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.     

Ca2+-dependent K+ transport in the Ehrlich ascites tumor cell

The possible presence and properties of the Ca2+-dependent K+ channel have been investigated in the Ehrlich ascites tumor cell. The treatment with ionophore A23187 + CA2+, propranolol or the electron donor system ascorbate-phenazine methosulphate, all of which activate that transport system in the human erythrocyte, produces in the Ehrlich cell a net loss of K+ (balanced by the uptake of Na+) and a stimulation of both the influx and the efflux of 86Rb. These effects were antagonized by quinine, a known inhibitor of the Ca2+-dependent K+ channel in other cell systems, and by the addition of EGTA to the incubation medium. Ouabain did not have an inhibitory effect. These results suggests that the Ehrlich cell possesses a Ca2+-dependent K+ channel whose characteristics are similar to those described in other cell systems.     

Kinetic comparison of ouabain-resistant K:CI fluxes (K:Cl [Co]-transport) stimulated in sheep erythrocytes by membrane thiol oxidation and alkylation

Summary The stimulatory effects of two thiol (SH) group oxidants, methylmethane thiosulfonate (MMTS) and diazene dicarboxylic acid bis [N,N-dimethylamide] (diamide), on the kinetics of ouabain-resistant (OR) K:Cl [co]-transport in low K (LK) sheep red blood cells were compared with the effects of alkylating agents, notably N-ethylmaleimide (NEM). At low concentrations, both MMTS and diamide stimulated K:CI [co]-transportv and with a latency period, as measured by OR zero-trans K efflux and OR uptake of external Rb, Rb_o, as K congener in Cl and NO₃ media. At high concentrations the effect of diamide saturated, and that of MMTS disappeared. The stimulatory effect of MMTS was partially reversed by the reducing agent dithiothreitol (DTT) known to fully restore the diamide-activated K flux (Lauf, J. Memb. Biol. 101:179–188, 1988). In diamide pre-equilibrated LK sheep red cells, the K_m of K:Cl [co]-transport for external Cl, Cl_o, was 84.3 mM, and 18.7 mM for Rb_o, with nearly identical V_max values around 4 mmol Rb/L cells × h for K (Rb) fluxes in Cl and after correction for the small Cl-independent component. Zero net K (Rb) flux existed at K_c (cell K)/Rb_o concentration ratios, [K]_c/[Rb]_c, of 0.8 i.e. when the electrochemical driving forces across the membrane were about equal. The measured K efflux/Rb influx ratios were almost twice those predicted from [K]_c/[Rb]_o and the Cl equilibrium potential suggesting that the diamide-stimulated K (Rb) flux may occur through non-diffusional, carrier-mediated transport. The effects of NEM and of A23187 plus/minus Ca or chelators on K: [co]Cl-transport (Lauf, Am. J. Physiol. 249:C271–278, 1985) consisted primarily of V_max changes. Thus, all chemical interventions resulted in an increase of the number of actively transporting K:Cl [co]-transport units or an augmented turnover number per existing site.     

Protein kinase C: subcellular redistribution by increased Ca2+ influx. Evidence that Ca2+-dependent subcellular redistribution of protein kinase C is involved in potentiation of beta-adrenergic stimulation of pineal cAMP and cGMP by K+ and A23187

Phenylephrine is known to stimulate translocation of protein kinase C in rat pinealocytes (Sugden, D., Vanecek, J., Klein, D.C., Thomas, T.P., and Anderson, W. B. (1985) Nature 314, 359-361). In the present study, the receptor mediating this effect was found to belong to the alpha 1-adrenoceptor subclass. Activation of this receptor is also known to produce a sustained increase in [Ca2+]i by increasing net influx (Sugden, A. L., Sugden, D., and Klein, D. C. (1985) J. Biol. Chem. 261, 11608-11612), which points to the possible importance of Ca2+ influx in the subcellular redistribution (activation) of protein kinase C in intact cells. This possibility was investigated by reducing extracellular Ca2+ ((Ca2+]o) with EGTA or by inhibiting Ca2+ influx with inorganic Ca2+ blockers. These treatments reduced alpha 1-adrenoceptor-mediated translocation of protein kinase C. This suggested that elevation of Ca2+ influx alone triggers activation of protein kinase C. In support of this, it was found that treatments which elevate Ca2+ influx, including increased extracellular K+ and addition of the Ca2+ ionophore A23187, cause redistribution of protein kinase C. The effect of K+ was blocked by nifedipine and that of A23187 by EGTA, indicating that effects of these agents are Ca2+-dependent. The possible role of phospholipase C activation in these effects was examined by measuring the formation of [3H]diacylglycerol by cells labeled with [3H]arachidonic acid. Although [3H]diacylglycerol formation was easily detected in the presence or absence of an effective concentration of an inhibitor of diacylglycerol kinase, none of the agents which cause rapid translocation of protein kinase C were found to cause a rapid increase in the generation of [3H]diacylglycerol. These findings establish that an increase in Ca2+ influx is sufficient to trigger translocation of protein kinase C. In addition, we found that a very close correlation exists between translocation of protein kinase C by phenylephrine, K+, and A23187 and their ability to potentiate beta-adrenergic stimulation of cAMP and cGMP accumulation. This provides strong support to the proposal that translocation of protein kinase C is required for potentiation of beta-adrenergic stimulation of pinealocyte cAMP and cGMP accumulation.     

K+ transport in 'tight' epithelial monolayers of MDCK cells. Evidence for a calcium-activated K+ channel

Measurements of 86Rb efflux across the apical and basal-lateral aspects of intact monolayers of 'high-resistance' MDCK cells mounted in Ussing chambers have been made. A transient increase in 86Rb efflux across both epithelial borders upon stimulation with adrenalineeeeeee or ionophore A23187 is observed. The increased 86Rb across the basal cell aspects is of greatest quantitative importance. Measurements of total cellular K+ contents by flame photometry of tissue extracts indicate a net loss of K+ following adrenalin addition. The effects of adrenalin and ionophore A23187 upon 86Rb efflux are abolished in 'Ca2+ -free' media. The properties of the Ca2+ -dependent increase in 86Rb efflux show similarities to Ca2+ -activated K+ conductances in other tissues, notably human red cells, including inhibition by quinine (1 mM), tetraethylammonium (25 mM) and insensitivity to bee venom toxin (apamin) (25 nM). Adrenalin is only effective when applied to the basal bathing solution suggesting that the receptors mediating adrenalin action are located upon the basal-lateral membranes. Half maximal stimulation of 86Rb efflux by adrenalin is observed at 9.1 X 10(-7) M. The action of various adrenergic receptor agonists and antagonists are consistent with adrenalin action being mediated by an alpha-adrenergic receptor.     

Cyclic AMP and Ca2+-activated K+ transport in a human colonic epithelial cell line

Addition of either vasoactive intestinal peptide (VIP) or the Ca2+ ionophore, A23187, to confluent monolayers of the T84 epithelial cell line derived from a human colon carcinoma increased the rate of 86Rb+ or 42K+ efflux from preloaded cells. Stimulation of the rate of efflux by VIP and A23187 still occurred in the presence of ouabain and bumetanide, inhibitors of the Na+,K+-ATPase and Na+,K+,Cl- cotransport, respectively. The effect of A23187 required extracellular Ca2+, while that of VIP correlated with its known effect on cyclic AMP production. Other agents which increased cyclic AMP production or mimicked its effect also increased 86Rb+ efflux. VIP- or A23187-stimulated efflux was inhibited by 5 mM Ba2+ or 1 mM quinidine, but not by 20 mM tetraethylammonium, 4 mM 4-aminopyridine, or 1 microM apamin. Under appropriate conditions, VIP and A23187 also increased the rate of 86Rb+ or 42K+ uptake. Stimulation of the initial rate of uptake by either agent required high intracellular K+ and was not markedly affected by the imposition of transcellular pH gradients. The effect of A23187, but not VIP or dibutyryl cyclic AMP, was refractory to depletion of cellular energy stores. A23187-stimulated uptake was not significantly affected by anion substitution, however, stimulation of uptake by VIP required the presence of a permeant anion. This result may be due to the simultaneous activation of a cyclic AMP-dependent Cl- transport system. The kinetics of both VIP- and A23187-stimulated uptake and efflux were consistent with a channel-rather than a carrier-mediated K+ transport mechanism. The results also suggest that cyclic AMP and Ca2+ may activate two different kinds of K+ transport systems. Finally, both transport systems have been localized to the basolateral membrane of T84 monolayers, a result compatible with their possible regulatory role in hormone-activated electrogenic Cl- secretion.     

Na+ K+ pump and passive K+ transport in large and small red cell populations of anemic high and low K+ sheep

Reticulocytes, isolated by centrifugal elutriation from massively bled sheep and identified by cytometric techniques, were analyzed with respect to their cation transport properties. In sheep with genetically high K+ (HK) or low K+ (LK) red cells, two reticulocyte types were distinguished by conventional or fluorescence-staining techniques 5-6 days after hemorrhage: Large reticulocytes as part of a newly formed macrocytic (M) erythrocyte population, and small reticulocytes present among the adult red cell population (volume population III of normal sheep blood, Valet et al., 1978). Although cellular reticulin disappeared within a few days, the M-cell population persisted throughout weeks in the peripheral circulation permitting a transport study of in vivo maturation. At all times, M cells of LK sheep had lower K+ and higher Na+ contents than M cells of HK sheep. Regardless of the sheep genotypes, M cells apparently reduced their volume during their first days in circulation; however, throughout the observation period, they did not attain that characteristic for adult red cells. Both ouabain-sensitive K+ pump and ouabain-insensitive K+ leak fluxes were elevated in M cells of both HK and LK sheep. The increased K+ pump flux was mainly due to higher K+ pump turnover rather than to the modestly increased number of pumps as measured by [3H]ouabain binding. In contrast, small reticulocytes enriched from separated volume population III cells by a Percoll-density gradient exhibited transport parameters close to their prospective mature HK or LK red cells. The data support the concept that the M cells derived from emergency reticulocytes while the small reticulocytes represented precursors of normal red cell maturation. The Na+ and K+ composition found in M cells of HK and LK sheep, respectively, suggest development of the LK steady state at or prior to the reticulocyte state, a finding consistent with that of Lee and Kirk (1982) on low K+ dog red cells.     

Trans to cis proton concentration gradients accelerate ionophore A23187-mediated net fluxes of Ca2+ across the human red cell membrane

Ionophore A23187-mediated net influx of Ca2+ in ATP-depleted human red cells was studied as a function of the pH and the proton concentration gradient across the membranes. Utilizing the Ca2+-induced increase in K+ conductance of the cell membranes, various CCCP-mediated proton gradients were raised across the membranes of cells suspended in unbuffered salt solutions with different K+ concentrations. In ionophore-mediated equilibrium the concentration ratios of ionized Ca between ATP-depleted, DIDS-treated cells and their suspension medium were equal to the concentration ratios of protons raised to the second power. With no proton concentration gradient across the membranes the net influxes of Ca2+ as a function of pH resembled a titration curve of a weak acid, with half maximal net influx at pH 7.3, at 100 microM extracellular Ca2+. With cellular pH fixed at various values, the net influx of Ca2+ was determined as a function of the proton concentration gradient. A linear relationship between the logarithm of net influx and the difference between extracellular and cellular pH was found at all cellular pH values tested, but the proton concentration gradient acceleration was a function of the cellular pH. Accelerations between 10- and 40- times per unit delta pH were found and net effluxes were correspondingly decreased. The results are discussed in relation to present models of the mechanism of ionophore A23187-mediated Ca2+ transport. The importance of the proton concentration gradient dependency is discussed in relation to the induced oscillations in K+-conductance of human red cell membranes previously reported (Vestergaard-Bogind and Bennekou (1982) Biochim. Biophys. Acta 688, 37-44).     

Androgenic stimulation of endocytosis, amino acid and hexose transport in mouse kidney cortex involves increased calcium fluxes

Testosterone was previously shown to induce an early (less than 1 min) receptor-dependent stimulation of endocytosis, hexose and amino acid transport in mouse kidney cortex (Koenig, H., Goldstone, A. and Lu, C.Y. (1982) Biochem. Biophys. Res. Commun. 104, 165-172). Testosterone (10(-8) M) has now been found to stimulate rapidly (less than 30 s) the influx and efflux of 45Ca2+ in cortex slices. Testosterone also decreased mitochondrial 45Ca and augmented soluble 45Ca, indicating a mobilization of intracellular calcium. Incubation of cortex slices in calcium-free medium without or with 2.5 mM EGTA decreased basal endocytosis, hexose and amino acid transport and blocked the hormonal response. 100 microM verapamil blocked the hormonal response without affecting basal transport. The calcium ionophore A23187 rapidly stimulated endocytosis, hexose and amino acid transport. These data indicate that androgenic stimulation of membrane transport functions involves an increased influx of extracellular calcium and a mobilization of intracellular calcium. Increased cytosolic Ca2+ is probably the regulatory signal for these transport processes.     

Ca2+-activated K+ channels in human red cells. Comparison of single-channel currents with ion fluxes

Exposure of the inner surface of intact red cells or red cell ghosts to Ca2+ evokes unitary currents that can be measured in cell-attached and cell-free membrane patches. The currents are preferentially carried by K+ (PK/PNa 17) and show rectification. Increasing the Ca2+ concentration from 0 to 5 microM increases the probability of the open state of the channels parallel to the change of K+ permeability as observed in suspensions of red cell ghosts. Prolonged incubation of red cell ghosts in the absence of external K+ prevents the Ca2+ from increasing K+ permeability. Similarly, the probability to find Ca2+-activated unitary currents in membrane patches is drastically reduced. These observations suggest that the Ca2+-induced changes of K+ permeability observed in red cell suspensions are causally related to the appearance of the unitary K+ currents. Attempts to determine the number of K+ channels per cell were made by comparing fluxes measured in suspensions of red cells with the unitary currents in membrane patches as determined under comparable ionic conditions. At 100 mM KCl in the external medium, where no net movements of K+ occur, the time course of equilibration of 86Rb+ does not follow a single exponential. This indicates a heterogeneity of the response to Ca2+ of the cells in the population. The data are compatible with the assumption that 25% of the cells respond with Pk = 33.2 X 10(-14)cm3/s and 75% with Pk = 3.1 X 10(-14)cm3/s. At 100 mM external K+ the zero current permeability of a single channel is 6.1 X 10(-14)cm3/s (corresponding to a conductance of 22 pS).(ABSTRACT TRUNCATED AT 250 WORDS)     

Ca2+-dependent K+ transport in lymphocytes

The treatment of rat thymocytes with A23187 + Ca2+, ascorbate-phenazine methosulphate or propranolol induced quinine-sensitive fluxes of K+ (Rb+) suggesting the presence in the cell membrane of Ca2+-dependent K+ channels. Concanavalin A induced K+ channel activation only at very high doses (13 micrograms/ml). Neither quinine nor the increase of the K+ concentration in the medium to 30 mM prevented the stimulation of amino acid transport induced by concanavalin A, suggesting that the Ca2+-dependent K+ channel is not involved in the early phenomena of lymphocyte activation.     

Presence of a Ca2+-dependent K+ channel in brown adipocytes. Possible role in maintenance of alpha 1-adrenergic stimulation

We have previously demonstrated mobilization of Ca2+ in and 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 the alpha 1-subtype. Further, we have found that the Rb+ (K+) efflux was dependent upon a primary Ca2+ mobilization event in response to the alpha 1-adrenergic stimulation, since the Rb+ efflux could also be demonstrated by the addition of the Ca2+ 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 alpha 1-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 to alpha 1-adrenergic stimulation is discussed.     

The modulation of rat brain Na(+)-Ca2+ exchange by K+

The involvement of potassium ions in the Na(+)-Ca2+ exchange process was studied in rat brain synaptic plasma membrane (SPM) vesicles. Addition of equimolar [K+] to the intravesicular and the extravesicular medium led to a stimulation of the Na+ gradient-dependent Ca2+ influx; this stimulation was noticeable already at 0.5 mM and reached its maximum at 2 mM K+. The magnitude of the K+ stimulation was between 1.3-2.5-fold in different SPM preparations. K+ ions also stimulated the Na(+)-dependent Ca2+ efflux. K+ stimulation of Na(+)-Ca2+ exchange is of considerable specificity, since it is not mimicked by either Li+ or H+. The following lines of evidence suggest that K+ modulation of Na(+)-Ca2+ exchange involves the catalytic moiety of the transporter itself and not an unrelated K+ channel which modulates the membrane potential. 1) K+ stimulation of the transport process was conserved following reconstitution of the transporter into phospholipid-rich liposomes, an experimental condition which presumably separates the native membrane proteins among different vesicular structures. 2) K+ stimulation of Na+ gradient-dependent Ca2+ influx persists also when the build up of negative inside membrane potential is prevented by addition of carbonyl cyanide p-trifluoromethoxy phenylhydrazone which renders the membrane highly permeable to protons both in the native and the reconstituted preparation. 3) K+ stimulation of Na+ gradient-dependent Ca2+ influx is obtained also when tetraethylammonium chloride, 2,3-diaminopyridine and Cs+ are added to the Ca2+ uptake medium. Reconstituted SPM vesicles take up 86Rb+ in response to activation of Na+ gradient-dependent Ca2+ influx. The ratio of Ca2+ taken up by SPM vesicles in a Na+ gradient-dependent manner to the corresponding amounts of Rb+ taken up varies between 8-5 in different SPM preparations. If the stoichiometry of the process is 1 Rb+/1 Ca2+, then Rb+ cotransport is mediated by 10-20% of the transporters present in the preparation.     

Stable membrane potentials and mechanical K+ responses activated by internal Ca2+ in HeLa cells

Stable membrane potentials have been measured with microelectrodes in HeLa cells and their average value (-38 mV) compares well with the Cl- electrochemical potential (-34 mV). A hyperpolarizing response is observed upon microelectrode penetrations and following a mechanical or electrical shock; this response is due to a large increase in the K+ permeability, which can also be triggered by Ca2+ injection or blocked by EGTA injection through the microelectrode. Stable hyperpolarizations are obtained with the Ca2+ ionophore A 23187.     

Unidirectional K+ fluxes in rat thymocytes stimulated by concanavalin A.

Unidirectional K+ fluxes were estimated in isolated rat thymocytes by 42K exchange kinetics. The cells were either preloaded with isotope and the release of it measured during incubation for one hour at 38 degrees C, or the cellular uptake of isotope during a similar incubation was measured. The influx rate of untreated thymocytes was: 2.3-10(-12) moles cm-2-s-1 and efflux rate: 1.8-10(-12) moles cm-2-s-1. When con A was added to the cells, influx was raised 74% and efflux 65%. Maximal effect was obtained when the concentration of con A was 15 mug/ml, but concentrations as low as 0.75 mug/ml were effective. Hydrocortisone resistant thymocytes responded at least was well as untreated cells to con A, which also raised RNA synthesis rate in the former cells 2.5 times. Using an extracellular marker, 51CrEDTA, intracellular concentrations of some ions was estimated in the thymocytes after one hour incubation: Na+: 30 mmoles/kg water, K+: 177 mmoles/kg water and Cl-:43 mmoles/kg water. Cellular water content: 69%. These values were not found significantly altered when con A was present. Since con A raised influx and efflux to the same extent and no net flux of K+ could be detected, it is proposed that both active and passive transport of K+ was increased by con A. The increased fluxes induced by con A, can apparently not be reversed by removal of con A from the incubation medium or by addition of the inhibiting hapten, alpha-methyl-D-mannoside.     

Delayed activation of calcium pump during transient increases in cellular Ca2+ concentration and K+ conductance in hyperpolarizing human red cells

The net Ca2+ influx was increased in human red cells in suspension by adding moderate concentrations of the Ca2+ ionophore A23187, and due to the increased cellular Ca2+ concentration [( Ca]i) the K+ channels opened (the 'Gardos effect'). At low K+ concentration and with the protonophore CCCP in the buffer-free medium the cells hyperpolarized and the extracellular pH (pH0) increased, enhancing the A23187-mediated net Ca2+ influx. This elicited a prolonged response, viz. a primary transient increase of pH0 and [Ca]i followed by one or more spontaneous pH0 and [Ca]i transients. We explored the pump-mediated Ca2+ efflux by blocking the A23187-mediated Ca2+ flux with CoCl2 at appropriate times during the prolonged response. The Ca2+ pumping was higher during the descendent than during the ascendent phase of the primary transient at equal values of [Ca]i. The data were analyzed using a mathematical model that accounts for the prolonged oscillatory response, including pH0 and [Ca]i. In conclusion, the activation of the Ca2+ pump is delayed due to slow binding of cellular calmodulin, which is a hysteretic response to a rapid increase of the cellular Ca2+ concentration. This mechanism may be important for generation and execution of transient signals in other types of cell.     

Biochemical studies of the excitable membrane of Paramecium aurelia. I. 45Ca2+ fluxes across resting and excited membrane.

The characteristics of Ca2+ transport across the excitable membrane of Paramecium aurelia were studied by measuring 45Ca2+ influx and efflux. The intracellular concentration of free Ca2+ in resting P. aurelia was at least ten times less than the extracellular concentration. Ca2+ influx was easily measurable at 0 degrees C, but not at 23 degrees C. The influx of 45Ca2+ was stimulated by the same conditions which cause membrane depolarization and ciliary reversal. Addition of Na+ and K+ (which stimulate ciliary reversal) resulted in a 10-fold increase in the rate of Ca2+ influx. An externally applied, pulsed, electric field (1-2 mA/cm2 of electrode surface), caused the rate of Ca2+ influx to increase 3-5 times, with the extent of stimulation dependent on the current density and the pulse width. Ca2+ influx had the characteristics of a passive transport system and was associated with the chemically or electrically triggered Ca2+ "gating" mechanism, which has been studied electrophysiologically. In contrast, Ca2+ efflux appeared to be catalyzed by an active transport system. With cells previously loaded at 0 degrees C with 45Ca2+, Ca2+ efflux was rapid at 23 degrees C, but did not occur at 0 degrees C. This active Ca2+ efflux mechanism is probably responsible for maintaining the low internal Ca2+ levels in unstimulated cells.     

Induction of 86Rb fluxes by Ca2+ and volume changes in thymocytes and their isolated membranes

Cell swelling and elevated intracellular Ca2+ increase K+ permeability in lymphocytes. Experiments were performed to test whether these effects can also be elicited in isolated plasma membrane vesicles. Rabbit thymocytes, used as a source of membrane vesicles, were found to regain their volume after swelling in hypotonic, low-K+ media. This regulatory volume decrease (RVD) was inhibited by quinine and trifluoperazine, but not affected by ouabain. Both efflux and uptake of K+ (86Rb) were stimulated by hypotonicity. Addition of A23187 plus Ca2+ also increased 86Rb fluxes. Ca2+- and volume-induced 86Rb fluxes were also studied in isolated membranes. A plasma membrane-rich vesicle fraction, enriched over 11-fold in 5'-nucleotidase, was isolated from thymocytes. The vesicles were about 35% inside-out and trapped 86Rb in an osmotically active compartment of approximately 1.3 microliter/mg protein. Equilibrium exchange fluxes of 86Rb in the vesicles were unaffected by Ca2+ with or without A23187. Calmodulin had no effect on 86Rb permeability but stimulated ATP-dependent Ca2+ accumulation. Hypotonic swelling increased both uptake and efflux of 86Rb from vesicles. However, this increase was not blocked by either quinine or trifluoperazine, was not specific for K+ (86Rb), and is probably unrelated to RVD. It is concluded that components essential for the volume- and Ca2+-induced changes in K+ permeability are lost or inactivated during membrane isolation. An intact cytoarchitecture may be required for RVD.     

Quinine and quinidine inhibit and reveal heterogeneity of K-Cl cotransport in low K sheep erythrocytes

Low K (LK) sheep red blood cells (SRBCs) serve as a model to study K-Cl cotransport which plays an important role in cellular dehydration in human erythrocytes homozygous for hemoglobin S. Cinchona bark derivatives, such as quinine (Q) and quinidine (QD), are effectively used in the treatment of malaria. In the present study, we investigated in LK SRBCs, the effect of various concentrations of Q and QD on Cl-dependent K efflux and Rb influx (K(Rb)-Cl flux), activated by either swelling in hyposmotic media, thiol alkylation with N-ethylmaleimide (NEM), or by cellular Mg (Mg _i ) removal through A23187 in the presence of external chelators. K efflux or Rb influx were determined in Cl and NO₃ medium and K(Rb)-Cl flux was defined as the Cl-dependent (Cl minus NO₃) component. K(Rb)-Cl flux stimulated by all three interventions was inhibited by both Q and QD in a dose-dependent manner. Maximum inhibition of K(Rb)-Cl flux occurred at Q and QD concentrations ?1 mm. The inhibitory effect of Q was manifested in Cl, but not in NO₃, whereas QD reduced K and Rb fluxes both in Cl and NO₃ media. The mean 50% inhibitory concentration (IC⁵⁰) of Q and QD to inhibit K(Rb)-Cl flux varied between 0.23 and 2.24 mm. From determinations of the percentages of inhibition of the different components of K and Rb fluxes, we found that SRBCs possess a Cl-dependent QD-sensitive and a Cl-dependent QD-insensitive K efflux and Rb influx. These two components vary in magnitude depending on the manipulation and directional flux, but in average they are about 50% of the total Cl-dependent flux. This study raises the possibility that, in SRBCs, the Cl-dependent K(Rb) fluxes are heterogeneous. This work was supported by a grant from the National Institutes of Health (NIH DK5RO1 37,160).     

Modulation of Ca2+-mediated K+-gating of erythrocyte ghosts by external Ca-EGTA

Using 86Rb+ as a marker for K+ permeability, we find that extracellular Ca-EGTA influences the rate of 86Rb+ efflux from erythrocyte ghosts preloaded with 86Rb+ and "buffered" Ca2+. At an internal free Ca2+, where the rate of 86Rb+ efflux is minimal and uninfluenced by either external EGTA or external Ca2+, external Ca-EGTA at 0.2-0.5 mM can raise the flux rate to as high as can be attained by raising internal Ca2+, in the presence of an excess externally either of Ca2+ or of EGTA. Higher concentrations of Ca-EGTA (up to 1-2 mM) diminish the flux rate. External Ca-EDTA or Mg-EDTA can substitute for Ca-EGTA in enhancing and suppressing flux rate. The peak rate is insensitive to external free Ca2+ but depends on internal Ca2+; internal Mg-EDTA does not substitute for internal Ca-EGTA. Thus, the erythrocyte membrane is asymmetric with respect to its interaction with Ca2+ and Ca-EGTA. Also, 22Na+ does not substitute for 86Rb+. The peak rate of 86Rb+ flux produced by external Ca-EGTA is diminished by chlorpromazine (0.1 mM) and augmented by 1-propranolol (25 microM), in the same way as the rate produced by increasing internal Ca2+. The results suggest that external Ca-EGTA enhances the affinity of internal Ca2+ for its receptor(s) which operate the K+-gate at the inner surface of the membrane. At external concentrations of Ca-EGTA above 1-2 mM, 86Rb+ flux rate again rises with increase of Ca-EGTA. This phenomenon does not depend upon internal Ca2+, is not affected by chlorpromazine or by 1-propranolol, and is associated with an enhanced permeability to 22Na+, inulin, and haemoglobin.