The effect of salts on catecholamine fluxes and adenosine triphosphatase activity in storage vesicles from the adrenal medulla

1. Influx and efflux of catecholamine and adenosine triphosphatase activity in storage vesicles from the adrenal medulla were studied with dl-[(14)C]adrenaline in different media. 2. The lowest values for flux and adenosine triphosphatase activity were observed in sucrose media in which an ATP-dependent influx of catecholamine compensated for an efflux of the same magnitude. Efflux in the presence or absence of ATP was similar. 3. In media containing sodium succinate or glutarate adenosine triphosphatase activity was higher and the ATP-dependent influx of catecholamine was about twice that observed in iso-osmotic sucrose medium. In the presence of ATP influx and efflux of catecholamine were balanced; in its absence there was a net release of catecholamine, since efflux was more than twice the influx. Efflux in the presence or absence of ATP was similar. 4. In media containing sodium or potassium chloride and in the presence of ATP influx and adenosine triphosphatase activity were further enhanced, but in the absence of ATP there was no further increase in influx, since catecholamine was released with or without ATP at the same rate. Efflux was therefore twice as high in the presence of ATP as in its absence. 5. Sodium nitrate suppressed the ATP-dependent influx nearly completely, but caused a greatly enhanced efflux, which was twice as high in the presence of ATP as in its absence. 6. The extinction of vesicular suspensions remained unchanged in the presence of ATP under conditions where the catecholamine efflux was balanced by the influx. Under conditions where the efflux was not compensated by influx, the extinction of the suspensions decreased in the presence of ATP more than in its absence.     

Influence of nucleotides, cations and nucleoside triphosphatase inhibitors on the release of ribonucleic acid from isolated rat liver nuclei.

The reasons underlying reported discrepancies in the effects of ATP, ADP, adenosine 5'-[beta gamma-methylene]triphosphate, AMP + PPi, P-chloromercuribenzoate and F- on RNA efflux from isolated rat liver nuclei and on nuclear envelope nucleoside triphosphatase activity were investigated. The stimulatory effect of ADP was attributed to myokinase activity associated with the nuclei; this activity was eluted on repeated washing with nuclear incubation medium. In the absence of Ca2+ and Mn2+, ATP, adenosine 5'[beta gamma-methylene]triphosphate and AMP +PPi were found to promote release of both DNA and RNA. In the presence of 0.5 mM-Ca2+ and 9.3 mM-Mn2+, only ATP promoted RNA efflux to a significant extent. In the absence of spermidine, Ca2+ and Mn2+, nuclei released large quantities of DNA and RNA into the medium; this effect was promoted by p-chloromereuribenzoate. In the presence of the three cations, however, p-chloromercuribenzoate inhibited RNA efflux. F- caused a slight leakage of DNA from nuclei. The results are discussed in terms of models for the effects of ATP and analogues on RNA efflux and nuclear stability.     

The influence of external sodium ions on the sodium pump in erythrocytes

1. A study has been made of the interaction between Na(+) and K(+) on the adenosine triphosphatase activity of erythrocyte ;ghosts', and on the K(+) influx and Na(+) efflux of intact erythrocytes. The adenosine triphosphatase activity and the ion movements were greater at a low external K(+) concentration in the absence of Na(+) than they were in the presence of 150mm-Na(+). The inhibition by external Na(+) of K(+) influx had an inhibitory constant of 5-10mm. 2. Activation by K(+) of kidney microsomal adenosine triphosphatase was retarded by Na(+), and activation by Na(+) was retarded by K(+). Fragmented erythrocyte membranes behaved similarly. 3. These observations suggest that there is competition between Na(+) and K(+) at the K(+)-sensitive site of the membrane.     

The effect of a cross-bridging thiol reagent on the catecholamine fluxes of adrenal medulla vesicles

The thiol groups of the vesicular protein of bovine adrenal medulla were allowed to react with the bifunctional thiol reagent bis-(N-maleimidomethyl) ether and with the monofunctional thiol reagent N-ethylmaleimide, and the ATP-dependent and -independent catecholamine fluxes of the modified preparations were studied. 1. During the initial phase of the reaction bis-(N-maleimidomethyl) ether blocks twice as many thiol groups as does N-ethylmaleimide at equimolar concentrations. 2. Labelling of the bis-(N-maleimidomethyl) ether-protein compound with [(14)C]-cysteine shows that 70-80% of the blocked thiol groups are interconnected by the bifunctional thiol reagent. 3. At a low extent of reaction (1.5mol of thiol groups/10(6)g of protein) the catecholamine efflux is diminished. If more than 2mol of thiol groups/10(6)g of protein are blocked, the efflux is enhanced whichever thiol reagent is applied. 4. If 2-4mol of thiol groups/10(6)g of protein are blocked the inhibition of the catecholamine influx increases linearly with the proportion of the thiol groups blocked. 5. ATP protects the catecholamine influx and the adenosine triphosphatase activity against bis-(N-maleimidomethyl) ether poisoning somewhat less effectively than against N-ethylmaleimide poisoning.     

Influence of anion flux upon mitochondrial oxidative phosphorylation

We have measured mitochondrial ATP synthesis during passive anion influx and find that influx of phosphate leads to diminished efficiency (as reflected in the ATP:0 ratio) whereas influx of acetate produced enhanced efficiency. The anions, sulfate, propionate, and thiocyanate, are without influence on the ATP:0 ratio. It is likely that the opposite effects of phosphate and acetate on the ATP:0 ratio reflect phosphate-acetate exchange, and that acetate influx produces its positive effect on ATP synthesis by promoting phosphate efflux. Thus, phosphate efflux may be associated with increased, and phosphate influx, with decreased energy conservation.     

Adenosine triphosphatase activity of mycoplasma membranes

Rottem, Shlomo (Hebrew University, Jerusalem, Israel), and Shmuel Razin. Adenosine triphosphatase activity of mycoplasma membranes. J. Bacteriol. 92:714-722. 1966.-Adenosine triphosphatase activity of Mycoplasma laidlawii, M. gallisepticum, and Mycoplasma sp. strain 14 was confined to the cell membrane. The enzymatic activity was dependent on magnesium, but was not activated by sodium and potassium. Ouabain did not inhibit the adenosine triphosphatase activity of the mycoplasmas, and did not interfere with the active accumulation of potassium by M. laidlawii cells. Sulfhydryl-blocking reagents and fluoride inhibited the enzymatic activity, whereas 2,4-dinitrophenol was without any effect. Membranes of M. laidlawii hydrolyzed other nucleotide triphosphates and adenosine diphosphate (ADP), but at a lower rate than adenosine triphosphate (ATP). Nucleoside-2'-(3')-phosphates, ribose-5-phosphate, glucose-6-phosphate, and pyrophosphate were not hydrolyzed by the membrane preparations. It seems that the enzyme(s) involved in ATP hydrolysis by M. laidlawii membranes is strongly bound to the membrane subunits, which would account for the failure to purify the enzyme by protein fractionation techniques. The adenosine triphosphatase activity of mycoplasma membranes resembles in its properties that of similar enzymes studied in bacteria. The mycoplasma enzyme(s) seems to differ from the adenosine triphosphatase associated with ion transport in mammalian cell membranes and from mitochondrial adenosine triphosphatase.     

Autocrine regulation of volume-sensitive anion channels in airway epithelial cells by adenosine

The activity of volume-sensitive Cl- channels was studied in human tracheal epithelial cells (9HTEo-) by taurine efflux experiments. The efflux elicited by a hypotonic shock was partially inhibited by adenosine receptor antagonists, by alpha,beta-methyleneadenosine 5'-diphosphate (alphabetaMeADP), an inhibitor of the 5'-ectonucleotidase, and by adenosine deaminase. On the other hand, dipyridamole, a nucleoside transporter inhibitor, increased the swelling-induced taurine efflux. Extracellular ATP and adenosine increased taurine efflux by potentiating the effect of hypotonic shock. alphabetaMeADP strongly inhibited the effect of extracellular ATP but not that of adenosine. These results suggest that anion channel activation involves the release of intracellular ATP, which is then degraded to adenosine by specific ectoenzymes. Adenosine then binds to purinergic receptors, causing the activation of the channels. To directly demonstrate ATP efflux, cells were loaded with [3H]AMP, and the release of radiolabeled molecules was analyzed by high performance liquid chromatography. During hypotonic shock, cell supernatants showed the presence of ATP, ADP, and adenosine. alphabetaMeADP inhibited adenosine formation and caused the appearance of AMP. Under hypotonic conditions, elevation of intracellular Ca2+ by ionomycin caused an increase of ATP and adenosine in the extracellular solution. Our results demonstrate that volume-sensitive anion channels are regulated with an autocrine mechanism involving swelling-induced ATP release and then hydrolysis to adenosine.     

Inhibition of ribonucleic acid efflux from isolated SV40-3T3 cell nuclei by 3''-deoxyadenosine (cordycepin).

The effect of 3'-deoxyadenosine (cordycepin) on mRNA efflux from isolated SV40-3T3 cell nuclei has been studied and compared with its effect on the nucleoside triphosphatase activity in the isolated nuclear envelope. Inhibition of mRNA efflux occurs rapidly, but is dependent on the presence of ATP. Half-maximal inhibition occurs with 40 microM-cordycepin. The effect is not simulated by 2'-deoxyadenosine or by actinomycin D, and adenosine provides a substantial degree of protection against it. Cordycepin does not directly inhibit the nucleoside triphosphatase. The stimulation of this enzyme by poly(A) is not affected unless the poly(A) and cordycepin are incubated together with nuclear lysate in the presence of ATP; in this case the stimulation is significantly reduced. Possible interpretations of these results and their relevance for understanding the system in vivo for nucleo-cytoplasmic messenger transport are discussed.     

ATP as a source of interstitial adenosine in the rat heart.

The contribution of neuronal ATP to interstitial adenosine levels was investigated in isolated perfused rat hearts. Ventricular surface transudates, representing interstitial fluid, were analyzed for norepinephrine, ATP, and adenosine. Exocytotic release of norepinephrine was induced by electrical stimulation of cardiac efferents emanating from the stellate ganglion. Ganglion stimulation increased contractility, interstitial norepinephrine, ATP, and adenosine. Interstitial adenosine was 11- to 27-fold higher than interstitial ATP, suggesting that the released ATP is unlikely the only source of adenosine. In the presence of AOPCP (alpha,beta-methyleneadenosine 5'-diphosphate), an ecto-5'-nucleotidase inhibitor, the ganglion-stimulated increase in interstitial ATP and adenosine reached levels similar to those in the absence of AOPCP, also suggesting that adenosine does not derive from extracellular ATP. The perfusate Ca2+ was raised from 1 to 4 mM to determine the importance of the enhanced contractile function on the levels of norepinephrine, ATP, and adenosine. The results were increases in contractility and interstitial norepinephrine, ATP, and adenosine, which were not suppressed with atenolol, indicating a norepinephrine-independent release of ATP and adenosine. Reserpine treatment and administration of guanethidine depleted the catecholamine stores and diminished the catecholamine release, respectively. However, neither agent altered Ca2+-induced increases in ATP and adenosine. It is concluded that the amount of neuronal-derived ATP is low and most likely does not contribute significantly to interstitial levels of adenosine. Furthermore, elevations in interstitial norepinephrine, ATP, and adenosine are associated with neuronal-independent increases in contractile function.     

An active-site-directed adenosine triphosphate analogue binds to the beta-subunits of factor F1 mitochondrial adenosine triphosphatase with its triphosphate moiety.

The reaction of the mixed anhydride of [3H]ATP and mesitylenecarboxylic acid and soluble mitochondrial adenosine triphosphatase is accompanied by the covalent binding of one molecule of the inhibitor to a molecule of the enzyme and results in the inhibition of adenosine triphosphatase activity by more than 90%. The electrophoresis of adenosine triphosphatase modified by reaction with the mixed anhydride of [3H]ATP and mesitylenecarboxylic acid in polyacrylamide gel in the presence of sodium dodecyl sulphate showed that the inhibitor is bound to the beta-subunit of the enzyme. The results suggest that ATP may also bind to the beta-subunit of the adenosine triphosphatase with its triphosphate moiety.     

Kinetic studies on rat liver and beef heart mitochondrial adenosine triphosphatase: the effects of the chromium complexes of adenosine triposphate and adenosine diphosphate on the kinetic properties.

The kinetics of isolated rat liver and beef heart mitochondrial adenosine triphosphatase (ATPase) were studied by using the chromium ATP and ADP complexes as substrate analogs. It was found that both chromium ATP (CrATP) and chromium ADP (CrADP) are competitive inhibitors of ATP hydrolysis. The presence or absence of ATPase-activating anions, e.g., bisulfite, had little effect on the type or potency of the inhibition by these chromium complexes. Both CrADP and CrATP were noncompetitive inhibitors of the hydrolysis of ITP with both the heart and liver-derived enzymes. It was also found that CrADP was a consistently more effective inhibitor than the ATP complex with the beef heart enzyme. These results are consistent with the existence of two types of nucleotide binding sites on mitochondrial ATPases: One site is regulatory and is rather specific for adenosine polyphosphates, while the other site is relatively nonspecific and serves as the hydrolytic site.     

Catalytic properties of the F1-adenosine triphosphatase from Escherichia coli K-12 and its genetic variants as revealed by 18O exchanges

We have examined intermediate Pi-water oxygen exchange during [gamma-18O]ATP hydrolysis by the F1 adenosine triphosphatase from Escherichia coli K-12. Water oxygen incorporation into each Pi released was increased as ATP concentration was lowered as observed previously for the same reaction catalyzed by the enzyme from eukaryotic sources. Heterogeneous distributions of 18O in product Pi were produced by coexisting epsilon subunit-replete and epsilon subunit-depleted enzyme molecules. The epsilon-replete enzyme showed a much higher probability for oxygen exchange. These data imply that the epsilon subunit inhibits net ATP hydrolysis by imposing conformational constraints which reduce the cooperative conformational interactions that promote ADP and Pi release. Four enzyme variants altered in alpha or beta subunit structure with reduced net hydrolytic activity showed sharply increased oxygen exchange during ATP hydrolysis. Heterogeneity was apparent in the 18O distribution of the product Pi, however. That behavior could reflect hindered conformational interactions and/or increased affinity of the alpha 3 beta 3 gamma delta complex for the epsilon subunit. In contrast, enzyme from mutant uncA401 showed very little oxygen exchange accompanying hydrolysis of 20 microM ATP. This is the only enzyme so far reported with this unusual property. Its rate limitation appears to be in the hydrolytic rather than the product release step of the catalytic sequence.     

Extracellular ATP induces ion fluxes and inhibits growth of Friend erythroleukemia cells

Extracellular ATP (1 mM) inhibited the growth of Friend virus-infected murine erythroleukemia cells (MEL cells) but had no effect on dimethyl sulfoxide-induced differentiation. ATP (1 mM) also caused changes in the permeability of MEL cells to ions. There was an increased influx of 45Ca2+ from a basal level of 5 pmol/min to 18 pmol/min/10(6) cells to achieve a 2-fold increase in steady-state Ca2+ as measured at isotopic equilibration. Ca2+ influx was blocked by diisothiocyanostilbene disulfonate (DIDS), an inhibitor of anion transport. ATP also stimulated Cl- uptake, and this flux was inhibited by DIDS. The ratio of ATP stimulated Cl- to Ca2+ uptake was 1.6:1. K+ and Na+ influx were also stimulated by ATP, but phosphate uptake was inhibited; the Na+ influx dissipated the Na+ gradient and thus inhibited nutrient uptake. ATP-stimulated K+ influx was ouabain inhibitable; however, the total cellular K+ decreased due to an ATP-stimulated ouabain-resistant K+ efflux. Na+ influx and Ca2+ influx occurred by separate independent routes, since Na+ influx was not inhibited by DIDS. The effects observed were specific for ATP *K1/2 MgATP = 0.7 mM) since AMP, GTP, adenosine, and the slowly hydrolyzable ATP analogue adenyl-5'-yl imidodiphosphate were without effect. The major ionic changes in the cell were a decrease in K+ and increase in Na+; cytoplasmic pH and free Ca2+ did not change appreciably. These ATP-induced changes in ion flux are considered to be responsible for growth inhibition.     

Phospholipid-protein interactions in the Ca2+-adenosine triphosphatase of sarcoplasmic reticulum.

Ca2+-adenosine triphosphatase from sarcoplasmic reticulum has been delipidated by gel filtration through a Sephadex G-200 column equilibrated with buffer containing cholate. The delipidated Ca2+-adenosine triphosphatase had negligible adenosine triphosphatase activity, but up to 50% of the ATPase activity was restored when the delipidated enzyme was recombined with phosphilipids. It was shown with the delipidated preparation that the phosphorylation of the enzyme by either ATP or Pi was entirely dependent on phospholipids. Among the purified phospholipids, phosphatidylcholine reactivated the adenosine triphosphatase activity better than phosphatidylethanolamine. Vesicles capable of translocating Ca2+ were reconstituted from delipidated Ca2+-adenosine triphosphatase and phosphatidylethanolamine, but not with phosphatidylcholine alone. We conclude that the firmly bound phospholipids which are purified together with the adenosine triphosphatase protein are not essential for the pump since they can be substituted by phosphatidylethanolamine isolated from soybeans.     

Adenosine triphosphate consumption by bacterial arginyl-transfer ribonucleic acid synthetases.

ATP consumption by arginyl-tRNA synthetases from Escherichia coli and Bacillus stearothermophilus has been investigated by the firefly luciferin--luciferase assay. Arginyl-tRNA synthetase from E. coli utilizes ATP only for aminocylation of tRNA with a 1:1 stoicheiometry. In contrast, we have shown an adenosine triphosphatase activity of arginyl-tRNA synthetase from B. stearothermophilus in the absence of tRNAArg. Dowex chromatography revealed the formation of ADP by the thermophile enzyme; under aminoacylation conditions, AMP was also formed in amounts stoicheiometric with arginyl-tRNA formation.     

Tumor-associated a2 vacuolar ATPase acts as a key mediator of cancer-related inflammation by inducing pro-tumorigenic properties in monocytes

Cancer-related inflammation profoundly affects tumor progression. Tumor-associated macrophages (TAMs) are known regulators of that inflammation, but the factors that initiate cancer-related inflammation are poorly understood. Tumor invasiveness and poor clinical outcome are linked to increased expression of cell surface-associated vacuolar adenosine triphosphatases. The a2 isoform vacuolar adenosine triphosphatase is found on the surface on many solid tumors, and we have identified a peptide cleaved from a2 isoform vacuolar adenosine triphosphatase called a2NTD. a2NTD has properties necessary to induce monocytes into a pro-oncogenic TAM phenotype. The peptide upregulated both pro- and anti-inflammatory mediators. These included IL-1β and IL-10, which are important in promoting inflammation and immune escape by tumor cells. The secretion of inflammatory cytokine IL-1β was dependent on ATP, K(+) efflux, and reactive oxygen species, all mediators that activate the inflammasome. These findings describe a mechanism by which tumor cells affect the maturation of TAMs via a nontraditional cytokine-like signal, the a2NTD peptide.     

Carboxyatractyloside-insensitive influx and efflux of adenine nucleotides in rat liver mitochondria

Unidirectional transport (influx and efflux) of adenine nucleotides in rat liver mitochondria was examined using carboxyatractyloside to inhibit rapid exchange of matrix and external adenine nucleotides via the adenine nucleotide translocase. Influx of adenine nucleotides was concentration-dependent. ATP was the preferred substrate with a Km of 2.67 mM and V of the preferred substrate with a Km of 2.67 mM and V of 8.33 nmol/min/mg of protein. For ADP, the Km was 14.7 mM and V was 10.8 nmol/min/mg of protein. Efflux of adenine nucleotides was also concentration-dependent, varying directly as a function of the matrix adenine nucleotide pool size. Any increase in the influx of adenine nucleotides was coupled to an increase in efflux. However, as the external ATP concentration was increased, influx was stimulated to a much greater extent than was efflux. This imbalance suggested that under certain conditions adenine nucleotide movement might be coupled to the movement of an alternate anion such as phosphate. Adenine nucleotide efflux increased as the external phosphate concentration was varied from 0.5 to 4 mM. Also, increasing the external phosphate concentration caused adenine nucleotide influx to decrease, suggesting competition. In the absence of external adenines and phosphate, no efflux occurred. Both adenine nucleotide influx and efflux were depressed if Mg2+ was omitted. Adenine nucleotide efflux in the presence of external phosphate was inhibited much less by lack of Mg2+ than was efflux in the presence of external ATP. This evidence supports a model in which either adenine nucleotides (probably with Mg2+) or phosphate can move across the mitochondrial membrane on a single carrier. Net adenine nucleotide movements can occur when adenine nucleotide movement is coupled to the movement of phosphate in the opposite direction.     

Extracellular ATP4- promotes cation fluxes in the J774 mouse macrophage cell line

Extracellular ATP stimulates transmembrane ion fluxes in the mouse macrophage cell line J774. In the presence of Mg2+, nonhydrolyzable ATP analogs and other purine and pyrimidine nucleotides do not elicit this response, suggesting the presence of a specific receptor for ATP on the macrophage plasma membrane. One candidate for such a receptor is the ecto-ATPase expressed on these cells. We, therefore, investigated the role of this enzyme in ATP-induced 86Rb+ efflux in J774 cells. The ecto-ATPase had a broad nucleotide specificity and did not hydrolyze extracellular ATP in the absence of divalent cations. 86Rb+ efflux was not blocked by inhibition of the ecto-ATPase and did not require Ca2+ or Mg2+. In fact, ATP-stimulated 86Rb+ efflux was inhibited by Mg2+ and correlated with the availability of ATP4- in the medium. In the absence of divalent cations, the slowly hydrolyzable ATP analogs adenosine 5'-(beta, gamma-imido)triphosphate (AMP-PNP) and adenosine 5'-O-(3-thio)triphosphate (ATP-gamma-S) also stimulated 86Rb+ efflux, albeit at higher concentrations than that required for ATP4-. Exposure of J774 cells to 10 mM ATP for 45 min caused death of 95% of cells. By this means we selected variant J774 cells that did not exhibit 86Rb+ efflux in the presence of extracellular ATP but retained ecto-ATPase activity. These results show that the ecto-ATPase of J774 cells does not mediate the effects of ATP on these cells; that ATP4- and not MgATP2- promotes 86Rb+ efflux from these cells; and that hydrolysis of ATP is not required to effect this change in membrane permeability. These findings suggest that J774 cells possess a plasma membrane receptor which binds ATP4-, AMP-PNP, and ATP-gamma-S, and that the ecto-ATPase limits the effects of ATP on these cells by hydrolyzing Mg-ATP2-.     

Simultaneous bindings of ATP and vanadate to (Na+ + K+)-ATPase. Implications for the reaction mechanism of the enzyme

Inhibition of (Na+ + K+)-dependent adenosine triphosphatase phosphatase by vanadate is thought to occur through the tight binding of vanadate to the same site from which Pi is released. To see if ATP binds to [48V] vanadate-enzyme complex, just as it does to the phosphoenzyme, the effects of Na+, K+, and ATP on the dissociation rate of the complex at 10 degrees C were studied. The rate constant was increased by Na+, and this increase was blocked by K+, indicating that either Na+ or K+ binds to the complex. ATP alone, or in combination with K+, had no effect on the rate constant. In the presence of Na+, however, ATP caused a further increase in the rate constant. The value of K0.5 of Na+ was the same in the presence or absence of ATP; K0.5 of ATP (0.2 mM) did not seem to change significantly when Na+ concentration was varied, and K0.5 of K+, at a constant Na+ concentration, was the same in the presence or absence of ATP. The data indicate that ATP binds to the enzyme-vanadate complex regardless of the presence or absence of Na+ or K+, but it affects the dissociation rate only when Na+ is bound simultaneously. The value of K0.5 of Na+ decreased as pH was increased in the range of 6.5-7.8, but K0.5 of ATP was independent of pH. Demonstration of ATP binding to the enzyme-vanadate complex provides further support for the suggestion that the oligomeric enzyme contains a low-affinity regulatory site for ATP that is distinct from the interacting high-affinity catalytic sites.     

Functional characterization of the uncoupler-insensitive Na+ pump of the halotolerant bacterium,Ba1

Respiration initiates Na⁺ efflux from Na⁺-preloaded cells of the halotolerant bacterium, Ba₁. This efflux can take place against the concentration and electrochemical gradients. Since it is not inhibited by carbonylcyanide-p-trifluoromethoxyphenyl-hydrazone or N'N'-dicyclohexylcarbodiimide, it seems unlikely that either Δp (electrochemical potential difference of H⁺ across the membrane) generated by the primary proton pump or ATP play a role in the transduction of the energy supplied by electron transport. The electrogenic extrusion of Na⁺ causes passive counterflow of protons and/or simultaneous flux of permeant anions. In the absence of permeant anions the charge compensation attained by influx of protons is not complete. The membrane potential which persists in this case is inside negative and insensitive to uncoupler. The influx of protons builds up a ΔpH of reversed sign (more acid inside), which is insensitive to uncoupler. The simultaneous efflux of Na⁺ and permeant anions diminishes the intracellular salt content and, as a corollary, causes volume contraction. Thus, the respiration-linked, uncoupler-insensitive Na⁺ pump may play a role in the regulation of the intracellular salt content.