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.     

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.     

Biochemical characterization of the Na+/K+/Cl- co-transport in chick cardiac cells

Cultured chick cardiac cells possess a Na+K+Cl-co-transport system that is inhibited by the "loop diuretics" benzmetanide (IC50 = 0.3 microM), bumetanide (IC50 = 0.6 microM), piretanide (IC50 = 1.5 microM) and furosemide (IC50 = 5 microM). The K0.5 values for Cl- and Na+ activation of the bumetanide-sensitive 86Rb+ uptake are 59 mM and 40mM respectively. Bumetanide also inhibits a 22Na+ uptake component that is suppressed when external Cl- or K+ are substituted by impermeant ions. The ratio of bumetanide-sensitive 86Rb+ to 22Na+ uptake is close to 1. The cardiac Na+/K+/Cl- cotransport is a major uptake pathway for Na+ and K+. It accounts for 50% of the initial rate of 86Rb+ uptake and 17% of the initial rate of 22Na+ uptake by chick cardiac cells. It is activated two-fold by an hyperosmotic shock produced with 200 mM mannitol.     

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.     

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.     

Volume-regulating behavior of human platelets

Human platelets exposed to hypotonic media undergo an initial swelling followed by shrinking (regulatory volume decrease [RVD]). If the RVD is blocked, the degree of swelling is in accord with osmotic behavior. The cells could swell at least threefold without significant lysis. Two methods were used to follow the volume changes, electronic sizing and turbidimetry. Changes in shape produced only limited contribution to the measurements. The RVD was very rapid, essentially complete in 2 to 8 minutes, with a rate proportional to the degree of initial cell swelling. RVD involved a loss of KCl via volume-activated conductive permeability pathways for K+ and anions, presumably Cl-. In media containing greater than 50 mM KCl, the shrinking was inhibited and with higher concentrations was reversed (secondary swelling), suggesting that it is driven by the net gradient of K+ plus Cl-. The K+ pathway was specific for Rb+ and K+ compared to Li+ and Na+. The Cl- pathway accepted NO-3 and SCN- but not citrate or SO4(2-). In isotonic medium, the permeability of platelets to Cl- appeared to be low compared to that of K+. After hypotonic swelling both permeabilities were increased, but the Cl- permeability exceeded that of K+. The Cl- conductive pathway remained open as long as the cells were swollen. RVD was incomplete unless amiloride, an inhibitor of Na+/H+ exchange, was present or unless Na+ was replaced by an impermeant cation. In addition, acidification of the cytoplasm occurred upon cell swelling. This reduction in pHi appeared to activate Na+/H+ exchange, with a resultant uptake of Na+ and reduction in the rate and amount of shrinking. Like other cells, platelets responded to hypertonic shrinking with activation of Na+/H+ exchange, but regulatory volume increase was not detectable.     

Bumetanide-sensitive potassium transport and volume regulation in turkey erythrocytes

The bumetanide-sensitive (K+ + Na+ + 2Cl-)-cotransport system in turkey erythrocytes is activated by either of two treatments: addition of epinephrine or an increase in osmolarity. At elevated (20 mM) K+ concentration, cotransport activity induced by epinephrine slowly (within 90 min) declines to background level again. This time-dependent inactivation has been linked to bumetanide-sensitive cell swelling. We have compared both the initial rate of cotransport activity and its time dependence after induction by either epinephrine, increased osmolarity or a combination of the two treatments. As a measure of cotransport activity we took the bumetanide-sensitive fraction of 86Rb+ influx. Immediately after activation, several kinetic characteristics of this flux (Vmax; Km towards K+; Ki towards bumetanide; pH profile) were identical in cells activated by either treatment. By contrast, cotransport activated by hypertonicity was significantly more resistant towards subsequent inactivation. We show this to be due to the increase in intracellular ion concentrations brought about by hypertonic cell shrinkage. This tended to reverse the driving force for cotransport, and thereby prevented the bumetanide-sensitive swelling associated with inactivation. Our data support the notion that cell volume plays a key role both in the activation and in the time-dependent inactivation of bumetanide-sensitive transport.     

Growth factors activate the bumetanide-sensitive Na+/K+/Cl-cotransport in hamster fibroblasts

alpha-Thrombin, a potent mitogen for the hamster fibroblast cell line CCL 39, stimulates by approximately 3-fold 86Rb+ uptake in a mutant lacking the Na+/H+ antiport activity (PS 120). The major component of this stimulated 86Rb+ (K+) uptake is a bumetanide-sensitive flux (IC50 = 0.4 microM), which accounts for 50% of total K+ uptake in Go-arrested cells and is approximately 4-fold stimulated by maximal thrombin concentrations (EC50 = 5 X 10(-4) units/ml). This bumetanide-sensitive 86Rb+ uptake represents a Na+/K+/Cl- cotransport, as indicated by its dependence on extracellular Na+ and Cl- and by the existence in PS 120 cells of a bumetanide-sensitive K+-dependent 22Na+ uptake. The stimulation reaches its maximum within 2 min, is reduced at acidic intracellular pH values (half-maximal at pHi = 6.8), and can also be induced, to a lesser extent, by EGF and the phorbol ester 12-O-tetradecanoylphorbol 13-acetate, the effects of which are nearly additive. In contrast, preincubation with 12-O-tetradecanoylphorbol 13-acetate results in inhibition of thrombin- and EGF-induced stimulations, suggesting that activated protein kinase C might exert a feedback inhibitory control. This study clearly demonstrates that the growth factor-induced activation of the Na+/K+/Cl- cotransport is separated from the activation of the Na+/H+ antiport. However, activation of this cotransporter does not seem to play a major role in the mitogenic signaling pathway since its complete inhibition with bumetanide reduces only by 25-30% reinitiation of DNA synthesis.     

Hypotonic shock activated Cl- and K+ pathways in human fibroblasts.

The exposure of human fibroblasts to hypotonic medium (200 mosmolal) evoked the activation of both 36Cl- influx and efflux, which were insensitive to inhibitors of the anion exchanger and of the anion/cation cotransport, and conversely were inhibited by the Cl(-)-channel blocker 5-nitro-2-(3-phenylpropylamino)benzoic acid (NPPB). 36Cl- efflux was linked to a parallel efflux of 86Rb+; thus conductive K+ and Cl- pathways are activated during volume regulation in human fibroblasts. This conclusion is supported by evidence that, in hypotonic medium, 36Cl- influx and 86Rb+ efflux were both enhanced by depolarization of the plasma membrane. Depletion of the intracellular K+ content, obtained by preincubation with the ionophore gramicidin in Na(+)-free medium, had no effect on Cl- efflux in hypotonic medium. This result has been interpreted as evidence for independent activation of K+ and Cl- pathways. It is also concluded that the anion permeability is the rate-limiting factor in the response of human fibroblasts to hypotonic stress.     

Characterization of a Na(+)-K(+)-2Cl- cotransport system in oocytes from Xenopus laevis

In order to characterize the transport systems mediating K+ uptake into oocytes, flux studies employing 86Rb were performed on Xenopus oocytes stripped of follicular cells by pretreatment with Ca2(+)-Mg2(+)-free Barth's medium. Total Rb+ uptake consisted of an ouabain-sensitive and an ouabain-insensitive flux. In the presence of 100 mmol/l NaCl and 0.1 mmol/l ouabain the ouabain-insensitive flux amounted to 754.7 +/- 59.9 pmol/oocyte per h (n = 30 cells, i.e., 10 cells each from three different animals). In the absence of Na+ (Na+ substituted by N-methylglucamine) or when Cl- was replaced by NO3- the ouabain-insensitive flux was reduced to 84.4 +/- 42.9 and 79.2 +/- 12.1 pmol/oocyte per h, respectively (n = 50 cells). Furthermore, this Na(+)- and Cl(-)-dependent flux was completely inhibited by 10(-4) mol/l bumetanide, a specific inhibitor of the Na(+)-K(+)-2Cl- cotransport system. These results suggest that K+ uptake via a bumetanide-sensitive Na(+)-K(+)-2Cl- cotransport system represents a major K+ pathway in oocytes.     

Activation of NKCC1 by hyperosmotic stress in human tracheal epithelial cells involves PKC-delta and ERK

Hyperosmotic stress activates Na+-K+-2Cl- cotransport (NKCC1) in secretory epithelia of the airways. NKCC1 activation was studied as uptake of 36Cl or 86Rb in human tracheal epithelial cells (HTEC). Application of hypertonic sucrose or NaCl increased bumetanide-sensitive ion uptake but did not affect Na+/H+ and Cl-/OH-(HCO3-) exchange carriers. Hyperosmolarity decreased intracellular volume (Vi) after 10 min from 7.8 to 5.4 microl/mg protein and increased intracellular Cl- (Cl-i) from 353 to 532 nmol/mg protein. Treatment with an alpha-adrenergic agent rapidly increased Cl-i and Vi in a bumetanide-sensitive manner, indicating uptake of ions by NKCC1 followed by osmotically obligated water. These results indicate that HTEC act as osmometers but lose intracellular water slowly. Hyperosmotic stress also increased the activity of PKC-delta and of the extracellular signal-regulated kinase ERK subgroup of the MAPK family. Activity of stress-activated protein kinase JNK was not affected by hyperosmolarity. PD-98059, an inhibitor of the ERK cascade, reduced ERK activity and bumetanide-sensitive 36Cl uptake. PKC inhibitors blocked activation of ERK indicating that PKC may be a downstream activator of ERK. The results indicate that hyperosmotic stress activates NKCC1 and this activation is regulated by PKC-delta and ERK.     

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)     

Volume Regulation of Nerve Terminals

Pinched-off presynaptic nerve terminals (synaptosomes) possess significant regulatory volume increase (RVI) and regulatory volume decrease (RVD) capabilities. Following a swelling induced by a hypotonic challenge, the synaptosomes regulate their volume and adjust it, in 2 min, to within 5% of its initial value (RVD) at an initial rate of -0.77 +/- 0.10%/s (mean +/- SEM). Following a shrinking induced by a hypertonic challenge, the synaptosomes also regulate their volume at an initial rate of 0.18 +/- 0.02%/s (RVI), resulting in a new steady state, reached within 5-10 min, with a synaptosomal volume below the original volume. The omission of Na+ or K+ ions from the extrasynaptosomal medium reduces the initial rate of RVI by 72.5 and 66.5%, respectively. The "loop diuretics" bumetanide and furosemide significantly inhibited the RVI of the synaptosomes. In contrast, ouabain, amiloride, or 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid did not have any significant effect on RVI parameters. Furthermore, bumetanide-sensitive 86Rb uptake by rat brain synaptosomes was stimulated threefold by a hypertonic perturbation of 30%. Thus we conclude that the RVI of synaptosomes is mainly due to a stimulation of the Na+, K+, Cl- co-transport system induced by the synaptosomal shrinking following the hypertonic challenge.     

Volume-induced increase of anion permeability in human lymphocytes

Peripheral blood mononuclear cells (PBM) readjust their volumes after swelling in hypotonic media. This regulatory volume decrease (RVD) is associated with a loss of cellular K+ and is thought to be promoted by an increased permeability to this ion. In contrast, no change in volume was observed when K+ permeability of PBM in isotonic media was increased to comparable or higher levels using valinomycin. Moreover, valinomycin-induced 86Rb+ loss in K+-free medium was considerably slower than in K+-rich medium. These results suggest that anion conductance limits net salt loss in isotonic media. Direct measurements of relative conductance confirmed that in volume-static cells, anion conductance is lower than that of K+. In volume-regulating cells depolarization occurred presumably as a result of increased anion conductance. Accordingly, the efflux of 36Cl from PBM was markedly increased by hypotonic stress. Since both membrane potential and intracellular 36Cl concentration are reduced in hypotonically swollen cells, the increased efflux is probably due to a change in Cl- permeability. Anions and cations seem to move independently through the volume-induced pathways: the initial rate of 86Rb uptake in swollen cells was not affected by replacement of external Cl- by SO=4; conversely, 36Cl fluxes were unaffected by substitution of K+ by Na+. The data indicate that anion conductance is rate-determining in salt and water loss from PBM. An increase in anion conductance is suggested to be the critical step of RVD of human PBM.     

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 Na+,K+,2Cl- -cotransport system in HeLa cells and HeLa cell mutants exhibiting an altered efflux pathway

We have investigated the characteristics of a transport system in HeLa cells, which turned out to be very similar to a previously described Na+, K+, 2Cl- -cotransport system. For further understanding about the physiological role of the cotransporter, we have mutagenized HeLa cells and selected progeny cells for growth in low potassium (0.2 mM) medium. The selected HeLa cells (LK1) exhibited alterations in the Na+,K+,2Cl- -cotransport system. LK1 cells showed a remarkable reduction of 86Rb+ efflux via the cotransporter when compared to the parental HeLa cells. In contrast, bumetanide-sensitive potassium influx, measured by 86Rb+ uptake, was increased in the LK1 cells (increase in Vmax). Km values of the cotransporter in HeLa cells and LK1 mutants revealed similar properties for 86Rb+ and 22Na+ uptake. In addition, (3H)-bumetanide binding studies were carried out on intact HeLa cells; 1.7 pmol/mg protein (3H)-bumetanide was specifically bound to HeLa parental cells, which could be calculated to a number of 103,000 binding sites/cell. LK1 cells present, 1.44 pmol/mg protein, specifically bound (3H)-bumetanide and, respectively, 137,000 binding sites/cell. The LK1 cells also exhibited an increase in the number of (3H)-ouabain binding sites as well as an increase in the activity of the Na+,K+-ATPase, expressed as a function of ouabain-sensitive 86Rb+ uptake. Furthermore, LK1 cells were different in the concentrations of intracellular Na+ (increases) and K+ (decreases) when compared to the HeLa parental cells. When grown in low K+ medium (0.2 mM K+), protein content and cell volume were increased in the LK1 cells, while the DNA content was not significantly different between both cell lines.     

Regulatory volume decrease in the presence of HCO3- by single osteosarcoma cells UMR-106-01.

The technique for the simultaneous recording of cell volume changes and pHi in single cells was used to study the role of HCO3- in regulatory volume decrease (RVD) by the osteosarcoma cells UMR-106-01. In the presence of HCO3-, steady state pHi is regulated by Na+/H+ exchange, Na+ (HCO3-)3 cotransport and Na(+)-independent Cl-/HCO3- exchange. Following swelling in hypotonic medium, pHi was reduced from 7.16 +/- 0.02 to 6.48 +/- 0.02 within 3.4 +/- 0.28 min. During this period of time, the cells performed RVD until cell volume was decreased by 31 +/- 5% beyond that of control cells (RVD overshoot). Subsequently, while the cells were still in hypotonic medium, pHi slowly increased from 6.48 +/- 0.02 to 6.75 +/- 0.02. This increase in pHi coincided with an increase in cell volume back to normal (recovery from RVD overshoot or hypotonic regulatory volume increase (RVI)). The same profound changes in cell volume and pHi after cell swelling were observed in the complete absence of Cl- or Na+, providing HCO3- was present. On the other hand, depolarizing the cells by increasing external K+ or by inhibition of K+ channels with quinidine, Ba2+ or tetraethylammonium prevented the changes in pHi and RVD. These findings suggest that in the presence of HCO3-, RVD in UMR-106-01 cells is largely mediated by the conductive efflux of K+ and HCO3-. Removal of external Na+ but not Cl- prevented the hypotonic RVI that occurred after the overshoot in RVD. Amiloride had no effect, whereas pretreatment with 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS) strongly inhibited hypotonic RVI. Thus, hypotonic RVI is mediated by a Na+(out)-dependent, Cl(-)-independent and DIDS-inhibitable mechanism, which is indicative of a Na+(HCO3-)3 cotransporter. This is the first evidence for the involvement of this transporter in cell volume regulation. The present results also stress the power of the new technique used in delineating complicated cell volume regulatory mechanisms in attached single cells.     

Characterization of Na+/K+/Cl- cotransport in cultured HT29 human colonic adenocarcinoma cells

A Na+/K+/Cl- cotransport pathway has been examined in the HT29 human colonic adenocarcinoma cell line using 86Rb as the K congener. Ouabain-resistant bumetanide-sensitive (OR-BS) K+ influx in attached HT29 cells was 17.9 +/- 0.9 nmol/min per mg protein at 25 degrees C. The identity of this pathway as a Na+/K+/Cl- cotransporter has been deduced from the following findings: (a) OR-BS K+ influx ceased if the external Cl- (Cl-o) was replaced by NO3- or the external Na+ (Na+o) by choline; (b) neither OR-BS 24Na+ nor 36Cl- influx was detectable in the absence of external K+ (K+o); and (c) concomitant measurements of 86Rb+, 22Na+, and 36Cl- influx indicated that the stoichiometry of the cotransport system approached a ratio of 1N+:1K+:2Cl-. In addition, OR-BS K+ influx was exquisitely sensitive to cellular ATP levels. Depletion of the normal ATP content of 35-40 nmol/mg protein to 10-15 nmol/mg protein, a concentration at which the ouabain-sensitive K+ influx was unaffected, completely abolished K+ cotransport. OR-BS K+ influx was slightly reduced by the divalent cations Ca2+, Ba2+, Mg2+ and Mn2+. Although changes in cell volume, whether shrinking or swelling, did not influence OR-BS K+ influx, ouabain-sensitive K+ influx was activated by cell swelling. As in T84 cells, we found that the OR-BS K+ influx in HT29 cells was stimulated by exogenous cyclic AMP analogues and by augmented cyclic AMP content in response to vasoactive intestinal peptide, forskolin, norepinephrine and forskolin or prostaglandin E1.     

Phorbol ester TPA inhibits the stimulation of bumetanide-sensitive Na+/K+/Cl- transporter by different mitogens in quiescent BALB/c 3T3 mouse fibroblasts

In this study we examined the effect of the phorbol ester 12-O-tetradecanoyl-phorbol-13-acetate (TPA) on the bumetanide-sensitive Na+/K+/Cl- transporter in quiescent BALB/c 3T3 cells. We have shown that exposure of quiescent BALB/c 3T3 cultures to phorbol ester did not inhibit the basal bumetanide-sensitive Rb+ influx or efflux. In fact, at high concentration (100 ng/ml), TPA slightly stimulated the bumetanide-sensitive Rb+ influx and efflux. However, when the quiescent cultures were stimulated by serum or by defined growth factors, the stimulated fraction of the bumetanide-sensitive Rb+ influx was drastically inhibited by exposure of the cells to the phorbol ester TPA. Based on the above findings, we propose that activation of protein kinase C by the phorbol ester TPA does not inhibit the Na+/K+/Cl- cotransport activity; however it does suppress only the growth-factors-stimulated fraction of the cotransport in quiescent BALB/c 3T3 cells. These data propose that activation of kinase C has a regulatory feedback effect on the stimulation of the Na+/K+/Cl- cotransport activity by growth factors.     

Role of the Na+/K+/Cl- transporter in the positive inotropic effect of ouabain in cardiac myocytes

In this study we have characterized the bumetanide-sensitive K+/Na+/Cl- cotransport in cultured rat cardiac myocytes. 1) It carries about 10% of the total K+ influx. 2) It is sensitive to furosemide (Ki0.5 = 10(-6)M) and bumetanide (Ki0.5 = 10(-7)M). 3) It is strongly dependent on the extracellular concentrations of Na+ and Cl-. 4) It carries out influx of both ions, K+ and Na+. A therapeutic concentration of ouabain (10(-7) M) stimulated the bumetanide-sensitive K+ influx (as measured by 86Rb+), in the cultured myocytes, with no effect on the bumetanide-resistant K+ influx, which was mediated mostly by the Na+/K+ pump. Stimulation of the bumetanide-sensitive Rb+ influx by a low ouabain concentration was strongly dependent on Na+ and Cl- in the extracellular medium. A low concentration of ouabain (10(-7) M) was found to increase the steady-state level of cytosolic Na+ by 15%. This increase was abolished by the addition of bumetanide or furosemide. These findings suggest that ouabain, at a low (10(-7) M) concentration, induced its positive inotropic effect in rat cardiac myocytes by increasing Na+ influx into the cells through the bumetanide-sensitive Na+/K+/Cl- cotransporter. In order to examine this hypothesis, we measured the effect of bumetanide on the increased amplitude of systolic cell motion induced by ouabain. Bumetanide or furosemide, added to cultured cardiac myocytes, inhibited the increased amplitude of systolic cell motion induced by ouabain. Neither bumetanide nor furosemide alone has any significant effect on the basal amplitude of systolic cell motion. We propose that stimulation of bumetanide-sensitive Na+ influx plays an essential role in the positive inotropic effect in rat cardiac myocytes induced by low concentration of ouabain.