Effect of ethidium bromide on Ca2+ uptake by yeast

Summary Ethidium bromide and other cationic dyes have been found to inhibit movalent cation uptake. This dye also produces in a K⁺-free medium an efflux of K⁺ which could be of the electrogenic type.The study of the effects of the same cationic dyes on Ca²⁺ uptake showed a large stimulation of the uptake rate of the divalent cation of more than tenfold.The analysis of the effects of one of the cationic dyes on Ca²⁺ uptake indicated that the efflux of K⁺ is of the electrogenic type and can drive the uptake of the divalent cation.Kinetic data on Ca²⁺ uptake indicate that, both under normal or under stimulated conditions, the divalent cation is taken up by the same transport system.The addition of ethidium bromide, besides, can stimulate the uptake of Mn²⁺ and¹⁴C-glycine and could be a good weapon to magnify and study some of the characteristics of ion transport systems in yeast.     

Effect of ionophore A23187 upon membrane function and ion movement in human and toad erythrocytes

Summary Addition of 0.1–0.3 m A23187, a divalent cation ionophore, to human erythrocytes suspended in a 1.0mm ⁴⁵Ca²⁺-containing buffer results in a small ( two fold) increase in [Ca²⁺] _i , a significant decrease in osmotic fragility, and a decrease in intracellular K⁺ (100 mmoles/liter of cells to 70 mmoles/liter cells) without significant alteration of intracellular [Na⁺]. This decrease in [K⁺] _i is associated with a significant decrease in packed cell volume and correlates directly with the observed alteration is osmotic fragility. Increasing extracellular K⁺ to 125mm prevents the A23187-induced changes in osmotic fragility, K⁺ content and cell volume, but does not prevent the ionophore-induced uptake of⁴⁵Ca²⁺. Addition of 0.1–0.3 m A23187 to toad erythrocytes leads to an increase in⁴⁵Ca²⁺ uptake comparable to that observed in human erythrocytes, but does not alter osmotic fragility, cell volume or K⁺ content. Higher concentrations of ionophore (3.0–10.0 m) cause a 30- to 50-fold increase in⁴⁵Ca²⁺ uptake and concomitant change in K⁺ content, cell volume and osmotic fragility. These changes in cell properties can be prevented by increasing extracellular [K⁺] to 90mm. The difference in sensitivity of the two cell types to A23187 is attributed to the presence of additional intracellular calcium pools within toad erythrocytes that prevent an increase in cytoplasmic Ca²⁺ until Ca²⁺ uptake is increased substantially at the higher concentrations of A23187.     

Characterization of inside-out oriented H+-ATPases in cholate-pretreated renal brush-border membrane vesicles

Summary Exposure of porcine renal brush-border membrane vesicles to 1.2% cholate and subsequent detergent removal by dialysis reorients almost all N-ethylmaleimide (NEM)-sensitive ATPases from the vesicle inside to the outside. ATP addition to cholate-pretreated, but not to intact, vesicles causes H⁺ uptake as visualized by the pH indicator, acridine organge. The reoriented H⁺-pump is electrogenic because permeant extravesicular anions or intravesicular K⁺ plus valinomycin enhance H⁺ transport. ATP stimulates H⁺ uptake with an apparentK _m of 93 m. Support of H⁺ uptake andP _i liberation by ATP>GTPITP> UTP indicates a preference for ATP and utilization of other nucleotides at lower efficiency. ADP is a potent, competitive inhibitor of ATP-driven H⁺ uptake,(K _i , 24 m). Mg²⁺ and Mn^2– support ATP-driven H⁺ uptake, but Ca²⁺, Ba²⁺ and Zn²⁺ do not. Imm Zn²⁺ inhibits MgATP-driven H⁺ transport completely. NEM-sensitiveP _i liberation is stimulated by Mg²⁺ and Mg^2– and, unlike H⁺ uptake, also by Ca²⁺ suggesting Ca²⁺-dependent ATP hydrolysis unrelated to H⁺ transport. The inside-out oriented H⁺-pump is relatively insensitive toward oligomycin, azide, N,N-dicyclohexylcarbodiimide (DCCD) and vanadate, but efficiently inhibited by NEM (apparentK _i , 0.77 m), and 4-chloro-7-nitro-benzoxa-1,3-diazole (NBD-Cl; apparentK _i , 0.39 m). Taken together, the H⁺-ATPase of proximal tubular brush-border membranes exhibits characteristics very similar to those of vacuolar type (V-type) H⁺-ATPases. Hence,V-type H⁺-ATPases occur not only in intracellular organelles but also in specialized plasma membrane areas.     

Modulation of the serotonin-activated K+ channel by G protein subunits and nucleotides in rat hippocampal neurons

In hippocampal neurons, 5-hydroxytryptamine (5-HT) activates an inwardly rectifying K⁺ current via G protein. We identified the K⁺ channel activated by 5-HT (K_5-HT channel) and studied the effects of G protein subunits and nucleotides on the K⁺ channel kinetics in adult rat hippocampal neurons. In inside-out patches with 10 m 5-HT in the pipette, application of GTP (100 m) to the cytoplasmic side of the membrane activated an inwardly rectifying K⁺ channel with a slope conductance of 36±1 pS (symmetrical 140 mm K⁺) at –60 mV and a mean open time of 1.1±0.1 msec (n=5). Transducin activated the (K_5-HT) channels and this was reversed by -GDP. Whether the K_5-HT channel was activated endogenously (GTP, GTPS) or exogenously (), the presence of 1 mm ATP resulted in a 4-fold increase in channel activity due in large part to the prolongation of the open time duration. These effects of ATP were irreversible and not mimicked by AMPPMP, suggesting that phosphorylation might be involved. However, inhibitors of protein kinases A and C (H-7, staurosporine) and tyrosine kinase (tyrphostin 25) failed to block the effect of ATP. These results show that G activates the G protein-gated K⁺ channel in hippocampal neurons, and that ATP modifies the gating kinetics of the channel, resulting in increased open probability via as yet unknown pathways.     

Examination of the substrate stoichiometry of the intestinal Na+/phosphate cotransporter

Summary The substrate stoichiometry of the intestinal Na⁺/phosphate cotransporter was examined using two measures of Na⁺-dependent phosphate uptake: initial rates of uptake with [³²P] phosphate and phosphate-induced membrane depolarization using the potential-sensitive dye diSC₃(5). Isotopic phosphate measures electrogenic and electroneutral Na⁺-dependent phosphate uptake, while phosphate-induced membrane depolarization measures electrogenic phosphate uptake. Using these measures of Na-dependent phosphate uptake, three parameters were compared: substrate affinity; phenylglyoxal sensitivity and labeling; and inhibiton by mono- and di-fluorophosphates. Na⁺/phosphate cotransport was found to have similar Na⁺ activations (apparentK _0.5's of 28 and 25mm), apparentK _m 's for phosphate (100 and 410 m), andK _0.5's for inhibition by phenylglyoxal (70 and 90 m) using isotopic phosphate, uptake and membrane depolarization, respectively. Only difluorophosphate inhibited Na⁺-dependent phosphate uptake below 1mm at pH 7.4.Difluorophosphate also protected a 130-kDa polypeptide from FITC-PG labeling in the presence of Na⁺ with apparentK _0.5 for phosphate of 200 m; similar to the apparentK _m for phosphate uptake, andK _0.5 for phosphate protection against FITC-PG inhibition of Na⁺-dependent phosphate uptake and FITC-PG labeling of the 130-kDa polypeptide. These results indicate that the intestinal Na⁺/phosphate cotransporter is electrogenic at pH 7.4, that H₂PO ₄ ^– is the transport-competent species, and that the 130-kDa polypeptide is an excellent candidate for the intestinal Na⁺/phosphate cotransporter.     

Ca2+ pumping ATPase of cardiac sarcolemma is insensitive to membrane potential produced by K+ and Cl− gradients but requires a source of counter-transportable H+

Summary The sensitivity of the Ca²⁺ pumping ATPase of bovine cardiac sarcolemma (SL) to changes in membrane potential was studied in a preparation of sealed SL vesicles. Membrane potential was imposed by preincubating the vesicles in media of defined ion composition (K⁺, Cl^–, choline⁺ and gluconate^–) and diluting into media of differing ion composition. The durations of the ion gradients and relative ion permeabilities were determined in separate experiments by the dependence of the half time for net K⁺ (or choline⁺) movement coupled with these anions (Cl^– or gluconate^–), registered by the fluorescence of 1-anilino-8-naphthalene sulfonate (Chiu, V.C.K., Jaumes. D.H. 1980.J. Membrane Biol. 56:203–218). Relative permeabilities were: 1.0, K⁺, 10.0, 1 m valinomycin-K⁺; 4.0, Cl^–, 0.66, choline⁺; 0.38, gluconate^–. Durations of the gradients ranged between 17 sec (KCl, valinomycin) to 195 sec (K⁺-gluconate^–). In separate experiments. active Ca²⁺ uptake was monitored using chlorotetracycline (CTC) fluorescence, a technique validated by 45-Ca²⁺ measureaments (Dixon, D., Brandt, N., Haynes, D.H. 1984.J. Biol. Chem. 259:13737–13741). Active Ca²⁺ uptake was initiated in the presence of monovalent ion gradients. The values of the membrane potentials (E _m ) imposed by the monovalent ion gradients were calculated using the ion concentrations, their relative permeabilities and the Goldman-Hodgkin-Katz equation. No effect of membrane potential on transport rate was observed (4%, for 5–7%sd) for imposed potentials as extreme as +71 and –67 mV. Formal analysis shows that the above observations are not compatible with models in which the Ca²⁺ pumping ATPase functions in an electrogenic or charge-uncompensated fashion. Further experimentation showed that the pump rate is slowed when uptake is measured at less-than-adequate concentrations of buffer (5vs. 25mm HEPES/Tris). This, together with further control experiments using nigericin and FCCP, gave evidence that the pump requires a source of counter-transportable H⁺ in the vesicle lumen. The above experimentation also underlines the need for control of internal pH to obviate erroneous interpretation of ion perturbation experiments. The results are compared with results obtained with the Ca²⁺ ATPase pump of skeletal sarcoplasmic reticulum.     

Role of the sodium pump and the background K+ channel in passive K+(Rb+) uptake by isolated cardiac sarcolemmal vesicles

Summary A simple procedure was developed for the isolation of a sarcolemma-enriched membrane preparation from homogenates of bullfrog (Rana catesbeiana) heart. Crude microsomes obtained by differential centrifugation were fractionated in Hypaque density gradients. The fraction enriched in surface membrane markers consisted of 87% tightly sealed vesicles. The uptake of⁸⁶Rb⁺ by the preparation was measured in the presence of an opposing K⁺ gradient using a rapid ion exchange technique. At low extravesicular Rb⁺ concentrations, at least 50% of the uptake was blocked by addition of 1mm ouabain to the assay medium. Orthovanadate (50 m), ADP (2.5mm), or Mg (1mm) were also partial inhibitors of Rb⁺ uptake under these conditions, and produced a complete block of Rb⁺ influx in the presence of 1mm ouabain. When⁸⁶Rb⁺ was used as a tracer of extravesicular K⁺ (Rb ₀ ⁺ 40 m K ₀ ⁺ =0.1–5mm) a distinct uptake pathway emerged, as detected by its inhibition by 1mm Ba²⁺ (K _0.5=20 m). At a constant internal K⁺ concentration (K _in ⁺ =50mm) the magnitude of the Ba²⁺-sensitive K⁺ uptake was found to depend on K ₀ ⁺ in a manner that closely resembles the K⁺ concentration dependence of the background K⁺ conductance (I _Kl) observed electrophysiologically in intact cardiac cells. We conclude that K⁺ permeates passively this preparation through two distinct pathways, the sodium pump and a system identifiable as the background potassium channel.     

Rabit distal colon epithelium: II. Characterization of (Na+, K+, Cl−)-cotransport and [3H]-bumetanide binding

Summary Loop diuretic-sensitive (Na⁺,K⁺,Cl^–)-cotransport activity was found to be present in basolateral membrane vesicles of surface and crypt cells of rabbit distal colon epithelium. The presence of grandients of all three ions was essential for optimal transport activity (Na⁺,K⁺) gradien-driven³⁶Cl fluxes weree half-maximally inhibited by 0.14 m bumetanide and 44 m furosimide. While⁸⁶Rb uptake rates showed hyperbolic dependencies on Na⁺ and K⁺ concentrations with Hill coefficients of 0.8 and 0.9, respectively, uptakes were sigmoidally related to the Cl^– concentration, Hill coefficient 1.8, indicating a 1 Na⁺: 1 K⁺:2 Cl^– stoichiometry of ion transport.The interaction of putative (Na⁺, K⁺, Cl^–)-cotransport proteins with loop diuretics was studied from equilibrium-binding experiments using [³H]-bumetanide. The requirement for the simulataneous presence of Na⁺,K⁺, and Cl^–, saturability, reversibility, and specificity for diuretics suggest specific binding to the (Na⁺, K⁺, Cl^–)-cotransporter. [³H]-bumetanide recognizes a minimum of two classes of diuretic receptors sites. high-affinity (K _D1=0.13 m;B _max1 =6.4 pmol/mg of protein) and low-affinity (K _D2=34 m;B _max2=153 pmol/mg of protein) sites. The specific binding to the high-affinity receptor was found to be linearly competitive with Cl^– (K ₁=60mm), whereas low-affinity sites seem to be unaffected by Cl^–. We have shown that only high-affinity [³H]-bumetanide binding correlates with transport inhibition raising questions on the physiological significance of diuretic receptor site heterogeneity observed in rabbit distal colon epithelium.     

Malic enzyme of Chromatium vinosum

Malic enzyme of the phototrophic bacterium Chromatium vinosum strain D that lacks malate dehydrogenase was partially purified yielding a specific activity of 55 units/mg protein. The constitutive enzyme with a molecular weight of 110,000 and a pH optimum of 8.0 was absolutely dependent on the presence of a monovalent cation (NH ₄ ⁺ , K⁺, Cs⁺, or Rb⁺) as well as a divalent cation (Mn²⁺, or Mg²⁺). The enzyme was inhibited by oxaloacetate, glyoxylate, and NADPH. The K _0.5 value for L-malate and the inhibition constants for oxaloacetate and glyoxylate are dependent on the concentration of the monovalent cation, whereas the K _m value for NADP (18 M) and the K ₁ value for NADPH (42 M) are independent. Throughout all kinetic measurements hyperbolic saturation curves and linear double reciprocal plots were obtained.Abbreviations OAA oxaloacetate - OD optical density     

High-affinity sodium-dependent uptake of ascorbic acid by rat osteoblasts

Summary Ascorbic acid is essential for the formation of bone by osteoblasts, but the mechanism by which osteoblasts transport ascorbate has not been investigated previously. We examined the uptake ofl-[¹⁴C]ascorbate by a rat osteoblast-like cell line (ROS 17/2.8) and by primary cultures of rat calvaria cells. In both systems, cells accumulatedl-[¹⁴C]ascorbate during incubations of 1–30 min at 37°C. Unlike propionic acid, which diffuses across membranes in protonated form, ascorbic acid did not markedly alter cytosolic pH. Initial ascorbate uptake rate saturated with increasing substrate concentration, reflecting a high-affinity interaction that could be described by Michaelis-Menten kinetics (apparentK _m =30±2 m andV _max=1460±140 nmol ascorbate/g protein/min in ROS 17/2.8 cells incubated with 138mm extracellular Na⁺). Consistent with a stereoselective carrier-mediated mechanism, unlabeledl-ascorbate was a more potent inhibitor (IC₅₀=30±5 m) ofl-[¹⁴C]ascorbate transport than wasd-isoascorbate (IC₅₀=380±55 m). Uptake was dependent on both temperature and Na⁺, since it was inhibited by cooling to 4°C and by substitution of K⁺, Li⁺ or N-methyl-d-glucamine for extracellular Na⁺. Decreasing the external Na⁺ concentration lowered both the affinity of the transporter for ascorbate and the apparent maximum velocity of transport. We conclude that osteoblasts possess a stereoselective, high-affinity, Na⁺-dependent transport system for ascorbate. This system may play a role in the regulation of bone formation.     

[3H]Histamine uptake and release by astrocytes from rat brain: Effects of sodium deprivation,high potassium,and potassium channel blockers

Histamine transport has been characterized in cultured astroglial cells of rat brain. The kinetics of [³H]-histamine uptake yielded a K_m of 0.19±0.03 M and a V_max of 3.12±0.75 pmol×mg protein^–1×min^–1. Transport system revealed high affinity for histamine and an approximately ten times higher capacity than that shown in cultured glial cells of chick embryonic brain. Ouabain which interferes with utilization of ATP to generate ion gradients, and the replacement of Na⁺ with choline inhibited the initial rate of uptake showing a strong Na⁺-dependency and suggesting the presence of a tightly coupled sodium/histamine symporter. Dissipation of K⁺-gradient (in>out) by high K⁺ or by K⁺-channel blockers, BaCl₂, (100 M), quinine (100 M) or Sparteine (20 M) produced also remarkable inhibitions in the uptake of [³H]-histamine. Impromidine, a structural histamine-analogue could inhibit the uptake non-competitively in a range of concentrations of 1 to 10 M with a K_i value of 2.8 M, indicating the specificity of the uptake. [³H]histamine uptake measurements carried out by using a suspension of dissociated hypothalamic cells, of rat brain showed a strong gliotoxin-sensitivity and yielded a Km of 0.33±0.08 M; and a V_max of 2.65±0.35 pmoles×mg protein^–1×min^–1. The uptake could be reversed by incubating the cells in histamine-free Krebs medium. The [³H]histamine efflux was sensitive to Na⁺ omission, ouabain treatment and high K⁺ or K⁺ channel blockers, resulting in marked elevations in the efflux. Data indicate that glial uptake of histamine is a high affinity, Na⁺-dependent and electrogenic, driven by an inward-oriented sodium ion gradient and an outward-oriented potassium ion gradient and functions as part of histamine inactivation, at least in a shunt mechanism.Abbreviations used HA histamine - [³H]HA [2.5-³H]-histamine - dl--aAA dl-alpha-aminoadipic acid - (Na⁺+K⁺) ATP-ase sodium and potassium activated adenosine triphosphatase - SAH S-Adenosyl-d-Homocysteine - HNMT histamine-N-methyltransferase     

Riboflavin uptake by rat small intestinal brush border membrane vesicles: A dual mechanism involving specific membrane binding

The first step of riboflavin absorption was studied by determining the uptake of the vitamin by rat small intestinal brush border membrane vesicles. Vesicles were incubated at 25°C in the presence of [³H]-riboflavin at concentrations within the physiological intraluminal range for rat. The time course of [³H]-riboflavin uptake was unaffected by Na⁺ or K⁺ gradients. The 5 sec uptake rate plotted as a function of the initial concentration of [³H]-riboflavin in the medium (0.125 to 7.5 m) revealed the presence of a dual mechanism, with a saturable component (apparent kinetic constants: 0.12 m for K _m and 0.36 pmol · mg^-1 protein · 5 sec^-1 for J _max) prevailing at low concentrations (<2 m), and a nonsaturable component prevailing at higher concentrations. The presence of a carrier-mediated system for riboflavin was validated by counter-transport experiments. At equilibrium, uptake was almost completely accounted for by membrane binding, whereas at earlier times the transport component accounted for about 30% of total uptake. The plot of [³H]-riboflavin binding at equilibrium as a function of its concentration in the medium was quite similar to that of the 5 sec uptake rate in both intact and osmotically shocked vesicles and demonstrated the occurrence of a saturable component: binding constants were 0.07 (K _d) in m), 0.54 (B _max in pmol · mg^-1 protein), and 0.11 (K _d), 1.13 (B _max, respectively, indicating the existence of specific riboflavin binding sites. The specificity of riboflavin binding to the membrane was confirmed by preliminary studies with structural analogues. Specific binding could represent the first step of a specific riboflavin entry mechanism in enterocytes.This research was supported by grants from Italian MPI 60% (1989, 1992) and CNR n. 90, 02467 CT 04. We wish to express our gratitude to Prof. E. Perucca (Department of Internal Medicine, Clinical Pharmacology Unit, University of Pavia) for revising the English, and to Mrs. M. Agrati Greco and Mrs. P. Vai Gatti for secretarial assistance and excellent typing.     

Evidence for two K+ currents activated upon hyperpolarization ofParamecium tetraurelia

Summary Hyperpolarization of voltage-clampedParamecium tetraurelia in K⁺ solutions elicits a complex of Ca²⁺ and K⁺ currents. The tail current that accompanies a return to holding potential (–40 mV) contains two K⁺ components. The tail current elicited by a step to –110 mV of 50-msec duration contains fast-decaying (3.5 msec) and slow-decaying (20 msec) components. The reversal potential of both components shifts by 55–57 mV/10-fold change in external [K⁺], suggesting that they represent pure K⁺ currents. The dependence of the relative amplitudes of the two tail currents on duration of hyperpolarization suggests that the slow K⁺ current activates slowly and is sustained, whereas the fast current activates rapidly during hyperpolarization and then rapidly inactivates. Iontophoretic injection of a Ca²⁺ chelator, EGTA, specifically reduces slow tail-current amplitude without affecting the fast tail component. Both K⁺ currents are inhibited by extracellular TEA⁺ in a concentration-dependent, noncooperative manner, whereas the fast K⁺ current alone is inhibited by 0.7mm quinidine.     

Alanine and taurine transport by the gill epithelium of a marine bivalve: Effect of sodium on influx

Summary Marine mussels can accumulate amino acids from seawater into the epithelial cells of the gill against chemical gradients in excess of 5×10⁶ to 1. Uptake of both alanine and taurine into gill tissue isolated fromMytilus californianus was found to be dependent upon Na⁺ in the external solution. Uptake of these amino acids was described by Michaelis-Menten kinetics, and a reduction in external [Na⁺] (from 425 to 213mm) increased the apparent Michaelis constants (alanine, from 8 to 17 m; taurine, from 4 to 39 m) without a significant influence on theJ _max's of these processes. Fivemm harmaline, an inhibitor of Na-cotransport processes in many systems, reduced both alanine and taurine uptake by more than 95%; this inhibition appeared to be competitive in nature, with an apparentK _i of 43 m for the interaction with alanine uptake. Increasing the external [Na⁺] from 0 to 510mm produced a sigmoid activation of alanine and taurine uptake withK _Na's of approximately 325mm. The apparent Hill coefficients for this activation were 7.3 and 7.4 for alanine and taurine, respectively. These data are consistent with uptake mechanisms which require comparatively high concentrations of Na⁺ to activate transport, and which couple several Na⁺ ions to the transport of each amino acid. These characteristics, in conjunction with the previously demonstrated low passive permeability of the apical membrane to amino acids, result in systems capable of i) accumulating amino acids from seawater to help meet the nutritional needs of this animal, and ii) maintaining the high intracellular amino-acid concentrations associated with volume regulation in the gill.     

A cation channel in frog lens epithelia responsive to pressure and calcium

Summary Patch-clamp recording from the apical surface of the epithelium of frog lens reveals a cation-selective channel after pressure (about ±30 mm Hg) is applied to the pipette. The open state of this channel has a conductance of some 50 pS near the resting potential (–56.1±2.3 mV) when 107mm NaCl and 10 HEPES (pH 7.3) is outside the channel. The probability of the channel being open depends strongly on pressure but the current-voltage relation of the open state does not. With minimal Ca²⁺ (55±2 m) outside the channel, the current-voltage relation is nonlinear even in symmetrical salt solutions, allowing more current to flow into the cell than out. The channel, in minimal Ca²⁺ solution, is selective among the monovalent cations in the following sequence K⁺>Rb⁺>Cs⁺>Na⁺>Li⁺. The conductance depends monotonically on the mole fraction of K⁺ when the other ion present is Li⁺ or Na⁺. The single-channel current is a saturating function of [K⁺] when K⁺ is the permeant ion, for [K⁺]214mm. When [Ca²⁺]=2mm, the currentvoltage relation is linearized and the channel cannot distinguish Na⁺ and K⁺.     

Role of the furosemide-sensitive Na+/K+ transport system in determining the steady-state Na+ and K+ content and volume of human erythrocytesin vitro andin vivo

Summary To study the physiological role of the bidirectionally operating, furosemide-sensitive Na⁺/K⁺ transport system of human erythrocytes, the effect of furosemide on red cell cation and hemoglobin content was determined in cells incubated for 24 hr with ouabain in 145mm NaCl media containing 0 to 10mm K⁺ or Rb⁺. In pure Na⁺ media, furosemide accelerated cell Na⁺ gain and retarded cellular K⁺ loss. External K⁺ (5mm) had an effect similar to furosemide and markedly reduced the action of the drug on cellular cation content. External Rb⁺ accelerated the Na⁺ gain like K⁺, but did not affect the K⁺ retention induced by furosemide. The data are interpreted to indicate that the furosemide-sensitive Na⁺/K⁺ transport system of human erythrocytes mediates an equimolar extrusion of Na⁺ and K⁺ in Na⁺ media (Na⁺/K⁺ cotransport), a 1:1 K⁺/K⁺ (K⁺/Rb⁺) and Na⁺/Na⁺ exchange progressively appearing upon increasing external K⁺ (Rb⁺) concentrations to 5mm. The effect of furosemide (or external K⁺/Rb⁺) on cation contents was associated with a prevention of the cell shrinkage seen in pure Na⁺ media, or with a cell swelling, indicating that the furosemide-sensitive Na⁺/K⁺ transport system is involved in the control of cell volume of human erythrocytes. The action of furosemide on cellular volume and cation content tended to disappear at 5mm external K⁺ or Rb⁺. Thein vivo red cell K⁺ content was negatively correlated to the rate of furosemide-sensitive K⁺ (Rb⁺) uptake, and a positive correlation was seen between mean cellular hemoglobin content and furosemide-sensitive transport activity. The transport system possibly functions as a K⁺ and waterextruding mechanism under physiological conditiosin vivo. The red cell Na⁺ content showed no correlation to the activity of the furosemide-sensitive transport system.     

Ba2+-Induced conductance fluctuations of spontaneously fluctuating K+ channels in the apical membrane of frog skin (Rana temporaria)

Summary We studied the influence of mucosal Ba²⁺ ions on the recently described (Zeiske & Van Driessche, 1979a, J. Membrane Biol. 47:77) transepithelial, mucosa towards serosa directed K⁺ transport in the skin ofRana temporaria. The transport parametersG (conductance), PD (potential difference),I _sc (short-circuit current, K⁺ current), as well as the noise ofI _sc were recorded. Addition of millimolar concentrations of Ba²⁺ to the mucosal K⁺-containing solution resulted in a sudden but quickly reversible drop inI _sc.G andI _sc decreased continuously with increasing Ba²⁺ concentration, (Ba²⁺) _o . The apparent Michaelis constant of the inhibition by Ba²⁺ lies within the range 40–80 m. The apical membrane seems to remain permselective for K⁺ up to 500 m (Ba²⁺) _o . Higher (Ba²⁺) _o , however, appears to induce a shunt (PD falls,G increases). This finding made an accurate determination of the nature of the inhibition difficult but our results tend to suggest a K⁺-channel block by K⁺–Ba²⁺ competition. In the presence of Ba²⁺, the power spectrum of the K⁺ current shows a second Lorentzian component in the low-frequency range, in addition to the high-frequency Lorentzian caused by spontaneous K⁺-channel fluctuations (Van Driessche & Zeiske, 1980). Both Lorentzian components are only present with mucosal K⁺ and can be depressed by addition of Cs⁺ ions, thus indicating that Ba²⁺ ions induce K⁺-channel fluctuations. The dependence of the parameters of the induced Lorentzian on (Ba²⁺) _o , shows a rise in the plateau values to a maximum around 60 m (Ba²⁺) _o , followed by a sharp and progressive decrease to very low values. The corner frequency which reflects the rate of the Ba²⁺-induced fluctuations, however, increases quasi-linearly up to 1mm (Ba²⁺) _o with a tendency to saturate at higher (Ba²⁺) _o . Based on a three-state model for the K⁺ channel (having one open state, one closed by the spontaneous fluctuation and one blocked by Ba²⁺) computer calculations compared favorably with our results. The effect of Ba²⁺ could be explained by assuming reversible binding at the outer side of the apical K⁺ channel, thereby blocking the open channel in competition with K⁺. The association-dissociation of Ba²⁺ at its receptor site is thought to cause a chopping of the K⁺ current, resulting in modulated current fluctuations.     

Effect of calcium on the H+/K+ ATPase of hog gastric microsomes

Summary The K⁺-stimulated, ouabain-insensitive ATPase activity present in vesicles of microsomal fractions from hog gastric mucosa can be demonstrated in fresh preparations by adding Ca²⁺ (M range) to the incubation medium. Ca²⁺ effect is similar but not additive to the effect of gramicidin or freezing. High Ca²⁺ concentrations (1 mM) produce an inhibotory effect on the K⁺-stimulated ATPase activity. This effect, is not seen in the presence of gramicidin. Calcium increases the magnitude of ATP-driven H⁺ uptake in vesicles exposed to K⁺ for periods of time up to 60 min. At longer times of exposure (120 min) the response does not differ from controls. It is concluded that Ca²⁺ at low concentrations (m range) enhances the K⁺ permeability of the vesicular membrane. At higher concentrations (mm range), Ca²⁺ becomes inhibitory to the K⁺ permeability. A role for Ca²⁺ as a second messenger in stimulus-secretion coupling in the parietal cell is discussed.     

Ouabain resistance of the epithelial cell line (Ma104) is not due to lack of affinity of its pumps for the drug

Na⁺, K⁺-pumps of most eukaryotic animal cells bind ouabain with high affinity, stop pumping, and consequently loose K⁺, detach from each other and from the substrate, and die. Lack of affinity for the drug results in ouabain resistance. In this work, we report that Ma104 cells (epithelial from Rhesus monkey kidney) have a novel form of ouabain-resistance: they bind the drug with high affinity (Km about 4×10^–8 m), they loose their K⁺ and stop proliferating but, in spite of these, up to 100% of the cells remain attached in 1.0 m ouabain, and 53% in 1.0 mm. When 4 days later ouabain is removed from the culture medium, cells regain K⁺ and resume proliferation. Strophanthidin, a drug that attaches less firmly than ouabain, produces a similar phenomenon, but allows a considerably faster recovery. This reversal may be associated to the fact that, while in ouabain-sensitive MDCK cells Na⁺, K⁺-ATPases blocked by the drug are retrieved from the plasma membrane, those in Ma104 cells remain at the cell-cell border, as if they were cell-cell attaching molecules. Cycloheximide (10 g/ml) and chloroquine (10 m) impair this recovery, suggesting that it also depends on the synthesis and insertion of a crucial protein component, that may be different from the pump itself. Therefore ouabain resistance of Ma104 cells is not due to a lack of affinity for the drug, but to a failure of its Na⁺, K⁺-ATPases to detach from the plasma membrane in spite of being blocked by ouabain.We wish to thank Dr. E. Rodríguez-Boulán for the generous supply of Ma104 cells, as well as acknowledge the generous economic support of the National Research Council (CONACYT) of Mexico. Confocal experiments were performed in the Confocal Microscopy Unit of the Physiology Department, CINVESTAV.     

Ionic effects on bumetanide binding to the activated Na/K/2Cl cotransporter: Selectivity and kinetic properties of ion binding sites

Summary The loop diuretic bumetanide binds specifically to the Na/K/2Cl cotransporter of many cell types including duck erythrocytes. Membranes isolated from these erythrocytes retain the ability to bind bumetanide when cells are exposed to cotransport activity stimuli prior to membrane isolation. An extensive study of the effects of ions on specific [³H]bumetanide binding to such membranes is presented here and compared to the activity of these ions in supporting transport function in intact cells. Both Na⁺ and K⁺ enhanced bumetanide binding in a saturable manner consistent with a single-site interaction. The K _m for each ion was dependent on the concentration of the other cation suggesting heterotropic cooperative interactions between the Na⁺ and K⁺ binding sites. Na⁺ and K⁺ were partially replaceable, with the selectivity of the Na⁺ site being Na⁺ > Li⁺ > NH ₄ ⁺ ; N-methyl-d-glucamine⁺, choline⁺ and tetramethylammonium⁺ also supported a small amount of specific binding when substituted for Na⁺. The selectivity of the K⁺ site was K⁺ Rb⁺ > NH ₄ ⁺ > Cs⁺; N-methyl-d-glucamine⁺, choline⁺ and tetramethylammonium⁺ were inactive at this site. The results of transport experiments revealed a slightly different pattern. Li⁺ could partially substitute for Na⁺ in supporting coteansport, but other monovalent cations were completely inactive. The order of potency at the K⁺ site was NH ₄ ⁺ > K⁺ Rb⁺ > Cs⁺ other monovalent cations. The effect of Cl^- on bumetanide binding was biphasic, being stimulatory at low [Cl^-] but inhibitory at high [Cl^-]. As this implies the existence of two Cl^- binding sites (termed Cl _H and Cl _L for the high- and low- affinity sites, respectively) each phase was examined individually. Cl^- binding to Cl _H could be described by a rectangular hyperbola with a K _m of 2.5 mm, while kinetic analysis of the inhibition of bumetanide binding at high [Cl^-] revealed that it was of a noncompetitive type (K _i = 112.9 mm). The selectivity of anion binding to the two sites was distinct. Cl _H was highly selective with Cl^- > SCN^- > Br^-; F^-, NO ₃ ^- , ClO ₄ ^- , MeSO ₄ ^- , gluconate^- and SO ₄ ^2- were inactive. The efficacy of anion inhibition of binding to Cl _L was ClO ₄ ^- > I^- > SCN^- > NO₃ > Cl^-; F^-, MeSO ₄ ^- , gluconate^-, and SO ₄ ^2- were inactive. Thus, Cl _H is much more selective than Cl _L and largely accounts for the specificity of the system with respect to anion transport. SO ₄ ^- , NO ₃ ^- , I^-, SCN^- and ClO ₄ ^- did not support cotransport when bound to Cl _L and the latter three anions were inhibitory. Mg²⁺ was found to stimulate binding at a narrowly defined peak around 1.5 mm, but was inhibitory at higher concentrations. Other divalent cations caused a similar inhibition of bumetanide binding but did not exert a stimulatory effect at 1.5 mm. Divalent cations have little effect on cotransport in intact cells at concentrations up to 20 mm, suggesting that their effects on diuretic binding reflect interactions at internally disposed sites. Bumetanide binding was optimal at a pH of 7.8–8.1 and declined sharply as the pH was lowered towards 6. The titration curve correlated well with the effect of pH on cotransport in intact cells; the inhibitory effect of low pH suggests that protonation of the cotransporter may inhibit its function.We thank Drs. Brad Pewitt, John Westley and Mrinalini Rao for discussion, Sara Leung and Artelia Watson for their excellent technical assistance, and Dr. R.J. Turner for his gift of [³H] bumetanide. This work was supported in part by Cystic Fibrosis Center grant #CF RO11 7-04.