Characterization of ATP transport into chromaffin granule ghosts. Synergy of ATP and serotonin accumulation in chromaffin granule ghosts.

ATP is an excitatory neurotransmitter that is stored and cosecreted with catecholamines from cells of the adrenal medulla. While the transport of catecholamines into chromaffin granule ghosts has been extensively characterized, there is little information on the mechanism of ATP transport into these structures. Here we show that ATP transport is driven by the electrical component of the electrochemical proton gradient created by the chromaffin granule membrane H+-ATPase, and that the accumulated nucleotide is released from the vesicles by inhibition of the H+-ATPase. GTP and UTP are also substrates for this transporter, distinguishing it from the mitochondrial ADP/ATP exchanger. Accumulation of ADP and ATP (rather than exchange with intravesicular ATP) is demonstrated by high pressure liquid chromatography measurements. The anion transport inhibitor 4,4-diisothiocyanatostilbene-2,2-disulfonic acid (Ki = 27 μM) inhibits ATP transport, while atractyloside, the inhibitor of the mitochondrial ATP/ADP exchanger, is a very poor inhibitor. Finally, we have demonstrated a synergy between the accumulation of ATP and that of serotonin (i.e. more of each solute accumulates when the two are accumulated together), supporting the view that there is an interaction between serotonin and ATP that reduces their effective concentration within the ghosts.

ATP is an excitatory neurotransmitter that is stored and cosecreted with catecholamines from cells of the adrenal medulla. While the transport of catecholamines into chromaffin granule ghosts has been extensively characterized, there is little information on the mechanism of ATP transport into these structures. Here we show that ATP transport is driven by the electrical component of the electrochemical proton gradient created by the chromaffin granule membrane H ؉ -ATPase, and that the accumulated nucleotide is released from the vesicles by inhibition of the H ؉ -ATPase. GTP and UTP are also substrates for this transporter, distinguishing it from the mitochondrial ADP/ATP exchanger. Accumulation of ADP and ATP (rather than exchange with intravesicular ATP) is demonstrated by high pressure liquid chromatography measurements. The anion transport inhibitor 4,4-diisothiocyanatostilbene-2,2-disulfonic acid (K i ‫؍‬ 27 M) inhibits ATP transport, while atractyloside, the inhibitor of the mitochondrial ATP/ADP exchanger, is a very poor inhibitor. Finally, we have demonstrated a synergy between the accumulation of ATP and that of serotonin (i.e. more of each solute accumulates when the two are accumulated together), supporting the view that there is an interaction between serotonin and ATP that reduces their effective concentration within the ghosts.
ATP is an excitatory neurotransmitter with the unique feature that its final degradation product extracellularly is an inhibitory neurotransmitter, adenosine (1)(2)(3). In order to control transmitter release, neurons and other secretory cells store neurotransmitters in specialized vesicles, called synaptic vesicles, large dense core vesicles, and chromaffin granules. ATP is stored and cosecreted with positively charged, amine neurotransmitters such as the catecholamines and acetylcholine (4). Chromaffin cells of the adrenal medulla store catecholamines, together with ATP, at high concentrations (approximately 500 and 150 mM, respectively) in secretory granules called chromaffin granules (5)(6)(7). It has been proposed that costorage of ATP and catecholamines reduces the effective intragranular osmolarity to that of the cytosol, preventing lysis (8). An interaction between catecholamines and ATP has also been suggested by experiments showing that depletion of intragranular catecholamine by treatment of chromaffin cells with the granule amine transporter inhibitor, reserpine, causes the depletion of stored ATP (9).
Since intact granules contain large amounts of ATP, transport into granules cannot be distinguished from exchange with intragranular ATP. Therefore, we decided to use chromaffin granule ghosts to characterize the vectorial transport of ATP.
In this paper, we show that the vectorial transport of ATP into chromaffin granule ghosts results in the accumulation of ATP, and that the transport is not due to exchange with intravesicular ATP. Our results contradict an earlier report describing transport into chromaffin granule ghosts, in which it was concluded that chromaffin granule membranes do not contain a membrane potential-dependent ATP transporter (19). We present a biochemical characterization of this ATP transport and propose a mechanism for the transport of ATP into acidified storage vesicles. We also demonstrate that there is a synergy between the accumulation of ATP and that of 5-HT. Preparation of Chromaffin Granule Ghosts-Chromaffin granule ghosts were purified essentially as described by Phillips and Apps (20). Dissected medullae from 10 -20 bovine adrenal glands were minced thoroughly and collected in 1-2 ml of buffer (0.3 M sucrose, 10 mM HEPES, pH 7, 1 mM EDTA, 5 g/ml leupeptin, and 2 g/ml pepstatin)/g of tissue. Next, they were minced in a blender for 30 s and then homogenized in 3-5 strokes with a glass/Teflon homogenizer. The homogenate was spun at 1000 rpm in a GSA rotor for 10 min at 4°C, and the supernatant was then spun at 11,000 rpm in a GSA rotor for 20 min at 4°C. Pellets were suspended in a small volume of buffer (0.3 M sucrose, 10 mM HEPES, pH 7, and 1 mM EDTA) and then lysed by diluting 50 -100-fold in low ionic strength buffer (5 mM HEPES, 5 mM EDTA). After lysing the granules for 1 h on ice, sucrose was added to 0.3 M and membranes were collected (41,000 ϫ g, 30 min). Membranes were suspended in 1-2 ml of buffer (0.3 M sucrose, 10 mM HEPES, pH 7, and 1 mM EDTA)/g of medullae. Five milliliters of membranes were loaded onto step gradients: 4.5 ml of 0.4 M sucrose, 10 mM HEPES, 1 mM EDTA on top of 2.5 ml of 0.4 M sucrose, 10 mM HEPES, 1 mM EDTA in D 2 O, and spun in a Beckman SW 40 at 40,000 rpm for 30 min. Chromaffin membranes, marked by their pink color, have a high lipid to protein ratio and band at the sucrose/sucrose D 2 O interface, while the brown mitochondrial membranes pellet. Ghosts were aliquoted, frozen in liquid N 2 at 5-10 mg/ml, and stored at Ϫ70°C for up to 1 year without loss of activity.

Materials-Bovine
The catecholamine content of the ghosts was determined and found to be 13-16 nmol/mg protein, indicating that greater than 99% of the chromaffin granule contents had been removed from the preparation (intact granules contain approximately 2200 nmol/mg of catecholamine; Ref. 21).
Transport Assays-Transport assays were all done in essentially the same way: 200 g of membrane protein were mixed with ATP, MgCl 2 (or MgATP), an ATP regenerating system (100 g/ml creatine kinase and 5 mM phosphocreatine), and in some cases with serotonin (5-HT) and 1-2 Ci of radioactive substrate ([␣-32 P]ATP, [ 3 H]5-HT) in the buffers indicated. Reactions were done at 37°C, and the reaction was stopped by transfer to an ice bath. 100 l of reaction mix was diluted into 2 ml of ice-cold buffer, and chromaffin granule ghosts containing trapped substrate were collected on 0.45-m nitrocellulose filters, using a 10-manifold vacuum filter apparatus. The filters were then washed with 3 ϫ 2 ml of ice-cold buffer (300 mM sucrose and 10 mM HEPES), dried, immersed in scintillation fluid and counted for radioactivity. When tritiated substrate was used, filters sat in scintillant overnight prior to counting.
Measurement of ⌬pH-14 C-MeNH 2 distribution across the chromaffin granule membrane, as described by Johnson and Scarpa (15), was used to determine the pH gradient. Chromaffin granule ghosts containing trapped MeNH 2 were collected on nitrocellulose filters as described above for the other transport assays. The pH gradient is given by the expressions Measurement of ⌬⌿-[ 14 C]SCN distribution across the chromaffin granule membrane, as described by Johnson and Scarpa (15), was used to determine the membrane potential. Chromaffin granule ghosts containing trapped [ 14 C]SCN Ϫ were collected on nitrocellulose filters as described above for the other transport assays.
HPLC Assays-A Beckman HPLC was equipped with an anion exchange column. Samples were prepared for HPLC and eluted with a gradient of NH 4 H 2 PO 4 and KCl as described by Pogolotti and Santi (22). Eluted material was detected by absorbance at 259 nm. Fig.  1 shows the time dependence of bafilomycin A 1 -sensitive ATP transport into chromaffin granule ghosts (bafilomycin A 1 is a specific inhibitor of the chromaffin granule membrane, V-type, H ϩ -ATPase (23)) in a sucrose buffer containing 6 mM ATP and 1 mM MgCl 2 . A total of approximately 6 nmol of ATP associates with 1 mg of the ghost protein under these conditions (Fig. 1A). Bafilomycin A 1 inhibits a fraction of the ATP that associates with the ghosts: 0.5 Ϯ 0.2 nmol/mg at 5 min and 2.0 Ϯ 0.4 nmol/mg at 40 min; bafilomycin A 1 -sensitive transport is shown in Fig. 1B. Direct measurement of ATP accumulated within chromaffin granule ghosts (HPLC experiments described below) suggests that most of the bafilomycin A 1 -insensitive association is due to the binding of ATP to ghost membranes and to nitrocellulose filters. Oligomycin, which inhibits the mitochondrial H ϩ -ATPase (24), has no effect on the association of ATP (data not shown); therefore, the observed transport is into chromaffin granule ghosts and not into contaminat-ing mitochondria.

Active ATP Transport into Chromaffin Granule Ghosts-
The concentration dependence of bafilomycin A 1 -sensitive ATP transport at 10 min is shown in Fig. 2A. An Eadie-Hofstee plot of the data is shown in Fig. 2B: K m(app) ϭ 2.9 Ϯ 1.1 mM and V max ϭ 1.2 Ϯ 0.3 nmol/mg/10 min.
GTP and UTP Are Also Transported into Chromaffin Granule Ghosts- Fig. 3 shows that ATP, GTP, and UTP are all substrates for the chromaffin granule nucleotide transporter. This lack of specificity for nucleotides was reported previously for transport into intact chromaffin granules (18) and distinguishes the chromaffin granule system from the ATP/ADP exchanger of mitochondria, which is highly selective for ATP and ADP and will not transport other nucleotides (25).
Direct Measurement of Transported Nucleotide by HPLC Demonstrates That ADP and ATP Are Accumulated and That Transport Depends on the Electrical Component of the Electrochemical Proton Gradient-In order to demonstrate that the observed transport was due to the accumulation of ATP as opposed to exchange with residual ATP in the preparation, the FIG. 1. Time course of ATP uptake into chromaffin granule ghosts. Chromaffin granule ghosts were incubated in buffer containing 300 mM sucrose, 10 mM HEPES, pH 7, 6 mM ATP, 1 mM MgCl 2 , 5 mM creatine phosphate, and 100 g/ml creatine kinase for 5-60 min at 37°C, as described under "Experimental Procedures." A, the data shown, expressed as nmol/mg are the averages of four measurements Ϯ S.E. Ⅺ, no bafilomycin A 1 ; f, ϩ 10 M bafilomycin A 1 . B, the data shown are the difference Ϯ 10 M bafilomycin A 1 , between the points shown in A.
amounts of ADP and ATP in chromaffin granule ghosts incubated under various conditions were analyzed by anion exchange HPLC (Table I). Both ADP and ATP accumulated within the ghosts in a bafilomycin A 1 -sensitive manner. Although only ATP was added, ADP was also found, presumably as a result of hydrolysis by the H ϩ -ATPase and other ATPases in the preparation. The accumulation of ADP within the ghosts suggests that it is also a substrate of the ATP transporter. Approximately 1.02 Ϯ 0.25 nmol of ADP and ATP/mg of protein are present in the chromaffin granule ghost preparation, a small fraction of the 531 Ϯ 66 nmol/mg of protein that are associated with intact chromaffin granules (21). As shown, the amount of ADP and ATP associated with the ghosts does not increase upon the addition of 6 mM ATP at 0°C (1.10 Ϯ 0.21 nmol/mg). However, upon incubation at 37°C, 6.55 Ϯ 1.02 nmol/mg of ADP and ATP associate with chromaffin granule ghosts. When the H ϩ -ATPase is inhibited by bafilomycin A 1 , the amount of nucleotide associated with the ghosts is 2.14 Ϯ  blocks transport. The amount of ADP and ATP accumulated in the presence of 20 mM KSCN (1.79 Ϯ 0.08 nmol/mg) is roughly equivalent to the amount of accumulation in the presence of bafilomycin A 1 (2.19 Ϯ 0.65 nmol/mg). The conclusion is that ATP transport is energized by the positive inside membrane potential generated by the V-type, H ϩ -ATPase.
In addition, the amount of ADP and ATP that accumulate increases to 9.21 Ϯ 0.23 nmol/mg when serotonin (5-HT) is added to the reaction. This effect of 5-HT on ATP accumulation is discussed in detail below.
Chloride Stimulates ATP Transport-It has been shown (15), and we have confirmed (Fig. 4A) that chloride increases the pH gradient and decreases the membrane potential of chromaffin granule ghosts, suggesting that the anion readily enters the ghosts. Since chloride lowers the membrane potential, we assumed that the presence of chloride would decrease ATP uptake. Surprisingly, as shown in Fig. 4B, chloride stimulates the transport of ATP The inhibitory effect of 180 mM KCl is presumably due to a reduction in the membrane potential. Evidently, at the high KCl concentration ATP transport that is energized only by the membrane potential is completely inhibited, whereas 5-HT transport that is energized by both the membrane potential and the pH gradient is only partially inhibited. In order to verify that this stimulation of ATP transport was a specific effect of chloride, we tested the effect of 20 mM potassium gluconate and found that it did not stimulate ATP transport (0.67 Ϯ 0.22 nmol/ mg/40 min in the presence of 20 mM potassium gluconate versus 1.07 Ϯ 0.21 nmol/mg/40 min in sucrose buffer). This stimulation by chloride is either due to its ability to stimulate the chromaffin granule membrane H ϩ -ATPase (Moriyama and Nelson (26) have shown that the purified, V-type, H ϩ -ATPase from chromaffin granule membranes is stimulated by chloride) or to a direct effect on the nucleotide transporter itself.
ATP Associated with Granule Ghosts Is Releasable- Table II shows that most (Ͼ99%) of the 4.23 nmol/mg of ATP transported into ghosts in 30 min is released when the ghosts are diluted 10-fold in buffer containing 10 M bafilomycin A 1 to inhibit the V-type, H ϩ -ATPase. Most (ϳ85%) of the accumulated serotonin (5-HT) is also released from ghosts under the same conditions. Approximately 50% of the accumulated ATP (2.27 nmol/mg) and 20% of the accumulated 5-HT (2.40 nmol/ mg) are released from the ghosts upon dilution alone. The fact that the transported solutes can be released from the granule ghosts suggests that their uptake is energized by the electrochemical proton gradient and is not due to exchange with intravesicular substrates.
DIDS Inhibition of ATP Transport-Since DIDS is an inhibitor of anion transporters such as Band 3 (27,28), we looked at the ability of DIDS to inhibit ATP transport into chromaffin granule ghosts. Fig. 5 shows the dose response of ATP transport activity to DIDS (K i ϭ 27.3 Ϯ 9.6 M). Since DIDS does not significantly inhibit the formation of the pH gradient (⌬pH ϭ 0.89 in the presence of 20 M DIDS and ⌬pH ϭ 0.96 in the absence of DIDS; DIDS inhibits ATP transport approximately 50% at this concentration), we conclude that it inhibits ATP transport preferentially as compared to the H ϩ -ATPase.
Atractyloside Inhibition of ATP Transport-It has been reported that atractyloside, an inhibitor of the mitochondrial ATP/ADP exchanger (25), inhibits nucleotide uptake by intact granules (17). We find that the effect of atractyloside depends on the relative concentrations of ATP and Mg 2ϩ . As is shown in with both 2 mM and 6 mM ATP and 1 mM Mg 2ϩ (0.43 Ϯ 0.08 nmol/mg/40 min for 2 mM ATP and 2.18 Ϯ 0.18 nmol/mg/40 min for 6 mM ATP), there is no effect of atractyloside on ATP transport. Interestingly, the amount of ATP transported at a given concentration of ATP is greater when the Mg 2ϩ concentration is 1 mM rather than equal to the concentration of ATP.
Synergy of ATP and Serotonin Transport into Chromaffin Granule Ghosts-Since the non-ideal behavior of solutions of catecholamines and ATP suggests that they interact (8), we wondered whether the accumulation of serotonin (5-HT) and ATP might be coupled, such that larger amounts of both solutes accumulate when they are taken up together. Fig. 7A shows that the uptake of ATP at 6 mM ATP increases approximately 2-fold as the concentration of 5-HT increases from 0 to 400 M; under the same conditions, the uptake of 5-HT increases from 0 to 25 nmol/mg/40 min (Fig. 7B). Thus at 50 M 5-HT, 3.0 Ϯ 0.1 nmol/mg/40 min of ATP and 8.2 Ϯ 0.3 nmol/mg/40 min of 5-HT are transported, while at 400 M 5-HT, 5.9 Ϯ 0.6 nmol/mg/40 min of ATP and 24.8 Ϯ 0.8 nmol/mg/40 min of 5-HT are trans-ported. It is interesting to note that the ratio of ATP to 5-HT within the ghosts decreases from 0.38 to 0.24 as the extravesicular concentration of 5-HT increases from 50 to 400 M. A likely explanation for this decrease is the fact that the pH within the ghosts rises from pH 5.5 (a pH at which ATP 3Ϫ would predominate) at 50 M 5-HT to pH 7 (a pH at which ATP 4Ϫ would predominate) at 400 M 5-HT (data not shown).
Table III also illustrates the relationship between ATP and 5-HT transport into chromaffin granule ghosts. With 6 mM ATP in the buffer, addition of 200 M 5-HT increases ATP uptake approximately 2-fold from 2.4 Ϯ 0.4 to 4.8 Ϯ 0.7 nmol/mg/40 min. Similarly, with 200 M 5-HT in the buffer, increasing the [ATP] from 2 mM to 6 mM increases 5-HT accumulation 2-fold from 10.0 Ϯ 1.6 to 20.8 Ϯ 2.0 nmol/mg/40 min. The evidence is that ATP and 5-HT accumulate in the ghosts to higher levels when both are present together, suggesting that there is an interaction between accumulated ATP and serotonin that reduces the "free" concentration of solutes within the ghosts. DISCUSSION We show here that: 1) ATP is transported into chromaffin granule ghosts (K m ϭ 2.9 mM and V max ϭ 1.2 nmol/mg/10 min, measured at 2 mM chloride) (Figs. 1 and 2); 2) this transport is due to the accumulation of ADP and ATP and is not due to exchange with intravesicular nucleotide (Table I); 3) the transport requires the membrane potential but not the proton gradient set up by the proton pump (Table I); 4) the trapped nucleotides can be released (Table II); 5) GTP and UTP are also substrates for the transporter (Fig. 3); 6) chloride stimulates ATP transport (Fig. 4); 7) DIDS (K i ϭ 26 M) inhibits this ATP transporter (Fig. 5), while atractyloside is a weak inhibitor (Fig. 6); 8) although transport requires magnesium, magnesium concentrations that are equal to or greater than the concentration of ATP inhibit ATP transport (Fig. 6); and 9) the amount of ATP transported can be substantial (6 nmol/mg), up to 38% of the amount of accumulated 5-HT (Fig. 7). These TABLE II Reversibility of ATP and 5-HT uptake Chromaffin granule ghosts were incubated with 200 mM 5-HT and 6 mM ATP in buffer containing 300 mM sucrose, 20 mM KCl, 10 mM HEPES, pH 7, 1 mM MgCl 2 , 5 mM creatine phosphate, and 100 g/ml creatine kinase for 30 min at 37°C. To test for the reversibility of transport, samples were then diluted 1:10 in sucrose buffer with or without 10 mM bafilomycin A 1 , and incubated an additional 30 min at 37°C. The data shown are the averages of four measurements Ϯ S.E. The amount associated with ghosts in the presence of 10 mM bafilomycin A 1 , determined as the average of four measurements Ϯ S.E., has been subtracted from each value shown. features are considerably different from those of the ATP transport systems in yeast endoplasmic reticulum vesicles (29) (K m ϭ 10 M and V max ϭ 1.2 nmol/mg/min), rat liver endoplasmic reticulum vesicles (30) (K m ϭ 4 M and V max ϭ 6.6 pmol/mg/ min) and rat liver Golgi vesicles (31) (K m ϭ 0.9 M and V max ϭ 58 pmol/mg/min).
Saturable uptake of nucleotides into intact chromaffin granules has been reported previously (17,18,32). However, this work has been brought into question for two reasons. First, intact granules contain large amounts of ATP; therefore, transport into granules cannot be distinguished from exchange with intragranular ATP. Second, the only report describing ATP transport into chromaffin granule ghosts (which do not contain appreciable amounts of ATP) concluded that chromaffin granule membranes do not contain an ATP transporter and that ATP enters chromaffin granules and ghosts by passive diffusion (19). Our clear demonstration of vectorial transport of ATP into chromaffin granule ghosts, resulting in the accumulation of nucleotide, refutes the latter argument. The common characteristics of ATP uptake into chromaffin granules and transport into ghosts are: 1) ATP, GTP, and UTP are all substrates; 2) transport is inhibited by proton ionophores and SCN Ϫ and not by ammonium ion; and 3) transport is inhibited by DIDS.
Although transport of ATP into chromaffin granule ghosts can be detected in the absence of chloride, and high concentrations of chloride inhibit ATP transport by lowering the membrane potential, chloride stimulates transport at concentrations up to 100 mM (Fig. 4B). One possibility is that chloride stimulates the activity of the V-type, H ϩ -ATPase (26). Alternatively, chloride may stimulate the ATP transporter directly.
ATP transport into chromaffin granule ghosts is similar to that seen for the other negatively charged neurotransmitter, L-glutamate (K m ϭ 1.6 mM and V max ϭ 13 nmol/mg/min) (33)(34)(35)(36)(37). Like ATP transport, L-glutamate transport into synaptic vesicles is energized by the membrane potential generated by the synaptic vesicle membrane, V-type, H ϩ -ATPase, and it is inhibited by DIDS. In addition, low concentrations of chloride stimulate L-glutamate transport, while high concentrations of chloride inhibit it. While this effect of chloride may be due to the stimulation of the V-type, H ϩ -ATPase (26), chloride does not have this dramatic effect on the transport of other neurotransmitters that are also coupled to the V-type, H ϩ -ATPases (see, for example, the effect of chloride on 5-HT transport into chromaffin granule ghosts; Fig. 4B). Therefore, we propose that the effects of chloride are direct effects on the transporters for ATP and L-glutamate.
Since the uptake measured in these experiments is inhibited by bafilomycin A 1 and not by oligomycin, we are confident that there is no contribution from the mitochondrial ATP/ADP exchanger. Furthermore, GTP and UTP are also transported into chromaffin granule ghosts; these nucleotides are not substrates for the mitochondrial exchanger (25). However, both transporters can be inhibited by atractyloside, although the chromaffin granule system is only very weakly inhibited. In addition, like the ATP transport systems of yeast endoplasmic reticulum and rat liver Golgi vesicles, the chromaffin granule ATP transporter is inhibited by DIDS (K i ϭ 26 M) (Fig. 5).
Importantly, there is a synergy between ATP and serotonin (5-HT) accumulation in chromaffin granule ghosts ( Fig. 7 and Table III). At a given [ATP], there is a proportional increase in ATP accumulation with increasing extravesicular ; similarly, at one , there is an increase in 5-HT accumulation with an increase in extravesicular [ATP]. Evidently, the amounts of 5-HT and ATP taken up by chromaffin granule ghosts, under the proper conditions, are balanced so that one molecule of ATP with 3-4 negative charges is capable of neutralizing the positive charge of 3-4 5-HT molecules. If ATP 4Ϫ is the substrate of the transporter, a possibility compatible with the effect of Mg 2ϩ on the transport activity, at an intravesicular TABLE III Synergy of ATP and 5-HT uptake Chromaffin granule ghosts were incubated with 2 mM or 6 mM ATP and with or without 200 M 5-HT in buffer containing 300 mM sucrose, 20 mM KCl, 10 mM HEPES, pH 7, 2 mM MgCl 2 , 5 mM creatine phosphate, and 100 g/ml creatine kinase for 40 min at 37°C. The data shown are the averages of four measurements Ϯ S.E. The amount associated with ghosts in the presence of 10 M bafilomycin A 1 , determined as the average of four measurements Ϯ S.E., has been subtracted from each value shown.  7. Effect of 5-HT transport on ATP transport into chromaffin granule ghosts. Chromaffin granule ghosts were incubated with increasing concentrations of 5-HT in buffer containing 300 mM sucrose, 20 mM KCl, 10 mM HEPES, pH 7, 6 mM ATP, 1 mM MgCl 2 , 5 mM creatine phosphate, and 100 g/ml creatine kinase for 40 min at 37°C, as described under "Experimental Procedures." The data, ATP transport (A) and 5-HT transport (B), shown, expressed as nmol/mg/40 min, are the average of four measurements Ϯ S.E. The amount associated in the presence of 10 M bafilomycin A 1 , determined as the average of four measurements Ϯ S.E., was subtracted from each value shown. pH of 5.5 the accumulated ATP will have three negative charges. This preferential neutralization of accumulated ATP by 5-HT as opposed to protons suggests that there is an interaction between ATP and 5-HT that reduces the effective concentration of the two within the ghosts. The idea that biogenic amines and ATP interact is further supported by the observation that the osmolarity of a solution of 0.6 M epinephrine and 0.15 M ATP is 250 mosm, one third the expected osmolarity (8).