AtNHX8, a member of the monovalent cation: proton antiporter-1 family in Arabidopsis thaliana, encodes a putative Li/H antiporter

The Arabidopsis monovalent cation:proton antiporter-1 (CPA1) family includes eight members, AtNHX1-8. AtNHX1 and AtNHX7/SOS1 have been well characterized as tonoplast and plasma membrane Na+/H+ antiporters, respectively. The proteins AtNHX2-6 have been phylogenetically linked to AtNHX1, while AtNHX8 appears to be related to AtNHX7/SOS1. Here we report functional characterization of AtNHX8. AtNHX8 T-DNA insertion mutants are hypersensitive to lithium ions (Li+) relative to wild-type plants, but not to the other metal ions such as sodium (Na+), potassium (K+) and caesium (Cs+). AtNHX8 overexpression in a triple-deletion yeast mutant AXT3 that exhibits defective Na+/Li+ transport specifically suppresses sensitivity to Li+, but does not affect Na+ sensitivity. Likewise, AtNHX8 overexpression complemented sensitivity to Li+, but not Na+, in sos1-1 mutant seedlings, and increased Li+ tolerance of both the sos1-1 mutant and wild-type seedlings. Results of Li+ and K+ measurement of loss-of-function and gain-of-function mutants indicate that AtNHX8 may be responsible for Li+ extrusion, and may be able to maintain K+ acquisition and intracellular ion homeostasis. Subcellular localization of the AtNHX8-enhanced green fluorescent protein (EGFP) fusion protein suggested that AtNHX8 protein is targeted to the plasma membrane. Taken together, our findings suggest that AtNHX8 encodes a putative plasma membrane Li+/H+ antiporter that functions in Li detoxification and ion homeostasis in Arabidopsis.     

Ion specificity of cardiac sarcolemmal Na+/H+ antiporter

In bovine cardiac sarcolemmal vesicles, an outward H+ gradient stimulated the initial rate of amiloride-sensitive uptake of 22Na+, 42K+, or 86Rb+. Release of H+ from the vesicles was stimulated by extravesicular Na+, K+, Rb+, or Li+ but not by choline or N-methylglucamine. Uptakes of Na+ and Rb+ were half-saturated at 3 mM Na+ and 3 mM Rb+, but the maximal velocity of Na+ uptake was 1.5 times that of Rb+ uptake. Na+ uptake was inhibited by extravesicular K+, Rb+, or Li+, and Rb+ uptake was inhibited by extravesicular Na+ or Li+. Amiloride-sensitive uptake of Na+ or Rb+ increased with increase in extravesicular pH and decrease in intravesicular pH. In the absence of pH gradient, there were stimulations of Na+ uptake by intravesicular Na+ and K+ and of Rb+ uptake by intravesicular Rb+ and Na+. Similarly, there were trans stimulations of Na+ and Rb+ efflux by extravesicular alkali cations. The data suggest the existence of a nonselective antiporter catalyzing either alkali cation/H+ exchange or alkali cation/alkali cation exchange. Since increasing Na+ caused complete inhibition of Rb+/H+ exchange, but saturating K+ caused partial inhibitions of Na+/H+ exchange and Na+/Na+ exchange, the presence of a Na(+)-selective antiporter is also indicated. Although both antiporters may be involved in pH homeostasis, a role of the nonselective antiporter may be in the control of Na+/K+ exchange across the cardiac sarcolemma.     

Identification and characterization of Vnx1p, a novel type of vacuolar monovalent cation/H+ antiporter of Saccharomyces cerevisiae

We identified and characterized Vnx1p, a novel vacuolar monovalent cation/H+ antiporter encoded by the open reading frame YNL321w from Saccharomyces cerevisiae. Despite the homology of Vnx1p with other members of the CAX (calcium exchanger) family of transporters, Vnx1p is unable to mediate Ca2+ transport but is a low affinity Na+/H+ and K+/H+ anti-porter with a Km of 22.4 and 82.2 mm for Na+ and K+, respectively. Sequence analyses of Vnx1p revealed the absence of key amino acids shown to be essential for Ca2+/H+ exchange. vnx1Delta cells displayed growth inhibition when grown in the presence of hygromycin B or NaCl. Vnx1p activity was found in the vacuoles and shown to be dependent on the electrochemical potential gradient of H+ generated by the action of the V-type H+-ATPase. The presence of Vnx1p at the vacuolar membrane was further confirmed with cells expressing a VNX1::GFP chimeric gene. Similar to Nhx1p, the prevacuolar compartment-bound Na+/H+ antiporter, the vacuole-bound Vnx1p appears to play roles in the regulation of ion homeostasis and cellular pH.     

Mixed type inhibition of the renal Na+/H+ antiporter by Li+ and amiloride. Evidence for a modifier site

We studied the interactions of Na+, Li+, and amiloride on the Na+/H+ antiporter in brush-border membrane vesicles from rabbit renal cortex. Cation-mediated collapse of an outwardly directed proton gradient (pHin = 6.0; pHout = 7.5) was monitored with the fluorescent amine, acridine orange. Proton efflux resulting from external addition of Na+ or Li+ exhibited simple saturation kinetics with Hill coefficients of 1.0. However, kinetic parameters for Na+ and Li+ differed (Km for Li+ = 1.2 +/- 0.1 mM; Km for Na+ = 14.3 +/- 0.8 mM; Vmax for Li+ = 2.40 +/- 0.07 fluorescence units/s/mg of protein; Vmax for Na+ = 7.10 +/- 0.24 fluorescence units/s/mg of protein). Inhibition of Na+/H+ exchange by Li+ and amiloride was also studied. Li+ inhibited the Na+/H+ antiporter by two mechanisms. Na+ and Li+ competed with each other at the cation transport site. However, when [Na+] was markedly higher than [Li+], [( Na+] = 90 mM; [Li+] less than 1 mM), we observed noncompetitive inhibition (Vmax for Na+/H+ exchange reduced by 25%). The apparent Ki for this noncompetitive inhibition was congruent to 50 microM. In addition, 2-30 mM intravesicular Li+, but not Na+, resulted in trans inhibition of Na+/H+ exchange. Amiloride was a mixed inhibitor of Na+/H+ exchange (Ki = 30 microM, Ki' = 90 microM) but was only a simple competitive inhibitor of Li+/H+ exchange (Ki = 10 microM). At [Li] = 1 mM and [amiloride] less than 100 microM, inhibition of Na+/H+ exchange by a combination of the two inhibitors was always less than additive. These results suggest the presence of a cation-binding site (separate from the cation-transport site) which could be a modifier site of the Na+/H+ antiporter.     

Escherichia coli mutants with altered cation recognition by the melibiose carrier.

Revertants that showed normal cation recognition for melibiose transport were isolated from mutants with altered cation recognition (W3133-2S and W3133-2T) of Escherichia coli. Although the original two mutants possessed a second alteration, an increased activity of the Na+(Li+)/H+ antiporter, the revertants, which possessed the normal melibiose carrier, still showed altered properties of the Na+(Li+)/H+ antiporter. These results support the view that the alterations in the melibiose carrier and in the Na+(Li+)/H+ antiporter, observed in the mutants, are not genetically linked.     

Characterization of a multigene-encoded sodium/hydrogen antiporter (sha) from Pseudomonas aeruginosa: its involvement in pathogenesis

Sha (also known as Mrp/Mnh/Pha) is a Na+/H+ antiporter encoded by a cluster of six or seven genes that probably form a multisubunit transport complex. The Sha system is important for the homeostasis of H+, Na+, and other monovalent cations and plays a critical role in various functions, including alkaliphily, sporulation, and symbiosis. Here, we characterized the sha homologue genes from the opportunistic pathogen Pseudomonas aeruginosa, which exist as a cluster of six genes (PA1054 to PA1059). The gene cluster PA1054 to PA1059, but not the cluster with a deletion of PA1054, complemented a growth defect in the presence of 0.2 M NaCl and a defect in Na+/H+ antiport activity of the Escherichia coli TO114 mutant lacking the three major Na+/H+ antiporters, indicating that genes PA1054 to PA1059 are responsible for Na+/H+ antiport activity. We disrupted PA1054 (a shaA homologue gene) and determined its effect on Na+ tolerance during growth, Na+ efflux, and pathogenicity in mice. Disruption of PA1054 resulted in severe Na+ sensitivity during growth and decreased Na+ efflux activity. In mice, the deletion mutant of PA1054 also exhibited an attenuated virulence in systemic, pulmonary, and urinary tract infections and also a decrease in colonization of the infected organs. From these results, we conclude that the genes PA1054 to PA1059 encode a Na+/H+ antiporter that is largely responsible for Na+ extrusion in P. aeruginosa and has a role in the infection of the pathogen. We propose to designate PA1054 to PA1059 as the sha (sodium hydrogen antiporter) genes, shaABCDEFG.     

Insights into the biochemistry of the ubiquitous NhaP family of cation/H+ antiporters

Na+/H+ antiporters are integral membrane proteins that exchange Na+ for H+ across the cytoplasmic or organellar membranes of virtually all living cells. They are essential for control of cellular pH, volume homeostasis, and regulation of Na+ levels. Na+/H+ antiporters have become increasingly characterized and are now becoming important drug targets. The recently identified NhaP family of Na+/H+ antiporters, from the CPA1 superfamily, contains proteins with a surprisingly broad collective range of transported cations, exchanging protons for alkali cations such as Na+, Li+, K+, or Rb+ as well as for Ca2+ and, possibly, NH4+. Questions about ion selectivity and the physiological impact of each particular NhaP antiporter are far from trivial. For example, Vc-NhaP2 from Vibrio cholerae has recently been shown to function in vivo as a specific K+/H+ antiporter while retaining the ability to exchange H+ for Na+ and bind (but not exchange with H+) Li+ in a competitive manner. These and other findings reviewed in this communication make antiporters of the NhaP type attractive systems to study intimate molecular mechanisms of cation exchange. In an evolutionary perspective, the NhaP family seems to be a phylogenetic entity undergoing active divergent evolution. In this minireview, to rationalize peculiarities of the cation specificity in the NhaP family, the "size-exclusion principle" and the idea of "ligand shading" are discussed.     

Gill (Na+ + K+)- and Na+-stimulated Mg2+-dependent ATPase activities in the gilthead bream (Sparus auratus L.)

1. Gilthead gill 10(-3) M ouabain-inhibited (Na+ + K+)-ATPase and 10(-2) M ouabain-insensitive Na+-ATPase require the optimal conditions of pH 7.0, 160 mM Na+, 20 mM K+, 5 mM MgATP and pH 4.8-5.2, 75 mM Na+, 2.5 mM Mg2+, 1.0 mM ATP, respectively. 2. The main distinctive features between the two activities are confirmed to be optimal pH, the ouabain-sensitivity and the monovalent cation requirement, Na+ plus another cationic species (K+, Rb+, Cs+, NH4+) in the (Na+ + K+)-ATPase and only one species (Na+, K+, Li+, Rb+, Cs+, NH4+ or choline+) in the Na+-ATPase. 3. The aspecific Na+-ATPase activation by monovalent cations, as well as by nucleotide triphosphates, opposed to the (Na+ + K+)-ATPase specificity for ATP and Na+, relates gilthead gill ATPases to lower organism ATPases and differentiates them from mammalian ones. 4. The discrimination between the two activities by the sensitivity to ethacrynic acid, vanadate, furosemide and Ca2+ only partially agrees with the literature. 5. Present findings are viewed on the basis of the ATPase's presumptive physiological role(s) and mutual relationship.     

Na+/Ca2+ antiport in cultured arterial smooth muscle cells. Inhibition by magnesium and other divalent cations

Cultured smooth muscle cells from rat aorta were loaded with Na+, and Na+/Ca2+ antiport was assayed by measuring the initial rates of 45Ca2+ influx and 22Na+ efflux, which were inhibitable by 2',4'-dimethylbenzamil. The replacement of extracellular Na+ with other monovalent ions (K+, Li+, choline, or N-methyl-D-glucamine) was essential for obtaining significant antiport activity. Mg2+ competitively inhibited 45Ca2+ influx via the antiporter (Ki = 93 +/- 7 microM). External Ca2+ or Sr2+ stimulated 22Na+ efflux as would be expected for antiport activity. Mg2+ did not stimulate 22Na+ efflux, which indicates that Mg2+ is probably not transported by the antiporter under the conditions of these experiments. Mg2+ inhibited Ca2+-stimulated 22Na+ efflux as expected from the 45Ca2+ influx data. The replacement of external N-methyl-D-glucamine with K+, but not other monovalent ions (choline, Li+), decreased the potency of Mg2+ as an inhibitor of Na+/Ca2+ antiport 6.7-fold. Other divalent cations (Co2+, Mn2+, Cd2+, Ba2+) also inhibited Na+/Ca2+ antiport activity, and high external potassium decreased the potency of each by 4.3-8.6-fold. The order of effectiveness of the divalent cations as inhibitors of Na+/Ca2+ antiport (Cd2+ greater than Mn2+ greater than Co2+ greater than Ba2+ greater than Mg2+) correlated with the closeness of the crystal ionic radius to that of Ca2+.     

Functional expression of the multi-subunit type calcium/proton antiporter from Thermomicrobium roseum

Multiple resistance and pH adaptation (Mrp) antiporters are widely distributed in various prokaryotes and have been reported to function as a hetero-oligomeric monovalent cation/proton antiporter, which exchanges a cytoplasmic monovalent cation (Na(+) , Li(+) , and/or K(+) ) with extracellular H(+) . In many organisms, they are essential for survival in alkaline or saline environments. Here, we report that the Mrp antiporter from the thermophilic gram-negative bacterium, Thermomicrobium roseum, does not catalyze monovalent cation/proton antiport like the Mrp antiporters studied to date, but catalyzes Ca(2+) /H(+) antiport in Escherichia coli membrane vesicles.     

Na(+)-H+ antiport detected through hydrogen ion currents in rat alveolar epithelial cells and human neutrophils

Voltage-activated H(+)-selective currents were studied in cultured adult rat alveolar epithelial cells and in human neutrophils using the whole-cell configuration of the patch-clamp technique. The H+ conductance, gH, although highly selective for protons, was modulated by monovalent cations. In Na+ and to a smaller extent in Li+ solutions, H+ currents were depressed substantially and the voltage dependence of activation of the gH shifted to more positive potentials, when compared with the "inert" cation tetramethylammonium (TMA+). The reversal potential of the gH, Vrev, was more positive in Na+ solutions than in inert ion solutions. Amiloride at 100 microM inhibited H+ currents in the presence of all cations studied except Li+ and Na+, in which it increased H+ currents and shifted their voltage-dependence and Vrev to more negative potentials. The more specific Na(+)-H+ exchange inhibitor dimethylamiloride (DMA) at 10 microM similarly reversed most of the suppression of the gH by Na+ and Li+. Neither 500 microM amiloride nor 200 microM DMA added internally via the pipette solution were effective. Distinct inhibition of the gH was observed with 1% [Na+]o, indicating a mechanism with high sensitivity. Finally, the effects of Na+ and their reversal by amiloride were large when the proton gradient was outward (pHo parallel pHi 7 parallel 5.5), smaller when the proton gradient was abolished (pH 7 parallel 7), and absent when the proton gradient was inward (pH 6 parallel 7). We propose that the effects of Na+ and Li+ are due to their transport by the Na(+)-H+ antiporter, which is present in both cell types studied. Electrically silent H+ efflux through the antiporter would increase pHi and possibly decrease local pHo, both of which modulate the gH in a similar manner: reducing the H+ currents at a given potential and shifting their voltage- dependence to more positive potentials. A simple diffusion model suggests that Na(+)-H+ antiport could deplete intracellular protonated buffer to the extent observed. Evidently the Na(+)-H+ antiporter functions in perfused cells, and its operation results in pH changes which can be detected using the gH as a physiological sensor. Thus, the properties of the gH can be exploited to study Na(+)-H+ antiport in single cells under controlled conditions.     

Glycine uptake in brush border vesicles isolated from the rat intestinal cells

The uptake of glycine in osmotically active brush border membrane vesicles (obtained by the Mg++ precipitation method) has been studied and a partial characterization of its transport system has been established. The glycine uptake in these vesicles was stimulated by the presence of sodium and in the presence of an inwardly directed Na+ -gradient glycine was accumulated inside the vesicles. The effect of Na+ was specific; other monovalent cation as Li+, K+, Rb+ and choline were uneffective in the stimulation of glycine uptake, under the same experimental conditions. Preliminary experiments show an important role of some anions on the glycine uptake. A strong inhibition in the uptake rate was found when the measurements were carried out in the presence of sodium cyclamate, while in the presence of NaSCN the measured uptake values were similar to those observed in the presence of NaCl.     

Enzymatic and energetic properties of the aerobic respiratory chain-linked NADH oxidase system in the marine bacterium Pseudomonas nautica

Membranes of Pseudomonas nautica, grown aerobically on a complex medium, oxidized both NADH and deamino-NADH as substrates. The activity of membrane-bound NADH oxidase was activated by monovalent cations including Na+, Li+, and K+. The activation by Na+ was higher than that by Li+ and K+. The maximum activity of NADH oxidase was obtained at about pH 9.0 in the presence of 0.08 M NaCl. The NADH oxidase activity was completely inhibited by 60 microM 2-heptyl-4-hydroxyquinoline-N-oxide (HQNO), while the NADH:quinone oxidoreductase activity was about 37% inhibited by 60 microM HQNO. The activities of NADH oxidase and NADH:quinone oxidoreductase were about 40% inhibited by 60 microM rotenone. The fluorescence quenching technique revealed that electron transfer from NADH to ubiquinone-1 (Q-1) or oxygen generated a membrane potential (deltapsi) which was larger and more stable in the presence of Na+ than in the absence of Na+. However, the All was highly sensitive to a protonophore, carbonyl-cyanide m-chlorophenylhydrazone (CCCP) even at alkaline pH.     

Basolateral Na(+)-H+ antiporter. Mechanisms of electroneutral and conductive ion transport

The basolateral Na-H antiporter of the turtle colon exhibits both conductive and electroneutral Na+ transport (Post and Dawson. 1992. American Journal of Physiology. 262:C1089-C1094). To explore the mechanism of antiporter-mediated current flow, we compared the conditions necessary to evoke conduction and exchange, and determined the kinetics of activation for both processes. Outward (cell to extracellular fluid) but not inward (extracellular fluid to cell) Na+ or Li+ gradients promoted antiporter-mediated Na+ or Li+ currents, whereas an outwardly directed proton gradient drove inward Na+ or Li+ currents. Proton gradient-driven, "counterflow" current is strong evidence for an exchange stoichiometry of > 1 Na+ or Li+ per proton. Consistent with this notion, outward Na+ and Li+ currents generated by outward Na+ or Li+ gradients displayed sigmoidal activation kinetics. Antiporter-mediated proton currents were never observed, suggesting that only a single proton was transported per turnover of the antiporter. In contrast to Na+ conduction, Na+ exchange was driven by either outwardly or inwardly directed Na+, Li+, or H+ gradients, and the activation of Na+/Na+ exchange was consistent with Michaelis-Menten kinetics (K1/2 = 5 mM). Raising the extracellular fluid Na+ or Li+ concentration, but not extracellular fluid proton concentration, inhibited antiporter-mediated conduction and activated Na+ exchange. These results are consistent with a model for the Na-H antiporter in which the binding of Na+ or Li+ to a high-affinity site gives rise to one-for-one cation exchange, but the binding of Na+ or Li+ ions to other, lower-affinity sites can give rise to a nonunity, cation exchange stoichiometry and, hence, the net translocation of charge. The relative proportion of conductive and nonconductive events is determined by the magnitude and orientation of the substrate gradient and by the serosal concentration of Na+ or Li+.     

Influence of temperature on ionic sparing effect and cell-associated cations in the moderate halophile, Micrococcus varians var. halophilus

Cells of the moderately halophilic Micrococcus varians var. halophilus grew well in a chemically defined medium containing 1 to 3 M NaCl and 0.0103 M K+. The requirement for NaCl could be partially replaced by K+,:Li+ and Cs+. The efficiency of the sparing effect of these cations for NaCl was in order of K+ GReater than Li+ greater than Cs+. Increase in growth temperature was found to enchance the sparing effect of Li+ and Cs+ but not that of K+. Over the range of NaCl concentrations in which the cells grew well, cell-Na+ concentrations were similar to the medium NaCl concentrations while cellK+ concentrations were several-fold that in the medium. Cell-bound Na+ and K+ concentrations increased proportionally with medium NaCl concentration and growth temperature. The temperature-dependent cation accumulation was more obvious with K+ than Na+. The cell-associated Na+ + K+ concentrations were almost as high as or slightly higher than the external media which contained appropriate levels of NaCl regardless of the growth temperature.     

Dependence of Na+-Ca2+ exchange and Ca2+-Ca2+ exchange on monovalent cations

Two mechanisms of passive Ca2+ transport, Na+-Ca2+ exchange and Ca2+-Ca2+ exchange, were studied using highly-purified dog heart sarcolemmal vesicles. About 80% of the Ca2+ accumulated by Na+-Ca2+ exchange or Ca2+-Ca2+ exchange could be released as free Ca2+, while up to 20% was probably bound. Na+-Ca2+ exchange was simultaneous, coupled countertransport of Na+ and Ca2+. The movement of anions during Na+-Ca2+ exchange did not limit the initial rate of Na+-Ca2+ exchange. Na+-Ca2+ exchange was electrogenic, with a reversal potential of about -105 mV. The apparent flux ratio of Na+-Ca2+ exchange was 4 Na+:1 Ca2+. Coupled cation countertransport by the Na+-Ca2+ exchange mechanism required a monovalent cation gradient with the following sequence of ion activation: Na+ much greater than Li+ greater than Cs+ greater than K+ greater than Rb+. In contrast to Na+-Ca2+ exchange, Ca2+-Ca2+ exchange did not require a monovalent cation gradient, but required the presence of Ca2+ plus a monovalent cation on both sides of the vesicle membrane. The sequence of ion activation of Ca2+-Ca2+ exchange was: K+ much greater than Rb+ greater than Na+ greater than Li+ greater than Cs+. Na+ inhibited Ca2+-Ca2+ exchange when Ca2+-Ca2+ exchange was supported by another monovalent cation. Both Na+-Ca2+ exchange and Ca2+-Ca2+ exchange were inhibited, but with different sensitivities, by external MgCl2, quinidine, or verapamil.     

Roles of K+ and Na+ in pH homeostasis and growth of the marine bacterium Vibrio alginolyticus.

The marine bacterium Vibrio alginolyticus, containing 470 mM-K+ and 70 mM-Na+ inside its cells, was able to regulate the cytoplasmic pH (pH(in)) in the narrow range 7.6-7.8 over the external pH (pH(out)) range 6.0-9.0 in the presence of 400 mM-Na+ and 10 mM-K+. In the absence of external K+, however, pHin was regulated only at alkaline pH(out) values above 7.6. When the cells were incubated in the presence of unusually high K+ (400 mM) and 4 mM Na+, the pH(in) was regulated only at acidic pH(out) values below 7.6. These results could be explained by postulating a K+/H+ antiporter as the regulator of pH(in) over the pH(out) range 6.0-9.0. When Na(+)-loaded/K(+)-depleted cells were incubated in 400 mM-Na+ in the absence of K+, an inside acidic delta pH was generated at pH(out) values above 7.0. After addition of diethanolamine the inside acidic delta pH collapsed transiently and then returned to the original value concomitant with the extrusion of Na+, suggesting the participation of a Na+/H+ antiporter for the generation of an inside acidic delta pH. In the presence of 400 mM-K+, at least 5 mM-Na+ was required to support cell growth at pH(out) below 7.5. An increase in Na+ concentration allowed the cells to grow at a more alkaline pH(out). Furthermore, cells containing more Na+ inside could more easily adapt to grow at alkaline pH(out). These results indicated the importance of Na+ in acidification of the cell interior via a Na+/H+ antiporter in order to support cell growth at alkaline pH(out) under conditions where the activity of a K+/H+ antiporter is marginal.     

Sodium ion and the neutrotransmitter-stimulated 32P labelling of phosphoinositides and other phospholipids in the iris muscle

The effects of Na+, other cations and the neurotransmitters, acetylcholine and norepinephrine on 32Pi incorporation into phospholipids of the rabbit iris smooth muscle were investigated [1]. The basal 32P-labelling of phospholipids including phosphatidic acid, phosphatidylinositol, phosphatidylcholine, phosphatidylethanolamine and the polyphosphoinositides increased with Na+ concentration [2]. The neurotransmitter-stimulated 32P labelling of phosphatidic acid, phosphatidylinositol and phosphatidylcholine is dependent on the presence of extracellular Na+ [3]. The monovalent cation requirement for Na+ specific. Of the monovalent cations Li+, NH+4, K+, Choline+ and Tris, only Li+ partially substituted for Na+ [4]. A significant decrease in 32P labelling of phospholipids in response to acetylcholine was observed when Ca2+ and/or K+ were added to an isoosmotic medium deficient of Na+ [5]. Ouabain, which blocks the Na+-pump, inhibited the basal 32Pi incorporation into phosphatidylcholine and the acetylcholine-stimulated 32P labelling of phosphatidic acid, phosphatidylinositol and phosphatidylcholine [6]. It was suggested that phosphoinositide breakdown is associated with Ca2+ influx as we have previously reported (Akhtar, R.A. and Abdel-Latif, A.A. (1978) J. Pharmacol. Exp. Ther. 204, 655-668) and that the enhanced 32P-labelling of phosphoinositides could be associated with Na+ outflux, via the Na+-pump mechanism.