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.     

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).     

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.     

Anion-dependent cation transport in erythrocytes

A selective survey of the literature reveals at least three major anion-dependent cation transport systems, defined as Na+ + Cl-, K+ + Cl- and Na+ + K+ + Cl- respectively. In human red cells, kinetic data on the fraction of K+ and Na+ influx inhibitable by bumetanide are presented to indicate an Na+:K+ stoichiometry of 1:2. For LK sheep red cells the large Cl- -dependent K+ leak induced by swelling is shown to share many characteristics with that induced by N-ethylmaleimide (NEM) treatment. NEM has complex effects, both inhibiting and then activating Cl- -dependent K+ fluxes dependent on NEM concentration. The alloantibody anti-L can prevent the action of NEM. In human red cells NEM induces a large Cl- -dependent specific K+ flux, which shows saturation kinetics. Its anion preference is Cl- greater than Br- greater than SCN- greater than I- greater than NO3- greater than MeSO4-. This transport pathway is not inhibited by oligomycin or SITS, although phloretin and high concentrations of furosemide and bumetanide (over 0.3 mM) do inhibit. Quinine (0.5 mM) is also an inhibitor. It is concluded that at least two distinct Cl- -dependent transport pathways for K+ are inducible in mammalian red cells, although the evidence for their separation is not absolute.     

Barium mimics the effect of D-glucose on 86Rb+ fluxes in mouse pancreatic beta-cells

The interaction between Ba2+, furosemide and D-glucose on 86Rb+ fluxes in ob/ob mouse islets was investigated. Ba2+ (2 mM) significantly reduced the ouabain-resistant 86Rb+ influx, without affecting the ouabain-sensitive influx. D-Glucose (20 mM) reduced the 86Rb+ influx in the absence of Ba2+ (2 mM) but not in the presence of the cation. Furosemide, an inhibitor of Na+, K+, Cl- co-transport, reduced the 86Rb+ influx and the effect was partly additive to the effect of 2 mM Ba2+. When the islets were preincubated with Ba2+ (2 mM) the specific effect of 1 mM furosemide on the 86Rb+ influx was reduced, whereas, in acute experiments, Ba2+ (2 mM) did not affect the specific effect of furosemide on 86Rb+ influx. 86Rb+ efflux from preloaded islets was significantly reduced by 2 mM Ba2+ and during the first 5 min of ion efflux the effect of the combination of 2 mM Ba2+ and 1 mM furosemide was stronger than the effect of Ba2+ alone. The data show that Ba2+ reduces 86Rb+ fluxes in the beta-cells and suggest that this is mainly mediated by inhibition of K+ channels in the beta-cell plasma membrane. Long-term exposure to Ba2+ may also reduce the activity of the Na+, K+, Cl- co-transport system. The effect of Ba2+ on K+ channels may help to explain the stimulatory effect on insulin release in the absence of nutrient secretagogues.     

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.     

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.     

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.     

Extracellular ATP increases cation fluxes in human erythrocytes by activation of the P2X7 receptor

Canine erythrocytes are known to undergo a reversible increase in cation permeability when incubated with extracellular ATP. We have examined the expression and function of P2X receptors on human erythrocytes using confocal microscopy and a panel of anti-P2X(1-7) antibodies and have measured monovalent cation fluxes in the presence of various nucleotide agonists. Human erythrocytes expressed P2X7 receptors on all cells examined from eight of eight subjects, as well as P2X2 at a far lower staining intensity in six of eight subjects. ATP stimulated the efflux of 86Rb+ (K+) from human erythrocytes in a dose-dependent fashion with an EC50 of approximately 95 microM. Other nucleotides also induced an efflux of 86Rb+ from erythrocytes with an order of agonist potency of 2'- and 3'-O(4-benzoylbenzoyl) ATP (BzATP) > ATP > 2-methylthio-ATP (2MeSATP) > adenosine 5'-O-(3-thiotriphosphate) (ATPgammaS), whereas ADP or UTP had no effect. ATP-induced efflux of 86Rb+ from erythrocytes was inhibited by extracellular Na+ and oxidized ATP, as well as by KN-62, an antagonist specific for the human P2X7 receptor. When erythrocytes were incubated in isotonic KCl medium, the addition of ATP stimulated an 86Rb+ influx approximately equal in magnitude to ATP-stimulated 86Rb+ efflux from the same cells. BzATP also stimulated the influx of 22Na+ into erythrocytes incubated in isotonic NaCl medium. Both ATP-induced efflux and influx of 86Rb+ and 22Na+ were impaired in erythrocytes from subjects who had inherited loss-of-function polymorphisms in the P2X7 receptor. These results suggest that the reversible permeabilization of erythrocytes by extracellular ATP is mediated by the P2X7 receptor.     

Na+/K+/Cl- cotransport in resealed ghosts from erythrocytes of the Milan hypertensive rats.

The erythrocytes (RBC) of the Milan hypertensive rats (MHS) have a smaller volume and faster Na+/K+/Cl- cotransport than RBC from normotensive controls (MNS). The difference in Na+/K+/Cl- cotransport is no longer present in inside-out Vesicles (IOV) of RBC membrane. To differentiate between cytoplasmic or membrane skeleton abnormalities as possible causes of these differences. Resealed ghosts (RG) were used to measure ion transport systems. The following results have been obtained: (1) RG from MHS have a smaller volume than MNS (mean +/- S.E. 20.7 +/- 0.45 vs. 22.09 +/- 0.42 fl, P < 0.05). (2) RG showed a bumetanide-sensitive Na efflux that retains the characteristics of the Na+/K+/Cl- cotransport of the original RBC: it is K(+)- and Cl(-)-sensitive and dependent on the intracellular Na+ concentration. (3) The Na+/K+/Cl- cotransport was faster in RG from MHS than in those from MNS (mean +/- S.E. 0.095 +/- 0.01 vs. 0.066 +/- 0.01 rate constant h-1, P < 0.01). These results, together with those of IOV, support the hypothesis that an abnormality in the membrane skeletal proteins may play a role in the different Na+/K+/Cl- cotransport modulation between MHS and MNS erythrocytes.     

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.     

Ca2+-activated Na+ fluxes in human red cells. Amiloride sensitivity

The effect of Ca2+ on the ouabain- and bumetanide-resistant Na+ fluxes in intact red cells was studied at relatively constant internal Ca2+, membrane potential, and cell volume. The red cell calcium concentration was modified using the ionophore A23187. In fresh red cells, the Na+ influx and efflux (1.2 +/- 0.13 and 0.26 +/- 0.07 mmol/liter cells x h, respectively) were not affected by amiloride (1 mM). When external Ca2+ was raised from 0 to 150 microM, in the presence of A23187, both the Na+ influx and efflux were stimulated (about 3.5-fold). The Ca2+-activated Na+ efflux and influx had an apparent Km for activation by Ca2+o of about 25 microM. The Ca2+-dependent Na+ transport was inhibited 30-60% by amiloride (ID50 = 17.3 +/- 8 microM). Amiloride, however, had no effect on the Ca2+-dependent K+ influx. The amiloride-sensitive (AS) transport pathway was a linear function of the Na+o concentration in the range from 0 to 75 mM. The Ca2+i activation seems to depend on the metabolic integrity of red cells. 1) It does not take place in ATP-depleted red cells; 2) ATP-repletion of ATP-depleted red cells fully restored AS Na influx; and 3) ATP-enrichment (ATP-red cells) enhanced the AS Na influx by about 100%. The Ca2+-activated AS Na+ influx was not affected by either DIDS or trifluoperazine. The present results indicate that in human erythrocytes an increase in internal Ca2+ activates on otherwise silent AS Na+-transport system, which is dependent on the metabolic integrity of the red cells.     

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.     

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.     

Taurine and cell volume maintenance in the shark rectal gland: cellular fluxes and kinetics

Tissue slices of shark rectal gland are studied to examine the kinetics of the cellular fluxes of taurine, a major intracellular osmolyte in this organ. Maintenance of high steady-state cell taurine (50 mM) is achieved by a ouabain-sensitive active Na+-dependent uptake process and a relatively slow efflux. Uptake kinetics are described by two saturable taurine transport components (high-affinity, Km 60 microM; and low-affinity, Km 9 mM). [14C]Taurine uptake is enhanced by external Cl-, inhibited by beta-alanine and unaffected by inhibitors of the Na+/K+/2Cl- co-transport system. Two cellular efflux components of taurine are documented. Incubation of slices in p-chloromercuribenzene sulfonate (1 mM) reduces taurine uptake, increases efflux of taurine and induces cell swelling. Studies of efflux in isotonic media with various cation and anion substitutions demonstrate that high-K+ markedly enhances taurine efflux irrespective of cell volume changes (i.e. membrane stretching is not involved). Moreover, iso-osmotic cell swelling induced in media containing propionate is not associated with enhanced efflux of taurine from the cells. It is suggested that external K+ exerts a specific effect on the cytoplasmic membrane to increase its permeability to taurine.     

Swelling-activated K+ efflux and regulatory volume decrease efficiency in human bronchial epithelial cells

This study describes the correlation between cell swelling-induced K+ efflux and volume regulation efficiency evaluated with agents known to modulate ion channel activity and/or intracellular signaling processes in a human bronchial epithelial cell line, 16HBE14o(-1). Cells on permeable filter supports, differentiated into polarized monolayers, were monitored continuously at room temperature for changes in cell height (T(c)), as an index of cell volume, whereas (86)Rb efflux was assessed for K+ channel activity. The sudden reduction in osmolality of both the apical and basolateral perfusates (from 290 to 170 mosmol/kg H(2)O) evoked a rapid increase in cell volume by 35%. Subsequently, the regulatory volume decrease (RVD) restored cell volume almost completely (to 94% of the isosmotic value). The basolateral (86)Rb efflux markedly increased during the hyposmotic shock, from 0.50 +/- 0.03 min(-1) to a peak value of 6.32 +/- 0.07 min(-1), while apical (86)Rb efflux was negligible. Channel blockers, such as GdCl(3) (0.5 mM), quinine (0.5 mM) and 5-nitro-2-(3-phenyl-propylamino) benzoic acid (NPPB, 100 microM), abolished the RVD. The protein tyrosine kinase inhibitors tyrphostin 23 (100 microM) and genistein (150 microM) attenuated the RVD. All agents decreased variably the hyposmosis-induced elevation in (86)Rb efflux, whereas NPPB induced a complete block, suggesting a link between basolateral K(+) and Cl(-1) efflux. Forskolin-mediated activation of adenylyl cyclase stimulated the RVD with a concomitant increase in basolateral (86)Rb efflux. These data suggest that the basolateral extrusion of K+ and Cl(-1) from 16HBE14o(-1) cells in response to cell swelling determines RVD efficiency.     

Ca-induced K transport in human red blood cell ghosts containing arsenazo III. Transmembrane interactions of Na, K, and Ca and the relationship to the functioning Na-K pump

Increasing free intracellular Ca (Cai) from less than 0.1 microM to 10 microM by means of A23187 activated Ca-stimulated K transport and inhibited the Na-K pump in resealed human red cell ghosts. These ghosts contained 2 mM ATP, which was maintained by a regenerating system, and arsenazo III to measure Cai. Ca-stimulated K transport was activated 50% at 2-3 microM free Cai and the Na-K pump was inhibited 50% by 5-10 microM free Cai. Free Cai from 1 to 8 microM stimulated K efflux before it inhibited the Na-K pump, dissociating the effect of Ca on the two systems. 3 microM trifluoperazine inhibited Ca-stimulated K efflux and 0.5 mM quinidine reduced Na-K pumping by 50%. In other studies, incubating fresh intact cells in solutions containing Ca and 0.5 microM A23187 caused the cells to lose K heterogeneously. Under the same conditions, increasing A23187 to 10 microM initiated a homogeneous loss of K. In ATP-deficient ghosts containing Cai equilibrated with A23187, K transport was activated at the same free Cai as in the ghosts containing 2 mM ATP. Neither Cao nor the presence of an inward Ca gradient altered the effect of free Cai on the permeability to K. In these ghosts, transmembrane interactions of Na and K influenced the rate of Ca-stimulated K efflux independent of Na- and K-induced changes in free Cai or sensitivity to Cai. At constant free Cai, increasing Ko from 0.1 to 3 mM stimulated K efflux, whereas further increasing Ko inhibited it. Increasing Nai at constant Ki and free Cai markedly decreased the rate of efflux at 2 mM Ko, but had no effect when Ko was greater than or equal to 20 mM. These transmembrane interactions indicate that the mechanism underlying Ca-stimulated K transport is mediated. Since these interactions from either side of the membrane are independent of free Cai, activation of the transport mechanism by Cai must be at a site that is independent of those responsible for the interaction of Na and K. In the presence of A23187, this activating site is half-maximally stimulated by approximately 2 microM free Ca and is not influenced by the concentration of ATP. The partial inhibition of Ca-stimulated K efflux by trifluoperazine in ghosts containing ATP suggests that calmodulin could be involved in the activation of K transport by Cai.(ABSTRACT TRUNCATED AT 400 WORDS)     

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)     

Glucose reduces both Rb+ influx and efflux in pancreatic islet cells

Microdissected, beta-cell-rich pancreatic islets from ob/ob mice were used in studies of 86Rb+ transport. D-Glucose (20 mM) induced a biphasic reduction in 86Rb+ efflux. The reduction stabilized within 10 min at 34% of the efflux rate at zero glucose. The initial 86Rb+ uptake (5 min) was dose-dependently reduced by ouabain with maximum inhibition at 1 mM. D-Glucose (20 mM) did not affect the ouabain-sensitive 86Rb+ influx but markedly reduced (48%) the ouabain-resistant isotope influx. The results suggest that D-glucose does not affect the Na+/K+ pump in pancreatic beta-cells and that the glucose-sensitive K+-transporting modalities (K+ channels) in the beta-cells can mediate both inward and outward K+ flux.     

Transport of univalent cations across the erythrocyte membrane of hypertensive rats of various ages

In the erythrocytes incubated at low temperature (3-6 degrees C), the uptake of Li+ in 6- and 16-week old spontaneously hypertensive rats (SHR) was significantly higher than in the normotensive rats (WKY) of the same age. During the incubation of cells at 37 degrees C no difference occurred in either ouabain-sensitive or ouabain-resistant fluxes of Rb+, Na+ and Li+ between the 16-week old SHR and the WKY. K+ efflux from the erythrocytes at 3 degrees C was consistently stimulated after addition to the incubation medium of 1 mmol/l Ca2+. The value of Ca2+-dependent K+-transport was significantly elevated in 16-week old SHR than in the WKY, but there was no difference in 6-week old rats. Propranolol-induced Ca2+-dependent K+ efflux from the cells at 22 degrees C was markedly higher in 6- and 16-week old SHR as compared with the WKY. The results provide a further evidence in favor of the hypothesis on the existence of a "membrane defect" in red blood cells in the SHR.