Intracellular Na+ and K+ activities and membrane conductances in the collecting tubule of Amphiuma

Membrane potentials and conductances, and intracellular ionic activities were studied in isolated perfused collecting tubules of K+-adapted Amphiuma. Intracellular Na+ (aNai) and K+ (aKi) activities were measured, using liquid ion-exchanger double-barreled microelectrodes. Apical and basolateral membrane conductances were estimated by cable analysis. The effects of inhibition of the apical conductance by amiloride (10(-5) M) and of inhibition of the basolateral Na-K pump by either a low K+ (0.1 mM) bath or by ouabain (10(-4) M) were studied. Under control conditions, aNai was 8.4 +/- 1.9 mM and aKi 56 +/- 3 mM. With luminal amiloride, aNai decreased to 2.2 +/- 0.4 mM and aKi increased to 66 +/- 3 mM. Ouabain produced an increase of aNai to 44 +/- 4 mM, and a decrease of aKi to 22 +/- 6, and similar changes were observed when the tubule was exposed to a low K+ bath solution. During pump inhibition, there was a progressive decrease of the K+-selective basolateral membrane conductance and of the Na+ permeability of the apical membrane. A similar inhibition of both membrane conductances was observed after pump inhibition by low K+ solution. Upon reintroduction of K+, a basolateral membrane hyperpolarization of -23 +/- 4 mV was observed, indicating an immediate reactivation of the electrogenic Na-K pump. However, the recovery of the membrane conductances occurred over a slower time course. These data imply that both membrane conductances are regulated according to the intracellular ionic composition, but that the basolateral K+ conductance is not directly linked to the pump activity.     

Independence of apical membrane Na+ and Cl- entry in Necturus gallbladder epithelium

Transepithelial fluid transport (Jv) and intracellular Na+ and Cl- activities (aNai, aCli) were measured in isolated Necturus gallbladders to establish the contribution of different proposed apical membrane entry mechanisms to transepithelial salt transport. In 10 mM HCO3- Ringer's, Jv was 13.5 +/- 1.1 microliter X cm-2 X h-1, and was significantly reduced by a low bicarbonate medium and by addition of amiloride (10(-3)M) or SITS (0.5 X 10(-3)M) to the mucosal bathing solution. Bumetanide (10(-5)M) was ineffective. Bilateral Na+ removal abolished Jv. The hypothesis of NaCl cotransport was rejected on the basis of the following results, all obtained during mucosal bathing solution changes: during Na+ removal, aNai fell 4.3 times faster than aCli; during Cl- removal, aCli fell 7.5 times faster than aNai; amiloride (10(-3) M) reduced aNai at a rate of 2.4 +/- 0.3 mM/min, whereas aCli was not changed; bumetanide (10(-5) M) had no significant effects on Jv or aCli. The hypothesis of Na-K-Cl cotransport was rejected for the same reasons; in addition, K+ removal from the mucosal bathing solution (with concomitant Ba2+ addition) did not alter aNai or aCli. The average rate of NaCl entry under normal transporting conditions, estimated from Jv, assuming that the transported fluid is an isosmotic NaCl solution, was 22.5 nmol X cm-2 X min-1. Upon sudden cessation of NaCl entry, assuming no cell volume changes, aNai and aCli should fall at an average rate of 4.8 mM/min. To compare this rate with the rates of Na+ and Cl- entry by ion exchange, the Na+ or Cl- concentration in the mucosal bathing solution was reduced rapidly to levels such that electroneutral cation or anion exchange, respectively, should cease. The rate of Na+ or Cl- entry before this maneuver was estimated from the initial rate of fall of the respective intracellular ionic activity upon the mucosal solution substitution. aNai and aCli decreased at initial rates of 3.7 +/- 0.4 and 5.9 +/- 0.8 mM/min, respectively. The rate of fall of aNai upon reduction of external [Na] was not affected by amiloride (10(-3) M), and the rate of fall of aCli upon reduction of external [Cl] was unchanged by SITS (0.5 X 10(-3) M), which indicates that net cation or anion exchange was, in fact, abolished by the changes in Na+ and Cl- gradients, respectively. I conclude that double exchange (Na+/H+ and Cl-/HCO-3) is the predominant or sole mechanism of apical membrane NaCl entry in this epithelium.     

Cellular mechanisms of potassium homeostasis in the mammalian nervous system

Double-barrelled ion-sensitive microelectrodes were used to measure changes in the intracellular activities of K+, Na+, and Cl- (aKi, aNai, aCli) in neurones of rat sympathetic ganglia and in glial cells of slices from guinea-pig olfactory cortex. In sympathetic neurones, carbachol and gamma-aminobutyric acid (GABA) produced a reversible decrease of aKi. The decrease of aKi during carbachol was accompanied by a rise of aNai, whereas in the presence of GABA decreases of aKi and aCli were seen. The reuptake of K+ released during the action of carbachol was completely blocked by ouabain, whereas furosemide inhibited the aKi recovery after the action of GABA. In glial cells, in contrast to the observations in the sympathetic neurones, aKi and aCli increased, whereas aNai decreased when neuronal activity was enhanced by repetitive stimulation of the lateral olfactory tract. It was found that barium ions and ouabain strongly reduced the activity-related rise of intraglial aKi in slices of guinea-pig olfactory cortex. These data show that mammalian neurones as well as glial cells possess several K+ uptake mechanisms that contribute to potassium homeostasis. Ouabain, furosemide, and Ba2+ are useful pharmacological tools to separate these mechanisms.     

Effects of L-alanine on membrane potential, potassium (86Rb) permeability and cell volume in hepatocytes from Raja erinacea

Isolated hepatocytes from the elasmobranch Raja erinacea were examined for their regulatory responses to a solute load following electrogenic uptake of L-alanine. The transmembrane potential (Vm) was measured with glass microelectrodes filled with 0.5 M KCl (75 to 208 M omega in elasmobranch Ringer's solution) and averaged -61 +/- 16 mV (S.D.; n = 68). L-Alanine decreased (depolarized) Vm by 7 +/- 3 and 18 +/- 2 mV at concentrations of 1 and 10 mM, respectively. Vm did not repolarize to control values during the 5-10 min impalements, unless the amino acid was washed away from the hepatocytes. The depolarizing effect of L-alanine was dependent on external Na+, and was specific for the L-isomer of alanine, as D- and beta-alanine had no effect. Hepatocyte Vm also depolarized on addition of KCN or ouabain, or when external K+ was increased. Rates of 86Rb+ uptake and efflux were measured to assess the effects of L-alanine on Na+/K+-ATPase activity and K+ permeability, respectively. Greater than 80% of the 86Rb+ uptake was inhibited by 2 mM ouabain, or by substitution of choline+ for Na+ in the incubation media. L-Alanine (10 mM) increased 86Rb+ uptake by 18-49%, consistent with an increase in Na+/K+ pump activity, but had no effect on rubidium efflux. L-Alanine, at concentrations up to 20 mM, also had no measurable effect on cell volume as determined by 3H2O and [14C]inulin distribution. These results indicate that Na+-coupled uptake of L-alanine by skate hepatocytes is rheogenic, as previously observed in other cell systems. However, in contrast to mammalian hepatocytes, Vm does not repolarize for at least 10 min after the administration of L-alanine, and changes in cell volume and potassium permeability are also not observed.     

Stoichiometry and voltage dependence of the sodium pump in voltage- clamped, internally dialyzed squid giant axon

The stoichiometry and voltage dependence of the Na/K pump were studied in internally dialyzed, voltage-clamped squid giant axons by simultaneously measuring, at various membrane potentials, the changes in Na efflux (delta phi Na) and holding current (delta I) induced by dihydrodigitoxigenin (H2DTG). H2DTG stops the Na/K pump without directly affecting other current pathways: (a) it causes no delta I when the pump lacks Na, K, Mg, or ATP, and (b) ouabain causes no delta I or delta phi Na in the presence of saturating H2DTG. External K (Ko) activates Na efflux with Michaelis-Menten kinetics (Km = 0.45 +/- 0.06 mM [SEM]) in Na-free seawater (SW), but with sigmoid kinetics in approximately 400 mM Na SW (Hill coefficient = 1.53 +/- 0.08, K1/2 = 3.92 +/- 0.29 mM). H2DTG inhibits less strongly (Ki = 6.1 +/- 0.3 microM) in 1 or 10 mM K Na-free SW than in 10 mM K, 390 mM Na SW (1.8 +/- 0.2 microM). Dialysis with 5 mM each ATP, phosphoenolpyruvate, and phosphoarginine reduced Na/Na exchange to at most 2% of the H2DTG-sensitive Na efflux. H2DTG sensitive but nonpump current caused by periaxonal K accumulation upon stopping the pump, was minimized by the K channel blockers 3,4-diaminopyridine (1 mM), tetraethylammonium (approximately 200 mM), and phenylpropyltriethylammonium (20-25 mM) whose adequacy was tested by varying [K]o (0-10 mM) with H2DTG present. Two ancillary clamp circuits suppressed stray current from the axon ends. Current and flux measured from the center pool derive from the same membrane area since, over the voltage range -60 to +20 mV, tetrodotoxin-sensitive current and Na efflux into Na-free SW, under K-free conditions, were equal. The stoichiometry and voltage dependence of pump Na/K exchange were examined at near-saturating [ATP], [K]o and [Na]i in both Na-free and 390 mM Na SW. The H2DTG-sensitive F delta phi Na/delta I ratio (F is Faraday's constant) of paired measurements corrected for membrane area match, was 2.86 +/- 0.09 (n = 8) at 0 mV and 3.05 +/- 0.13 (n = 6) at -60 to -90 mV in Na-free SW, and 2.72 +/- 0.09 (n = 7) at 0 mV and 2.91 +/- 0.21 (n = 4) at -60 mV in 390 mM Na SW. Its overall mean value was 2.87 +/- 0.07 (n = 25), which was not significantly different from the 3.0 expected of a 3 Na/2 K pump.(ABSTRACT TRUNCATED AT 400 WORDS)     

The Resting Potential of Mouse Leydig Cells: Role of an Electrogenic Na+/K+ Pump

Resting potentials (Vm) were measured in mouse Leydig cells, using the whole-cell patch-clamp technique. In contrast to conventional microelectrode measurements, where a biphasic potential was observed, we recorded a stable Vm around -32.2 +/- 1.2 mV (mean +/- SEM, n = 159), at 25 degrees C, and an input resistance larger than 2.7 x 109 W. Although Vm is sensitive to changes in the extracellular concentrations of potassium and chloride, the relationship between Vm and these ions' concentrations cannot be described by either the Goldman-Hodgkin-Katz or the Nernst equation. Perifusing cells with potassium-free solution or 10?3 M ouabain induced a marked depolarization averaging 20.1 +/- 3.2 mV (n = 9) and 23.1 +/- 2.8 mV, (n = 7), respectively. Removal of potassium or addition of ouabain with the cell voltage-clamped at its Vm, resulted in an inwardly directed current, due to inhibition of the Na+K+ATPase. The pump current increased with temperature with a Q10 coefficient of 2.3 and had an average value of -6.5 +/- 0.4 pA (n = 21) at 25 degrees C. Vm also varied strongly with temperature, reaching values as low as -9.2 +/- 1.2 mV (n = 22) at 15 degrees C. Taking the pump current at 25 degrees C and a minimum estimate for the membrane input resistance, we can see that the Na+K+ATPase could directly contribute with 17.7 mV to the Vm of Leydig cells, which is a major fraction of the ?32.2 +/- 1.2 mV (n = 159) observed.     

Delta endotoxin is a potent inhibitor of the (Na,K)-ATPase

A 68-kDa protein, delta endotoxin, produced by Bacillus thuringiensis ssp. Kurstaki inhibits ion transport, (Na,K)-ATPase, and K+-p-nitrophenylphosphatase activity catalyzed by the Na+ pump. The Ki for inhibition of the K+-p-nitrophenylphosphatase activity of purified dog kidney (Na,K)-ATPase was approximately 0.37 microM. Delta endotoxin had a similar Ki for inhibition of (Na,K)-ATPase activity when assayed at low Na+ concentration (10 mM) but the inhibition was reversed when high concentrations of Na+ (100 mM NaCl) were added to the assay. Phosphorylation of the active site aspartyl residue with 32PO3-4 was also blocked by delta endotoxin. Ouabain-sensitive 86Rb+ uptake into intact human red blood cells was not inhibited by externally added toxin; however, strophanthidin-inhibitable 22Na+ uptake into inside-out vesicles from red blood cells was completely blocked by delta endotoxin (Ki = 0.73 microM). These data suggest that delta endotoxin must enter the cell before it can inhibit the Na+ pump.     

Intracellular Na+, K+, and C1- activities in Balanus photoreceptors

Ion-sensitive microelectrodes were used to measure intracellular activities (aix) of Na+, K+, and C-1 in Balanus photoreceptors. Average values of aiNa, aiK, and aiCl were 28 mM, 120 mM, and 65 mM, respectively. Equilibrium potentials calculated from these average values were: Na+ +64 mV, K+ - 77 mV, and and Cl- -42 mV; ther average value of the resting potential for all cells examined was -41 mV. Long exposure to intense illumination produced measurable increases in aiNa. Classical Na+ - K+ reciprocal dilution experiments were analyzed with and without observed changes in aiK. As aoK was increased, the membrane depolarized, and aiK increased. Better agreement was found between the membrane potential and the directly determined EK than expected from the standard relation between Em and aoK. The latter produced pNa:pK estimates of the resting photoreceptor membrane that were higher than estimates based on data from the ion electrodes. Generally, Em was more negative than EK as aoK was increased. This is consistent with a significant chloride permeability in the dark-adapted photoreceptor.     

细胞外钠离子浓度对羊浦肯野纤维膜钠泵活动的影响

采用离子选择电极测量羊浦肯野纤维细胞膜内钠离子活度(~(ai)N_a),细胞间钾离子活度(a~ok)及细胞膜电位(v_m),观察不同浓度低钠,无钙液对其影响,在无钙低钠液中,细胞内Na~+逐出,α~iNa 降低,其变化速率,幅值与[Na]_o 相关,同时也受细胞 a~iNa 初始水平(aiNa(o))的影响。aiNa 下降6min 时的稳态水平与[Na]_o 呈直线正相关,这些结果表明,[Na]_o 降低时,细胞膜钠泵活动加强,细胞内 Na~+逐出增加,其最终结果是使 Na+跨膜梯度维持相对稳定,因而可以认为是 Na~+跨膜梯度而不是单纯的细胞内 Na~+控制膜钠泵活动。在低 Na~+液引起细胞内 Na~+主动逐出增加的同时,细胞膜出现超极化,[Na]_o 愈低,膜超极化程度愈高,从低钠液引起的 a~i_(Na),V_m,α~o_k 变化之间的时程关系看,膜超极化主要由加大的外向泵电流引起,同时发生的细胞间 K~+浓度变化对其也有一定影响。     

Measurement with microelectrodes of intracellular Na+, K+, H+ and Cl- activities and of membrane potential in normal rat liver slices and during the 4-dimethylaminoazobenzene-induced rat hepatocarcinogenesis

Steady-state membrane potentials (Vm) and intracellular Na+ (aiNa), K+ (aiK), H+ (aiH) and Cl- (aiCl) activities were measured with double-barrelled ion-selective microelectrodes in liver slices from normal rats and during the 4-dimethylaminoazobenzene-induced (DAB) hepatocarcinogenesis. Rats fed with the experimental regimen without the carcinogen were used as control animals. In Krebs-Henseleit bicarbonate saline containing 5.5 mM glucose as bathing solution at 37 degrees C, Vm was found to be significantly lower in neoplastic hepatocytes, compared to normal liver cells. Vm decreased also in control rat liver cells. Increased Na+/K+ ratios and Na+ + K+ activities were found in cancerous hepatocytes whereas H+ and Cl- activities decreased. Therefore, the intracellular pH increased significantly in neoplastic cells, compared to normal and control cells. This could reflect activation of the Na+/H+ exchange system during the DAB-induced hepatocarcinogenesis, leading to a stimulation of cell metabolism with increased rate of protein and DNA synthesis and loss of growth control, under these conditions.     

Effects of hyperosmotic medium on hepatocyte volume, transmembrane potential and intracellular K+ activity

Hepatocyte transmembrane potential (Vm) behaves as an osmometer and varies with changes in extracellular osmotic pressure created by altering the NaCl concentration in the external medium (Howard, L.D. and Wondergem, R. (1987) J. Membr. Biol. 100, 53). We now have demonstrated similar effects on Vm by increasing external osmolality with added sucrose and not altering ionic strength. We also have demonstrated that hyperosmotic stress-induced depolarization of Vm results from changes in membrane K+ conductance, gK, rather than from changes in the K+ equilibrium potential. Vm and aKi of hepatocytes in liver slices were measured by conventional and ion-sensitive microelectrodes, respectively. Cell water vols. were estimated by differences in wet and dry weights of liver slices after 10-min incubations. Effect of hyperosmotic medium on membrane transference number for K+, tK, was measured by effects on Vm of step-changes in external [K+]. Hepatocyte Vm decreased 34, 52 and 54% when tissue was superfused with medium made hyperosmotic with added sucrose (50, 100 and 150 mM). Correspondingly, aKi increased 10, 18 and 29% with this hyperosmotic stress of added sucrose. Tissue water of 2.92 +/- 0.10 kg H2O/kg dry weight in control solution decreased to 2.60 +/- 0.05, 2.25 +/- 0.06 and 2.22 +/- 0.05 kg H2O/kg dry weight with additions to medium of 50, 100 and 150 mM sucrose, respectively. Adding 50 mM sucrose to medium decreased tK from 0.20 +/- 0.01 to 0.05 +/- 0.01. Depolarization by 50% with hyperosmotic stress (100 mM sucrose) also occurred in Cl-free medium where Cl- was substituted with gluconate. We conclude that hepatocytes shrink during hyperosmotic stress, and the aKi increases. The accompanying decrease in Vm is opposite to that expected by an increase in aKi, and at least in part results from a concomitant decrease in gK. Changes in membrane Cl- conductance most likely do not contribute to osmotic stress-induced depolarization, since equivalent decreases in Vm occurred with added sucrose in cells depleted of Cl- by superfusing tissue with Cl-free medium.     

Effect of stimulation frequency on intracellular Na+ activity in rat atrial muscle

Mean intracellular Na+ activity (aNai) was measured in rat left atrial muscle stimulated at increasing frequencies between 0 and 12 Hz. Low-pass filtered signals from conventional and ion-selective microelectrodes were used to determine aNai. Preparations were bathed in a low Ca2+ (0.1 mM) Krebs-Henseleit buffer containing 1.0 mM Mn2+ to abolish contractile motion and permit stable impalements. Under these conditions, aNai increased progressively with frequency from 5.8 +/- 1.5 mM at 0 Hz to a maximum of 12.7 +/- 2.1 mM, which was observed at 10, 11, or 12 Hz. Further increases in frequency exceeded the effective refractory period, and aNai tended to decrease. These data suggest that aNai can be approximately doubled in rat atrial muscle by increasing the depolarization rate from 0 to 10-12 Hz, a range that has been shown to elicit a two- to three-fold elevation in Na+-pump activity in similar preparations.     

Transmembrane Na+ and Ca2+ electrochemical gradients in cardiac muscle and their relationship to force development

Na+- and CA2+-sensitive microelectrodes were used to measure intracellular Na+ and Ca2+ activities (alpha iCa) of sheep ventricular muscle and Purkinje strands to study the interrelationship between Na+ and Ca2+ electrochemical gradients (delta muNa and delta muCa) under various conditions. In ventricular muscle, alpha iNa was 6.4 +/- 1.2 mM and alpha iCa was 87 +/- 20 nM ([Ca/+] = 272 nM). A graded decrease of external Na+ activity (alpha oNa) resulted in decrease of alpha iNa, and increase of alpha iCa. There was increase of twitch tension in low- alpha oNa solutions, and occasional increase of resting tension in 40% alpha oNa. Increase of external Ca2+ (alpha oCa) resulted in increase of alpha iCa and decrease of alpha iNa. Decrease of alpha oCa resulted in decrease of alpha iCa and increase of alpha iNa. The apparent resting Na-Ca energy ratio (delta muCa/delta muNa) was between 2.43 and 2.63. When the membrane potential (Vm) was depolarized by 50 mM K+ in ventricular muscle, Vm depolarized by 50 mV, alpha iNa decreased, and alpha iCa increased, with the development of a contracture. The apparent energy coupling ratio did not change with depolarization. 5 x 10(-6) M ouabain induced a large increase in alpha iNa ad alpha iCa, accompanied by an increase in twitch and resting tension. Under the conditions we have studied, delta muNa and delta muCa appeared to be coupled and n was nearly constant at 2.5, as would be expected if the Na-Ca exchange system was able to set the steady level of alpha iCa. Tension threshold was about 230 nM alpha iCa. The magnitude of twitch tension was directly related to alpha iCa.     

Charge translocation by the Na+/K+ pump under Na+/Na+ exchange conditions: intracellular Na+ dependence

The effect of intracellular (i) and extracellular (o) Na+ on pre-steady-state transient current associated with Na+/Na+ exchange by the Na+/K+ pump was investigated in the vegetal pole of Xenopus oocytes. Current records in response to 40-ms voltage pulses from -180 to +100 mV in the absence of external Na+ were subtracted from current records obtained under Na+/Na+ exchange conditions. Na+-sensitive transient current and dihydroouabain-sensitive current were equivalent. The quantity of charge moved (Q) and the relaxation rate coefficient (ktot) of the slow component of the Nao+-sensitive transient current were measured for steps to various voltages (V). The data were analyzed using a four-state kinetic model describing the Na+ binding, occlusion, conformational change, and release steps of the transport cycle. The apparent valence of the Q vs. V relationship was near 1.0 for all experimental conditions. When extracellular Na+ was halved, the midpoint voltage of the charge distribution (Vq) shifted -25.3+/-0.4 mV, which can be accounted for by the presence of an extracellular ion-well having a dielectric distance delta=0.69+/-0.01. The effect of changes of Nai+ on Nao+-sensitive transient current was investigated. The midpoint voltage (Vq) of the charge distribution curve was not affected over the Nao+ concentration range 3.13-50 mM. As Nai+ was decreased, the amount of charge measured and its relaxation rate coefficient decreased with an apparent Km of 3.2+/-0.2 mM. The effects of lowering Nai+ on pre-steady-state transient current can be accounted for by decreasing the charge available to participate in the fast extracellular Na+ release steps, by a slowly equilibrating (phosphorylation/occlusion) step intervening between intracellular Na+ binding and extracellular Na+ release.     

Apical membrane Na+/H+ exchange in Necturus gallbladder epithelium. Its dependence on extracellular and intracellular pH and on external Na+ concentration

Intracellular microelectrode techniques and extracellular pH measurements were used to study the dependence of apical Na+/H+ exchange on mucosal and intracellular pH and on mucosal solution Na+ concentration ([Na+]o). When mucosal solution pH (pHo) was decreased in gallbladders bathed in Na(+)-containing solutions, aNai fell. The effect of pHo is consistent with titration of a single site with an apparent pK of 6.29. In Na(+)-depleted tissues, increasing [Na+]o from 0 to values ranging from 2.5 to 110 mM increased aNai; the relationship was well described by Michaelis-Menten kinetics. The apparent Km was 15 mM at pHo 7.5 and increased to 134 mM at pHo 6.5, without change in Vmax. In Na(+)-depleted gallbladders, elevating [Na+]o from 0 to 25 mM increased aNai and pHi and caused acidification of a poorly buffered mucosal solution upon stopping the superfusion; lowering pHo inhibited both apical Na+ entry and mucosal solution acidification. Both effects can be ascribed to titration of a single site; the apparent pK's were 7.2 and 7.4, respectively. Diethylpyrocarbonate (DEPC), a histidine-specific reagent, reduced mucosal acidification by 58 +/- 4 or 39 +/- 6% when exposure to the drug was at pHo 7.5 or 6.5, respectively. Amiloride (1 mM) did not protect against the DEPC inhibition, but reduced both apical Na+ entry and mucosal acidification by 63 +/- 5 and 65 +/- 9%, respectively. In the Na(+)-depleted tissues mean pHi was 6.7. Cells were alkalinized by exposure to mucosal solutions containing high concentrations of nicotine or methylamine. Estimates of apical Na+ entry at varying pHi, upon increasing [Na+]o from 0 to 25 mM, indicate that Na+/H+ exchange is active at pHi 7.4. Intracellular H+ stimulated apical Na+ entry by titration of more than one site (apparent pK 7.1, Hill coefficient 1.7). The results suggest that external Na+ and H+ interact with one site of the Na+/H+ exchanger and that cytoplasmic H+ acts on at least two sites. The external titratable group seems to be an imidazolium, which is apparently different from the amiloride-binding site. The dependence of Na+ entry on pHi supports the notion that the Na+/H+ exchanger is operational under normal transport conditions.     

Respiratory-driven Na+ electrical potential in the bacterium Vitreoscilla

Vitreoscilla is a Gram-negative bacterium with unique respiratory physiology in which Na+ was implicated as a coupling cation for the generation of a transmembrane electrical gradient (delta psi). Thus, cells respiring in the presence of 110 mM Na+ generated a delta psi of -142 mV compared to only -42 and -56 mV for Li+ and choline, respectively, and even the -42 and -56 mV were insensitive to the protonophore 3,5-di-tert-butyl-4-hydroxybenzaldehyde (DTHB). The kinetics of delta psi formation and collapse correlated well with the kinetics of Na+ fluxes but not with those of H+ fluxes. Cyanide inhibited respiration, Na+ extrusion, and delta psi formation 81% or more, indicating that delta psi formation and Na+ extrusion were coupled to respiration. Experiments were performed to distinguish among three possible transport systems for this coupling: (1) a Na(+)-transporting ATPase; (2) an electrogenic Na+/H+ antiport system; (3) a primary Na+ pump directly driven by the free energy of electron transport. DCCD and arsenate decreased cellular ATP up to 86% but had no effect on delta psi, evidence against a Na(+)-transporting ATPase. Low concentrations of DTHB had no effect on delta psi; high concentrations transiently collapsed delta psi, but led to a stimulation of Na+ extrusion, the opposite of that expected for a Na+/H+ antiport system. Potassium ion, which collapses delta psi, also stimulated Na+ extrusion. The experimental evidence is against Na+ extrusion by mechanisms 1 and 2 and supports the existence of a respiratory-driven primary Na+ pump for generating delta psi in Vitreoscilla.     

Na/Ca exchange and Na/K-ATPase function are equally concentrated in transverse tubules of rat ventricular myocytes

Formamide-induced detubulation of rat ventricular myocytes was used to investigate the functional distribution of the Na/Ca exchanger (NCX) and Na/K-ATPase between the t-tubules and external sarcolemma. Detubulation resulted in a 32% decrease in cell capacitance, whereas cell volume was unchanged. Thus, the surface-to-volume ratio was used to assess the success of detubulation. NCX current (I(NCX)) and Na/K pump current (I(pump)) were recorded using whole-cell patch clamp, as Cd-sensitive and K-activated currents, respectively. Both inward and outward I(NCX) density was significantly reduced by approximately 40% in detubulated cells. I(NCX) density at 0 mV decreased from 0.19 +/- 0.03 to 0.10 +/- 0.03 pA/pF upon detubulation. I(pump) density was also lower in detubulated myocytes over the range of voltages (-50 to +100 mV) and internal [Na] ([Na](i)) investigated (7-22 mM). At [Na](i) = 10 mM and -20 mV, I(pump) density was reduced by 39% in detubulated myocytes (0.28 +/- 0.02 vs. 0.17 +/- 0.03 pA/pF), but the apparent K(m) for [Na](i) was unchanged (16.9 +/- 0.4 vs. 17.0 +/- 0.3 mM). These results indicate that although thet-tubules represent only approximately 32% of the total sarcolemma, they contribute approximately 60% to the total I(NCX) and I(pump). Thus, the functional density of NCX and Na/K pump in the t-tubules is 3-3.5-fold higher than in the external sarcolemma.     

Insulin affects the sodium affinity of the rat adipocyte (Na+,K+)-ATPase

The K0.5 for intracellular sodium of the two forms of (Na+,K+)-ATPase which exist in rat adipocytes (Lytton, J., Lin, J. C., and Guidotti, G. (1985) J. Biol. Chem. 260, 1177-1184) has been determined by incubating the cells in the absence of potassium in buffers of varying sodium concentration; these conditions shut off the Na+ pump and allow sodium to equilibrate into the cell. The activity of Na+,K+)-ATPase was then monitored with 86Rb+/K+ pumping which was initiated by adding isotope and KCl to 5 mM, followed by a 3-min uptake period. Atomic absorption and 22Na+ tracer equilibration were used to determine the actual intracellular [Na+] under the different conditions. The K0.5 values thus obtained were 17 mM for alpha and 52 mM for alpha(+). Insulin treatment of rat adipocytes had no effect on the intracellular [Na+] nor on the Vmax of 86Rb+/K+ pumping, but did produce a shift in the sodium ion K0.5 values to 14 mM for alpha (p less than 0.025 versus control) and 33 mM for alpha(+) (p less than 0.005 versus control). This change in affinity can explain the selective stimulation of alpha(+) by insulin under normal incubation conditions. Measurement of the K0.5 for sodium ion of (Na+,K+)-ATPase in membranes isolated from adipocytes revealed only a single component of activation with a low K0.5 of 3.5 or 12 mM in the presence of 10 or 100 mM KCl, respectively. Insulin treatment of the isolated membranes or of the cells prior to membrane separation had no effect on these values.     

Hemisodium, a novel selective Na ionophore. Effect on normal human erythrocytes.

Hemisodium is a novel Na ionophore that belongs to the class of compounds called cryptands. These compounds possess an electron-rich cavity for binding of cations and are conformationally organized during synthesis to favor the selective binding of one cation over another. In media containing 145 mM NaCl and 5 mM KCl, hemisodium (10(-5) M) increased erythrocyte Na content from 23 to 345 mmol/kg.dry cell solid (dcs) over 4 h and increased water content from 1.8 to 3.5 liter/kg.dcs over the same period. K content decreased somewhat over the same time period, but this fall in K content was prevented entirely by incubation in either low Na media (to prevent net Na entry) or in Cl free media. Thus, the decrease in K content in high NaCl media was due to cell swelling, which activated KCl cotransport, and not due to a direct action of hemisodium on K permeability. Hemisodium-mediated Na transport was conductive, because erythrocyte membrane potential (Vm), determined by diS-C3-5 fluorescence, changed from -9 to +22 mV in high Na media in the presence of hemisodium and DIDS. In cells equilibrated with sulfamate, an anion with low conductive permeability, Vm changed 54 mV per 10-fold change in external Na concentration with the addition of hemisodium. In contrast, a 10-fold change in the external concentration of K, Rb, Cs, or T1 failed to alter Vm in the presence of hemisodium, suggesting a high Na specificity of the ionophore. Na conductance determined from net fluxes increased from 0.04 to 5.2 microS/cm2 with 10 microM hemisodium, and with that concentration the ratio of Na to K conductance was 45:1. Among the Na ionophores available so far, hemisodium appears to have the greatest specificity. Hemisodium may be a valuable tool in membrane transport studies.     

Ionic selectivity, saturation, and block in a K+-selective channel from sarcoplasmic reticulum

The open-channel conductance properties of a voltage-gated channel from sarcoplasmic reticulum were studied in planar phospholipid membranes. The channel is ideally selective for K+ over Cl- and for K+ over Ca++. In symmetrical 1 M solutions, the single-channel conductance (in pmho) falls in the order: K+ (214) > NH4+ (157) > Rb+ (125) > Na+ (72) > La+ (8.1) > Cs+ (< 3). In neutral bilayers, the channel conductance saturates with ion activity according to a rectangular hyperbolic relation, with half-saturation activities of 54 mM for K+ and 34 mM for Na+. Under symmetrical salt conditions, the K+:Na+ channel conductance ratio increases with salt activity, but the permeability ratio, measured by single-channel bi-ionic potentials, is constant between 20 mM and 2.5 M salt; the permeability ratio is equal to the conductance ratio in the limit of low-salt concentration. The channel conductance varies < 5% in the voltage range -100 to +70 mV. The maximum conductance varies K+ and Na+ is only weakly temperature dependent (delta H++ = 4.6 and 5.3 kcal/mol, respectively), but that of Li+ varies strongly with temperature (delta H++ = 13 kcal/mol). The channel's K+ conductance is blocked asymmetrically by Cs+, and this block is competitive with K+. The results are consistent with an Eyring-type barriers as it permeates the channel. The data conform to Lüger's (1973. Biochem. Biophys. Acta. 311:423-441) predictions for a "pure" single-ion channel.