Sodium-calcium ion exchange in skeletal muscle sarcolemmal vesicles

Summary The Ca²⁺ permeability of rabbit skeletal muscle sarcolemmal vesicles was investigated by means of radioisotope flux measurements. A membrane vesicle fraction highly enriched in sarcolemma, as revealed by enzymatic markers, was obtained from the 22–27% region of sucrose gradients after isopycnic centrifugation. The ability of sarcolemmal vesicles to exchange Na⁺ for Ca²⁺ was investigated by measuring Ca²⁺ influx into and efflux from sarcolemmal vesicles in the presence and absence of a Na⁺ gradient. It was found that Ca²⁺ movements were enhanced in the direction of the higher Na⁺ concentration. When intra- and extravesicular Na⁺ concentrations were high, Na⁺–Na⁺ exchange predominated and Na⁺–Ca²⁺ exchange was low or absent. The presence of the Ca²⁺ ionophore A23187 in the dilution medium resulted in the rapid release of Ca²⁺ and the elimination of the Na⁺-enhanced efflux of Ca²⁺, suggesting that internal rather than bound external Ca²⁺ was exchanged with Na⁺. La³⁺ abolished Na⁺–Ca²⁺ exchange and decreased overall membrane permeability. Na⁺–Ca²⁺ exchange was not due to sarcoplasmic reticulum or mitochondrial contaminants. This investigation suggests that skeletal muscle, like cardiac muscle and neurons, is capable of a transmembranous Na⁺–Ca²⁺ exchange.     

Cation transport properties of a synthetic Ca2+-selective peptide ionophore in phospholipid and sarcoplasmic reticulum vesicles

Transport by the synthetic cyclic peptide ionophore CYCLEX-2E (Deber, C.M., Young, M.E.M., and Tom-Kun, J. (1980) Biochemistry 19, 6194–6198), which in contrast to Ca²⁺ ionophore A23187 contains no ionizable protons, has been studied with respect to Ca²⁺ and Na⁺ transport, and the involvement of exchanged, or counter-transported ions during the transport process. CYCLEX-2E was found to equilibrate Na⁺ and Ca²⁺ gradients across phospholipid vesicle membranes. Experiments using the indicator dye Arsenazo III established that calcium ions were indeed reaching the aqueous intravesicular compartments. Absence of metal cations in the external buffer slowed, but did not eliminate, the efflux of Ca²⁺ from phosphatidylcholine vesicles. As an example of its activity in a biological membrane, CYCLEX-2E was shown to be capable of producing Ca²⁺ efflux from sarcoplasmic reticulum vesicles which had been loaded with Ca²⁺ in an ATP-dependent manner. The overall results suggest that in transport by synthetic peptide ionophores typified by CYCLEX-2E, electroneutrality is achieved either through (a) peptide-mediated compensating (but not coupled) fluxes of other cations, or where this is not an option, by (b) transmembrane diffusion of permeant ions such as H⁺, OH^?, or Cl^?.     

Acetylcholine receptor-mediated sodium ion efflux after rapid hypoosmotic loading of radiotracer

Torpedo membrane vesicles (microsacs) enriched in acetylcholine receptors can be loaded with radiotracer permeants such as ²²Na⁺ and [³H]sucrose in only a few minutes. The fast loading technique employs exposure of vesicles to hypoosmotic solutions containing the radiotracers. Subsequent to loading of permeants, their efflux from the vesicles can be determined by a Millipore filtration technique. Efflux rates following hypoosmotic loading are comparable to those following the conventional loading technique which employs isotonic solutions. Hence, efflux measurements can be begun immediately after vesicle preparation. Furthermore, simultaneous loading of ²²Na⁺ and [³H]sucrose allows a normalization of ²²Na⁺ efflux with respect to vesicle volume, since ²²Na⁺, but not [³H]sucrose, is released specifically by cholinergic effectors. Such double-isotope measurements increase the reliability of the data derived.     

Characterization of Exchange Inhibitory Peptide Effects on Na+/Ca2+ Exchange in Rat and Human Brain Plasma Membrane Vesicles

Abstract: The inhibitory effects of Na⁺/Ca²⁺ exchange inhibitory peptide (XIP), which corresponds to residues 219–238 of the Na⁺/Ca²⁺ exchange protein from canine heart, were studied in both rat and human brain plasma membrane vesicles. XIP had very high potency with respect to the inhibition of the initial velocity of intravesicular Na⁺-dependent Ca²⁺ uptake in both rat brain [IC₅₀ = 3.05 ± 0.69 µM (mean ± SE)] and human brain (IC₅₀ = 3.58 ± 0.58 µM). The maximal inhibition seen in rat brain vesicles was ～80%, whereas human brain vesicles were inhibited 100%. XIP also inhibited extravesicular Na⁺-dependent Ca²⁺ release, and the inhibitory effect was enhanced by increasing the extravesicular Na⁺ concentration. In contrast, the inhibitory effect of bepridil was competitive with respect to extravesicular Na⁺. When XIP was added at steady state (5 min after the initiation of intravesicular Na⁺-dependent Ca²⁺ uptake), it was found that the intravesicular Ca²⁺ content declined with time. Analysis of unidirectional fluxes for Ca²⁺ at steady state showed that 50 µM XIP inhibited Ca²⁺ influx and efflux ～85 and 70%, respectively. This result suggested that XIP inhibited both Na⁺/Ca²⁺ exchange and Ca²⁺/Ca²⁺ exchange but had no effect on the passive release pathway for Ca²⁺. The results suggest structural homology among cardiac, rat, and human brain exchangers in the XIP binding domain and that the binding of Na⁺ or other monovalent cations, e.g., K⁺, is required for XIP to have its inhibitory effect on Ca²⁺ transport.     

Modulation of Na+-Ca2+ exchange in cardiac sarcolemmal vesicles by Ca2+ antagonists

Summary The purpose of this study was to examine the effect of three classes of Ca²⁺ antagonists, diltiazem, verapamil and nifedipine on Na⁺-Ca²⁺ exchange mechanism in the sarcolemmal vesicles isolated from canine heart. Na⁺-Ca²⁺ exchange and Ca²⁺ pump (ATP-dependent Ca²⁺ uptake) activities were assessed using the Millipore filtration technique. sarcolemmal vesicles used in this study are estimated to consist of several subpopulations wherein 23% are inside-out and 55% are right side-out sealed vesicles in orientation. The affect of each Ca²⁺ antagonist on the Na⁺-dependent Ca²⁺ uptake was studied in the total population of sarcolemmal vesicles, in which none of the agents depressed the initial rate of Ca²⁺ uptake until concentrations of 10 M were incubated in the incubation medium. However, when sarcolemmal vesicles were preloaded with Ca²⁺ via ATP-dependent Ca²⁺ uptake, cellular Ca²⁺ influx was depressed only by verapamil (28%) at 1 M in the efflux medium with 8 mM Na⁺. Furthermore, inhibition of Ca²⁺ efflux by verapamil was more pronounced in the presence of 16 mM Na⁺ in the efflux medium. The order of inhibition was; verapamil > diltiazem > nifedipine. These results indicate that same forms of Ca²⁺-antagonist drugs may affect the Na⁺-Ca²⁺ exchange mechanism in the cardiac sarcolemmal vesicles and therefore we suggest this site of action may contribute to their effects on the myocardium.     

Ba2+-inhibitable86Rb+ fluxes across membranes of vesicles from toad urinary bladder

Summary ⁸⁶Rb⁺ fluxes have been measured in suspensions of vesicles prepared from the epithelium of toad urinary bladder. A readily measurable barium-sensitive, ouabain-insensitive component has been identified; the concentration of external Ba²⁺ required for half-maximal inhibition was 0.6mm. The effects of externally added cations on⁸⁶Rb⁺ influx and efflux have established that this pathway is conductive, with a selectivity for K⁺, Rb⁺ and Cs⁺ over Na⁺ and Li⁺. the Rb⁺ uptake is inversely dependent on external pH, but not significantly affected by internal Ca²⁺ or external amiloride, quinine, quinidine or lidocaine. It is likely, albeit not yet certain, that the conductive Rb⁺ pathway is incorporated in basolateral vesicles oriented right-side-out. It is also not yet clear whether this pathway comprises the principle basolateral K⁺ channel in vivo, and that its properties have been unchanged during the preparative procedures. Subject to these caveats, the data suggest that the inhibition by quinidine of Na⁺ transport across toad bladder does not arise primarily from membrane depolarization produced by a direct blockage of the basolateral channels. It now seems more likely that the quinidine-induced elevation of intracellular Ca²⁺ activity directly blocks apical Na⁺ entry.     

Ca++ fluxes in resealed synaptic plasma membrane vesicles

The effect of the monovalent cations Na⁺, Li⁺, and K⁺ on Ca⁺⁺ fluxes has been determined in resealed synaptic plasma membrane vesicle preparations from rat brain. Freshly isolated synaptic membranes, as well as synaptic membranes which were frozen (?80°C), rapidly thawed, and passively loaded with K₂/succinate and ⁴⁵CaCl₂, rapidly released approximately 60% of the intravesicular Ca⁺⁺ when exposed to NaCl or to the Ca⁺⁺ ionophore A 23187. Incubation of these vesicles with LiCl caused a lesser release of Ca⁺⁺. The EC₅₀ for Na⁺ activation of Ca⁺⁺ efflux from the vesicles was approximately 6.6mM. exposure of the Ca⁺⁺-loaded vesicles to 150 mM KCl produced a very rapid (?1 sec) loss of Ca⁺⁺ from the vesicles, but the Na⁺-induced efflux could still be detected above this K⁺ - sensitive effect. Vesicles pre-loaded with NaCl (150 mM) exhibited rapid ⁴⁵Ca uptake with an estimated EC₅₀ for Ca⁺⁺ of 7–10 μM. This Ca⁺⁺ uptake was blocked by dissipation of the Na⁺ gradient. These observations are suggestive of the preservation in these purified frozen synaptic membrane preparations of the basic properties of the Na⁺Ca⁺⁺ exchange process and of a K⁺ - sensitive Ca⁺⁺ flux across the membranes.     

Influence of vanadate on calcium fluxes and net movement of calcium in intact squid axons

Intracellular vanadate at a concentration of 100 μM inhibits the uncoupled efflux of Ca²⁺ from intact axons but has little effect on the exchange fluxes and on the Ca²⁺-dependent Na⁺ efflux. External vanadate has no effect on the Ca²⁺ efflux. In addition and most importantly intracellular vanadate inhibits the Ca²⁺ efflux in the presence of external Na⁺ and Ca²⁺ suggesting that the uncoupled efflux is operative under physiological conditions. Measurements of the net movements of Ca²⁺ under near physiological conditions have confirmed this conclusion.     

Ca2+ control of electrolyte permeability in plasma membrane vesicles from cat pancreas

Summary The influence of Ca²⁺ and other cations on electrolyte permeability has been studied in isolated membrane vesicles from cat pancreas.Ca²⁺ in the micromolar to millimolar concentration range, as well as Mg²⁺, Sr²⁺, Mn²⁺ and La³⁺ at a tested concentration of 10^–4 m, increased Na⁺ permeability when applied at the vesicle inside. When added to the vesicle outside, however, they decreased Na⁺ permeability. Ba²⁺ was effective from the outside but not from the vesicle inside.When Ca²⁺ was present at both sides of the membrane, Na⁺ efflux was not affected as compared to that in the absence of Ca²⁺. Monovalent cations such as Rb⁺, Cs⁺, K⁺, Tris⁺ and choline⁺ decreased Na⁺ permeability when present at the vesicle outside at a concentration range of 10 to 100mm. Increasing Na⁺ concentrations from 10 to 100mm at the vesicle inside increased Na⁺ permeability.The temperature dependence of Na⁺ efflux revealed that the activation energy increased in the lower temperature range (0 to 10°C) when Ca²⁺ was present at the outside or at both sides, but not when present at the vesicle inside only or in the absence of Ca²⁺.The results suggest that the Ca²⁺ outside effect is due to binding of calcium to negatively charged phospholipids with a consequent reduction of both fluidity and Na⁺ permeability of the membrane. The Ca²⁺-inside effect most likely involves interaction with proteins with consequent increase in Na⁺ permeability.The data are consistent with current hypotheses on secretagogue-induced fluid secretion in acinar cells of the pancreas according to which secretagogues elicit NaCl and fluid secretion by liberating Ca²⁺ from cellular membranes and by stimulating Ca²⁺ influx into the cell. The increased intracellular Ca²⁺ concentration in turn increases the contraluminal Na⁺ permeability which leads to NaCl influx. The luminal sodium pump finally transports Na⁺ ions into the lumen.     

Calcium efflux from sarcoplasmic reticulum vesicles

Ca²⁺ efflux was studied in sarcoplasmic reticulum vesicles isolated from rabbit skeletal muscle. In experimental conditions in which the Ca²⁺ pump is reversed, the rate of Ca^2? efflux varies with the ADP, orthophosphate and Mg²⁺ concentrations of the assay medium and is inhibited by Na⁺.     

Cationic interactions with Na+-H+ exchange and passive Na+ flux in cardiac sarcolemmal vesicles

Na⁺-H⁺ exchange and passive Na⁺ flux were investigated in cardiac sarcolemmal vesicles as a function of changing the ionic composition of the reaction media. The inclusion of EGTA in the reaction medium resulted in a potent stumulation of Na⁺ uptake by Na⁺-H⁺ exchange. It was found that millimolar concentrations of Mg²⁺ and Li⁺ were capable of inhibiting Na⁺-H⁺ exchange by 80%. One mechanism by which these ions may inhibit intravesicular Na⁺ accumulation by Na⁺-H⁺ exchange is via an increase in Na⁺ efflux. An examination of Na⁺ efflux kinetics from vesicles pre-loaded with Na⁺ revealed that Na⁺, Ca²⁺, Mg²⁺ and Li⁺ could stimulate Na⁺ efflux. Na⁺-H⁺ exchange was potently inhibited by an organic divalent cation, dimenthonium, which screens membrane surface charge. This would suggest that Na⁺-H⁺ exchange occurs in the diffuse double layer region of cardiac sarcolemma and this phenomenon is distinctly different from other Na⁺ transport processes. The results in this study indicate that in addition to a stimulation of Na⁺ efflux, the inhibitory effects of Mg²⁺, Ca²⁺ and Li⁺ on Na⁺-H⁺ exchange may also involve a charge dependent screening of Na⁺ interactions with the membrane.     

Regulatory Processes on the Cytoplasmic Surface of the Na+/Ca2+ Exchanger from Lobster Exoskeletal Muscle

A partially purified preparation of the lobster muscle Na⁺/Ca²⁺ exchanger was reconstituted with, presumably, random orientation in liposomes. Ca²⁺ efflux from ⁴⁵Ca-loaded vesicles was studied in exchanger molecules in which the transporter cytoplasmic surface was exposed to the extravesicular (ev) medium. Extravesicular Na⁺ (Na _ev )-dependent Ca²⁺ efflux depended directly upon the extravesicular Ca²⁺ concentration ([Ca²⁺] _ev ) with a half-maximal activation at [Ca²⁺] _ev = 0.6 μm. This suggests that the lobster muscle exchanger is catalytically upregulated by cytoplasmic Ca²⁺, as in most other species. In contrast, at low [Na⁺] _ev , the Ca _ev -binding site (i.e., on the cytoplasmic surface) for Ca²⁺ transported via Ca²⁺/Ca²⁺ exchange was half-maximally activated by about 7.5 μm Ca²⁺. Mild proteolysis of the Na⁺/Ca²⁺ exchanger by α-chymotrypsin also upregulated the Na _ev -dependent Ca²⁺ efflux. Following proteolytic digestion in Ca-free medium, the exchanger was no longer regulated by nontransported ev Ca²⁺. Proteolytic digestion in the presence of 1.9 μm free ev Ca²⁺, however, induced only a 1.6-fold augmentation of Ca²⁺ efflux, whereas, after digestion in nominally Ca-free medium, a 2.3-fold augmentation was observed; Ca²⁺ also inhibited proteolytic degradation of the Na⁺/Ca²⁺ exchanger measured by immunoblotting. These data suggest that Ca²⁺, bound to a high affinity binding site, protects against the activation of the Na⁺/Ca²⁺ exchanger by α-chymotrypsin. Additionally, we observed a 6-fold increase in the Na⁺/Ca²⁺ exchange rate, on average, when the intra- and extravesicular salt concentrations were increased from 160 to 450 mm, suggesting that the lobster muscle exchanger is optimized for transport at the high salt concentration present in lobster body fluids. Received: 20 October 1999/Revised: 13 January 2000     

Demarcation of Ca2+ transport processes in guinea pig stomach smooth muscle

Summary Microsomal fractions were isolated from gastric antrum and fundus smooth muscle of guinea pigs. Ca²⁺ uptake into and Ca²⁺ release from the membrane vesicles were studied by a rapid filtration method, and Ca²⁺ transport properties of the different regions of the stomach were compared. ATP-dependent Ca²⁺ uptake was similar in microsomes isolated from both regions. This uptake was increased by oxalate and was not affected by NaN₃. Oxalate affected Ca²⁺ permeability of both antrum and fundus microsome vesicles similarly. Fundus microsome vesicles preincubated in 100mm NaCl and then diluted to 1/20 concentration with Na⁺-free medium had significantly higher ATP-independent Ca²⁺ uptake than vesicles preincubated in 100mm KCl and treated the same way. This was not true for antrum vesicles. Monensin abolished Na⁺-dependent Ca²⁺ uptake, and NaCl enhanced Ca²⁺ efflux from fundus microsome vesicles. The halflife values of Ca²⁺ loss from fundus vesicles in the presence of NaCl were significantly smaller than those in the presence of KCl. The release of Ca²⁺ from the vesicles within the first 3 min was accelerated by NaCl to three times that by KCl. However, NaCl had ro effect on Ca²⁺ release from antrum microsome vesicles.Results suggest two distinct mechanisms of stomach membrane Ca²⁺ transport: (1) ATP-dependent Ca²⁺ uptake and (2) Na⁺–Ca²⁺ exchange; the latter in the fundus only.     

Na+-driven Ca2+ transport in alkalophilic Bacillus

Ca²⁺ transport was studied in membrane vesicles of alkalophilic Bacillus. When Na⁺-loaded membrane vesicles were suspended in KHCO₃/KOH buffer (pH 10) containing Ca²⁺, rapid uptake of Ca²⁺ was observed. The apparent $\text{K}{}_{\mathrm{<mtext>m</mtext>}}$ value for Ca²⁺ measured at pH 10 was about 7 μM, and the $\text{K}{}_{\mathrm{<mtext>m</mtext>}}$ value shifted to 24 μM when measured at pH 7.4. The efflux of Ca²⁺ was studied with Ca²⁺-loaded vesicles. Ca²⁺ was released when Ca²⁺-loaded vesicles were suspended in medium containing 0.4 M Na⁺.Ca²⁺ was also transported in membrane vesicles driven by an artificial pH gradient and by a membrane potential generated by K⁺-valinomycin in the presence of Na⁺.These results indicate the presence of Ca²⁺/Na⁺ and H⁺/Na⁺ antiporters in the alkalophilic Bacillus A-007.     

Permeability of reconstituted sarcoplasmic reticulum vesicles: Reconstitution of the K+, Na+ channel

Permeability properties of reconstituted rabbit skeletal muscle sarcoplasmic reticulum vesicles were characterized by measuring efflux rates of [³H]inulin, [³H]choline⁺, ⁸⁶Rb⁺, and ²²Na⁺, as well as membrane potential changes using the voltage-sensitive probe, 3,3′-dipentyl-2,2′-oxacarbocyanine. Native vesicles were dissociated with deoxycholate and were reconstituted by dialysis. Energized Ca²⁺ accumulation was partially restored. About $\text{1}\text{2}$ of the reconstituted vesicles were found to be ‘leaky’, i.e., permeable to choline⁺ or Tris⁺ but not to inulin. The remaining reconstituted vesicles were ‘sealed’, i.e., impermeable to choline⁺, Tris⁺ and inulin. Sealed reconstituted vesicles could be further subdivided according to their K⁺, Na⁺ permeability. About $\text{1}\text{2}$, previously designated Type I, were readily permeable to K⁺ and Na⁺, indicating the presence of the K⁺, Na⁺ channel of sarcoplasmic reticulum. The remaining sealed vesicles (Type II) formed a permeability barrier to K⁺ and Na⁺, suggesting that they lacked the K⁺, Na⁺ channel. These studies show that the K⁺, Na⁺ channel of sarcoplasmic reticulum can be solubilized with detergent and reconstituted with retention of activity. Furthermore, our results suggest that part or all of the decreased Ca²⁺-loading efficiency of reconstituted vesicles may be due to the presence of a significant fraction of leaky vesicles.     

Na+ - and Ca2+ -dependent components in action potentials of the ovulation hormone producing caudo-dorsal cells in Lymnaea stagnalis (Gastropoda)

Action potentials in the afterdischarge of the ovulation hormone producing caudo–dorsal cells (CDC) of Lymnaea stagnalis are strikingly different from electrically evoked spikes in the silent resting and inhibited states of these cells. Spikes evoked in the silent states consist of one fast peak (80–100 mV; 10–15 ms). The overshoot is Na⁺ - and Ca²⁺ -dependent. Spikes are blocked in Na⁺ -free saline and by TTX. Repolarization is retarded by TEA. Co²⁺ increases the overshoot. Active state action potentials (60–80 mV) last up to 125 ms, due to activation of a slow component following the TTX-sensitive spike. The slow component is Na⁺ - and Ca²⁺ -dependent. In normal saline it is blocked by Co²⁺ and La³⁺. In Ca²⁺ -free saline the remaining part of the slow component is blocked by La³⁺ only. The slow component is voltage-dependent in a graded fashion. Activation is bound to the active state in which the CDC are depolarized by 20 mV. TEA and Ca²⁺ -free saline greatly increase spike duration in the active state. This suggests that, in addition to the classical TEA-sensitive channel, a Ca²⁺ -dependent K⁺ channel is involved in repolarization of active state action potentials. The underlying membrane properties and the functional significance are discussed in relation to the pacemaking mechanism of the CDC.     

An electrogenic Na+/Ca2+ antiporter in addition to the Ca2+ pump in cardiac sarcolemma

Vesicles isolated from rat heart, particularly enriched in sarcolemma markers, were examined for their sidedness by investigation of side-specific interactions of modulators with the asymmetric (Na⁺ + K⁺)-ATPase and adenylate cyclase complex. The membrane preparation with the properties expected for inside-out vesicles showed the highest rate of ATP-driven Ca²⁺ transport. The Ca²⁺ pump was stimulated 1.7- and 2.1-fold by external Na⁺ and K⁺, respectively, the half-maximal activation occurring at 35 mM monovalent cation concentration. In vesicles loaded with Ca²⁺ by pump action in a medium containing 160 mM KCl, a slow spontaneous release of Ca²⁺ started after 2 min. The rate of this release could be dramatically increased by the addition of 40 mM NaCl to the external medium. In contrast, 40 mM KCl exerted no appreciable effect on vesicles loaded with Ca²⁺ in a medium containing 160 mM NaCl. Ca²⁺ movements were also studied in the absence of ATP and Mg²⁺. Vesicles containing an outwardly directed Na⁺ gradient showed the highest Ca²⁺ uptake activity. These findings suggested the operation of a Ca²⁺/Na⁺ antiporter in addition to the active Ca²⁺ pump in these sarcolemmal vesicles. A valinomycin-induced inward K⁺-diffusion potential stimulated the Na⁺- Ca²⁺ exchange, suggesting its electrogenic nature. If in the absence of ATP and Mg²⁺ the transmembrane Na_i⁺/Na_o⁺ gradient exceeded 160/15 mM concentrations, Ca²⁺ uptake could be stimulated by the addition of 5 mM oxalate, indicating Na⁺ gradient-induced Ca²⁺ uptake to be a translocation of Ca²⁺ to the lumen of the vesicle. A sarcoplasmic reticulum contamination, removed by further sucrose gradient fractionation, contained rather low Na⁺-Ca²⁺ exchange activity. This result suggests that the activity can be entirely accounted for by the sarcolemmal content of the cardiac membrane preparation.     

Na+-transporting ATPase in the plasma membrane of halotolerant microalga Dunaliella maritima operates as a Na+ uniporter

A fraction of inside-out membrane vesicles enriched in plasma membranes (PM) was isolated from Dunaliella maritima cells. Attempts were made to reveal ATP-driven Na⁺-dependent H⁺ efflux from the PM vesicles to external medium, as detected by alkalization of the vesicle lumen. In parallel experiments, ATP-dependent Na⁺ uptake and electric potential generation in PM vesicles were investigated. The alkalization of the vesicle lumen was monitored with an impermeant pH-sensitive optical probe pyranine (8-hydroxy-1,3,6-pyrenetrisulfonic acid), which was loaded into vesicles during the isolation procedure. Sodium uptake was measured with ²²Na⁺ radioactive label. The generation of electric potential in PM vesicles (positive inside) was recorded with a voltage-sensitive probe oxonol VI. Appreciable Na⁺-and ATP-dependent alkalization of vesicle lumen was only observed in the presence of a protonophore CCCP (carbonyl cyanide-chlorophenylhydrazone). In parallel experiments, CCCP accelerated the ATP-dependent ²²Na⁺ uptake and abolished the electric potential generated by the Na⁺-ATPase at the vesicle membrane. A permeant anion NO^? ₃ accelerated ATP-dependent ²²Na⁺ uptake and promoted dissipation of the electric potential like CCCP did. At the same time, NO^? ₃ inhibited the ATP-and Na⁺-dependent alkalization of the vesicle lumen. The results clearly show that the ATP-and Na⁺-dependent H⁺ efflux from PM vesicles of D. maritima is driven by the electric potential generated at the vesicle membrane by the Na⁺-ATPase. Hence, the Na⁺-transporting ATPase of D. maritima carries only one ion species, i.e., Na⁺. Proton is not involved as a counter-ion in the catalytic cycle of this enzyme.     

Role of calcium and calmodulin in the regulation of the rabbit ileal brush-border membrane Na+/H+ antiporter

Summary In rabbit ileum, Ca²⁺/calmodulin (CaM) appears to be involved in physiologically inhibiting the linked NaCl absorptive process, since inhibitors of Ca²⁺/CaM stimulate linked Na⁺ and Cl^– absorption. The role of Ca²⁺/CaM-dependent phosphorylation in regulation of the brush-border Na⁺/H⁺ antiporter, which is believed to be part of the neutral linked NaCl absorptive process, was studied using purified brush-border membrane vesicles, which contain both the Na⁺/H⁺ antiporter and Ca²⁺/CaM-dependent protein kinase(s) and its phosphoprotein substrates. Rabbit ileal villus cell brush-border membrane vesicles were prepared by Mg precipitation and depleted of ATP. Using a freezethaw technique, the ATP-depleted vesicles were loaded with Ca²⁺, CaM, ATP and an ATP-regenerating system consisting of creatine kinase and creatine phosphate. The combination of Ca²⁺/CaM and ATP inhibited Na⁺/H⁺ exchange by 45±13%. This effect was specific since Ca²⁺/CaM and ATP did not alter diffusive Na⁺ uptake, Na⁺-dependent glucose entry, or Na⁺ or glucose equilibrium volumes. The inhibition of the Na⁺/H⁺ exchanger by Ca²⁺/CaM/ATP was due to an effect on theV _max and not on theK _m for Na⁺. In the presence of CaM and ATP, Ca²⁺ caused a concentration-dependent inhibition of Na⁺ uptake, with an effect 50% of maximum occurring at 120nm. This Ca²⁺ concentration dependence was similar to the Ca²⁺ concentration dependence of Ca²⁺/CaM-dependent phosphorylation of specific proteins in the vesicles. The Ca²⁺/CaM/ATP-inhibition of Na⁺/H⁺ exchange was reversed by W₁₃, a Ca²⁺/CaM antagonist, but not by a hydrophobic control, W₁₂, or by H-7, a protein kinase C antagonist. we conclude that Ca²⁺, acting through CaM, regulates ileal brush-border Na⁺/H⁺ exchange, and that this may be involved in the regulation of neutral linked NaCl absorption.     

Reaction of ozone with phospholipid vesicles and human erythrocyte ghosts

Phospholipid vesicles (unilamellar) and liposomes (multilamellar) made from egg phosphatidylcholine reacted similarly with ozone, producing hydrogen peroxide and malonaldehyde. On the basis of amount of ozone reacted, there was a 20% yield of hydrogen peroxide and 2.4% yield of malonaldehyde. The reactivity of the egg phosphatidylcholine membranes was a function of exposed membrane surface area. Large amounts of ozone caused no change in erythrocyte ghost phospholipid, fatty acid, or cholesterol composition. Thiobarbituric acid-positive material and conjugated dienes were present in very small quantities, suggesting some lipid oxidation which was below the limits of chromatographic detection. Ozone inhibited glyceraldehyde 3-phosphate dehydrogenase more than (Na⁺ + K⁺) adenosine triphosphate in exposed unsealed erythrocyte ghosts. The (Na⁺ + K⁺) adenosine triphosphatase activity sensitive to ozone was the ouabain-insensitive activity. Acetylcholinesterase activity was not significantly inhibited.