Characteristics of K+ uptake in S. enteritidis bacteria

Bacterial Salmonella enteritidis var. Issatchenko in media without exogenic energy source uptakes K+ in one step with Km 2.1 mM and Vmax 0.08 mM min-1/10(12) cells. This K+ uptake does not depend on pH and osmotic shock and is not inhibited by DCC. Endogenic energy source (glucose) leads to K+ uptake with Km 2.8 mM and Vmax 0.10 mM min-1/10(12) cells, and secretion of H+. The ratio of the DCC-sensitive fluxes of H+ to K+ equals 2. Arsenate and protonophores depress the K+ uptake. Valinomycin decreases the rate of K+ uptake. It is assumed that K+ uptake takes place via the Trk-like system, which works as a separate system as supercomplex with the H+-ATPase complex.     

The character of K+ uptake in anaerobically grown S. typhimurium

Tre character of K+ uptake in anaerobically grown S. typhimurium LT-2 is studied. In the alkaline media with glucose and moderate K+ activity these bacteria uptake K+ in two steps, the first of which has a high rate of K+ uptake, Km 2.1 mM and Vmax 0.44 mM/g. min and is sensitive to the medium osmolarity. Bacteria transfer from the media with high osmolarity to that with low one leads to a decrease of K+ uptake at the first step. The second increase of the medium osmolarity turns on the rapid K+ uptake only at alkaline pH. K+ uptake at the first step is inhibited by DCC and protonophores. In the absence of phosphate in the medium arsenate blocks K+ uptake at the first step, and when phosphate is available arsenate decreases K+ uptake. Valinomycin decreases the rate of K+ uptake. K+ uptake at the first step in S. typhimurium proceeds via Trk-like system which requires for K+ uptake both ATP and delta mu H+.     

Distinction of two components of passive Ca2+ transport into human erythrocytes by Ca2+ entry blockers

The nature of downhill Ca2+ net-transport into human erythrocytes was investigated using the experimental models of Ca2+ pump inhibition by vanadate and of intracellular chelation of Ca2+ by quin2. Ca2+ uptake by erythrocytes loaded with 0.5 mM vanadate and suspended in 145 mM Na+ -5 mM K+ media was reduced by about 60% when medium K+ was raised to 80 mM. Organic and inorganic Ca2+ entry blockers such as nifedipine (10(-5) M), verapamil (10(-4) M), diltiazem (10(-4) M), Co2+ (1.5 mM) and Cu2+ (0.1 mM) as well as the K+ channel blocker quinidine (1mM) inhibited Ca2+ uptake in 145 mM Na+ -5 mM K+ media by 60-75%. Flunarizine was less effective. In vanadate-loaded cells suspended in 70 mM Na+ -80 mM K+ media, in contrast, flunarizine exerted a dose-dependent inhibition of Ca2+ uptake by up to 80% at 10(-5) M, the other blockers being ineffective (except for verapamil at 10(-4) M). A similar pattern of inhibition was seen in quin2-loaded erythrocytes. The different susceptibility towards inhibitors may indicate that passive Ca2+ uptake by vanadate-loaded erythrocytes suspended in 145 mM Na+ -5 mM K+ media, on the one hand, and by vanadate-loaded erythrocytes suspended in 70 mM Na+ -80 mM K+ media as well as by quin2-loaded erythrocytes, on the other hand, is mediated by two different transport components.     

Copper ion redox state is critical for its effects on ion transport pathways and methaemoglobin formation in trout erythrocytes.

We have studied the mechanism of copper uptake by the cells, its oxidative action and effects on ion transport systems using rainbow trout erythrocytes. Cupric ions enter trout erythrocytes as negatively charged complexes with chloride and hydroxyl anions via the band 3-mediated Cl-/HCO3- exchanger. Replacement of Cl- by gluconate, and complexation of cupric ions with histidine abolish rapid Cu2+ uptake. Within the cell cupric ions interact with haemoglobin, causing methaemoglobin formation by direct electron transfer from heme Fe2+ to Cu2+, and consecutive proton release. Ascorbate-mediated reduction of cupric ions to cuprous decreases copper-induced metHb formation and proton release. Moreover, cuprous ions stimulate Na+H+ exchange and residual Na+ transport causing net Na+ accumulation in the cells. The effect requires copper binding to an externally facing thiol group. Copper-induced Na+ accumulation is accompanied by K+ loss occurring mainly via K+-Cl- cotransporter. Taurine efflux is also stimulated by copper exposure. However, net loss of osmolytes is not as pronounced as Na+ uptake and modest swelling of the cells occurs after 5 min of copper exposure. Taken together the results indicate that copper toxicity, including copper transport into the cells and its interactions with ion transport processes, depend on the valency and complex formation of copper ions.     

Copper uptake in wild type and copper metallothionein-deficient Saccharomyces cerevisiae. Kinetics and mechanism

The mechanism of copper uptake in Saccharomyces cerevisiae has been investigated using a combination of 64Cu2+ and atomic absorption spectrophotometry. A wild type copper-resistant CUP 1R-containing strain and a strain carrying a deletion of the CUP1 locus (yeast copper metallothionein) exhibited quantitatively similar saturable energy-dependent 64Cu2+ uptake when cultures were pregrown in copper-free media (medium [Cu] approximately 15 nM). The kinetic constants for uptake by the wild type strain were Vmax = 0.21 nmol of copper/min/mg of protein and Km = 4.4 microM. This accumulation of 64Cu2+ represented net uptake as confirmed by atomic absorption spectrophotometry. This uptake was not seen in glucose-starved cells, but was supported in glycerol- and ethanol-grown ones. Uptake was inhibited by both N3- and dinitrophenol and was barely detectable in cultures at 4 degrees C. When present at 50 microM, Zn2+ and Ni2+ inhibited by 50% indicating that this uptake process was relatively selective for Cu2+. 64Cu2+ accumulation was qualitatively and quantitatively different in cultures either grown in or preincubated with cold Cu2+. Either treatment resulted in the appearance of a fast phase (t 1/2 approximately 1 min) of 64Cu2+ accumulation which represented isotopic exchange since it did not lead to an increase in the mass of cell-associated copper; also, it was not energy-dependent. Exchange of 64Cu2+ into this pool was not inhibited by Zn2+. Pretreatment with Cu2+ caused a change in the rate of net accumulation as well; a 3-h incubation of cells in 5 microM medium Cu2+ caused a 1.6-fold increase in the velocity of energy-dependent uptake. Prior addition of cycloheximide abolished this Cu2(+)-dependent increase and, in fact, inhibited the 64Cu2+ uptake velocity by greater than 85%. The exchangeable pool was also absent in cycloheximide, Cu2(+)-treated cells suggesting that exchangeable Cu2+ derived from the copper taken up initially by the energy-dependent process. The thionein deletion mutant was similar to wild type in response to medium Cu2+ and cycloheximide indicating that copper metallothionein is not directly involved in Cu2+ uptake (as distinct from retention) in yeast.     

High K+ elevates cytosolic free Ca concentration due to mobilization from internal storage sites in rat parotid cells

1. Effects of high K+ on cytosolic free Ca concentration ([Ca2+]i) in rat parotid cells were studied using quin2. 2. High K+ elevated [Ca2+]i in a dose-dependent manner in normal and Ca-free media. The elevation of [Ca2+]i induced by high K+ was less in the latter medium. 3. High K+ depolarized the membrane in a dose-dependent manner in normal and Ca-free media. 4. Although monensin increased [Ca2+]i, high K+ did not affect 22Na uptake into cells. 5. After treatment with oligomycin, high K+ but not carbachol raised [Ca2+]i. 6. We suggest that high K+ increases [Ca2+]i due to mobilizing Ca2+ from the intracellular storage site which does not need energy.     

Changes of ATP, polyphosphate and K+ contents in Saccharomyces carlsbergensis during uptake of Mn2+ and glucose

The process of prolonged Mn2+ uptake by the yeast Saccharomyces carlsbergensis in the presence of 100 mM glucose and in the absence of phosphate can be divided into two steps. The first step (0-20 min) of Mn2+ uptake (4.3 mumol/g of wet cells) is characterized by an intense K+ efflux (23.8 mumol/g), synthesis of high molecular weight polyphosphate (HPP) (8.1 mumol/g) and decrease of ATP content (0.06 mumol/g). Simultaneously about 0.6 mumol of glucose is taken up and the level of low molecular weight polyphosphate (LPP) remains practically unchanged. The second step (20-120 min) of Mn2+ uptake (15.6 mumol/g) is characterized by a drop in HPP (16.6 mumol/g) and the synthesis of LPP (19.0 mumol/g). The ATP content decreases by 0.87 mumol/g as compared to the control, while that of K+ increases (5.7 mumol/g). During the first step of Mn2+ uptake the energy of the K+ concentration gradient may be used both for Mn2+ influx (2K+: 1Mn2+) and synthesis of HPP (1P:1.9K+). During the second step the Mn2+ accumulation is apparently driven by HPP conversion into LPP (1:1) and by ATPases serving the Mn2+/H+ exchange.     

Enhanced and selective adsorption of Pb2+ and Cu2+ by EDTAD-modified biomass of baker's yeast

Enhanced and selective removal of Pb2+ and Cu2+ in the presence of high concentration of K+, Na+, Ca2+ and Mg2+ were achieved by adsorption on biomass of baker's yeast modified with ethylenediaminetetraacetic dianhydride (EDTAD). The modified biomass was found to have high adsorption capacities and fast rates for Pb2+ and Cu2+, and it also displayed consistently high levels of metal uptake over the pH range from 2.7 to 6.0. From Langmuir isotherm, the adsorption capacities for Pb2+ and Cu2+ were found to be 192.3 and 65.0 mg g(-1), respectively, which are about 10 and 14 times higher than that of the unmodified biomass. Competitive biosorption experiments showed that the co-ions of K+, Na+, Ca2+ and Mg2+ had little effects on the uptake of Pb2+ and Cu2+ even at the concentration of 1.0 mol L(-1). The adsorbed Pb2+ and Cu2+ on the modified biomass could be effectively desorbed in an EDTA solution, and the regenerated biomass could be reused repeatedly with little loss of the adsorption capacity.     

Induced and spontaneous movements of potassium ions into mitochondria

1. Net movements of K(+) into metabolizing liver mitochondria before and after the addition of valinomycin have been measured by using selective glass electrodes. The advantage of using an automatic titrator to hold the K(+) concentration constant is demonstrated. 2. According to the energy source provided the induced movement after the addition of valinomycin can be either in or out. 3. Uptakes and rates of movement are higher in media containing acetate (20mm) than in media containing chloride (20mm). In each mixture comparisons were made at three pH values; at pH6.36 the induced rates are less than at pH7.0 or 7.8 but the final uptakes attained are increased. 4. The rate of uptake is increased by inorganic phosphate. 5. The presence of Mg(2+) slightly decreases the induced uptake and rate of movement; Ca(2+) can cause the induced movement of K(+) to be outward. 6. The rate of induced K(+) movement is related to the rate of extra oxygen consumption but with different factors in acetate (24 K(+) ions/oxygen molecule) and chloride media (10 K(+) ions/oxygen molecule). 7. The amount of K(+) gained is proportional to the loss of fluorescence of the suspension. 8. When K(+) moves there is a contrary movement of H(+) but the ratio depends on the conditions. At pH6.36 in chloride media the K(+)/H(+) ratio exceeded 10:1 and in no case did it fall to unity. 9. When K(+) is taken up there is a proportional diminution of light-scattering; it is inferred that swelling takes place along with K(+) accumulation. 10. It is shown by the use of tracer (42)K(+) that turnover of the ion in mitochondria is increased by valinomycin. 11. It is concluded that valinomycin both increases the permeability to K(+) and also, given an adequate energy supply, stimulates the K(+)-accumulating mechanism.     

Studies on the efflux of metalloporphyrin from rat liver mitochondria. Effect of K+ and other cations.

The mechanism by which metalloporphyrins synthesized within the mitochondria escape to the incubation medium was studied in isolated rat liver mitochondria. In a low-ionic-strength sucrose medium, the efflux of metalloporphyrins is markedly decreased when K+ (greater than 10 mM) is added. The effect of K+ is not dependent on the energy state of the mitochondria and it can in part be abolished by adding globin, but not albumin. K+ also decreases the uptake of porphyrins by the mitochondria and thereby the rate of synthesis of metalloporphyrins. Qualitatively similar results are found with Na+, Li+, Mg2+ and Ca2+. Quantitatively, however, the efficiency of cations to inhibit the release of metalloporphyrins decreases in the order: Mg2+ greater than Ca2+ greater than K+ greater than Li+ greater than Na+. Co-protoporhyrin behaves essentially as Co-deuteroporphyrin. The results provide further evidence that the efflux of metalloporphyrins from the mitochondria depends on haem-binding ligands of the suspending medium and also on the ionic strength of the incubation medium.     

Changes induced in the permeability barrier of the yeast plasma membrane by cupric ion.

A specific effect of Cu2+ eliciting selective changes in the permeability of intact Saccharomyces cerevisiae cells is described. When 100 microM CuCl2 was added to a cell suspension in a buffer of low ionic strength, the permeability barrier of the plasma membranes of the cells was lost within 2 min at 25 degrees C. The release of amino acids was partial, and the composition of the amino acids released was different from that of those retained in the cells. Mostly glutamate was released, but arginine was mainly retained in the cells. Cellular K+ was released rapidly after CuCl2 addition, but 30% of the total K+ was retained in the cells. These and other observations suggested that Cu2+ caused selective lesions of the permeability barrier of the plasma membrane but did not affect the permeability of the vacuolar membrane. These selective changes were not induced by the other divalent cations tested. A novel and simple method for differential extraction of vacuolar and cytosolic amino acid pools by Cu2+ treatment was established. When Ca2+ was added to Cu2+-treated cells, a large amount of Ca2+ was sequestered into vacuoles, with formation of an inclusion of a Ca2+-polyphosphate complex in the vacuoles. Cu2+-treated cells also showed enhanced uptake of basic amino acids and S-adenosylmethionine. The transport of these substrates showed saturable kinetics with low affinities, reflecting the vacuolar transport process in situ. With Cu2+ treatment, selective leakage of K+ from the cytosolic compartment appears to create a large concentration gradient of K+ across the vacuolar membrane and generates an inside-negative membrane potential, which may provide a driving force of uptake of positively charged substances into vacuoles. Cu2+ treatment provides a useful in situ method for investigating the mechanisms of differential solute pool formation and specific transport phenomena across the vacuolar membrane.     

Effect of temperature on H+-K+ exchange in Escherichia coli bacteria during their anaerobic growth

The H(+)-K(+)-exchange in E.coli grown under anaerobic conditions at temperatures from 17 to 37 degrees C was studied. The Arrhenius plots for both the N,N'-dicyclohexylcarbodiimide-sensitive release of H+ and K+ uptake by cells transferred into a fresh medium containing a carbon source (glucose) are nonlinear. The activation energy values for the transport of these cations at different temperatures significantly differ. It is shown that as the temperature decreases, the accumulation of K+ by cells is reduced. In this process, the initial rate of K+ absorption through the TrkA system, the time of accumulation of these cations by cells and the osmosensitivity of K+ uptake substantially decrease. At temperatures below 20 degrees C, the absorption becomes insensitive to the secondary osmoshock. However, the stoichiometry of N,N'-dicyclohexylcar-bodiimide-sensitive cation fluxes remains unchanged and is equal to 2H+:K+. It is assumed that the H(+)-K(+)-exchange proceeds by the operation of an ensemble of oligomers, formed from the protomers of F0F1 and TrkA, which rearrange by the action of temperature, whereas F0F1 and TrkA in each protomer do not change.     

Inhibition and stimulation of K+ transport across the frog erythrocyte membrane by furosemide, DIOA, DIDS and quinine

Frog erythrocytes were incubated in iso- or hypotonic media containing 10 mmol/l Rb+ and 0.1 mmol/l ouabain and both Rb+ uptake and K+ loss were measured simultaneously. Rb+ uptake by frog red cells in iso- and hypotonic media was reduced by 30-60% in the presence of 0.01-0.1 mmol/l [(dihydroindenyl)oxy] alkanoic acid (DIOA) or 0.5-1.0 mmol/l furosemide. Furosemide inhibited K+ loss from frog erythrocytes incubated in hypotonic media but did not affect it in isotonic media. DIOA at a concentration of 0.05 mmol/l inhibited of K+ loss from frog erythrocytes in both iso- and hypotonic media. At the concentrations of 0.01 and 0.02 mmol/l DIOA significantly suppressed K+ loss in a K+-free chloride medium but not in a K+-free nitrate medium. The Cl(-)-dependent K+ loss was completely blocked at a concentration of 0.1 mmol/l DIOA and the concentration required for 50% inhibition of K-Cl cotransport was approximately 0.015 mmol/l. However, the inhibitory effect of DIOA on K-Cl cotransport was masked by an opposite stimulatory effect on K+ transport which was also observed in nitrate medium. Quinine in a concentration of 0.2-1.0 mmol/l was able to inhibit Rb+ uptake and K+ loss only in hypotonic media. In isotonic media, quinine produced a stimulation of Rb+ uptake and K+ loss. A three to five-fold activation of Rb+ uptake and K+ loss was consistently observed in frog erythrocytes treated with 0.05-0.2 mmol/l 4,4'-diisothiocyanatostilbene-2,2'-disulphonic acid (DIDS). In contrast, another stilbene derivative 4-acetamido-4'-isothiocyanatostilbene-2,2'-disulphonic acid (SITS) had no effect on K+ transport in the cells. Thus, of these drugs tested in the present study only DIOA at low concentrations may be considered as a selective blocker of the K-Cl cotransporter in the frog red blood cells.     

星形神经胶质细胞摄取谷氨酸与环境因子和离子运输的关系

本文以星形神经胶质细胞为对象,用同位素示踪技术较详细地研究了介质中Na、、K~+和CL~-、不同浓度的卡因酸以及几种抑制剂对L-谷氨酸摄取的影响;并观察了L-谷氨酸对星形神经胶质细胞膜运输Na~+、K~+、Cl~-和Ca~(2+)等的作用.结果表明:L-谷氨酸的摄取依赖于介质中是否存在Na~+ ,在缺Na~+介质中对Cl~-的依赖性也较明显,但在正常Na~+浓度下,含Cl~_和缺Cl~_没有明显差别.当增加介质中K~+浓度引起膜的去极化时,则能降低L~_谷氨酸的摄取.反过来,L-谷氨酸的摄取也对Na~+、K~+、Cl~-等的运输起刺激作用.此外,卡因酸及所用的几种抑制剂对谷氨酸的摄取办有明显抑制作用.     

Uptake of glutamate in Corynebacterium glutamicum. 1. Kinetic properties and regulation by internal pH and potassium

The active uptake system for glutamate in Corynebacterium glutamicum is inducible by growth on glutamate as sole energy and carbon source and is also susceptible to catabolite repression by glucose. The basic level of uptake activity is low in glucose-grown cells (1.5 nmol.mg dry mass-1.min-1), it is intermediate when acetate is the carbon source (3.8 nmol.mg dry mass-1.min-1) and becomes fully induced by glutamate (15 nmol.mg dry mass-1.min-1). In all cases the uptake has, except for different Vmax values, identical kinetic and energetic properties, and is characterized by a low apparent Km value of 0.5-1.3 microM and by high substrate specificity. The transported substrate species is the deprotonated form which can also be concluded from the extremely high pH optimum of transport above pH 9. Glutamate uptake in cells grown in media with low K+ concentration is not influenced by external Na+ but is drastically stimulated by addition of K+. Stimulation by K+ could be separated into two different mechanisms. (a) Addition of K+ increases the internal pH, thereby stimulating glutamate uptake which is regulated by the internal pH in C. glutamicum. The apparent pK of the internal 'pH switch' is 6.6; below this value, uptake of glutamate is inhibited. (b) Internal K+ also directly promotes glutamate uptake. Effective uptake of glutamate can be observed only when the cytosolic K+ concentration exceeds a threshold value of about 200 mM. Stimulation of glutamate uptake by external K+ is not due to functional coupling of K+ and glutamate transport but reveals the necessity to replenish the internal K+ pool.     

Calcium uptake in rat liver mitochondria accompanied by activation of ATP-dependent potassium channel

The influence of potassium ions on calcium uptake in rat liver mitochondria is studied. It is shown that an increase in K+ and Ca2+ concentrations in the incubation medium leads to a decrease in calcium uptake in mitochondria together with a simultaneous increase in potassium uptake due to the potential-dependent transport of K+ in the mitochondrial matrix. Both effects are more pronounced in the presence of an ATP-dependent K+-channel (K+(ATP)-channel) opener, diazoxide (Dz). Activation of the K+(ATP)-channel by Dz alters the functional state of mitochondria and leads to an increase in the respiration rate in state 2 and a decrease in the oxygen uptake and the rate of ATP synthesis in state 3. The effect of Dz on oxygen consumption in state 3 is mimicked by valinomycin, but it is opposite to that of the classical protonophore uncoupler CCCP. It is concluded that the potential-dependent uptake of potassium is closely coupled to calcium transport and is an important parameter of energy coupling responsible for complex changes in oxygen consumption and Ca2+-transport properties of mitochondria.     

Active K+ transport in Mycoplasma mycoides var. Capri. Net and unidirectional K+ movements.

Analysis of the cation composition of growing Mycoplasma mycoides var. Capri indicates that these organisms have a high intracellular K+ concentration (Ki: 200--300 mM) which greatly exceeds that of the growth medium, and a low Na+ concentration (Na+i: 20 mM). Unlike Na+i,K+i varies with cell aging. The K+ transport properties studied in washed organisms resuspended in buffered saline solution show that cells maintain a steady and large K+ concentration gradient across their membrane at the expense of metabolic energy mainly derived from glycolysis. In starved cells, K+i decreases and is partially compensated by a gain in Na+. This substitution completely reverses when metabolic substrate is added (K+ reaccumulation process). Kinetic analysis of K+ movement in cells with steady K+ level shows that most of K+ influx is mediated by an autologous K+-K+ exchange mechanism. On the other hand, during K+ reaccumulation by K+-depleted cells, a different mechanism (a K+ uptake mechanism) with higher transport capacity and affinity drives the net K+ influx. Both mechanisms are energy-dependent. Ouabain and anoxia have no effect on K+ transport mechanisms; in contrast, both processes are completely blocked by dicyclohexylcarbodiimide, an inhibitor of the Mg2+ -dependent ATPase activity.     

The constitutive K+ pump in Serratia marcescens

Transport of K+ and H+ in the anaeronically and aerobically grown bacterium Serratia marcescens has been studied. The volumes of one cell of the anaerobically and aerobically grown bacterium were 3.7 X 10(-13) cm3 and 2.4 X 10(-13) cm3, respectively. Irrespective of the growth conditions the bacteria manifested the same respiration rate. However, the values of membrane potential for the anaerobically and aerobically grown bacterium were different and equal to -130 mV and -175 mV (interior negative), respectively, in the absence of an exogenic energy source. KCN + DCCD decreases delta psi down to almost zero in both species. DCCD alone decreases delta psi partially in anaerobes and increases delta psi in aerobes, whereas KCN alone reduces delta psi partially in both species. The introduction of glucose into the medium containing K+ reduces the absolute value of delta psi to [-160] mV in aerobes and to [-20] mV in anaerobes. The effect is not observed without external K+. In the presence of arsenate a delta psi is not reduced after the addition of glucose. At pH 7.5-7.8 the ATP level in aerobes grows notably faster than in anaerobes. The H+ extrusion becomes intensified when K+ uptake is activated by the increase in external osmotic pressure. Apparent Km and Vmax for K+ accumulation are 1.2 mM and 0.4 mM.min-1.g-1. The decrease of delta psi by glucose or KCN + DCCD have no effect on the K+ uptake whereas CCCP inhibits potassium accumulation. At the same time, arsenate stabilizes the delta psi value, but blocks K+ uptake. The accumulation of K+ correlates with the potassium equilibrium potential of -200 mV calculated according to the Nernst equation, whereas the delta psi measured was not more than [-25] mV. The calculated H+/ATP stoichiometry was 3.3 for aerobes. It was assumed that a constitutive K+ pump having a K+/ATP ratio equal to 2 or 3 operates in S. marcescens membranes.     

Potassium transport in nonpigmented epithelial cells of ocular ciliary body: inhibition of a Na+, K+, Cl- cotransporter by protein kinase C.

The mechanisms by which 86Rb+ (used as a tracer for K+) enters human nonpigmented ciliary epithelial cells were investigated. Ouabain-inhibitable bumetanide-insensitive 86Rb+ transport accounted for approximately 70-80% of total, whereas bumetanide-inhibitable ouabain-insensitive uptake accounted for 15-25% of total. K+ channel blockers such as BaCl2 reduced uptake by approximately 5%. Bumetanide inhibited 86Rb+ uptake with an IC50 of 0.5 microM, while furosemide inhibited with an IC50 of about 20 microM. Bumetanide-inhibitable 86Rb+ uptake was reduced in Na(+)-free or Cl(-)-free media, suggesting that Na+ and Cl- were required for optimal uptake via this mechanism. These characteristics are consistent with a Na+, K+, Cl- cotransporter in NPE cells. Treatment of NPE cells for 15 min with phorbol 12-myristate, 13-acetate (PMA), an activator of protein kinase C, caused a 50-70% decrease in 86Rb+ uptake via the Na+, K+, Cl- cotransporter. Other 86Rb+ uptake mechanisms were not affected. 86Rb+ uptake via the Na+, K+, Cl- cotransporter could be inhibited by other phorbol esters and by dioctanoylglycerol, an analog of diacylglycerol, but not by 4 alpha phorbol didecanoate, an ineffective activator of protein kinase C. Staurosporine, a protein kinase C inhibitor, blocked phorbol ester inhibition of 86Rb+ uptake. These data suggest that a Na+, K+, Cl- cotransporter in NPE cells is inhibited by activation of protein kinase C.     

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.