Specificity of the volume-activated amino acid efflux pathway in cultured human breast cancer cells

It has been shown that cell swelling stimulates the efflux of taurine from MCF-7 and MDA-MB-231 cells via a pathway which has channel-like properties. The purpose of this study was to examine the specificity of the volume-activated taurine efflux pathway in both cell lines. A hyposmotic shock increased the efflux of glycine, L-alanine, AIB (α-aminoisobutyric acid), D-aspartate but not L-leucine from MDA-MB-231 and MCF-7 cells. It was evident that the time course of activation/inactivation of those amino acids whose efflux was affected by cell swelling was similar to that of volume-activated taurine efflux. The effect of exogenous ATP on swelling-induced glycine, AIB and D-aspartate efflux from MDA-MB-231 cells was similar to that found on taurine efflux. In addition, volume-activated AIB efflux from MDA-MB-231 cells, like that of swelling-induced taurine efflux, was inhibited by diiodosalicylate. Tamoxifen inhibited volume-activated taurine release from both MDA-MB-231 and MCF-7 cells. The results suggest that neutral and anionic α-amino acids are able to utilize the volume-activated taurine efflux pathway in both cell lines. The effect of tamoxifen on breast cancer growth may, in part, be related to perturbations in cell volume regulation.     

Presence of a Ca2+-dependent K+ channel in brown adipocytes. Possible role in maintenance ofα1-adrenergic stimulation

We have previously demonstrated mobilization of Ca²⁺ in the efflux of Rb⁺ (K⁺) from isolated hamster brown adipocytes as a consequence of norepinephrine stimulation. We have now investigated the adrenoceptor subtype specificity of these responses and found them both to be of theα₁-subtype. Futher, we have found that the Rb⁺ (K⁺) effux was dependent upon a primary Ca²⁺mobilization event in response to the α₁-adrenergic stimulation, since the Rb⁺ efflux could also be demonstrated by the addition ionophore A23187 to the cells. The norepinephrine- and A23187-stimulated Rb⁺ effluxes were both inhibited by the Ca²⁺-dependent K⁺ -channel blocker apamin. Apamin also significantly attenuated Ca²⁺ mobilization in cells in response to a submaximal concentration of norepinephrine. We conclude that α₁-adrenergic stimulation of brown fat cells leads to a mobilization of intracellular Ca²⁺ which, in itself or via other mechanisms, leads to an increase in cytosolic Ca²⁺ concentration which, in turn, activates a Ca²⁺ -dependent K⁺ channel leading to a K⁺ release from these cells. A possible role for this channel to sustain and augment the response toα₁-adrenergic stimulation is discussed.     

Compartmental analysis of efflux of K+(86Rb+) and Rb+(86Rb+) from roots of intact plants of barley (Hordeum vulgare) of high and low K+ status, and after excision of the shoot

The classic compartment analysis of ion efflux from roots is often applied with the assumption that there is a system of 3 compartments in series. However, complex ion transport across the root tissues, as well as influences from the shoot, may complicate the picture. The present experiments were performed to study the immediate effects that excision of the shoot before the experiment exerts on the efflux of Rb⁺(⁸⁶Rb⁺) and of K⁺(⁸⁶Rb⁺) from 9-day-old roots of plants of barley (Hordeum vulgare L. cv. Salve). The efflux from high K⁺ and low K⁺ roots of intact and detopped plants were compared. After excision of the shoot of high K⁺ plants, a marked increase in efflux was observed after 2.5 h with a maximum at about 7 h. The increase in efflux was seen as a peak in plots of efflux versus time. Excision of the shoot from low K⁺ roots did not give rise to a consistent increase in efflux. Regular K⁺ ion efflux curves were observed from roots of intact plants of high or low K⁺ status. Furthermore, after a pulse treatment of 9-day-old roots of intact plants of high or low K⁺ status with a solution containing Rb⁺(⁸⁶Rb⁺), the Rb⁺(⁸⁶Rb⁺) transport to the shoots was not reduced during the following 3 h in unlabelled solution. It is suggested that both the peak appearing in the efflux plots and the maintained tracer transport to the shoots after transfer of the roots to an unlabelled solution indicate the existence of a K⁺/Rb⁺ transport system in the symplasm of the roots that has only a slow exchange with the bulk cytoplasm and vacuoles.     

Description and role in proliferation of iberiotoxin-sensitive currents in different human mammary epithelial normal and cancerous cells

Several studies suggested that potassium channels are involved in the proliferation of cancer cells but the involvement of the large conductance Ca²⁺-activated K⁺ channels (BK_Ca) in the cancerous phenomenon is still controversial. In the present study, we used iberiotoxin, a specific blocker of BK_Ca, and report the activity of an iberiotoxin-sensitive current in various human breast cancer cell lines (MCF-7, MDA-MB-231, MDA-MB-468 and MDA-MB-435s) as well as in normal mammary epithelial cells (HME).Iberiotoxin and NS1619, an activator of BK_Ca, did not interfere with either cell proliferation or with the invasive properties of the cells, under normal culture conditions. However, extracellular pulses of ATP, which induced transient increases in intracellular Ca²⁺ concentration, revealed a significant reduction effect of iberiotoxin on cell proliferation.We conclude that the iberiotoxin-sensitive current is not involved in cell proliferation in basal conditions but participates when the intracellular Ca²⁺ concentration is increased. These experiments also suggest that BK_Ca channels are not involved in the cancerous transformation and are probably a relic from normal cells.     

Aluminium interactions with K+(86Rb+) and 45Ca2+ fluxes in three cultivars of sugar beet (Beta vulgaris)

Three cultivars of sugar beet (Beta vulgaris L.), which are sensitive to aluminium (Al) in the order Primahill > Monohill > Regina, were grown in water culture for 2 weeks. Nutrients were supplied at 15% increase of amounts daily, corresponding to the nutrient demand for maximal growth. The 2.4-dinitrophenol (DNP)-sensitive (metabolic) and DNP-insensitive (non-metabolic) uptake of aluminium, phosphate. ⁴⁵Ca²⁺ and K⁺(⁸⁶Rb⁺) in roots were measured as well as transport to shoots of intact plants. All 3 cultivars absorbed more aluminium if DNP was present during the aluminium treatment than in its absence. It is suggested that sugar beets are able to extrude aluminium activity or that they possess an active mechanism to keep Al outside the cell. The presence of Al in the medium during the 1-h experiment affected the metabolic and non-metabolic fluxes of ⁴⁵Ca²⁺ and K⁺(⁸⁶Rb⁺) in different ways. In the presence of DNP, the influx of both ⁴⁵Ca²⁺ and K⁺(⁸⁶Rb⁺) and the efflux of ⁴⁵Ca²⁺ were inhibited by Al in a competitive way. At inhibition of ⁴⁵Ca²⁺ influx, 2 Al ions are probably bound per Ca²⁺ uptake site in cv. Regina (Al-tolerant), but in cvs Primahill and Monohill only one Al ion is bound (more Al sensitive). Aluminium competitively inhibited the active efflux of ⁴⁵Ca²⁺ (absence of DNP) in almost the same way in the 3 cultivars. In contrast, aluminium stimulated the influx of K⁺(⁸⁶Rb⁺) in cvs Primahill, Monohill and Regina in the absence of DNP. Thus, the Al effects on active and passive K⁺(⁸⁶Rb⁺) influx are different. The total influx of K⁺(⁸⁶Rb⁺) increased in the presence of Al and might be connected to an active exclusion of Al. Regina is the least Al-sensitive cultivar, probably because Al interferes less with the Ca²⁺ fluxes and because this cultivar actively excludes phosphate in the presence of Al. Thus Al-phosphate precipitation within the plant could be avoided.     

Oxidative stress increases potassium efflux from pancreatic islets by depletion of intracellular calcium stores

Oxidative stress to B-cells is thought to be of relevance in declining B-cell function and in the process of B-cell destruction. In other tissues including heart, brain and liver, oxidative stress has been shown to elevate the intracellular free calcium concentration and to provoke potassium efflux. We studied the effect of oxidative stress on Ca²⁺ and K⁺ (Rb⁺) outflow from pancreatic islets using the thiol oxidants DIP and BuOOH. Both compounds reversibly increased ⁸⁶Rb⁺ efflux in the presence of 3 and 16.7 mmol/l glucose. Stimulation of ⁸⁶Rb⁺ efflux was also evident in the absence of calcium. DIP evoked release of ⁴⁵Ca²⁺ from the pancreatic islets both in the presence or absence of extracellular calcium. Employing inhibitors of the calcium-activated potassium channel (K_Ca) and the high conductance K⁺-channel (BK_Ca), the effect of DIP on ⁸⁶Rb⁺ efflux was slightly diminished. Tolbutamide had no effect on ⁸⁶Rb⁺ efflux in the presence of DIP. On the other hand thapsigargin, a blocker of the Ca²⁺-ATPase of the endoplasmic reticulum, completely suppressed the DIP-mediated ⁸⁶Rb⁺ outflow. The data suggest that thiol oxidant-induced potassium efflux from pancreatic islets is mainly mediated through liberation of intracellular calcium and subsequent stimulation of calcium-activated potassium efflux.     

Inhibition of 86Rb+ and 22Na+ efflux of Ehrlich lettré tumor cells by Ca2+.

The mechanism of the protective effect of Ca²⁺ on cellular K⁺ content was studied by examination of the effect of Ca²⁺ on efflux of the K⁺ analog, ⁸⁶Rb⁺, from preloaded cells with the use of compounds which interfere with monovalent cation movements. Ca²⁺ decreased ⁸⁶Rb⁺ efflux to the same extent in the presence and absence of ouabain, suggesting that Ca²⁺ did not alter the activity of the (Na⁺ + K⁺)-adenosine triphosphatase pump. Ca²⁺ exerted a similar protective effect in the presence of furosemide, an inhibitor of K⁺-K⁺ exchange, indicative that Ca²⁺ was not inhibiting this pathway. Since Ca²⁺ did not influence these pathways, it is concluded that Ca²⁺ exerts its primary effect by slowing passive diffusion. In support of this, Ca²⁺ also slowed ²²Na⁺ efflux. In addition, ethanol-induced leakage of ⁸⁶Rb⁺ was reversed by extracellular Ca²⁺, suggestive of a Ca²⁺-membrane phospholipid interaction.     

Ouabain-resistant Na+, K+ transport system in mouse NIH 3T3 cells

Summary It is shown that the ouabain-resistant (OR) furosemide-sensitive K⁺(Rb⁺) transport system performs a net efflux of K⁺ in growing mouse 3T3 cells. This conclusion is based on the finding that under the same assay conditions the furosemidesensitive K⁺(Rb⁺) efflux was found to be two- to threefold higher than the ouabain-resistant furosemide-sensitive K⁺(Rb⁺) influx. The oubain-resistant furosemide-sensitive influxes of both²²Na and⁸⁶Rb appear to be Cl^– dependent, and the data are consistent with coupled unidirectional furosemide-sensitive influxes of Na⁺, K⁺ and Cl^– with a ratio of 1 1 2. However, the net efflux of K⁺ performed by this transport system cannot be coupled to a ouabain-resistant net efflux of Na⁺ since the unidirectional ouabain-resistant efflux of Na⁺ was found to be negligible under physiological conditions. This latter conclusion was based on the fact that practically all the Na⁺ efflux appears to be ouabainsensitive and sufficient to balance the Na⁺ influx under such steady-state conditions. Therefore, it is suggested that the ouabain-resistant furosemide-sensitive transport system in growing cells performs a facilitated diffusion of K⁺ and Na⁺, driven by their respective concentration gradients: a net K⁺ efflux and a net Na⁺ influx.     

l-Leucine transport in human breast cancer cells (MCF-7 and MDA-MB-231): kinetics, regulation by estrogen and molecular identity of the transporter

The transport of l-leucine by two human breast cancer cell lines has been examined. l-Leucine uptake by MDA-MB-231 and MCF-7 cells was via a BCH-sensitive, Na⁺-independent pathway. l-Leucine uptake by both cell lines was inhibited by l-alanine, d-leucine and to a lesser extent by l-lysine but not by l-proline. Estrogen (17β-estradiol) stimulated l-leucine uptake by MCF-7 but not by MDA-MB-231 cells. l-Leucine efflux from MDA-MB-231 and MCF-7 cells was trans-stimulated by BCH in a dose-dependent fashion. The effect of external BCH on l-leucine efflux from both cell types was almost abolished by reducing the temperature from 37 to 4 °C. There was, however, a significant efflux of l-leucine under zero-trans conditions which was also temperature-sensitive. l-Glutamine, l-leucine, d-leucine, l-alanine, AIB and l-lysine all trans-stimulated l-leucine release from MDA-MB-231 and MCF-7 cells. In contrast, d-alanine and l-proline had little or no effect. The anti-cancer agent melphalan inhibited l-leucine uptake by MDA-MB-231 cells but had no effect on l-leucine efflux. Quantitative real-time PCR revealed that LAT1 mRNA was approximately 200 times more abundant than LAT2 mRNA in MCF-7 cells and confirmed that MDA-MB-231 cells express LAT1 but not LAT2 mRNA. LAT1 mRNA levels were higher in MCF-7 cells than in MDA-MB-231 cells. Furthermore, LAT1 mRNA was more abundant than CD98hc mRNA in both MDA-MB-231 and MCF-7 cells. The results suggest that system L is the major transporter for l-leucine in both MDA-MB-231 and MCF-7 cells. It is possible that LAT1 may be the major molecular correlate of system L in both cell types. However, not all of the properties of system L reflected those of LAT1/LAT2/CD98hc.     

Turgor regulation inValonia macrophysa following acute osmotic shock

Summary The marine algaValonia macrophysa an inhabitant of shallow subtropical waters, is subjected to sudden dilutions of external seawater during rain showers. This study describes the mechanisms involved in turgor pressure regulation following acute hyposmotic shock. Turgor regulation is 88% effective and complete within 4 hr following hyposmotic shocks of up to –10 bar. Loss of vacuolar K⁺, Na⁺ and Cl^– accounts for the decrease in vacuolar osmotic pressure associated with turgor regulation. A novel mechanism of turgor regulation is exhibited byValonia macrophysa given hyposmotic shocks greater than about –4 bar. Such an osmotic shock causes cell wall tension to increase above a critical value of about 6×10⁵ dyne/cm, whereupon the protoplasm ruptures and the cell wall stretches irreversibly at a localized site. The protoplasm rupture is suggested by (1) a large abrupt increase in K⁺ efflux (as measured by⁸⁶Rb⁺), (2) a rapid decrease in turgor pressure as measured with a pressure probe, and (3) sudden depolarization of the vacuole potential. Evidence for an increase in cell wall permeability includes efflux from the vacuole of dextran (mol wt 70,000), which normally has a very low cell wall permeability, and scanning electron micrographs which show a trabeculated scar area in the cell wall. This mechanism of turgor regulation is physiologically important because 98% of the cells regained normal growth rate and turgor following acute osmotic shock.     

Osmoregulation of taurine efflux from cultured human breast cancer cells: comparison with volume activated Cl- efflux and regulation by extracellular ATP.

The properties and regulation of volume-activated taurine efflux from MDA-MB-231 and MCF-7 cells have been investigated. Volume-activated taurine release from both cell lines was almost completely inhibited by diidosalicylate. DIDS , was more effective at inhibiting swelling-induced taurine release from MCF-7 than from MDA-MB-231 cells. On the basis of comparing taurine, Cl(-) and I(-) efflux time courses, it appears that volume-activated taurine efflux does not utilize volume-sensitive anion channels in MDA-MB- 231 and MCF-7 cells. Extracellular ATP stimulated volume-activated taurine release from MDA-MB-231 cells but not from MCF-7 cells. The effect of ATP was mimicked by UTP and was dependent upon external calcium and inhibited by suramin. However, suramin inhibited volume-activated taurine efflux from both MDA-MB-231 and MCF-7 cells even in the absence of exogenously added ATP suggesting that it acts directly on the taurine efflux pathway and/or is inhibiting the effect of ATP released from the cells. Volume-activated taurine efflux from MDA-MB-231 cells was stimulated by ionomycin. In contrast, ionomycin had no effect on taurine release from MCF-7 cells. Adenosine also stimulated volume-activated taurine efflux from MDA-MB-231 cells. The results suggest that purines regulate taurine transport in MDA-MB- 231 cells via more than one type of receptor.     

Thiol-Dependent passive K/Cl transport in sheep red cells: IV. Furosemide inhibition as a function of external Rb+, Na+, and Cl−

Summary The effect of the loop diuretic furosemide (4-chloro-N-furfuryl-5-sulfamoyl-anthranilic acid) on the thiol-dependent, ouabain-insensitive K(Rb)/Cl transport in low K⁺ sheep red cells was studied at various concentrations of extracellular Rb⁺, Na⁺ and Cl^–. In Rb⁺-free NaCl media, 2×10^–3 m furosemide inhibited only one-half of thiol-dependent K⁺ efflux. In the presence of 23mm RbCl, however, the concentration of furosemide to produce 50% K⁺ efflux inhibition (IC₅₀) was 5×10^–5 m. In Rb⁺ containing NaCl media, the inhibitory effect of 10^–3 m furosemide was equal to that caused by NO ₃ ^– replacement of Cl^– in the medium. The apparent synergistic action of furosemide and external Rb⁺ on K⁺ efflux was also seen in the ouabain-insensitive Rb⁺ influx. A preliminary kinetic analysis suggests that furosemide binding alters both maximal K⁺(Rb⁺) transport and apparent external Rb⁺ affinity. In the presence of external Rb⁺, Na⁺ (as compared to choline) exerted a small but significant augmentation of the furosemide inhibition of K⁺(Rb⁺) fluxes. There was no effect of Cl^– on the IC₅₀ value of furosemide. As there is no evidence for coupled Na⁺K⁺ cotransport in low K⁺ sheep red cells, furosemide may modify thiol-dependent K⁺(Rb⁺/Cl flux or Rb⁺ (and to a slight degree Na⁺) modulate the effect of furosemide.     

Hyposmotically-Activated Efflux of L-Carnitine from a Human Mammary Cancer Cell Line

Cell-swelling, induced by a hyposmotic challenge, stimulated the efflux of L-carnitine from a human mammary cancer cell line, MDA-MB-231. The response was dependent upon the extent of the osmotic shock. Hyposmotically-activated L-carnitine efflux was inhibited by the anion transport blocker diiodosalicylate. The efflux of taurine from MDA-MB-231 cells was also stimulated by a hyposmotic shock via a pathway sensitive to diiodosalicylate. L-carnitine efflux from MDA-MB-231 cells was stimulated by isosmotic swelling in a manner which was inhibited by diiodosalicylate. The results suggest that L-carnitine may exit cells via a volume-sensitive pathway: it is possible that L-carnitine efflux may utilize the same pathway as amino acids. The efflux of L-carnitine via this route could have a major effect on the intracellular concentration of L-carnitine and could facilitate transepithelial L-carnitine transport.     

Effect of extracellular adenosine 5′-triphosphate on Ca2+ efflux from freshly isolated adult rat cardiomyocytes

The effect of extracellular adenosine 5′-triphosphate (ATP) on Ca²⁺ efflux from freshly isolated adult rat cardiomyocytes was examined. ATP stimulated the efflux of ⁴⁵Ca²⁺ from the cells in a concentration-dependent manner (0.01–1 mM). The ⁴⁵Ca²⁺ efflux from the cells was also stimulated by adenosine-5′-O-(3-thiotriphosphate) (ATP-γs) and α,β-methylene-ATP and adenosine 5′-diphosphate, but not by adenosine 5′-monophosphate and adenosine. The ATP-stimulated ⁴⁵Ca²⁺ efflux was not affected by deprivation of the extracellular Ca²⁺, but was dependent on the presence of extracellular Na⁺. These results indicate that ATP stimulates extracellular Na⁺-dependent ⁴⁵Ca²⁺ efflux from freshly isolated adult rat cardiomyocytes, probably through its stimulatory effect on the plasma membrane P₂ purinoceptors which may couple to Na⁺/Ca²⁺ exchange.     

Fractal analysis and ionic dependence of endocytotic membrane activity of human breast cancer cells

The endocytic membrane activities of two human breast cancer cell lines (MDA-MB-231 and MCF-7) of strong and weak metastatic potential, respectively, were studied in a comparative approach. Uptake of horseradish peroxidase was used to follow endocytosis. Dependence on ionic conditions and voltage-gated sodium channel (VGSC) activity were characterized. Fractal methods were used to analyze quantitative differences in vesicular patterning. Digital quantification showed that MDA-MB-231 cells took up more tracer (i.e., were more endocytic) than MCF-7 cells. For the former, uptake was totally dependent on extracellular Na⁺ and partially dependent on extracellular and intracellular Ca²⁺ and protein kinase activity. Analyzing the generalized fractal dimension (D _q ) and its Legendre transform f(α) revealed that under control conditions, all multifractal parameters determined had values greater for MDA-MB-231 compared with MCF-7 cells, consistent with endocytic/vesicular activity being more developed in the strongly metastatic cells. All fractal parameters studied were sensitive to the VGSC blocker tetrodotoxin (TTX). Some of the parameters had a “simple” dependence on VGSC activity, if present, whereby pretreatment with TTX reduced the values for the MDA-MB-231 cells and eliminated the differences between the two cell lines. For other parameters, however, there was a “complex” dependence on VGSC activity. The possible physical/physiological meaning of the mathematical parameters studied and the nature of involvement of VGSC activity in control of endocytosis/secretion are discussed.     

Bradykinin-induced potassium current in cultured bovine aortic endothelial cells

Summary Bovine aortic endothelial cells (BAECs) respond to bradykinin with an increase in cytosolic-free Ca²⁺ concentration, [Ca²⁺] _i , accompanied by an increase in surface membrane K⁺ permeability. In this study, electrophysiological measurement of K⁺ current was combined with⁸⁶Rb⁺ efflux measurements to characterize the K⁺ flux pathway in BAECs. Bradykinin- and Ca²⁺-activated K⁺ currents were identified and shown to be blocked by the alkylammonium compound, tetrabutylammonium chloride and by the scorpion toxin,noxiustoxin, but not by apamin or tetraethylammonium chloride. Whole-cell and single-channel current analysis suggest that the threshold for Ca²⁺ activation is in the range of 10 to 100nm [Ca²⁺] _i . The whole-cell current measurement show voltage sensitivity only at the membrane potentials more positive than 0 mV where significant current decay occurs during a sustained depolarizing pulse. Another K⁺ current present in control conditions, an inwardly rectifying K⁺ current, was blocked by Ba²⁺ and was not affected bynoxiustoxin or tetrabutylammonium chloride. Efflux of⁸⁶Rb^– from BAEC monolayers was stimulated by both bradykinin and ionomycin. Stimulated efflux was blocked by tetrabutyl- and tetrapentyl-ammonium chloride and bynoxiustoxin, but not by apamin or furosemide. Thus,⁸⁶Rb⁺ efflux stimulated by bradykinin and ionomycin has the same pharmacological sensitivity as the bradykinin- and Ca²⁺-activated membrane currents. The results confirm that bradykinin-stimulated⁸⁶Rb⁺ efflux occurs via Ca²⁺-activated K⁺ channels. The blocking agents identified may provide a means for interpreting the role of the Ca²⁺-activated K⁺ current in the response of BAECs to bradykinin.     

Effects of ionic strength and relative humidity on the efflux of K+ (86Rb) and Ca2+ (45Ca) from roots of intact seedlings of cucumber, oat and wheat

Spring wheat (Triticum aestivum L. cv. Svenno), oat (Avena sativa L. cv. Brighton) and glasshouse cucumber (Cucumis sativus L. cv. Bestseller F1) were cultured for a week after germination on complete nutrient solutions of three different dilutions (1, 25 and 50% of the full strength medium). K⁺(⁸⁶Rb) and ⁴⁵Ca were present during the whole culture period. Relative humidity (RH) was 50% except during the last day, when half the material was transferred to 90% RH. Efflux of labelled ions was then followed during eight hours on unlabelled solutions of the same composition as before, and at both 50% and 90% RH in the atmosphere. – Uptake of K⁺(⁸⁶Rb) during growth tended to be saturated in the 25% medium. Contrariwise, the level of Ca²⁺ in the roots increased continuously with strength of the medium. At low concentrations cucumber roots were higher in Ca²⁺ than roots of oat or wheat, whereas all three species showed similar levels of Ca²⁺ in 50% medium. – At the lowest ionic strength, smooth efflux curves were obtained that could be resolved according to the three-compartment theory. At higher ionic strength, irregularities were observed, and more for Ca²⁺ than for K⁺; but for practical purposes compartment analysis with the same time constants could be applied as for the lowest concentration. – Discrimination between K⁺ and Rb⁺ differed between the roots, but not much between the shoots of different species. The roots of oat and wheat took up Rb⁺ preferentially over K⁺ in the 25% and 50% media; whereas K⁺ was preferred over Rb⁺ or little discrimination made in 1% medium and for cucumber. The shoots generally showed less discrimination than the roots. The main variability in discrimination between K⁺ and Rb⁺ thus appears to be localized in the tonoplasts of the roots cells. – Low RH around the shoots increased efflux of K⁺(⁸⁶Rb) from the cytoplasm and vacuoles of the root cells as compared to the efflux at high RH. DNP (2,4-dinitrophenol) in the medium had the same effect as high RH around the shoots. The signal system that must exist between shoots and roots is discussed as a response to “drought” conditions. In relation to investigations of others, it is assumed that the effect of DNP may indicate that part of the chain between roots and shoots consists of metabolically influenced sites, whose output is influenced by the rate of water transport.     

Inhibition of Na+/K+-ATPase may be one mechanism contributing to potassium efflux and cell shrinkage in CD95-induced apoptosis

To investigate the involvement of K⁺ efflux in apoptotic cell shrinkage, we monitored efflux of the K⁺ congener,⁸⁶ Rb⁺, and cell volume during CD95-mediated apoptosis in Jurkat cells. An anti-CD95 antibody caused apoptosis associated with intracellular GSH depletion, a significant increase in ⁸⁶Rb⁺ efflux, and a decrease in cell volume compared with control cells. Preincubating Jurkat cells with Val-Ala-Asp-chloromethylketone (VAD-cmk), an inhibitor of caspase proteases, prevented the observed ⁸⁶Rb⁺ efflux and cell shrinkage induced by the anti- CD95 antibody. A wide range of inhibitors against most types of K⁺ channels could not inhibit CD95-mediated efflux of⁸⁶ Rb⁺, however, the uptake of⁸⁶ Rb⁺ by Jurkat cells was severely compromised when treated with anti-CD95 antibody. Uptake of⁸⁶ Rb⁺ in Jurkat cells was sensitive to ouabain (a specific Na⁺/K⁺-ATPase inhibitor), demonstrating Na⁺/K⁺-ATPase dependent K⁺ uptake. Ouabain induced significant⁸⁶ Rb⁺ efflux in untreated cells, as well as it seemed to compete with⁸⁶ Rb⁺ efflux induced by the anti-CD95 antibody, supporting a role for Na⁺/K⁺-ATPase in the CD95-mediated⁸⁶ Rb⁺ efflux. Ouabain treatment of Jurkat cells did not cause a reduction in cell volume, although together with the anti-CD95 antibody, ouabain potentiated CD95-mediated cell shrinkage. This suggests that the observed inhibition of Na⁺+/K⁺-ATPase during apoptosis may also facilitate apoptotic cell shrinkage.     

Valinomycin-induced potassium and rubidium permeability of intact cells of Acholeplasma laidlawii B

The valinomycin-induced K⁺ and Rb⁺ permeability in cells of Acholeplasma laidlawii B differing in fatty acid and cholesterol content was studied using three different techniques: (i) by following the swelling of the cells in potassium acetate optically; (iii) by recording the efflux of K⁺ using a potassium-selective glass electrode; and (ii) by measuring the efflux of Rb⁺ (after preincubation of the cells with ⁸⁶Rb⁺) with a filter technique.If unsaturation of the membrane lipids was increased, the permeability was found to increase. Cholesterol appeared to cause a slight decrease in permeability.The valinomycin-induced efflux of K⁺ is gradually reduced when the temperature is lowered and becomes zero below the gel-liquid crystalline phase transition.     

A single mechanism for the stimulation of insulin release and 86Rb+ efflux from rat islets by cationic amino acids

The mechanisms by which cationic amino acids influence pancreatic B-cell function have been studied by monitoring simultaneously ⁸⁶Rb⁺ efflux and insulin release from perifused rat islets. The effects of two reference amino acids arginine and lysine were compared with those of closely related substances to define the structural requirements for recognition of these molecules as secretagogues. Arginine accelerated ⁸⁶Rb⁺ efflux and increased insulin release in the absence or in the presence of 7mm-glucose. Its effects on efflux did not require the presence of extracellular Ca²⁺ or Na⁺, but its insulinotropic effects were suppressed in a Ca²⁺-free medium and inhibited in an Na⁺-free medium. Among arginine derivatives, only 2-amino-3-guanidinopropionic acid mimicked its effects on ⁸⁶Rb⁺ efflux and insulin release; citrulline, guanidinoacetic acid, 3-guanidinopropionic acid and guanidine were inactive. Norvaline and valine also increased ⁸⁶Rb⁺ efflux, but their effect required the presence of extracellular Na⁺; they did not stimulate insulin release. Lysine as well as the shorter-chain cationic amino acids ornithine and 2,4-diaminobutyric acid accelerated ⁸⁶Rb⁺ efflux in a Ca²⁺- and Na⁺-independent manner. Their stimulation of insulin release was suppressed by Ca²⁺ omission, but only partially inhibited in an Na⁺-free medium. The uncharged glutamine and norleucine increased the rate of ⁸⁶Rb⁺ efflux in the presence of glucose, only if extracellular Na⁺ was present. Norleucine slightly increased release in a Ca²⁺- and Na⁺-dependent manner. The effects of lysine on efflux and release were not mimicked by other related substances such as 1,5-diaminopentane and 6-aminohexanoic acid. The results suggest that the depolarizing effect of cationic amino acids is due to accumulation of these positively charged molecules in B-cells. This causes acceleration of the efflux of K⁺ (⁸⁶Rb⁺) and activation of the influx of Ca²⁺ (which triggers insulin release). The prerequisite for the stimulation of B-cells by this mechanism appears to be the presence of a positive charge on the side chain of the amino acid, rather than a specific group.