect of ouabain upon diuretic-sensitive K+ transport in cultured cells. Evidence for separate modes of operation of the transporter

(1) Unidirectional K+ (86Rb) influx and efflux were measured in subconfluent layers of MDCK renal epithelial cells and HeLa carcinoma cells. (2) In both MDCK and HeLa cells, the furosemide-inhibitable and chloride-dependent component of K+ influx/efflux was stimulated 2-fold by a 30 min incubation in 1 . 10(-3) M ouabain. (3) Measurements of net K+ loss and Na+ gain in ouabain-treated cells at 1 h failed to show any diuretic sensitive component, confirming the exchange character of the diuretic-sensitive fluxes. (4) Prolonged incubations for 2.5 h in ouabain revealed a furosemide- and anion-dependent K+ (Cl-) outward net flux uncoupled from net Na+ movement. Net K+ (Cl-) outward flux was half-maximally inhibited by 2 microM furosemide. (5) After 2.5 h ouabain treatment, the anion and cation dependence of the diuretic-sensitive K+ influx/efflux were essentially unchanged when compared to untreated controls.     

Effects of chloride deficiency on the pancreatic B-cell response to acetylcholine

Muscarinic stimulation of pancreatic B-cells markedly amplifies insulin secretion through complex mechanisms which involve changes in membrane potential and ionic fluxes. In this study, normal mouse islets were used to evaluate the role of Cl- ions in these effects of acetylcholine (ACh). Whatever the concentration of glucose, the rate of 36Cl- efflux from islet cells was unaffected by ACh. Replacement of Cl- by impermeant isethionate in a medium containing 15 mM glucose did not affect, or only slightly decreased, the ability of ACh to depolarize the B-cell membrane and increase electrical activity, to accelerate 45Ca2+ and 86Rb+ efflux from islet cells, and to amplify insulin release. In the absence of extracellular Ca2+, a high concentration of ACh (100 microM) mobilized intracellular Ca2+ and caused a transient release of insulin and a sustained acceleration of 86Rb+ efflux. None of these effects was affected by Cl- omission or by addition of furosemide, a blocker of the Na+, K+, 2Cl- cotransport. Isethionate substitution for Cl- in a medium containing a nonstimulatory concentration of glucose (3 mM) barely reduced the depolarization of B-cells by ACh, but inhibited the concomitant increase in 86Rb+ efflux. We have no explanation for the latter effect that was not mimicked by furosemide. In conclusion, ACh stimulation of pancreatic B-cells, unlike that of exocrine acinar cells, is largely independent of Cl- and is insensitive to furosemide. The acceleration of ionic fluxes produced by ACh does not involve the Na+, K+, 2Cl- cotransport system.     

Effects of fluoride and vanadate on K+ transport across the erythrocyte membrane of Rana temporaria

K-Cl cotransport activity in frog erythrocytes was estimated as a Cl- -dependent component of K+ efflux from cells incubated in Cl- - or NO3- -containing medium at 20 degrees C. Decreasing the osmolality of the medium resulted in an increase in K+ efflux from the cells in a Cl- medium but not in an NO3- medium. Treatment of red cells with 5 mM NaF caused a significant decrease (approximately 50%) in K+ loss from the cells in iso- and hypotonic Cl- media but only a small decrease in K+ loss in isotonic NO3- medium. Addition of 1 mM vanadate to an isotonic Cl- medium also led to a significant reduction in K+ efflux. Similar inhibitory effects of NaF and vanadate on K+ efflux in a Cl- medium, but not in an NO3- medium were observed when the incubation temperature was decreased from 20 to 5 degrees C. Thus, under various experimental conditions, NaF and vanadate inhibited about 50% of Cl- -dependent K+ efflux from frog red cells probably due to inhibition of protein phosphatases. Cl- -dependent K+ (86Rb) influx into frog erythrocytes was nearly completely blocked (approximately 94%) by 5 mM NaF. In a NO3- medium, K+ influx was mainly mediated by the Na+,K+ pump and was unchanged in the presence of 5 mM NaF, 0.03 mM Al3+ or their combination. These data indicate that G proteins or cAMP are not involved in the regulation of Na+,K+ pump activity which is activated by catecholamines and phosphodiesterase blockers in these cells.     

The action of epinephrine on Madin-Darby canine kidney cells

We have used cultured monolayers of Madin-Darby canine kidney (MDCK) cells, which form epithelial layers of high transepithelial resistance, grown on Millipore filters, for transport studies. In the absence of hormones net ion transport is of small magnitude and is consistent with a net absorptive flow (apical to basal) of Na+. Epinephrine, effective only from the basolateral cell surface, stimulates a net secretion (basal to apical) of Cl-. A substantial portion of net Cl- secretion is inhibited by loop diuretics such as furosemide applied to the basolateral cell aspects. The participation of a diuretic-sensitive cotransport system for Na+, K+, and Cl-, similar to that found in other cells, in transepithelial Cl- flux is postulated. The action of catecholamines on MDCK cell adenylate cyclase and on a Ca2+-activated K+ conductance is described.     

Characterisation of a Na+/K+/Cl- cotransporter in alkylating agent-sensitive L1210 murine leukemia cells

The mode of influx of 86Rb+, a K+ congener, to exponentially proliferating L1210 murine leukemia cells, incubated in a Krebs-Ringer buffer, has been characterised. The influx was composed of a ouabain-sensitive fraction (approx. 40%), a loop diuretic-sensitive fraction (approx. 40%) and a fraction which was insensitive to both types of inhibitor (approx. 15%). The fraction of ouabain-insensitive 86Rb+ influx, which was fully inhibited by furosemide (1 mM) or bumetanide (100 microM), was completely inhibited when Cl- was completely substituted by nitrate or gluconate ions, but was slightly (29 +/- 12%) stimulated if the Cl- was substituted by Br-. The substitution of Na+ by Li+, choline or tetramethylammonium ions inhibited the loop diuretic-sensitive fraction of 86Rb+ uptake. These results suggested that a component of 86Rb+ influx to L1210 cells was mediated via a Na+/K+/Cl- cotransporter. 86Rb+ efflux from L1210 cells which had been equilibrated with 86Rb+ and incubated in the presence or absence of 1 mM ouabain, was insensitive to the loop diuretics. Additionally, efflux rates were found to be independent of the external concentration of K+, suggesting that efflux was not mediated by K+-K+ exchange. The initial rate of 86Rb+ influx to L1210 cells in the plateau phase of growth was reduced to 44% of that of exponentially dividing cells, the reduction being accounted for by significant decreases in both ouabain- and loop diuretic-sensitive influx; these cells were reduced in volume compared to cells in the exponential phase of cell growth. In cells which had been deprived of serum for 18 h, and which showed an increase of the proportion of cells in the G1 phase of the cell cycle, the addition of serum stimulated an immediate increase in the furosemide-sensitive component of 86Rb+ influx. Diuretic-sensitive 86Rb+ influx was not altered by the incubation of the cells with 100 microM dibutyryl cyclic AMP, but was inhibited by 10 microM of the cross-linking agent nitrogen mustard (bis(2-chloro-ethyl)methylamine, HN2).     

Volume-dependent regulation of ion carriers in human and rat erythrocytes: role of cytoskeleton and protein phosphorylation

This study examines the effect of heat-induced cytoskeleton transitions and phosphoprotein phosphatase inhibitors on the activity of shrinkage-induced Na+, K+, 2Cl- cotransport and Na+/H+ exchange in rat erythrocytes and swelling-induced K+, Cl- cotransport in human and rat blood cells. Preincubation of human and rat erythrocytes at 49 degrees C drastically activated K+, Cl- cotransport and completely (rat) or partly (human) abolished its volume-dependent regulation. The same procedure did not affect basal activity of Na+, K+, 2Cl- cotransport but completely abolished its activation by shrinkage thus suggesting the involvement of a thermosensitive element of cytoskeleton network in the volume-dependent regulation of cotransporters. Both the shrinkage- and electrochemical proton gradient-induced Na+/H+ exchange was inhibited by the heat treatment to the same extent (50-70%), thus indicating the different signaling pathways involved in the activation of Na+, K+, 2Cl- cotransport and Na+/H+ exchange by cell shrinkage. This suggestion is in accordance with data on the different kinetics of volume-dependent activation and inactivation of these carriers as well as on their sensitivity to medium osmolality. Both swelling- and heat-induced increments of K+, Cl- cotransport activity were diminished by inhibitors of phosphoprotein phosphatases (okadaic acid and calyculin). In rat erythrocytes these compounds potentiate shrinkage-induced Na+/H+ exchange. On the contrary, neither basal nor shrinkage-induced Na+, K+, 2Cl- cotransport was affected by these compounds. Our results indicate a key role of cytoskeleton network in volume-dependent activation of K+, Cl- and Na+, K+, 2Cl- cotransport and the involvement of protein phosphorylation-dephosphorylation cycle in regulation of the activity of K+, Cl- cotransport and Na+/H+ exchange.     

Regulatory interaction of ATP Na+ and Cl- in the turnover cycle of the NaK2Cl cotransporter

To probe the mechanism by which intracellular ATP, Na+, and Cl- influence the activity of the NaK2Cl cotransporter, we measured bumetanide-sensitive (BS) 86Rb fluxes in the osteosarcoma cell line UMR- 106-01. Under physiological gradients of Na+, K+, and Cl-, depleting cellular ATP by incubation with deoxyglucose and antimycin A (DOG/AA) for 20 min at 37 degrees C reduced BS 86Rb uptake from 6 to 1 nmol/mg protein per min. Similar incubation with 0.5 mM ouabain to inhibit the Na+ pump had no effect on the uptake, excluding the possibility that DOG/AA inhibited the uptake by modifying the cellular Na+ and K+ gradients. Loading the cells with Na+ and depleting them of K+ by a 2-3- h incubation with ouabain or DOG/AA increased the rate of BS 86Rb uptake to approximately 12 nmol/mg protein per min. The unidirectional BS 86Rb influx into control cells was approximately 10 times faster than the unidirectional BS 86Rb efflux. On the other hand, at steady state the unidirectional BS 86Rb influx and efflux in ouabain-treated cells were similar, suggesting that most of the BS 86Rb uptake into the ouabain-treated cells is due to K+/K+ exchange. The entire BS 86Rb uptake into ouabain-treated cells was insensitive to depletion of cellular ATP. However, the influx could be converted to ATP-sensitive influx by reducing cellular Cl- and/or Na+ in ouabain-treated cells to impose conditions for net uptake of the ions. The BS 86Rb uptake in ouabain-treated cells required the presence of Na+, K+, and Cl- in the extracellular medium. Thus, loading the cells with Na+ induced rapid 86Rb (K+) influx and efflux which, unlike net uptake, were insensitive to cellular ATP. Therefore, we suggest that ATP regulates a step in the turnover cycle of the cotransporter that is required for net but not K+/K+ exchange fluxes. Depleting control cells of Cl- increased BS 86Rb uptake from medium-containing physiological Na+ and K+ concentrations from 6 to approximately 15 nmol/mg protein per min. The uptake was blocked by depletion of cellular ATP with DOG/AA and required the presence of all three ions in the external medium. Thus, intracellular Cl- appears to influence net uptake by the cotransporter. Depletion of intracellular Na+ was as effective as depletion of Cl- in stimulating BS 86Rb uptake.(ABSTRACT TRUNCATED AT 400 WORDS)     

Na+, K+ and Cl- transport in isolated small intestinal cells from guinea pig. Evidences for the existence of a second Na+ pump

Isolated small intestinal epithelial cells, after incubation at 4 degrees C for 30 min, reach ion concentrations (36 mM K+, 113 mM Na+ and 110 mM Cl-) very similar to those of the incubation medium. Upon rewarming to 37 degrees C, cells are able to extrude Na+, Cl- and water and to gain K+. Na+ extrusion is performed by two active mechanisms. The first mechanism, transporting Na+ by exchanging it for K+, is inhibited by ouabain and is insensitive to ethacrynic acid. It is the classical Na+ pump. The second mechanism transports Na+ with Cl- and water, is insensitive to ouabain but is inhibited by ethacrynic acid. Both mechanisms are inhibited by dinitrophenol and anoxia. The second Na+ extruding mechanism could be the Na+/K+/2Cl- cotransport system. However, this possibility can be ruled out because the force driving cotransport would work inwards, and because Na+ extrusion with water loss continues after substitution of Cl- by NO3-. We propose that enterocytes have a second Na+ pump, similar to that proposed in proximal tubular cells.     

Role of separate K+ and Cl- channels and of Na+/Cl- cotransport in volume regulation in Ehrlich cells

Cells resuspended in hypotonic medium initially swell as nearly perfect osmometers, but later recover their volume with an associated KCl loss. This regulatory volume decrease (RVD) is unaffected when nitrate is substituted for Cl- or if bumetanide or 4,4'-diisothiocyanostilbene-2,2'-disulfonate (DIDS) is added. It is inhibited by quinine, Ba2+, low pH, anticalmodulin drugs, and depletion of intracellular Ca2+. It is accelerated by the Ca2+ ionophore A23187, or by a sudden increase in external Ca2+ and at high pH. A net KCl loss is also seen after addition of ionophore A23187 in isotonic medium. Similarities are demonstrated between the KCl loss seen after addition of A23187 and the KCl loss seen during RVD. It is proposed that separate conductive K+ and Cl- channels are activated during RVD by release of Ca2+ from internal stores, and that the effect is mediated by calmodulin. After restoration of tonicity the cells shrink initially, but recover their volume with an associated KCl uptake. This regulatory volume increase (RVI) is inhibited when NO3- is substituted for Cl-, and is also inhibited by furosemide or bumetanide, but it is unaffected by DIDS. The unidirectional Cl-flux ratio is compatible with either a coupled uptake of Na+ and Cl-, or an uptake via a K+/Na+/2Cl- cotransport system. No K+ uptake was found, however, in ouabain-poisoned cells where a bumetanide-sensitive uptake of Na+ and Cl- in nearly equimolar amounts was demonstrated. Therefore, it is proposed that the primary process during RVI is an activation of an otherwise quiescent Na+/Cl- cotransport system with subsequent replacement of Na+ by K+ via the Na+/K+ pump. There is a marked increase in the rate of pump activity in the absence of a detectable increase in intracellular Na+ concentration.     

The Na+/K+/Cl- cotransport in C6 glioma cells. Properties and role in volume regulation

The role of the Na+/K+/Cl- cotransporter in the regulation of the volume of C6 astrocytoma cells was analyzed using isotopic fluxes and cell cytometry measurements of the cell volume. The system was inhibited by 'loop diuretics' with the following order of potency: benzmetanide greater than bumetanide greater than piretanide greater than furosemide. Under physiological conditions of osmolarity of the incubation media, equal rates of bumetanide-sensitive inward and outward K+ fluxes were observed. Blockade of the Na+/K+/Cl- cotransporter with bumetanide did not lead to a modification in the mean cell volume. When C6 cells were incubated in an hyperosmotic solution, a cell shrinkage was observed. It was accompanied by a twofold increase in the activity of the Na+/K+/Cl- cotransport, which then catalyzed the net influx of K+. In spite of this increased activity, no cell swelling could be measured. Incubation of the cells in an iso-osmotic medium deprived of either Na+, K+ or Cl- also produced cell shrinkage. Large activations (up to tenfold) of the Na+/K+/Cl- cotransport together with a cell swelling back to the normal volume were observed upon returning ion-deprived C6 cells to a physiological solution. This cell swelling was completely prevented in the presence of bumetanide. It is concluded that the Na+/K+/Cl- cotransport system is one of the transport systems involved in volume regulation of glial cells. The system can either be physiologically quiescent or active depending on the conditions used. A distinct volume regulating mechanism is the Na+/H+ exchange system.     

Volume regulatory activity of the Ehrlich ascites tumor cell and its relationship to ion transport

The volume regulatory response of the Ehrlich ascites tumor was studied in KCl-depleted, Na+-enriched cells. Subsequent incubation in K+-containing NaCl medium results in the reaccumulation of K+, Cl-, water and the extrusion of Na+. The establishment of the physiological steady state is due primarily to the activity of 2 transport systems. One is the Na/K pump (KM for K+o = 3.5 mM; Jmax = 30.1 mEq/kg dry min), which in these experiments was coupled 1K+/1 Na+. The second is the Cl--dependent (Na+ + K+) cotransport system (KM for K+o = 6.8 mM; Jmax = 20.8 mEq/kg dry min) which mediates, in addition to net ion uptake in the ratio of 1K+:1Na+:2Cl-, the exchange of K+i for K+o. The net passive driving force on the cotransport system is initially inwardly directed but does not decrease to zero at the steady state. This raises the possibility of the involvement of an additional source of energy. Although cell volume increases concomitant with net ion uptake, this change does not appear to be a major factor regulating the activity of the cotransport system.     

Furosemide-sensitive salt transport in the Madin-Darby canine kidney cell line. Evidence for the cotransport of Na+, K+, and Cl-

Confluent monolayer cultures of the Madin-Darby canine kidney (MDCK) cell line have been shown to possess a furosemide and bumetanide-sensitive (Na+,K+)-cotransport system. We have studied the effect of anion substitutions on (Na+,K+)-cotransport. In Na+-depleted cells, bumetanide-sensitive uptake of 22Na+ or 86Rb+ exhibited an absolute requirement for extracellular Cl-. Chloride could be replaced in the buffers by Br-, but not by F-, I-, acetate, nitrate, thiocyanate, sulfate, or gluconate. The effect of Cl- was saturating, and Na+-stimulated 86RB+ uptake as well as K+-stimulated 22Na+ uptake was shown to be dependent on the square of the Cl- concentration. The concentration of Cl- which gave half-maximal stimulation of cation cotransport varied between 58 and 70 mM. There was a small degree of cooperativity between the binding affinities for Cl- and K+ at constant Na+ concentrations. Bumetanide-sensitive 36Cl- uptake could be demonstrated when extracellular Na+ and K+ were present simultaneously. Uptake through this system was unaffected by changes in the membrane potential or by the imposition of pH gradients. Together these data strongly suggest that the bumetanide-sensitive transport system in Madin-Darby canine kidney cells co-transports Na+, K+, and Cl- in a ratio of 1:1:2.     

Characterization of a BALB/c 3T3 preadipose cell mutant with altered Na+K+Cl- cotransport activity

A BALB/c 3T3 cell mutant (3T3-E12) was isolated by its ability to survive at a low extracellular K+ concentration (0.14 mM). The growth rate of mutant cells was less dependent on external K+ than parental cells. Analysis of potassium transport revealed that 3T3-E12 cells have a decreased activity of the furosemide-sensitive Na+K+Cl- cotransport system, both in the efflux and influx modes. This is shown to be a result of a decrease in the apparent affinity of the transport system for K+ and Na+, but not Cl-. Upon exposure to the phorbol ester 12-0-tetradecanoyl-phorbol-13-acetate (TPA), BALB/c 3T3 cells exhibited a maximal volume decrease of 20%, while mutant cells shrunk by only 7%, suggesting that regulation of cell volume, at least four exposure to a tumor promoter, is impaired in mutant cells compared to parental 3T3 cells.     

Volume-dependent and NEM-stimulated K+,Cl- transport is elevated in oxygenated SS, SC and CC human red cells

Mechanisms involved in cell volume regulation are important in SS, SC cells as they might be involved in determining the extent of sickling and the generation of dense cells and irreversibly sickled cells. We have studied in these cells the response to cell swelling of the K+,Cl- transporter. We found that Hb SS, SC and CC red cells have higher values of a ouabain-resistant, chloride-dependent and NEM-stimulated K+ efflux than AA red cells. In contrast, the Na+,K+,Cl- cotransport estimated from the bumetanide-sensitive component of K+ efflux was not significantly different in SS, SC and CC red cells. The (ouabain + bumetanide)-resistant K+ efflux from SS, SC and CC red cells was stimulated by cell swelling induced by reduction of the osmotic pressure (300 to 220 mosmol/l) and pH (8 to 7) of the flux media (140 mM NaCl). The Cl--dependent K+ efflux stimulated by osmotic swelling highly correlated with the NEM-stimulated component (r = 0.8, p less than 0.001, n = 22) and the acid-pH-induced swelling (r = 0.969, p less than 0.001, n = 22), indicating that it is driven by the K+,Cl- transporter.     

Regulatory changes in the K+, Cl- and water contents of HeLa cells incubated in an isosmotic high K(+)-medium

HeLa cells had their normal medium replaced by an isosmotic medium containing 80 mM K+, 70 mM Na+ and 100 microM ouabain. The cellular contents of K+ first increased and then decreased to the original values, that is, the cells showed a regulatory decrease (RVD) in size. The initial increase was not inhibited by various agents except by substitution of medium Cl- with gluconate. In contrast, the regulatory decrease was inhibited strongly by addition of either 1 mM quinine, 10 microM BAPTA-AM without medium Ca2+, or 0.5 mM DIDS, and partly by either 1 mM EGTA without medium Ca2+, 10 microM trifluoperazine, or substitution of medium Cl- with NO3-. Addition of DIDS to the NO3(-)-substituted medium further suppressed the K+ loss but the effect was incomplete. Intracellular Ca2+ showed a transient increase after the medium replacement. These results suggest that the initial increase in cell K+ is a phenomenon related to osmotic water movement toward Donnan equilibrium, whereas the regulatory K+ decrease is caused by K+ efflux through Ca(2+)-dependent K+ channels. The K+ decrease induced a decrease in cellular water, i.e., RVD. The K+ efflux may be more selectively associated with Cl- efflux through DIDS-sensitive channels than the efflux of other anions.     

A phorbol ester-nonproliferative variant of Swiss 3T3 cells is deficient in Na+K+Cl- cotransport activity

The identity of the genetic defect(s) in Swiss 3T3 TNR-2 and TNR-9 that confers nonresponsiveness to the proliferative effect of 12-0-tetradecanoylphorbol-13-acetate (TPA) is not known. In BALB/c 3T3 cells, loss (via mutation) of a specific membrane ion transport system, the furosemide-sensitive Na+K+Cl- cotransporter, is associated with decreased responsiveness to TPA. In this study, the transport properties of parental Swiss 3T3 cells and the TPA-nonresponsive lines TNR-2 and TNR-9 were determined in the presence and absence of TPA. When the rate of 86Rb+ efflux (as a tracer for K+) was measured from each of the three cell lines, a furosemide- and TPA-inhibitable component of efflux was clearly evident in parental and TNR-9 cells but was virtually absent in TNR-2 cells. 86Rb+ influx measurements indicated the presence in parental 3T3 cells and the TNR-9 line of a substantial furosemide-sensitive flux that could be inhibited by TPA. In contrast, much less furosemide-sensitive influx was present in 3T3-TNR-2 cells and it was relatively unaffected by TPA. In both parental 3T3 and 3T3-TNR-2 cells, most of the furosemide-sensitive 86Rb+ influx is dependent on extracellular Na+ and Cl-. The apparent affinities of the transporter for these two ions, as well as for K+, were similar in both cell lines. In parental cells, the inhibition of furosemide-sensitive 86Rb+ influx was quite sensitive to TPA (K1/2 approximately equal to 1 nM) and occurred very rapidly after phorbol ester exposure. As expected because of its volume-regulatory role, inhibition of Na+K+Cl- cotransport by TPA in parental cells caused a substantial reduction in cell volume (25%). In contrast, because of the reduced level of cotransport activity in TNR-2 cells, TPA had only a slight effect on cell volume. These results suggest that the genetic defect in 3T3-TNR-2 cells (but not TNR-9 cells) responsible for nonresponsiveness to phorbol esters may be the reduction of Na+K+Cl- cotransport activity. Thus this membrane transport system may be an important component of the signal transduction pathway used by phorbol esters in 3T3 cells.