The effect of imidoesters,fluorodinitrobenzene and trinitrobenzenesulfonate on ion transport in human erythrocytes

Several amino-reactive chemical probes which differ in hydrophobicity and charge and in their ability to penetrate the red cell membrane were tested for their ability to modify K⁺ leak and inorganic phosphate (Pi) leak in intact human red cells. Methyl picolinimidate (MP), ethyl acetimidate (EA), methyl acetimidate (MA) are hydrophilic penetrating probes whereas isethionylacetimidate (IA) is a hydrophilic non-penetrating probe. The order of their effectiveness in inhibiting Pi leak was found to be MP>EA>MA>IA. This order is in decreasing hydrophobicity and suggests that some penetration into the bilayer or into hydrophobic domains of the anion transport protein is required to modify an amino group required for Pi permeability through the membrane. These imidoesters have little or no effect on K⁺ leak in the red cell.Trinitrobenzenesulfonate (TNBS) a relatively non-penetrating hydrophobic anionic probe and fluorodinitrobenzene (FDNB) a penetrating hydrophobic neutral probe have markedly different effects on K⁺ and Pi leak. TNBS has little effect on K⁺ leak but markedly inhibits Pi leak. The effect of TNBS on Pi leak is not blocked by prior treatment with IA suggesting that these probes sense different populations of amino groups in the membrane. FDNB nearly completely blocks Pi leak and markedly increases K⁺ leak. The results with TNBS and FDNB indicate an asymmetric arrangement of amino groups on the red cell membrane. Certain amino groups on the outer surface of the membrane regulate Pi permeability whereas certain amino groups on the inner surface of the membrane regulate K⁺ permeabilty. The data also suggest that these amino groups are in a hydrophobic domain.     

Ion permeability of rabbit intestinal brush border membrane vesicles

Summary The ion permeability of rabbit jejunal brush border membrane vesicles was studied by measuring unidirectional fluxes with radioactive tracers and bi-ionic diffusion potentials with the potential-sensitive fluorescent dye, diS–C₃-(5). Tracer measurements provide estimates of the absolute magnitudes of permeability coefficients, while fluorescence measurements provide estimates of relative and absolute ion permeabilities. The magnitudes of the permeability coefficients for Na⁺, K⁺, Rb⁺, and Br^– were approximately 5 nanoliters/(mg protein × sec) or 10^–5 cm/sec as determined by radioactive tracer measurements. The apparent selectivity sequence, relative to Na⁺, as determined by bi-ionic potential measurements was: F^–, isetheionate, gluconate, choline (<0.1)+(1.0)–(1.5)=NO ₃ ^– (1.5)–(2.3)+(2.4)+(2.5)+(2.6)+(3.9) 4 ^– +(12)–(40). The origin of this selectivity sequence and its relationship to the ion permeability of the brush border membrane in the intact epithelium are discussed.     

Effect of anisotonic media on volume,ion and amino-acid content and membrane potential of kidney cells (MDCK) in culture

Summary Effects of anisotonic media on a monolayer of confluent kidney cells in culture (MDCK) were studied by measuring: cell thickness and cross-section changes, ion and amino-acid content and membrane potential. The volume was also determined with cells in suspension. When cells in a monolayer were incubated in hypotonic media, the lateral and the apical membranes were rapidly stretched. Afterwards the lateral membranes returned to their initial state while the apical membranes remained stretched. This partial regulatory volume decrease (RVD) was verified with cells in suspension. RVD was accompanied by a loss of K⁺, Cl^– and amino acids, but there was no loss of inorganic phosphate. Also a transient hyperpolarization of the membrane potential was observed, suggesting an increase of the K⁺ conductance during RVD. Upon restoring the isotonic medium, a regulatory volume increase (RVI) was observed accompanied by a rapid Na⁺ and Cl^– increase and followed by a slow recovery of the initial K⁺ and Na⁺ content while amino acids remained at their reduced content. A transient depolarization of the membrane potential was measured during this RVI, suggesting that Na⁺ and Cl^– conductance could have increased. In hypertonic media, only a small and slow RVI was observed accompanied by an increase in K⁺ and Cl^– content but without any change of membrane potential. Quinine partly inhibited RVD in hypotonic media with cells in a monolayer while inhibiting RVD completely with cells in suspension. Incubation during four hours in a Ca²⁺ free medium had no effect on RVD. Furosemide and amiloride had no effect on RVD and RVI. Volume regulation, RVD or RVI, was not affected by replacing Cl^– by nitrate. When cells in a monolayer were incubated in a hypotonic K₂SO₄ medium, no RVD was observed. From these results, it seems that MDCK cells in a confluent monolayer regulate their volume by activating specific ion and amino-acid transport pathways. Selective K⁺ and Na⁺ conductances are activated during RVD and RVI, while the activated anion conductance has a low selectivity. The controlling mechanism might not be the free intracellular Ca²⁺ concentration.     

Charybdotoxin blocks with high affinity the Ca-activated K+ channel of Hb A and Hb S red cells: Individual differences in the number of channels

Summary We have investigated the effect of a purified preparation of Charybdotoxin (CTX) on the Ca-activated K⁺ (Ca–K) channel of human red cells (RBC). Cytosolic Ca²⁺ was increased either by ATP depletion or by the Ca ionophore A23187 and incubation in Na⁺ media containing CaCl₂. The Ca–K efflux activated by metabolic depletion was partially (77%) inhibited from 15.8±2.4 mmol/liter cell · hr, to 3.7±1.0 mmol/liter cell · hr by 6nm CTX (n=3). The kinetic of Ca–K efflux was studied by increasing cell ionized Ca²⁺ using A23187 (60 mol/liter cell), and buffering with EGTA or citrate; initial rates of net K⁺ efflux (90 mmol/liter cell K⁺) into Na⁺ medium containing glucose, ouabain, bumetanide at pH 7.4 were measured. Ca–K efflux increased in a sigmoidal fashion (n of Hill 1.8) when Ca²⁺ was raised, with aK _m of 0.37 m and saturating between 2 and 10 m Ca²⁺. Ca–K efflux was partially blocked (71±7.8%, mean ±sd,n=17) by CTX with high affinity (IC₅₀0.8nm), a finding suggesting that is a high affinity ligand of Ca–K channels. CTX also blocked 72% of the Ca-activated K⁺ efflux into 75mm K⁺ medium, which counteracted membrane hyperpolarization, cell acidification and cell shrinkage produced by opening of the K⁺ channel in Na⁺ media. CTX did not block Valinomycin-activated K⁺ efflux into Na⁺ or K⁺ medium and therefore it does not inhibit K⁺ movement coupled to anion conductive permeability.TheV _max, but not theK _m–Ca of Ca–K efflux showed large individual differences varying between 4.8 and 15.8 mmol/liter cell · min (FU). In red cells with Hb A,V _max was 9.36±3.0 FU (mean ±sd,n=17). TheV _max of the CTX-sensitive, Ca–K efflux was 6.27±2.5 FU (range 3.4 to 16.4 FU) in Hb A red cells and it was not significantly different in Hb S (6.75±3.2 FU,n=8). Since there is larger fraction of reticulocytes in Hb S red cells, this finding indicates that cell age might not be an important determinant of theV _max of Ca–K⁺ efflux.Estimation of the number of CTX-sensitive Ca-activated K⁺ channels per cell indicate that there are 1 to 3 channels/per cell either in Hb A or Hb S red cells. The CTX-insensitive K⁺ efflux (2.7±0.9 FU) may reflect the activity of a different channel, nonspecific changes in permeability or coupling to an anion conductive pathway.     

Membrane potential,anion and cation conductances in Ehrlich ascites tumor cell

Summary The fluorescence intensity of the dye 1,1-dipropyloxadicarbocyanine (DiOC₃-(5)) has been measured in suspensions of Ehrlich ascites tumor cells in an attempt to monitor their membrane potential (V _m ) under different ionic conditions, after treatment with cation ionophores and after hypotonic cell swelling. Calibration is performed with gramicidin in Na⁺-free K⁺/choline⁺ media, i.e., standard medium in which NaCl is replaced by KCl and cholineCl and where the sum of potassium and choline is kept constant at 155mm. Calibration by the valinomycin null point procedure described by Lariset al. (Laris, P.C., Pershadsingh, A., Johnstone, R.M., 1976,Biochim. Biophys. Acta 436:475–488) is shown to be valid only in the presence of the Cl^–-channel blocker indacrinone (MK196). Distribution of the lipophilic anion SCN^– as an indirect estimation of the membrane potential is found not to be applicable for the fast changes inV _m reported in this paper. Incubation with DiOC₃-(5) for 5 min is demenstrated to reduce the Cl^– permeability by 26±5% and the NO ₃ ^– permeability by 15±2%, while no significant effect of the probe could be demonstrated on the K⁺ permeability. Values forV _m , corrected for the inhibitory effect of the dye on the anion conductance, are estimated at –61±1 mV in isotonic standard NaCl medium, –78±3 mV in isotonic Na⁺-free choline medium and –46±1 mV in isotonic NaNO₃ medium. The cell membrane is depolarized by addition of the K⁺ channel inhibitor quinine and it is hyperpolarized when the cells are suspended in Na⁺-free choline medium, indicating thatV _m is generated partly by potassium and partly by sodium diffusion. Ehrlich cells have previously been shown to be more permeable to nitrate than to chloride. Substituting NO ₃ ^– for all cellular and extracellular Cl^– leads to a depolarization of the membrane, demonstrating thatV _m is also generated by the anions and that anions are above equilibrium. Taking the previously demonstrated single-file behavior of the K⁺ channels into consideration, the membrane conductances in Ehrlich cells are estimated at 10.4 S/cm² for K⁺, 3.0 S/cm² for Na⁺, 0.6 S/cm² for Cl^– and 8.7 S/cm² for NO ₃ ^– . Addition of the Ca²⁺-ionophore A23187 results in net loss of KCl and a hyperpolarization of the membrane, indicating that the K⁺ permeability exceeds the Cl^– permeability also after the addition of A23187. The K⁺ and Cl^– conductances in A23187-treated Ehrlich cells are estimated at 134 and 30 S/cm², respectively. The membrane potential is depolarized in hypotonically swollen cells, confirming that the increase in the Cl^– permeability following hypotonic exposure exceeds the concommitant increase in the K⁺ permeability. In control experiments where the membrane potentialV _m =E _K =E _Cl =E _Na , it is demonstrated that cell volume changes has no significant effect on the fluorescence signal, apparently because of a large intracellular buffering capacity. The increase in the Cl^– conductances is 68-fold when cells are transferred to a medium with half the osmolarity of the standard medium, as estimated from the net Cl^– efflux and the change inV _m . The concommitant increase in the K⁺ conductance, as estimated from the net K⁺ efflux, is only twofold.     

Ionic mechanisms underlying modulating effects of serotonin on acetylcholine-induced responses in Helix pomatia neurons

The ionic mechanisms underlying modulatory effects of serotonin on acetylcholine-response in identified and nonidentifiedHelix pomatia neurons were investigated using voltage-clamping techniques at the neuronal membrane. External application of 10^–5–10^–4 M serotonin to the membrane of neurons responding to application of acetylcholine depending on Na⁺ depolarization (D_Na response) reduced membrane conductivity during response to acetylcholine without changing reversal potential of acetylcholine-induced current. Acetylcholine (10^–6–10^–4 M) administration took place 1–3 min later. Neurons with response to acetylcholine application dependent on Cl⁺ depolarization (D_Cl response) or hyperpolarization (H_Cl response) behaved similarly. Analogous effects could be produced by external application of theophylline which, together with the latency and residual effect characteristic of serotonin action points to the participation of intracellular processes associated with the cellular cyclase system in the changes produced by serotonin in acetylcholineinduced response. Serotonin brought about a shift in reversal potential and an increase in the acetylcholine-induced current in those neurons where this response was associated with changed permeability at the membrane to certain types of ions. During two-stage acetylcholine-induced response of the D_Na-H_K type, serotonin inhibited the inward current stage. Mechanisms underlying modulatory serotonin action on acetylcholine-induced response in test neurons are discussed in the light of our findings.A. A. Bogomolets Institute of Physiology, Academy of Sciences of the Ukrainian SSR, Kiev. Translated from Neirofiziologiya, Vol. 20, No. 1, pp. 57–64, January–February, 1988.     

Silver ion (Ag+)-Induced increases in cell membrane K+ and Na+ permeability in the renal proximal tubule: Reversal by thiol reagents

Summary The initial mechanisms of injury to the proximal tubule following exposure to nephrotoxic heavy metals are not well established. We studied the immediate effects of silver (Ag⁺) on K⁺ transport and respiration with extracellular K⁺ and O₂ electrodes in suspensions of renal cortical tubules. Addition of silver nitrate (AgNO₃) to tubules suspended in bicarbonate Ringer's solution caused a rapid, dose-dependent net K⁺ efflux (K _m =10^–4 m,V _max=379 nmol K⁺/min/mg protein) which was not inhibited by furosemide, barium chloride, quinine, tetraethylammonium, or tolbutamide. An increase in the ouabain-sensitive oxygen consumption rate (QO₂) (13.9±1.1 to 25.7±4.4 nmol O₂/min/mg,P<0.001), was observed 19 sec after the K⁺ efflux induced by AgNO₃ (10^–4 m), suggesting a delayed increase in Na⁺ entry into the cell. Ouabain-insensitive QO₂, nystatin-stimulated QO₂, and CCCP-uncoupled QO₂ were not significantly affected, indicating preserved function of the Na⁺, K⁺-ATPase and mitochondria. External addition of the thiol reagents dithiothreitol (1mm) and reduced glutathione (1mm) prevented and/or immediately reversed the effects on K⁺ transport and QO₂. We conclude that Ag⁺ causes early changes in the permeability of the cell membrane to K⁺ and then to Na⁺ at concentrations that do not limit Na⁺, K⁺-ATPase activity or mitochondrial function. These alterations are likely the result of a reversible interaction of Ag⁺ with sulfhydryl groups of cell membrane proteins and may represent initial cytotoxic effects common to other sulfhydryl-reactive heavy metals on the proximal tubule.     

Passive H+/OH− permeability in epithelial brush border membranes

Passive H⁺/OH^– permeability across epithelial cell membranes is rapid and leads to partial dissipation of H⁺/OH^– gradients produced by H⁺ pumps and ion gradient-coupled H⁺/OH^– transporters. A heterogeneous set of H⁺/OH^– transport mechanisms exist in biological membranes: lipid solubility/diffusion, protein-mediated transport by specific proteins or by slippage through ion-coupled H⁺/OH^– transporters, and transport at the protein/lipid interface or through protein-dependent defects in the lipid structure. A variety of methods are available to study protein transport mechanisms accurately in cells and biomembrane vesicles including pH electrode recordings, pH-sensitive fluorescent and magnetic resonance probes, and potentiometric probes. In brush border vesicles from the renal proximal tubule, the characteristics of passive H⁺/OH^– permeability are quite similar to those reported for passive H⁺/OH^– permeability through pure lipid bilayers; slippage of protons through the brush border Na⁺/H⁺ antiporter or through brush border water channels is minimal. In contrast, passive H⁺/OH^– permeability in brush border vesicles from human placenta is mediated in part by a stilbene-sensitive membrane protein. To demonstrate the physiological significance of passive renal brush border H⁺/OH^– transport, proximal tubule acidification and cell pH regulation mechanisms are modeled mathematically for states of normal and altered H⁺/OH^– permeabilities.     

Cyanine dye fluorescence used to measure membrane potential changes due to the assembly of complement proteins C5b-9

Summary The fluorescent potentiometric indicator diS–C₃-(5) has been used to investigate changes in membrane potential due to assembly of the C5b-9 membrane attack complex of the complement system. EAC1-7 human red blood cells and resealed erythrocyte ghosts—bearing membrane-assembled C5b67 complexes—were generated by immune activation in C8-deficient human serum. Studies performed with these cellular intermediates revealed that the membrane potential of EAC1-7 red cells and ghosts is unchanged from control red cells (–7 mV) and ghosts (0 mV), respectively. Addition of complement proteins C8 and C9 to EAC1-7 red cells results in a dose-dependent depolarization of membrane potential which precedes hemolysis. This prelytic depolarization of membrane potential—and the consequent onset of hemolysis—is accelerated by raising external [K⁺], suggesting that the diffusional equilibration of transmembrane cation gradients is rate limiting to the cytolytic event. In the case of EAC1-7 resealed ghosts suspended at either high external [K⁺] or [Na⁺], no change in membrane potential (from 0 mV) could be detected after C8/C9 additions. When the membrane potential of the EAC1-7 ghost was displaced from 0 mV by selectively increasing the K⁺ conductance with valinomycin, a dose-dependent depolarization of the membrane was observed upon addition of C8 and C9. In these experiments, lytic breakdown of the ghost membranes was <5%. Conclusions derived from this study include: (i) measured prelytic depolarization of the red cell Donnan potential directly confirms the colloid-osmotic theory of immune cytolysis. (ii) The diffusional transmenbrane equilibration of Na⁺ and K⁺ through the C5b-9 pore results in a dose-dependent depolarization of the membrane potential (E _m ) which appears to be rate-limiting to cytolytic rupture of the target erythrocyte. (iii) Enhanced immune hemolysis observed in high K⁺ media cannot be attributed to cation-selective conductance across the C5b-9 pore, and is probably related to the nearequilibrium condition of potassium-containing red cells when suspended at high external K⁺. These experiments demonstrate that carbocyanine dye fluorescent indicators can be used to monitor electrochemical changes arising from immune damage to the plasma membrane under both cytolytic and noncytolytic conditions. Potential application of this method to the detection of sublytic pathophysiological changes in the plasma membrane of complement-damaged cells are discussed.     

Leukocytic functions in burn-injured patients

Anergy associated with an increase in suppressor helper T cell (T_c) ratio and a decrease in natural killer (NK) is one main cause of death following thermal injury (Tl). Recently, in vitro studies have shown that LTB₄ can induce human T_c to exert suppressor cell activity, and incubation of lymphocytes with LTB₄ for 24 hours significantly suppressed NK cell activity. Thus, we undertook an investigation of both AA metabolism and immunologic response in 20 patients who suffered 40–90% total body surface area (TBSA) burns. Cyclooxygenase (CO:RIA) and lipoxygenase (LO;HPLC det.) metabolites and superoxide (O₂^.−) production were measured in stimulated polymorphonuclear cells (PMNL) (A 23187 ± AA for icosanoid release; phorbol myristate acetate for O₂^.− production). Lyso-paf-acether (P-LPA) was measured in plasma samples. Ca²⁺-dependent K⁺ permeability in PMNL was measured by the cell K⁺ release induced by A 23187. T_c and T_c subsets were determined using monoclonal antibodies (OKT₃₊, OKT₄₊ and OKT₈₊). A biphasic sequential release of the different substances (leukocytic icosanoids and O₂^.− was monitored: increase ( 36–48 h after Tl) and decrease ( 72 h after Tl). The increase in AA stimulation was more transient than that of O₂^.−. The decline in the release of AA metabolites and O₂^.− production was associated with the anergic phase (decrease OKT₄₊/OKT₈₊ ratio) and with the clinical outcome of the patients. The decrease in LTB₄ and other LO metabolites could explain the impairment of neutrophil chemotaxis. Ca²⁺-dependent K⁺ permeability increased early up to 2 or 3 times normal.In order to go further with the mechanism of inhibition of LTB₄ and O₂^.− release, the effect of Tl plasma was assayed on normal leukocytes: a 10 min incubation with such plasma was sufficient to abolish LTB₄ secretion. A less important inhibition was observed with O₂^.− release (−32%) and Ca²⁺-dependent K⁺ permeability (−30%). Plasma inhibition seems to be due to a thermolabile factor(s) [protein(s): “suppressive factor(s) of membrane activation ”SFMA] which is (are) under active investigation using gel-filtration chromatography and fast protein liquid chromotography (FPLC). Among the SFMAs, certain acute phase proteins could play a key role: i.e., incubation (10 min) of normal PMNL with ceruloplasmin (1 mg/ml) abolished LO products and O₂^.− release.     

Role for sulfur-containing groups in the Na+−Ca2+ exchange of cardiac sarcolemmal vesicles

Summary Different amino acid residues in cardiac sarcolemmal vesicles were modified by incubation with various chemical reagents. The effects of these modifications on sarcolemmal Na⁺–Ca²⁺ exchange were examined. Dithiothreitol, an agent that maintains sulfur-containing residues in a reduced state, caused a time- and concentration-dependent decrease in Na⁺–Ca²⁺ exchange. The treatment with dithiothreitol resulted in a decrease inV _max values but did not alter theK _m for Ca²⁺ for the Na²⁺–Ca²⁺ exchange reaction. If Na⁺ replaced K⁺ as the ion present during the modification of sarcolemmal membranes with dithiothreitol, there was substantially less of an inhibitor effect on Na⁺–Ca²⁺ exchange. Similar results were obtained with reduced glutathione, a reagent that also maintains sulfur-containing residues in a reduced state. Two sulfhydryl modifying reagents, methylmethanethiosulfonate and N-ethylmaleimide, were capable of altering Na⁺–Ca²⁺ exchange, and the type of ion present during modification significantly affected the extent of this alteration. Almost all of the chemical reagents investigated that modified other amino acid resides (carboxyl, lysyl, histidyl, tyrosyl, tryptophanyl, arginyl and hydroxyl) had the capacity to alter Na⁺–Ca²⁺ exchange after preincubation with the sarcolemmal membrane vesicles. However, the sulfur residue-modifying reagents were the only compounds to exhibit significant differences in their action on Na⁺–Ca²⁺ exchange, depending on whether Na⁺ or K⁺ was present in the preincubation modification medium. The tryptophan modifier, N-bromosuccinimide, was the sole reagent that elicited a substantial increase in membrane permeability. The evidence is consistent with the hypothesis that sulfurcontaining residues interact with a Na⁺-binding site for Na⁺–Ca²⁺ exchange in cardiac sarcolemmal vesicles.     

Progesterone-induced down-regulation of an electrogenic Na+, K+-ATPase during the first meiotic division in amphibian oocytes

Summary Progesterone initiates the resumption of the meiotic divisions in the amphibian oocyte. Depolarization of theRana pipiens oocyte plasma membrane begins 6–10 hr after exposure to progesterone (1–2 hr before nuclear breakdown). The oocyte cytoplasm becomes essentially isopotential with the medium by the end of the first meiotic division (20–22 hr). Voltage-clamp studies indicate that the depolarization coincides with the disappearance of an electrogenic Na⁺, K⁺-pump, and other electrophysiological studies indicate a decrease in both K⁺ and Cl^– conductances of the oocyte plasma membrane. Measurement of [³H]-ouabain binding to the plasma-vitelline membrane complex indicates that there are high-affinity (K _d-4.2×10^–8 m), K⁺-sensitive ouabain-binding sites on the unstimulated (prophase-arrest) oocyte and that ouabain binding virtually disappears during membrane depolarization. [³H]-Leucine incorporation into the plasma-vitelline membrane complex increased ninefold during depolarization with no significant change in uptake or incorporation into cytoplasmic proteins or acid soluble pool(s). This together with previous findings suggests that progesterone acts at a translational level to produce a cytoplasmic factor(s) that down-regulates the membrane Na⁺, K⁺-ATPase and alters the ion permeability and transport properties of both nuclear and plasma membranes.     

pH equilibration in human erythrocyte suspensions

Summary A stopped-flow rapid reaction apparatus was used to study the rate of pH equilibration in human red cell suspensions. Flux of OH^– or H⁺ was determined over a wide range of extracellular pH (4–11) in CO₂-free erythrocyte suspensions. In these experiments, an erythrocyte suspension at pH 7.3 is rapidly mixed with an equal volume of NaCl solution at 3.0>pH>11.5. The pH of the extracellular fluid of the mixture changes rapidly as OH^– or H⁺ moves across the red cell membrane. Flux and velocity constants can be calculated from the initiald pH/dt using the known initial intra- and extracellular pH. It was found that the further the extracellular pH is from 7.3 (in either direction from 4–11), the greater the absolute value of total OH^– and/or H⁺ flux. Pretreatment with SITS (4-acetamido-4-isothiocyanostilbene-2,2-disulfonic acid), a potent anion exchange inhibitor, greatly reduces flux over the entire pH range, while exposure to valinomycin, a potassium ionophore, has no measurable effect. These data suggest that (i) both H⁺ and OH^– may be moving across the red cell membrane at all pH; (ii) the species dominating pH equilibration is probably dependent on the extracellular pH, which determines the magnitude of the driving gradient for each ion; and (iii) the rapid exchange pathway of the erythrocyte membrane may be utilized for both H⁺ and OH^– transport during CO₂-free pH equilibration.     

Anion-dependent transport of thallous ions through human erythrocyte membrane

Summary Unidirectional fluxes of ²⁰⁴Tl⁺ through the human red blood cell membrane were measured. The inward rate coefficient measured in a K⁺-free saline was 15.6±0.6 hr^–1. The influx of Tl⁺ could be partially inhibited with 0.1mm ouabain (by 28%), 0.1mm DIDS (by 50%) or 1mm furosemide (by 51%). The inhibitory effects of ouabain and DIDS or furosemide were additive. Half-maximal responses were seen at 0.72 m and 0.22mm concentrations of DIDS and furosemide, respectively. A similar action of these blockers on Tl⁺ influx was observed in the erythrocytes incubated in MgCl₂-sucrose media. The outward rate coefficient of ²⁰⁴Tl was also inhibited by DIDS and furosemide (by 65 and 52%, respectively). Rate coefficients of ²⁰⁴Tl influx and efflux decreased significantly in the red cells exposed to Cl^–-free media (NaNO₃ or Mg(NO₃)₂-sucrose). Under these conditions addition of DIDS and furosemide led to only a small inhibition of Tl⁺ fluxes. There was a linear increase in Tl⁺ influx with rising of external Cl^– concentration within 80–155mm or HCO ₃ ^– concentration from 20 to 40mm when the sum of anions was kept constant (155mm) with NO ₃ ^– . The HCO ₃ ^– -stimulated Tl⁺ influx was completely blocked by 0.05mm DIDS but only 67% by 1mm furosemide. The present study provides direct evidence for the occurrence of Cl^– (HCO ₃ ^– )-dependent, DIDS-sensitive movement of Tl⁺ across the human erythrocyte membrane in both directions. Under physiological conditions, about half of net Tl⁺ fluxes occurs due to an anion exchange mechanism. Our data fail to detect a contribution of the Na-K-Cl cotransport system to Tl⁺ transport in human erythrocytes.     

Effects of anions on cellular volume and transepithelial Na+ transport across toad urinary bladder

Summary The effects of complete substitution of gluconate for mucosal and/or serosal medium Cl^– on transepithelial Na⁺ transport have been studied using toad urinary bladder. With mucosal gluconate, transepithelial potential difference (V _T) decreased rapidly, transepithelial resistance (R _T) increased, and calculated short-circuit current (I _sc) decreased. CalculatedE _Na was unaffected, indicating that the inhibition of Na⁺ transport was a consequence of a decreased apical membrane Na⁺ conductance. This conclusion was supported by the finding that a higher amiloride concentration was required to inhibit the residual transport. With serosal gluconateV _T decreased,R _T increased andI _sc fell to a new steady-state value following an initial and variable transient increase in transport. Epithelial cells were shrunken markedly as judged histologically. CalculatedE _Na fell substantially (from 130 to 68 mV on average). Ba²⁺ (3mm) reduced calculatedE _Na in Cl^– Ringer's but not in gluconate Ringer's. With replacement of serosal Cl^– by acetate, transepithelial transport was stimulated, the decrease in cellular volume was prevented andE _Na did not fall. Replacement of serosal isosmotic Cl^– medium by a hypo-osmotic gluconate medium (one-half normal) also prevented cell shrinkage and did not result in inhibition of Na⁺ transport. Thus the inhibition of Na⁺ transport can be correlated with changes in cell volume rather than with the change in Cl^– per se. Nystatin virtually abolished the resistance of the apical plasma membrane as judged by measurement of tissue capacitance. With K⁺ gluconate mucosa, Na⁺ gluconate serosa, calculated basolateral membrane resistance was much greater, estimated basolateral emf was much lower, and the Na⁺/K⁺ basolateral permeability ratio was much higher than with acetate media. It is concluded the decrease in cellular volume associated with substitution of serosal gluconate for Cl^– results in a loss of highly specific Ba²⁺-sensitive K⁺ conductance channels from the basolateral plasma membrane. It is possible that the number of Na⁺ pump sites in this membrane is also decreased.     

Intracellular calcium content of human erythrocytes: Relation to sodium transport systems

Summary To study the possible role of intracellular Ca (Ca _i ) in controlling the activities of the Na⁺–K⁺ pump, the Na⁺–K⁺ cotransport and the Na⁺/Li⁺ exchange system of human erythrocytes, a method was developed to measure the amount of Ca embodied within the red cell. For complete removal of Ca associated with the outer aspect of the membrane, it proved to be essential to wash the cells in buffers containing less than 20nm Ca. Ca was extracted by HClO₄ in Teflon^® vessels boiled in acid to avoid Ca contaminations and quantitated by flameless atomic absorption. Ca _i of fresh human erythrocytes of apparently healthy donors ranged between 0.9 and 2.8 mol/liter cells. The mean value found in females was significantly higher than in males. The interindividual different Ca contents remained constant over periods of more than one year. Sixty to 90% of Ca _i could be removed by incubation of the cells with A23187 and EGTA. The activities of the Na⁺–K⁺ pump, of Na⁺–K⁺ cotransport and Na⁺/Li⁺ exchange and the mean cellular hemoglobin content fell with rising Ca _i ; the red cell Na⁺ and K⁺ contents rose with Ca _i . Ca depletion by A23187 plus EGTA as well as chelation of intracellular Ca²⁺ by quin-2 did not significantly enhance the transport rates. It is concluded that the large scatter of the values of Ca _i of normal human erythrocytes reported in the literature mainly results from a widely differing removal of Ca associated with the outer aspect of the membrane.     

Sulfhydryl-reactive heavy metals increase cell membrane K+ and Ca2+ transport in renal proximal tubule

Summary The cellular mechanisms by which nephrotoxic heavy metals injure the proximal tubule are incompletely defined. We used extracellular electrodes to measure the early effects of heavy metals and other sulfhydryl reagents on net K⁺ and Ca²⁺ transport and respiration (QO₂) of proximal tubule suspensions. Hg²⁺, Cu²⁺, and Au³⁺ (10^–4 m) each caused a rapid net K⁺ efflux and a delayed inhibition of QO₂. The Hg²⁺-induced net K⁺ release represented passive K⁺ transport and was not inhibited by barium, tetraethylammonium, or furosemide. Both Hg²⁺ and Ag⁺ promoted a net Ca²⁺ uptake that was nearly coincident with the onset of the net K⁺ efflux. A delayed inhibition of ouabainsensitive QO₂ and nystatin-stimulated QO₂, indicative of Na⁺, K⁺-ATPase inhibition, was observed after 30 sec of exposure to Hg²⁺. More prolonged treatment (2 min) of the tubules with Hg²⁺ resulted in a 40% reduction in the CCCP-uncoupled QO₂, indicating delayed injury to the mitochondria. The net K⁺ efflux was mimicked by the sulfhydryl reagents pCMBS and N-ethylmaleimide (10^–4 m) and prevented by dithiothreitol (DTT) or reduced glutathione (GSH) (10^–4 m). In addition, both DTT and GSH immediately reversed the Ag⁺-induced net Ca²⁺ uptake. Thus, sulfhydryl-reactive heavy metals cause rapid, dramatic changes in the membrane ionic permeability of the proximal tubule before disrupting Na⁺, K⁺-ATPase activity or mitochondrial function. These alterations appear to be the result of an interaction of the metal ions with sulfhydryl groups of cell membrane proteins responsible for the modulation of cation permeability.     

Characterization of cell ion exchange in the sea anemoneCondylactis gigantea

Summary Maintenance of intracellular ion contents and their relations to transmembrane potential were studied in tentacles ofCondylactis gigantea. Tentacles leached at 2°C in 10 mMK⁺ and 2 mM K⁺ artificial seawaters (10K ASW and 2K ASW) with and without 2 MM ouabain, and in 0K ASW, lost cell K⁺ and gained Na⁺. Rewarming to 25°C in 10K ASW resulted in a marked accumulation of K⁺ and extrusion of Na⁺ in tentacles leached in 10K ASW and in 0K ASW. Initial rate of Na⁺ extrusion was twice the initial rate of K⁺ accumulation, suggesting a pump coupling ratio of 2. In tentacles leached and rewarmed in 2K ASW, no net reaccumulation of K⁺ and little net extrusion of Na⁺ was observed; i.e., the pump just kept pace with the leaks. Ouabain inhibited K⁺ reaccumulation and Na⁺ extrusion. This effect was less marked in 10K ASW than in 2K ASW confirming, in anemone tentacles, the well documented ouabain-K⁺ antagonism observed in other systems. In no case did K _i ⁺ /K ₀ ⁺ equal Cl ₀ ^– /Cl _i ^– ; therefore, the distribution of these ions did not fit a Donnan distribution. Transmembrane potential difference was –22±3 mV in 10K ASW at 25°C. It fitted a modified Nernst equation which includes the pump coupling ratio and a Na⁺ to K⁺ permeability ratio of 0.31.A moderately high permeability of the cell membrane to Na⁺, and a ouabain and K⁺ sensitive ion pump, exchanging 2 Na⁺ for 1 K⁺, appear to be responsible for the observed ionic distribution and transmembrane potential in anemone cells.     

Volume regulation in the early proximal tubule of theNecturus kidney

Summary The ability of early proximal tubule cells of theNecturus kidney to regulate volume was evaluated using light microscopy, video analysis and conventional microelectrodes.Necturus proximal tubule cells regulate volume in both hyperand hyposmotic solutions. Volume regulation in hyperosmotic fluids is HCO ₃ ^– dependent and is associated with a decrease in the relative K⁺ conductance of the basolateral cell membrane and a decrease in the resistance ratio,R _a /R _bl . Volume regulation in hyposmotic solutions is also dependent upon the presence of HCO ₃ ^– but is also inhibited by 2mm Ba²⁺ in the basolateral solution. Hyposmotic regulation is accompanied by an increase in the relative K⁺ conductance of the basolateral cell membrane and an increase inR _a /R _bl . Neither hypo- nor hyposmotic regulation have any affect on the depolarization of the basolateral cell membrane potential induced by HCO ₃ ^– removal. We conclude that volume regulation in the early proximal tubule of the kidney involves both HCO ₃ ^– -dependent transport systems and the basolateral K⁺ conductance.     

Renal cortical basolateral Na+/HCO 3 − cotransporter: II. Detection of conformational changes with fluorescein isothiocyanate labeling

Fluorescein isothiocyanate (FITC) fluorescently labels amino groups and has been useful in detecting conformational changes in transport proteins through quenching or enhancement of the fluorescence signal upon exposure of protein to substrates. Solubilized renal basolateral membrane proteins, enriched in Na⁺/HCO ₃ ^– cotransporter activity, were reconstituted into liposomes and treated with FITC or its nonfluorescent analogue PITC (phenyl isothiocyanate). In the absence of Na⁺ and HCO ₃ ^– , incubation of proteoliposomes with PITC or FITC significantly inhibited cotransporter activity. However, in the presence of Na⁺ and HCO ₃ ^– during labeling both agents failed to inhibit cotransporter activity, indicating that these probes interact specifically with the cotransporter. In the presence of the substrates Na⁺ and HCO ₃ ^– , PITC binds covalently to amino groups unprotected by substrates leaving the Na⁺/HCO ₃ ^– cotransporter available for specific labeling with FITC. Addition of NaHCO₃ to FITC-labeled proteoliposomes resulted in a concentration-dependent enhancement of the fluorescence signal which was inhibited by pretreatment with 4,4-diisothiocyanostilbene 2,2-disulfonic acid (DIDS) prior to FITC labeling. SDS PAGE analysis of FITC-treated proteoliposomes showed the presence of two distinct fluorescent bands (approximate MW of 90 and 56 kD). In the presence of substrates, the fluorescence intensity of these bands was enhanced as confirmed by direct measurement of gel slice fluorescence. Thus, FITC detects conformational changes of the Na⁺/HCO ₃ ^– cotransporter and labels proteins which may represent the cotransporter or components of this cotransporter.This work was supported by the Merit Review Program from the Veterans Administration Central Office (J.A.L.A.), and the National Kidney Foundation of Illinois (A.A.B.).