Na+/K+/Cl- cotransport is stimulated by a Ca(++)-calmodulin-mediated pathway in BALB/c 3T3 fibroblasts.

In the present study, we investigated the role of intracellular Ca++ in the stimulation of the Na+/K+/Cl- cotransport in synchronized BALB/c 3T3 cells. The Na+/K+/Cl- cotransport was stimulated by the growth factors EGF, TGF-alpha, IGF-1, and IGF-2, which do not activate protein kinase C, but do induce a transient increase in free cytoplasmic Ca++. In addition, direct activation of protein kinase C by the phorbol ester 12-O-tetradecanoyl-phorbol-13-acetate (TPA) did not affect the Na+/K+/Cl- cotransport activity of quiescent cells. The Na+/K+/Cl- cotransport was also stimulated by the above mitogens in cells pretreated with the phorbol ester TPA. This treatment led to a progressive decline in the activity of cellular protein kinase C. This result implies that cells deficient in protein kinase C may still support stimulation of the Na+/K+/Cl- cotransport. Taken as a whole, these findings suggest that the Na+/K+/Cl- cotransport is stimulated predominantly by a protein kinase C-independent mechanism in BALB/c 3T3 fibroblasts. Both the intracellular Ca++ antagonist 8-(N,N-diethylamino)octyl-3,4,5-trimethoxybenzoate (TMB-8) and two potent calmodulin antagonists, trifluoperazine (TFP) and chloropromazine (CP), blocked serum- and mitogen-stimulated Na+/K+/Cl- cotransport. These results suggest that the Na+/K+/Cl- cotransport is stimulated by an increase of intracellular Ca++ and subsequently by a Ca(++)-calmodulin-mediated pathway in the synchronized BALB/c 3T3 fibroblasts.     

Protein kinase C and membrane transport: divergent responses of Na+/K+/Cl- cotransport and sugar transport to exogenous diacylglycerol

Even though the phorbol ester 12-O-tetradecanoylphorbol 13-acetate (TPA) is known to bind to and activate protein kinase C (PKC), it is still not certain that all cellular responses to phorbol esters are necessarily mediated by PKC. In BALB/c 3T3 preadipose cells, TPA has previously been shown to rapidly inhibit Na+K+Cl- -cotransport activity, stimulate 2-deoxyglucose uptake and induce ornithine decarboxylase activity. The cell-permeable diacylglycerol sn-1,2-dioctanoylglycerol (DiC8) was used in order to distinguish between PKC-dependent and -independent responses of BALB/c 3T3 cells. DiC8 modulated 86Rb+ fluxes in BALB/c 3T3 cells in the same manner as TPA: furosemide-sensitive 86Rb+ influx and efflux was inhibited, while in cotransport-defective cells no effect was observed. In contrast, DiC8 did not stimulate 2-deoxyglucose uptake in either parental or cotransport-defective cell lines, even though TPA is a very effective inducer of this transport system in both cell types. Pretreatment of cells with DiC8 did not substantially alter the subsequent induction of 2-deoxyglucose uptake by TPA, although a slight but reproducible reduction in the magnitude of the response was observed in DiC8-pretreated cells. The PKC-dependent phosphorylation of an acidic 80-kDa protein was stimulated by both TPA and DiC8 in parental and cotransport-defective cell lines, suggesting that a gross defect in the primary effector system used by both TPA and diacylglycerols cannot explain any of our results. Ornithine decarboxylase was induced by DiC8 and the K1/2 was approximately the same as that for inhibition of Na+/K+/Cl- cotransport in these cells. Thus, our results suggest that PKC is clearly essential for some phorbol ester membrane transport responses (such as inhibition of Na+/K+/Cl- cotransport), but our results do not allow us to conclude that other responses (such as stimulation of 2-deoxyglucose uptake) necessarily require PKC activation.     

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.     

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.     

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 esters and gene expression: the role of rapid changes in K+ transport in the induction of ornithine decarboxylase by 12-O- tetradecanoylphorbol-13-acetate in BALB/c 3T3 cells and a mutant cell line defective in Na+K+Cl- cotransport

A BALB/c 3T3 preadipose cell line defective in Na+K+Cl- cotransport (3T3-E12a cells) has been used to study the relationship between phorbol ester-induced rapid changes in cation fluxes and changes in expression of a gene known to be modulated by this agent. In contrast to its effect on parental 3T3 cells, 12-O-tetradecanoylphorbol-13-acetate (TPA) did not inhibit either furosemide-sensitive 86Rb+ influx or the rate of 86Rb+ efflux from preloaded mutant cells. TPA-induced changes in intracellular K+ content were diminished in 3T3-E12a cells as compared with parental cells. Thus, mutation of the Na+K+Cl- cotransport system renders overall potassium transport in mutant cells largely insensitive to modulation by TPA. The morphological and functional responses of 3T3 and 3T3-E12a cells to TPA were also compared. In contrast to the extensive and long-lasting changes in morphology of 3T3 cells after 0.16 microM TPA addition, only slight and shorter-lived morphological effects of TPA were observed in 3T3-E12a cells. The transport properties of mutant cells were not totally unresponsive to TPA since hexose transport (2-deoxyglucose uptake) could be stimulated in both cell types. To establish a possible link between early changes in cation fluxes and activation of gene expression by TPA, the induction of the enzyme ornithine decarboxylase (ODC) was studied in detail. Addition of fresh medium containing serum or exposure to hypoosmotic conditions resulted in the induction of ODC in both 3T3 and 3T3-E12a cells. However, TPA failed to cause an increase in ODC activity in mutant cells, although a substantial induction of the enzyme was seen in parental cells. These results suggest that rapid changes in ion fluxes mediated by the Na+K+Cl- cotransport system are necessary for at least one of the phorbol ester-induced changes in gene expression in responsive cells.     

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.     

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 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.     

Stimulation of bumetanide-sensitive Na+/K+/Cl- cotransport by different mitogens in synchronized human skin fibroblasts is essential for cell proliferation

In this study, we examined the role of the bumetanide-sensitive Na+/K+/Cl- cotransport in the mitogenic signal of human skin fibroblast proliferation. The Na+/K+/Cl- cotransport was dramatically stimulated by either fetal calf serum, or by recombinant growth factors, added to quiescent G0/G1 human skin fibroblasts. The following mitogens, FGF, PDGF, alpha-thrombin, insulin-like growth factor-1, transforming growth factor-alpha, and the phorbol ester, 12-O-tetradecanoyl-phorbol-13-acetate, all stimulated the Na+/K+/Cl- cotransport. In addition, all the above mitogens induced DNA synthesis in the synchronized human fibroblasts. In order to explore the role of the Na+/K+/Cl- cotransport in the mitogenic signal, the effect of two specific inhibitors of the cotransport, furosemide and bumetanide, was tested on cell proliferation induced by the above recombinant growth factors. Bumetanide and furosemide inhibited synchronized cell proliferation as was measured by (a) cell exit from the G0/G1 phase measured by the use of flow cytometry, (b) cell entering the S-phase, determined by DNA synthesis, and (c) cell growth, measured by counting the cells. The inhibition by furosemide and bumetanide was reversible, removal of these compounds, completely released the cells from the block of DNA synthesis. In addition, the two drugs inhibited DNA synthesis only when added within the first 2-6 h of cell release. These results indicate that the effect of these drugs is specific, and is not due to an indirect toxic effect. This study clearly demonstrates that the growth factor-induced activation of the Na+/K+/Cl- cotransport plays a major role in the mitogenic signaling pathway of the human fibroblasts.     

Phorbol esters and mitogenesis: comparison of the proliferative response of parental and Na+K+Cl- -cotransport-defective BALB/c 3T3 cells to 12-0-tetradecanoylphorbol-13-acetate

The ability of the phorbol ester 12-0-tetradecanoylphorbol-13-acetate (TPA) to stimulate the growth of quiescent BALB/c 3T3 cell lines lacking Na+K+Cl- cotransport activity was tested. We have previously isolated and characterized two mutant cell lines defective in this important ion transport system by mutagenesis and selection in medium containing low K+. To test our hypothesis that loss of this transport activity might abrogate the proliferative response to TPA, two kinds of mitogenesis assays were performed. First, the effect of 0.16 microM TPA on the saturation density of parental vs. mutant cell lines was determined. TPA caused a small but reproducible 30-35% increase in the saturation density of mutant cells compared to the 100-120% increase seen in parental cell lines. Second, the effect of TPA on the incorporation of 3H-thymidine into cell nuclei (labeling index) was measured. While some variability from experiment to experiment in the extent and time course of the response of mutant cells was noted, TPA either had no effect or only a small effect on the labeling index when compared to the response of parental cells. When a range of concentrations of TPA (0.016-1.6 microM) was tested, neither cell line exhibited a large response to any concentration. These results suggest that loss of Na+K+Cl- cotransport activity decreases the response of these cells to the mitogenic action of TPA.     

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.     

Fibroblast growth factor induces a transient net K+ influx carried by the bumetanide-sensitive transporter in quiescent BALB/c 3T3 fibroblasts

The bumetanide-sensitive transport system performed a net efflux of K+ in serum deprived quiescent cells. The addition of partially purified fibroblast growth factor (FGF) to G0/G1 phase 3T3 fibroblasts induced a transient net influx of K+, carried out by the bumetanide-sensitive transport system for 2-6 minutes. The stimulation of the bumetanide-sensitive K+ influx by FGF was followed by stimulation of the ouabain-sensitive K+ influx. In addition, both the bumetanide-sensitive and the ouabain-sensitive K+ influxes were found to be similarly stimulated when the G0/G1 3T3 cells were treated with insulin. These results suggest that growth factors such as FGF and insulin induce a change in the action of the bumetanide-sensitive transporter from performing net K+ efflux along its concentration gradient to an uphill transport pumping of K+ into the cell. We propose, therefore, that the bumetanide-sensitive transporter contributes to the increase in the intracellular K+ (and probable Na+) stimulated by growth factors such as FGF and insulin in early G1 phase of the cell cycle.     

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.     

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 resting pancreatic acinar cells

To understand the role of Na+, K+, and Cl- transporters in fluid and electrolyte secretion by pancreatic acinar cells, we studied the relationship between them in resting and stimulated cells. Measurements of [Cl-]i in resting cells showed that in HCO3(-)-buffered medium [Cl- ]i and Cl- fluxes are dominated by the Cl-/HCO3- exchanger. In the absence of HCO3-, [Cl-]i is regulated by NaCl and NaK2Cl cotransport systems. Measurements of [Na+]i showed that the Na(+)-coupled Cl- transporters contributed to the regulation of [Na+]i, but the major Na+ influx pathway in resting pancreatic acinar cells is the Na+/H+ exchanger. 86Rb influx measurements revealed that > 95% of K+ influx is mediated by the Na+ pump and the NaK2Cl cotransporter. In resting cells, the two transporters appear to be coupled through [K+]i in that inhibition of either transporter had small effect on 86Rb uptake, but inhibition of both transporters largely prevented 86Rb uptake. Another form of coupling occurs between the Na+ influx transporters and the Na+ pump. Thus, inhibition of NaK2Cl cotransport increased Na+ influx by the Na+/H+ exchanger to fuel the Na+ pump. Similarly, inhibition of Na+/H+ exchange increased the activity of the NaK2Cl cotransporter. The combined measurements of [Na+]i and 86Rb influx indicate that the Na+/H+ exchanger contributes twice more than the NaK2Cl cotransporter and three times more than the NaCl cotransporter and a tetraethylammonium-sensitive channel to Na+ influx in resting cells. These findings were used to develop a model for the relationship between the transporters in resting pancreatic acinar cells.     

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).     

Agonist stimulation of Na+/K+/Cl- cotransport in rat glomerular mesangial cells. Evidence for protein kinase C-dependent and Ca2+/calmodulin-dependent pathways

Studies were performed to investigate regulatory pathways of loop diuretic-sensitive Na+/K+/Cl- cotransport in cultured rat glomerular mesangial cells. Angiotensin II, alpha-thrombin, and epidermal growth factor (EGF) all stimulated Na+/K+/Cl- cotransport in a concentration-dependent manner. Pertussis toxin pretreatment reduced the effects of angiotensin II and alpha-thrombin but not that of EGF. Addition of the protein kinase C inhibitor staurosporine or down-regulation of protein kinase C by prolonged incubation with phorbol 12-myristate 13-acetate partially reduced the effects of angiotensin II and alpha-thrombin and completely blunted the phorbol 12-myristate 13-acetate-induced stimulation of Na+/K+/Cl- cotransport but did not affect EGF-induced stimulation. Exposure of cells to a calcium ionophore, A23187, resulted in a concentration-dependent stimulation of Na+/K+/Cl- cotransport, which was not significantly inhibited by down-regulation of protein kinase C but was completely inhibited by the calmodulin antagonist, N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide (W-7). Stimulation of the cotransport by angiotensin II or alpha-thrombin was also partially inhibited by W-7. Inhibitory effects of protein kinase C down-regulation and W-7 were additive and, when combined, produced a complete inhibition of angiotensin II-induced stimulation of Na+/K+/Cl- cotransport. In saponin-permeabilized mesangial cells, phosphorylation of a synthetic decapeptide substrate for Ca2+/calmodulin-dependent kinase II, Pro-Leu-Ser-Arg-Thr-Leu-Ser-Val-Ser-Ser-NH3, was demonstrated. Maximal activation of the decapeptide substrate phosphorylation required the presence of Ca2+ and calmodulin and was dependent on Ca2+ concentration. These findings indicate that stimulation of Na+/K+/Cl- cotransport by angiotensin II and alpha-thrombin is mediated by protein kinase C and Ca2+/calmodulin-dependent kinases whereas the action of EGF is mediated by other pathways.     

Effect of Na + flux inhibitors on induction of c-fos, c-myc, and ODC genes during cell cycle

The role of Na + transport systems in the mitogenic signal induced by growth factors was studied, and it was shown that two Na + transport systems contribute to the early increase in cytoplasmic Na + in response to serum growth factors, namely the amiloride-sensitive Na+/H+ antiport and the bumetanide-sensitive Na+/K+/Cl- cotransport. Bumetanide or amiloride, when added separately, inhibited part of the increase in cytoplasmic Na +, as a response to the addition of serum to quiescent BALB/c mouse 3T3 fibroblasts. Each drug also suppressed part of the stimulation of the ouabain-sensitive Rb + influx, which was controlled by intracellular Na +. However, when both drugs were added together with serum growth factors, a complete inhibition of the early increase in [Na +], and subsequently a complete blockage of Na+/K+ pump stimulation was obtained. Amiloride or bumetanide, when added separately, only partially inhibited DNA synthesis induced by serum, 24% and 8% respectively. However, when both drugs were added together, at the time of serum addition to the quiescent cells, cell entry into S-phase was completely inhibited. To investigate the mode of cell-cycle inhibition, analysis was done of the possible role of early Na + fluxes in the mitogenic signal transduced from cell membrane receptors to the nucleus. The effects of the two drugs amiloride and bumetanide on induction of three genes--c-fos, c-myc, and ornithin decarboxylase (ODC)--was measured during cell transition through the G1-phase. Amiloride and bumetanide, when added separately or in combination, did not inhibit the induction of c-fos, c-myc, and ODC mRNAs. These results suggest that stimulation of Na + fluxes by serum growth factors is essential for cell transition into the S-phase of cell cycle, but it plays no apparent role in the growth factor signal transduced from the cell surface to the interior of the cell, as manifested by c-fos, c-myc, and ODC genes induction.     

Early events elicited by bombesin and structurally related peptides in quiescent Swiss 3T3 cells. II. Changes in Na+ and Ca2+ fluxes, Na+/K+ pump activity, and intracellular pH

The amphibian tetradecapeptide, bombesin, and structurally related peptides caused a marked increase in ouabain-sensitive 86Rb+ uptake (a measure of Na+/K+ pump activity) in quiescent Swiss 3T3 cells. This effect occurred within seconds after the addition of the peptide and appeared to be mediated by an increase in Na+ entry into the cells. The effect of bombesin on Na+ entry and Na+/K+ pump activity was concentration dependent with half-maximal stimulation occurring at 0.3-0.4 nM. The structurally related peptides litorin, gastrin-releasing peptide, and neuromedin B also stimulated ouabain-sensitive 86Rb+ uptake; the relative potencies of these peptides in stimulating the Na+/K+ pump were comparable to their potencies in increasing DNA synthesis (Zachary, I., and E. Rozengurt, 1985, Proc. Natl. Acad. Sci. USA., 82:7616-7620). Bombesin increased Na+ influx, at least in part, through an Na+/H+ antiport. The peptide augmented intracellular pH and this effect was abolished in the absence of extracellular Na+. In addition to monovalent ion transport, bombesin and the structurally related peptides rapidly increased the efflux of 45Ca2+ from quiescent Swiss 3T3 cells. This Ca2+ came from an intracellular pool and the efflux was associated with a 50% decrease in total intracellular Ca2+. The peptides also caused a rapid increase in cytosolic free calcium concentration. Prolonged pretreatment of Swiss 3T3 cells with phorbol dibutyrate, which causes a loss of protein kinase C activity (Rodriguez-Pena, A., and E. Rozengurt, 1984, Biochem. Biophys. Res. Commun., 120:1053-1059), greatly decreased the stimulation of 86Rb+ uptake and Na+ entry by bombesin implicating this phosphotransferase system in the mediation of part of these responses to bombesin. Since some activation of monovalent ion transport by bombesin was seen in phorbol dibutyrate-pretreated cells, it is likely that the peptide also stimulates monovalent ion transport by a second mechanism.