Characteristics of Root Plasma Membrane ATPase and H+- extrusion in a K+- deficit Tolerant Rice Variety

The characteristics of root plasma membrane ATPase (PM-ATPase) of "Weiyou 49", a K+ -deficit tolerant rice (Oryza sativa L. ) variety and of "Yuanyou 1", a K+ -deficit non-tolerant rice variety, had some similarities:Their optimum pH value were both about 6.0; Their activities reached the maximum at ATP concentration of 3 mmol/L; Km was 0.85 mmol/L and external K+ stimulated their activities. However, when [K+ ] was less than or equal to 50 mmol/L in the medium, the increasing of K + stimulated the activity of the PM-ATPase of "Weiyou 49" much more than that of "Yuanyou 1". When [K+ ] was between 100 to 200 mmol/L, the difference of the PM-AT- Pase activities decreased between the two rice varieties caused by K + stimulation. The basic H + extrusion of the two varieties had no apparent difference, but the H + extrusion stimulated by K + was different. The H+ extrusion of "Weiyou 49" was relatively more sensitive to external K+ . The experiment using inhibitors showed that there were close relationship between the PM-ATPase activi- ties stimulated by K+ and K+ uptake in the two varieties. The inhibition of PM-ATPase activity and H+ -extrusion stimulated by K+ reduced the K+ uptake of the root segments in both varieties. So the possible reason for "Weiyou 49" growing well in the low external K+ was that its PM-ATPase and H+ extrusion was more sensitive to external K+ , especially when [K+ ] was low.     

Sodium ions as substitutes for protons in the gastric H,K-ATPase

In view of the striking homology among various ion-translocating ATPases including Na,K-ATPase, Ca-ATPase, and H,K-ATPase, and the recent evidence that protons can replace cytoplasmic sodium as well as potassium in the reaction mechanism of the Na,K-ATPase (Polvani, C., and Blostein, R. (1988) J. Biol. Chem. 263, 16757-16763), we studied the role of sodium as a substitute for protons in the H,K-ATPase reaction. Using hog gastric H,K-ATPase-rich inside-out membrane vesicles we observed 22Na+ influx which was stimulated by intravesicular potassium ions (K+i) at pH 8.5 but not at pH 7.1. This sodium influx was observed in medium containing ATP and was inhibited by vanadate and SCH28080, a selective inhibitor of the gastric H,K-ATPase. At least 2-fold accumulation of sodium was observed at pH 8.5. Experiments aimed to determine the sidedness of the alkaline pH requirement for K+i-dependent sodium influx showed that K+i-activated sodium influx depends on pHout and is unaffected by changes in pHin. These results support the conclusion that sodium ions substitute for protons in the H,K-ATPase reaction mechanism and provide evidence for a similarity in ion selectivity and/or binding domains of the Na,K-ATPase and the gastric H,K-ATPase enzymes.     

Relationship of Plasmalemma Redox Activity to K+ Uptake by Dunaliella salina Cells

The plasmalemma-bond redox system localized within the plasmalemma of unicellular green alga Dunaliella salina was studied. This system oxidized exogenous NADH, increased O2 consumption to 165 % and increased the pH of the external medium, while K+ influx was inhibited. With no NADH added, ferricyanide stimulated K+ uptake about 3 folds. In the presence of exogenous NADH, ferricyanide was rapidly reduced and the external medium was acidified, generating a greater electrochemical proton gradient across the plasmalemma, thus resulting an 6-fold increase of K+ influx. Typical inhibitors of plasmalemma H+-ATPase and redox system inhibited K+ uptake to different extent. That the inhibition of K+ uptake by vanadate could be resumed partly by addition of NADH and ferricyanide indicated that plasmalemma redox system operated in association with the H+-ATPase to exert an influence on K+ transportation. A model was presented in which the implication of two possible redox chains and H+-ATPase in generating an electrochemical potential gradient for protons (△uH+) was discussed.     

Studies on H+-ATPase in Cultured Rabbit Nonpigmented Ciliary Epithelium

Studies were conducted to examine the influence of the H⁺-ATPase inhibitor bafilomycin A₁ on cultured rabbit nonpigmented ciliary epithelial cells (NPE). Cytoplasmic pH and sodium concentrations were measured by digital fluorescence microscopy using BCECF and SBFI respectively. In some experiments, cell sodium content was measured by atomic absorption spectroscopy. Added alone, bafilomycin A₁ (100 nm) failed to change cytoplasmic pH but it caused an increase of cytoplasmic sodium concentration which occurred within 10 min. It is likely that the rise of cytoplasmic sodium concentration was responsible for the stimulation of active sodium-potassium transport which occurred in bafilomycin A₁-treated cells as judged by a 50% increase of ouabain sensitive potassium (⁸⁶Rb) uptake. In bafilomycin A₁-treated cells, but not in control cells, dimethylamiloride (DMA) inhibited ouabain-sensitive potassium (⁸⁶Rb) uptake in a dose-dependent manner with an IC₅₀ of ∼2 μm. DMA (10 μm) also prevented the increase of cytoplasmic sodium caused by bafilomycin A₁. Added alone, DMA (10 μm) failed to change cytoplasmic sodium content but reduced cytoplasmic pH by ∼0.4 pH units. In cells that first received 10 μm DMA, the subsequent addition of bafilomycin A₁ (100 nm) caused a further cytoplasmic pH reduction of ∼0.3 pH units. Taken together, the results suggest H⁺-ATPase might contribute to the regulation of basal cytoplasmic pH in cultured NPE. In the presence of bafilomycin A₁, Na-H exchanger activity appears to be stimulated, so stabilizing cytoplasmic pH but resulting in an increase of cytoplasmic sodium concentration and consequent stimulation of active sodium-potassium transport. Received: 19 March 1999/Revised: 20 September 1999     

Active extrusion of protons and exudation of carboxylic acids in response to iron deficiency by roots of chickpea (Cicer arietinum L.)

A chickpea cultivar, K-850, acidified the nutrient solution in response to iron deficiency, with subsequent re-greening of chlorotic leaves. No recovery of chlorosis was observed when the nutrient solution was buffered at a pH 6.3. During the period of acidification induced by iron deficiency, the roots of K-850 exuded more carboxylic acids than when supplied with sufficient iron. However, the rate of extrusion of protons was much higher than the rate of exudation of carboxylic acids during the acidification period. The extrusion of protons was inhibited by the addition of vanadate at the beginning of the decrease in pH. It appeared that acidification of the solution in response to iron deficiency was mediated by a proton-pumping ATPase, located at the plasma membrane. The presence of cations in the solution was essential for the extrusion of protons under iron deficiency, but the species of cation made no significant difference to the rate of extrusion of protons from roots. Therefore, we concluded that non-specific H+/cation antiport was involved in the acidification process.     

Presence of a Na+-stimulated P-type ATPase in the plasma membrane of the alkaliphilic halotolerant cyanobacterium Aphanothece halophytica

Aphanothece cells could take up Na(+) and this uptake was strongly inhibited by the protonophore, carbonyl cyanide m-chlorophenylhydrazone (CCCP). Cells preloaded with Na(+) exhibited Na(+) extrusion ability upon energizing with glucose. Na(+) was also taken up by the plasma membranes supplied with ATP and the uptake was abolished by gramicidin D, monensin or Na(+)-ionophore. Orthovanadate and CCCP strongly inhibited Na(+) uptake, whereas N, N'-dicyclohexylcarbodiimide (DCCD) slightly inhibited the uptake. Plasma membranes could hydrolyse ATP in the presence of Na(+) but not with K(+), Ca(2+) and Li(+). The K(m) values for ATP and Na(+) were 1.66+/-0.12 and 25.0+/-1.8 mM, respectively, whereas the V(max) value was 0.66+/-0.05 mumol min(-1) mg(-1). Mg(2+) was required for ATPase activity whose optimal pH was 7.5. The ATPase was insensitive to N-ethylmaleimide, nitrate, thiocyanate, azide and ouabain, but was substantially inhibited by orthovanadate and DCCD. Amiloride, a Na(+)/H(+) antiporter inhibitor, and CCCP showed little or no effect. Gramicidin D and monensin stimulated ATPase activity. All these results suggest the existence of a P-type Na(+)-stimulated ATPase in Aphanothece halophytica. Plasma membranes from cells grown under salt stress condition showed higher ATPase activity than those from cells grown under nonstress condition.     

(Na+ + K+)-ATPase in artificial lipid vesicles: influence of the concentration of mono- and divalent cations on the pumping rate

(Na+ + K+)-ATPase from kidney outer medulla was incorporated into artificial dioleoylphosphatidylcholine vesicles. Transport activity was induced by adding ATP to the external medium. A voltage-sensitive dye was used to detect the ATP-driven potassium extrusion in the presence of valinomycin. The observed substrate-protein interactions of the reconstituted (Na+ + K+)-ATPase largely agree with that from native tissues. An agreement between ATP hydrolysis and transport activity is given for concentration dependences of sodium, potassium, magnesium and calcium ions. The only significant deviations were observed in the influence of pH. Protons were found to have different influence on transport, enzymatic activity and phosphorylation of the enzyme. The transport studies showed a twofold interaction of protons with the protein: competition with sodium at the cytoplasmic ion binding sites, a non competitive inhibition of transport which is not correlated with protein phosphorylation.     

pH homeostasis and bioenergetic work in alkalophiles

Abstract A Na⁺/H⁺ antiporter catalyses coupled Na⁺ extrusion and H⁺ uptake across the membranes of extremely alkalophilic bacilli. This exchange is electrogenic, with H⁺ translocated inward > Na⁺ extruded. It is energized by the Δψ ² component of the ΔμH⁺ that is established during primary proton pumping by the alkalophile respiratory chain complexes. These complexes abound in the membranes of extreme alkalophiles. Combined activity of the respiratory chain, the antiporter, and solute transport systems that are coupled to Na⁺ re-entry, allow the alkalophiles to maintain a cytoplasmic pH that is several pH units more acidic than optimal external pH values for growth. There is no compelling evidence for a specific and necessary role for any ion other than sodium in pH homeostasis, and although there is very high cytoplasmic buffering capacity in the alkaline range, active mechanisms for pH homeostasis are crucial. Energization of the antiporter as well as the proton translocating F ₁ F ₀-ATPase that catalyses ATP synthesis in the extreme alkalophiles must accommodate the problem of the low net ΔμH⁺ and the very low concentrations of protons, per se, in the external medium. This problem is by-passed by other bioenergetic work functions, such as solute uptake or motility, that utilize sodium ions for energy-coupling in the place of protons.     

Effects of Cr on proton extrusion, potassium uptake and transmembrane electric potential in maize root segments

Abstract Proton extrusion of maize root Zea mays segments, was inhibited by the presence of Cr (o.n. + 6; present in solution as CrO₄^2-, Cr₂O₇^2-) in the incubation medium: the minimum inhibiting concentration was 2 × 10^?3 mol m^?3 and the inhibition progressively increased with Cr concentration. Cr inhibited proton extrusion. Also, when this activity was stimulated by the presence of K⁺ or fusicoccin (FC) in the incubation medium, the K⁺ and FC stimulating effect was still present when proton extrusion was inhibited by Cr. In addition, Cr inhibited K⁺ uptake. This inhibition was higher (50%) at K⁺ concentrations up to 1 mol m^?3 lower (15%) at higher K⁺ concentrations. This result indicates that the system responsible for K⁺ uptake operating at low K⁺ concentrations is more sensitive to Cr inhibition. Cr had no effect on transmembrane electric potential (PD). The depolarizing and hyper-polarizing effect of K+ and FC, respectively, were not affected by Cr; but Cr enhances the depolarizing effect of the uncoupler carbonylcyanide m-chlorophenylhydrazone (CCP). These results indicate that Cr inhibited the proton translocating mechanism coupled with K⁺ uptake, but did not change the net transport of charges through the plasmalemma. The Cr effect is discussed, taking into account the possibility of a direct effect of Cr at the membrane level or, alternatively, of an effect on some metabolic processes controlling membrane function.     

POTASSIUM EFFECTS ON ION TRANSPORT IN BRAIN SLICES

—(1) Fluxes of sodium, potassium, chloride and glutamate ions were studied in brain slices by aid of radio-isotopes. Desaturation curves showed the efflux to occur from at least two compartments with widely different kinetics. (2) The slowly exchanging component comprises from about 10 (sodium, potassium, chloride) to about 30 (glutamate) per cent of the radioactivity in the tissue. An energy-requiring uptake of potassium and extrusion of sodium seems to occur in this compartment, which probably includes the nerve cells. (3) A rather slow efflux of especially potassium ions from the rapidly exchanging fraction indicates that this component may not be purely extracellular, but also seems to include cells, which possibly are neuroglial. The hypothesis of a cellular origin is supported by the demonstration of an increase in the rate constant of the potassium efflux evoked in the presence of oxygen by high concentrations of potassium. (4) Evidence is presented that the increase in the rate constant of the potassium efflux is due to a potassium-induced stimulation of active transport. No coupling seems to occur between the stimulated potassium transport and movements of sodium, but potassium ions may be accompanied by glutamate ions.     

Active potassium extrusion regulated by intracellular pH in Streptococcus faecalis

Potassium extrusion in bacteria is thought to play a role in the regulation of the cytoplasmic pH; in several organisms, it has been ascribed to secondary antiport of K+ for protons. Streptococcus faecalis exhibited a distinctive pattern: potassium extrusion occurred only when the cytoplasmic pH was alkaline and required the generation of ATP. The key observation is that glycolyzing cells suspended in an alkaline medium extruded K+, even against a K+ concentration gradient, provided the medium contained a weak permeant base (e.g. diethanolamine or methylamine). The amines render the cytoplasmic pH alkaline; when conditions were arranged to keep the cytoplasm neutral, no K+ extrusion was seen. Potassium extrusion required the presence of either glucose or arginine and was unaffected by protonophores and by inhibition of the F1Fo-ATPase. When the medium contained [14C]methylamine, the cells accumulated the base to an extent stoichiometrically equivalent to the K+ lost. Concurrently, the cytoplasmic pH fell from 8.8 to 7.6, at which point K+ extrusion ceased. The results suggest that K+ extrusion is due to an ATP-driven transport system that expels K+ by exchange for H+ and is active only at alkaline cytoplasmic pH.     

K+ influx by Kup in Escherichia coli is accompanied by a decrease in H+ efflux

Escherichia coli accumulates K+ by means of multiple uptake systems of which Kup is the major transport system at acidic pH. In cells grown under fermentative conditions at pH 5.5, K+ influx by a wild-type strain upon hyper-osmotic stress at pH 5.5 was accompanied by a marked decrease in H+ efflux, with a 1:1 ratio of K+ to H+ fluxes. This was observed with cells treated with N,N'-dicyclohexylcarbodiimide. Similar results with a mutant defective in Kdp and TrkA but with a functional Kup system but not in a mutant defective in Kdp and Kup but having an active TrkA system suggest that Kup operates as a H+ -K+ -symporter.     

Futile cycling of ammonium ions via the high affinity potassium uptake system (Kdp) of Escherichia coli

Escherichia coli Frag1 was grown under various nutrient limitations in chemostat culture at a fixed temperature, dilution rate and pH both in the presence and the absence of a high concentration of ammonium ions by using either ammonium chloride or dl-alanine as the sole nitrogen source. The presence of high concentrations of ammonium ions in the extracellular fluids of potassium-limited cultures of E. coli Frag1 caused an increase of the specific rate of oxygen consumption of these cultures. In contrast, under phosphate-, sulphate- or magnesium-limited growth conditions no such increase could be observed. The presence of high concentrations of ammonium ions in potassium-limited cultures of E. coli Frag5, a mutant strain of E. coli Frag1 which lacks the high affinity potassium uptake system (Kdp), did not increase the specific rate of oxygen consumption.These results indicate that ammonium ions, very similar to potassium ions both in charge and size, are transported via the K dp leading to a futile cycle of ammonium ions and ammonia molecules (plus protons) across the cytoplasmic membrane. Both the uptake of ammonium ions and the extrusion of protons would increase the energy requirement of the cells and therefore increase their specific rate of oxygen consumption. The involvement of a (methyl)ammonium transport system in this futile cycle could be excluded.     

Cation transport in Escherichia coli. IX. Regulation of K transport

Kinetics of K exchange in the steady state and of net K uptake after osmotic upshock are reported for the four K transport systems of Escherichia coli: Kdp, TrkA, TrkD, and TrkF. Energy requirements for K exchange are reported for the Kdp and TrkA systems. For each system, kinetics of these two modes of K transport differ from those for net K uptake by K-depleted cells (Rhoads, D. B. F.B. Walters, and W. Epstein. 1976. J. Gen. Physiol. 67:325-341). The TrkA and TrkD systems are inhibited by high intracellular K, the TrkF system is stimulated by intracellular K, whereas the Kdp system is inhibited by external K when intracellular K is high. All four systems mediate net K uptake in response to osmotic upshock. Exchange by the Kdp and TrkA systems requires ATP but is not dependent on the protonmotive force. Energy requirements for the Kdp system are thus identical whether measured as net K uptake or K exchange, whereas the TrkA system differs in that it is dependent on the protonmotive force only for net K uptake. We suggest that in both the Kpd and TrkA systems formation of a phosphorylated intermediate is necessary for all K transport, although exchange transport may not consume energy. The protonmotive-force dependence of the TrkA system is interpreted as a regulatory influence, limiting this system to exchange except when the protonmotive force is high.     

Pincher,a pinocytic chaperone for nerve growth factor/TrkA signaling endosomes

A central tenet of nerve growth factor (NGF) action that is poorly understood is its ability to mediate cytoplasmic signaling, through its receptor TrkA, that is initiated at the nerve terminal and conveyed to the soma. We identified an NGF-induced protein that we termed Pincher (pinocytic chaperone) that mediates endocytosis and trafficking of NGF and its receptor TrkA. In PC12 cells, overexpression of Pincher dramatically stimulated NGF-induced endocytosis of TrkA, unexpectedly at sites of clathrin-independent macropinocytosis within cell surface ruffles. Subsequently, a system of Pincher-containing tubules mediated the delivery of NGF/TrkA-containing vesicles to cytoplasmic accumulations. These vesicles selectively and persistently mediated TrkA-erk5 mitogen-activated protein kinase signaling. A dominant inhibitory mutant form of Pincher inhibited the NGF-induced endocytosis of TrkA, and selectively blocked TrkA-mediated cytoplasmic signaling of erk5, but not erk1/2, kinases. Our results indicate that Pincher mediates pinocytic endocytosis of functionally specialized NGF/TrkA endosomes with persistent signaling potential.     

Effect of phenoxyethanol on the permeability of Escherichia coli NCTC 5933 to inorganic ions.

Concentrations of phenoxyethanol which retarded the growth rate of Escherichia coli NCTC 5933 in nutrient broth, stimulated the rates of respiration and total oxygen uptake of cell suspensions with glucose as carbon source, and were able to dissipate artificially induced membrane proton gradients. In cells with repressed oxidative phosphorylating activity, no stimulation of respiration was observed. These actions were characteristic of uncouples of oxidative phosphorylation. Similar concentrations of the drug caused additional increased permeability of the cytoplasmic membrane to K+ but not to Li+, Na+, Ca++, Mg++, NO-3, Cl-, So--4, or PO---4. Drug induced permeability of the membrane to protons and potassium ions were not found to be directly coupled.     

IONS AND THE TRANSPORT OF GAMMA-AMINOBUTYRIC ACID BY SYNAPTOSOMES

Abstract— The initial rate of uptake of [2,3-³H]gamma-aminobutyric acid by rat brain synaptosomes was studied under incubation conditions in which GABA metabolism was minimal. The presence of both sodium and potassium in the incubation medium was essential for sustained uptake. Uptake proceeded for a short period of time in the absence of potassium and then ceased. No uptake was observed when sodium chloride was completely replaced with sucrose or choline chloride. The sodium-dependence curve for GABA uptake was markedly sigmoid. The sigmoid character of the curve was not attributable to a lag phase in uptake at low sodium concentrations. Calcium strongly stimulated the initial rate of uptake at low sodium concentrations but had little effect at sodium concentrations above 100 m m and was not able to support uptake in the absence of sodium. The sigmoid character of thesodium-dependence curve was completely eliminated by 20 m m calcium ion. Magnesium and phosphate had little effect on the initial rate of GABA uptake.     

Aldosterone induction of electrogenic sodium transport in the apical membrane vesicles of rat distal colon

Na-H exchange is present in apical membrane vesicles (AMV) isolated from distal colon of normal rats. Because in intact tissue aldosterone both induces amiloride-sensitive electrogenic sodium transport and inhibits electroneutral sodium absorption, these studies with AMV were designed to establish the effect of aldosterone on sodium transport. An outward-directed proton gradient stimulated 22Na uptake in AMV isolated from distal colon of normal and dietary sodium depleted (with elevated aldosterone levels) experimental rats. Unlike normal AMV, proton gradient-dependent 22Na uptake in experimental AMV was inhibited when uptake was measured under voltage-clamped conditions. 10 microM amiloride inhibited the initial rate of proton gradient-dependent 22Na uptake in AMV of normal and experimental rats by 30 and 75%, respectively. In contrast, 1 mM amiloride produced comparable inhibition (90 and 80%) of 22Na uptake in normal and experimental AMV. Intravesicular-negative potential stimulated 22Na uptake in experimental but not in normal AMV. This increase was inhibited by 90% by 10 microM amiloride. An analogue of amiloride, 5-(N-ethylisopropyl) amiloride (1 microM), a potent inhibitor of electroneutral Na-H exchange in AMV of normal rat distal colon, did not alter potassium diffusion potential-dependent 22Na uptake. Increasing sodium concentration saturated proton gradient-dependent 22Na uptake in normal AMV. However, in experimental AMV, 22Na uptake stimulated by both proton gradient and potassium diffusion potential did not saturate as a function of increasing sodium concentration. We conclude from these results that an electrically sensitive conductive channel, not electroneutral Na-H exchange, mediates 22Na uptake in AMV isolated from the distal colon of aldosterone rats.     

THE EFFECT OF BENZIMIDAZOLE ON CATION UPTAKE BY PLANT ROOTS

Klingensmith , M. J. (Colgate U., Hamilton, N.Y.) The effect of benzimidazole on cation uptake by plant roots. Amer. Jour. Bot. 48(8): 711–716. Illus. 1961.—A number of benzazole compounds were examined for their effect on cation uptake. Benzimidazole was found to almost double the uptake of potassium by excised barley roots in a 6-hr period. Chlorobenzimidazole also enhanced the absorption of potassium but not to the same extent. This stimulation of potassium accumulation was found to be insensitive to cyanide but was dependent on the temperature of the ambient solution. There was also an increased accumulation of potassium by roots of intact barley plants with benzimidazole treatment without interference with subsequent transport of the potassium. Benzimidazole also stimulated uptake of sodium and calcium by excised barley roots but not at identical levels. Results are discussed in the light of various theories of ion absorption.     

Further Characteristics of the ATP-Stimulated Uptake of Calcium into Chromaffin Granules

Abstract: The ATP-stimulated uptake of ⁴⁵Ca²⁺ [and [³H](-)-noradrenaline ([³H]NA)] into chromaffin granules and that into mitochondria are driven by a protonic gradient ΔμH⁺, composed of the components ΔpH (concentration gradient of protons) and ΔΨ(electrical potential difference). The granular ATPase pumps protons into the matrix (ΔpH inside acid, ΔΨ positive), but the mitochondrial ATPase ejects protons from the matrix (ΔpH alkaline, ΔΨ negative inside). To show different driving forces of uptake, the rate of the ATP-stimulated uptake of ⁴⁵Ca²⁺ (and [³H]NA) into chromaffin granules was compared with the rate of the ATP-stimulated uptake of ⁴⁵Ca²⁺ into mitochondria (adrenomedullary or rat liver). In the presence of nitrate, the rate of the ATP-stimulated uptake of ⁴⁵Ca²⁺ into chromaffin granules is higher than in the presence of acetate, because the lyotropic anion nitrate stimulates the granular ATPase and increases ΔpH (acid inside). Compared with nitrate, the rate of the ATP-stimulated uptake of ⁴⁵Ca²⁺ into mitochondria is higher in the presence of the proton-carrying anion acetate, which, after permeation, provides protons for ejection by the ATPase. In the absence of ATP, a valinomycin-mediated potassium influx (ΔΨ inside positive) stimulates the granular uptake of [³H]NA, which has an electrogenic component, but not the granular uptake of ⁴⁵Ca²⁺, which is electroneutral. The electrogenic uptake of ⁴⁵Ca²⁺ into mitochondria is stimulated by a valinomycin-mediated potassium efflux (ΔΨ negative inside). The ATP-stimulated uptake of ⁴⁵Ca²⁺ into chromaffin granules is sensitive to ruthenium red, suggesting a carrier-mediated mechanism of uptake, and it is sensitive to atractyloside, indicating the simultaneous uptake of ATP. After collapse of ΔpH by ammonia, the ATP-stimulated uptake of ⁴⁵Ca²⁺ into chromaffin granules is abolished, but not that into mitochondria. In the presence of ammonia, the rate of the ATP-stimulated uptake of [³H]NA is very low, and an ATP-independent uptake of ⁴⁵Ca²⁺ into chromaffin granules is observed which is similar to the ATP-independent Ca²⁺/Na⁺ exchange at the granular membrane.