Amphetamine and other psychostimulants reduce pH gradients in midbrain dopaminergic neurons and chromaffin granules: A mechanism of action

Neuron,

Vol. 5, 797-808,

December,

1990, Copyright

0 1990 by Cell Press

Amphetamine and Other Psychostimulants Reduce pH Gradients in Midbrain Dopaminergic Neurons and Chromaffin Granules: A Mechanism of Action David Sulzer and Stephen Rayport Department of Psychiatry and Center for Neurobiology and Behavior Columbia University New York, New York 10032 Department of Neuropathology New York State Psychiatric Institute New York, New York 10032

Summary Rewarding properties of psychostimulants result from reduced uptake and/or increased release of dopamine at mesolimbic synapses. As exemplified by cocaine, many psychostimulants act by binding to the dopamine uptake transporter. However, this does not explain the action of other psychostimulants, including amphetamine. As most psychostimulants are weak bases and dopamine uptake into synaptic vesicles uses an interior-acidic pH gradient, we examined the possibility that psychostimulants might inhibit acidification. Pharmacologically relevant concentrations of amphetamine as well as cocaine and phencyclidine rapidly reduced pH gradients in cultured midbrain dopaminergic neurons. To examine direct effects on vesicles, we used chromaffin granules. The three psychostimulants, as well as fenfluramine, imipramine, and tyramine, reduced the pH gradient, resulting in reduced uptake and increased release of neurotransmitter. Inhibition of acidification by psychoactive amines contributes to their pharmacology and may provide a principal molecular mechanism of action of amphetamine. introduction Ventral tegmental area (VTA) dopaminergic neurons projecting to the nucleus accumbens are thought to mediate the rewarding effects of psychostimulants (Koob and Bloom, 1988). Amphetamine, cocaine, phencyclidine, and other major drugs of abuse act by increasing extraneuronal dopamine in this projection, as measured by in vivo microdialysis and voltammetry (Carboni et al., 1989; Di Chiara and Imperato, 1988; Hernandez et al., 1988; Mifsud et al., 1989). Animals self-administer cocaine and amphetamine directly into the nucleus accumbens, whereas lesioning or pharmacological blockade of dopaminergic transmission at this site blocks rewarding effects (Goeders and Smith, 1986; Hoebel et al., 1983). Anti-psychotics, which are dopamine receptor antagonists, abolish self-administration in animals and euphoria in man (Wise and Bozarth, 1987). Indeed, chronic or high doses of amphetamine produce symptoms behaviorally indistinguishable from schizophreniform psychosis (Ellinwood et al., 1973). The increased synaptic concentration of dopamine

produced by psychostimulants results from a reduction in presynaptic dopamine reuptake and/or increased release (Wise and Bozarth, 1987). While some psychostimulants, including cocaine, appear to increase extracellular dopamine by inhibiting dopamine reuptake at the plasma membrane dopamine transporter, this does not adequately explain the effects of other psychostimulants, particularly amphetamine. Amphetamine induces alterations in behavior and releases dopamine far in excess of what is predicted by its relatively weak binding to the dopamine transporter (Andersen, 1987; Ritz et al., 1987). The action of amphetamine is apparently neither dependent on activity nor due to dopamine receptor binding (Kuczenski et al., 1990; Shalaby et al., 1983). Indeed, amphetamine reduces accumulation of monoamines in synaptic vesicle preparations that lack the plasma membrane transporter (Knepper et al., 1988). Rather, amphetamine is thought to displace dopamine in synaptic vesicles, leading to increased synaptic levels; however, the molecular mechanism of amphetamine action has remained obscure (Carboni et al., 1989; Knepper et al., 1988; Liang and Rutledge, 1982; Zaczek et al., 1990). Catecholaminergic vesicles use an interior-acidic proton gradient for transmitter uptake. Amphetamine and several other psychostimulants are lipophilic weak bases (see Beckett and Moffat, 1969; Mack and Bonisch, 1979). We show here that such drugs perturb proton gradients in intracellular compartments in VTA dopaminergic neurons. The drugs work directly at thevesicular level, as shown in chromaffin granules used as a model vesicle preparation. This inhibition of proton gradients strongly affects neurotransmitter accumulation. We suggest that a weak base mechanism may in part explain how psychostimulants increase synaptic levels of dopamine, thereby mediating their euphoriant and psychotogenic effects. Results intracellular Acidification in Dopaminergic Cells VTA dopaminergic neurons give rise to the mesolimbic projection and are thus the cellular substrate for psychostimulant action. Cells from the VTA were isolated from postnatal rats and grown for 7-10 days in vitro on a glial monolayer. In culture, these neurons show most of the distinctive characteristics of dopaminergic cells in vivo or in krain slice preparations (Rayport et al., Sot. Neurosci., abstract, 1988, 1990).Asdopaminergicneuronsaretheonlycatecholaminergic cells in the ventral midbrain, we used 5-hydroxydopamine, which is specifically taken up by catecholaminergic synaptic vesicles and forms an electron-dense product (Tranzer and Thoenen, 1967), to examine them at the electron microscopic level. Two sizes of labeled vesicles were observed, chiefly

NC?UVJ” 798

A

Figure

1. Distribution

of Synaptic

Vesicles

in Cultured

VTA

Dopaminergic

Neurons

Cells were pretreated with 50 PM 5-hydroxydopamine to produce electrondense labeling of dopaminergic synaptic vesicles fTranzer and Thoenen, 1967). (A) A dopaminergic processes is shown abuting an unlabeled cell body. Dopaminergic vesicles (arrows) were frequently seen in axonal varicosities and observed occasionally along axonal processes. These vesicles fell into populations of two sizes, approximately 40-50 and 100 nm in diameter. (8) Vesicles of similar appearance (arrows) were frequently found lining the perimeter of the cell body, in particular between the usually eccentric nucleus and the plasmalemma, a region devoid of lysosomes. Bar, 500 nm (A); 1.5 pm (B).

in axonal varicosities (Figure IA) and closely apposing the plasma membrane of the cell body (Figure IB). Occasional vesicles were found in axonal processes, but never in dendrites. These observations suggest

Figure

2. Amphetamine

and NH&I

Alkalinize

IntracelluRrr

Acidic

that changes observed in are likely to parallel those We have recently shown stain with weak base vital

Compartments

in Midbrain

organelles in the cell body in vesicles at synaptic sites. that presynaptic structures dyes in situ, demonstrating

Dopaminergic

Neurons

Stained

with

NR

A representative field from a VTA culture that contained 37.5% + 1.5% dopaminergic cells (demonstrated by antigenicity to tyrosine hydroxylase) is shown. (A) NR (0.1%) was added to stain acidic compartments. After 30 min, discrete staining was seen in both neurons and glia. (B) After a 12 min incubation with 100 PM L-amphetamine free base, a gradual reduction in staining was apparent. A glial cell (C) was less affected by amphetamine than were the neurons. (C)After 1 min in 10 mM NHXI, most of the remaining staining was abolished. Similar results were obtained with 100 uM o-amphetamine sulfate, 100 uM cocaine, and 100 PM phencyclidine. Each drug was tested in triplicate. Bar, 10 pm.

Psychostimulants 799

Reduce

Intracellular

Figure 3. Amphetamine Reduces VTA Dopaminergic Neurons

pH Gradients

Proton

Gradients

in a Dose-Dependent

Manner

in AO-Stained

Intracellular

Compartments

of Cultured

A cell from a 3-day-old culture grown without a glial monolayer and later demonstrated to be dopaminergic (by tyrosine hydroxylase staining) is shown. Fluorescence excitation was attenuated with a neutral density filter to eliminate bleaching, and cells were observed with a low-light CCD camera. (A) A0 (100 nM) was added to stain acidic compartments. Staining was stable after 10 min. The condensed chromatin in the nucleolus was also stained, as a result of the well-known property of A0 to bind chromatin. (B) Five min later, after a 7 min incubation in 1 pM L-amphetamine free base, decreased staining of acidic organelles was apparent. Amphetamine (IO PM) added for 5 min caused a further reduction in staining (data not shown). (C) After a IO min incubation in 100 PM amphetamine, nearly all of the remaining staining was abolished. Note that staining of the nucleolusdid notdecreasewithamphetamineapplication. Undertheconditions used, no bleachingof fluorescencewas seen in control preparations after repeated exposures. Similar results were obtained with o-amphetamine sulfate and cocaine. Bar, 2 pm.

the presence of acidic intracellular compartments (Sulzer and Holtzman, 1989; Augenbraun et al., 1990). The stained compartments appear to be chiefly synaptic vesicles or related endocytic organelles involved in recycling of synaptic vesicle membrane. The staining can be abolished by exposure to metabolic poisons or weak bases such as ammonium, as would be expected of staining reflecting energy-dependent proton accumulation at intracellular sites. We stained VTA cultures with the fluorescent weak base vital dye acridine orange (AO) or with neutral red (NR), a weak base dye observed without fluorescence. Stained sites corresponded to synaptic vesicle-rich regions in processes and the cell body perimeter and to lysosomes, similar to the previous findings. The stained structures are also likely to include related organelles that maintain a low pH, such as endosomes and compartments involved in synaptic vesicle membrane recycling (Sulzer and Holtzman, 1989). All neurons displayed some staining, as did glial cells. As we noted previously for other neurons, the classic lipophilic weak base ammonium (here added as 10 mM NH4CI for 5 min) abolished intracellular staining.

Alkalinization of Intracellular Compartments in WA Neurons by Psychoactive Amines The psychostimulants tested produced results strikingly similar to those obtained with ammonium. In NR-treated VTA cultures (Figure ZA), addition of 100 PM amphetamine (L-amphetamine free base or D-amphetamine sulfate)caused markedly reduced intracel-

lular staining, both in processes and in the cell body (Figure 26); 10 mM ammonium completely abolished staining (Figure 2C). Similar results were obtained with 100 PM cocaine for 1 min or 100 PM phencyclidine for IO-15 min, both of which eliminated NR staining (data not shown). An appreciable reduction of staining was apparent at lower concentrations. These protocols were repeated with AO, yielding similar results. Differences in alkalinization between o-amphetamine sulfate and L-amphetamine free base were not evident using NR. To examine the effects of lower levels of the drugs and to observe alkalinization at higher resolution, we visualized the cultures with a chilled CCD camera and integrated low letiel fluorescence emission. VTA cells weregrown directlyon polyornithine-treated glass for 3 days, without a glial monolayer, to reduce background staining. Cultures were stained with 100 nM AO, producing punctate staining of acidic structures (Figure 3A). Exposure to 1 PM amphetamine for 12 min reduced A0 staining of intracellular compartments (Figure 3B). Treatment with 10 PM amphetamine for 5 min caused a further reductiQn (data not shown). Exposure to 100 PM amphetamine led to nearly complete loss of staining (Figure 3C). Similar results were obtained with either L-amphetamine free base or o-amphetamine sulfate. Partial loss of A0 staining was also seen after exposure to 1 PM cocaine for 5 min, and a further reduction was evident after 10 PM cocaine (data not shown). Intracellular compartments did not noticeably recover A0 or NR staining in the

presenceof the drugs, but underwent partial recovery after removal of the drugs. In control experiments repeated fluorescence excitation did not bleach A0 fluorescence. Reduced staining under these conditions demonstrates that psychoactive amines quite effectively inhibit intracellular proton gradients. As staining was eliminated from all intracellular compartments bysufficient concentrations of the drugs, the sites affected presumably include dopamine storage vesicles in the processes and in cell bodies as well as lysosomes and other organelles involved in endocytosis and secretion.

FCCP i

15

20

25

30

35

Time (min)

B 1.0

Alkalinization of Chromaffin Granule Preparations by Psychoactive Amines To examine the direct effects of weak base psychostimulants on synaptic vesicles, we used isolated bovine adrenal chromaffin granules and lysed, resealed vesicles prepared from the granules (ghosts) as model preparations. Chromaffin granules behave like monoaminergic synaptic vesicles in maintaining both an N-ethyl-maleimide (NEM)-sensitive, ATP-dependent transmembrane interior-acidic pH gradient and a reserpine-sensitive neurotransmitter uptake system (Johnson, 1988). As previously reported (Cidon et al., 1983), addition of ATP to this granule preparation resulted in an exponential acidification of the granule interior observed by quenching of A0 fluorescence, with a T, of 8 min (Figure 4A: ATP + Val). Using the weak base vital dye quinacrine in an established method (Schuldiner et al., 1972), we estimated the internal pH of the granules after incubation with ATP and valinomycin to be 5.64 f 0.03 (n = 4), a figure in the range of previous reports (Johnson, 1988). Addition of 100 uM amphetamine led to a 40% reduction in the fluorescence signal, reflecting alkalinization (Figure 4A, Amph). This effect was dose dependent (I& = 108 PM; Figure 4B). The pH gradient induced in whole granule or ghost preparations could be completely abolished by 1.3 uM p-trifluromethoxyphenylhydrazone carbonyl cyanide (FCCP), a protonophore (Figure 4A, FCCP), or was inhibited 90%-100% by 200 uM NEM, a granule ATPase inhibitor (see Nelson et al., 1988). To separate pH effects from those due to the transmembrane gradient (A@, the potassium ionophore valinomycin was used. Valinomycin increased the total fluorescence signal; however, 100 uM amphetamine caused proportionally the same quenching of fluorescence in preparations with or without valinomycin. To rule out a direct effect of amphetamine on the chromaffin granule proton ATPase, ATPase activity was assayed in the presence and absence of amphetamine. Granules were preincubated with valinomytin. After addition of 1 mM ATP for 12 min, ATPase activity was measured to be 323 f 20 nM ATP hydrolyzed per vg of protein per min (N = 7). With the inclusion of 100 uM n-amphetamine sulfate, activity measured 359 + 34 nM phosphate produced per ug

‘.

R = 0.99

I

0”

i

/ 10

Figure amine

4. Alkalinization

20 50 Amphetamine cont. of Chromaffin

100 (uM) Granules

200

500

by Amphet-

To determine intragranular pH, A0 fluorescence was measured spectrophotometrically. When A0 is taken up into acidic compartments the measured fluorescence decreases. The relative fluorescence represents the difference in fluorescence between the minimum fluorescence (096, after ATP-induced quenching; see Nelson et al., 1988) and the maximum fluorescence (10096, after FCCP, a protonophore); 0% thus represents the maximum pH gradient. (A) After a 15 min incubation in 0.67 PM AO, 1 mM ATP and 1 pg/ ml valinomycin were added, activating the granule proton pump and resulting in granular acidification observed by fluorescence quenching. The potassium ionophore valinomycin was used to promote acidification by shunting the transmembrane gradient (A@; similiar results were observed without valinomycin, but theoverall signal was smaller. Lhmphetaminefree base(100 PM) increased the A0 signal, indicating alkalinization. FCCP (1.3 FM) equilibrated the proton gradient. Reflecting their different mechanisms of action, acidification resumed after amphetamine, but not FCCP. (B) t-Amphetamine free base produces dose-dependent alkalinization of chromaffin granules. Experiments were conducted as above. Log[amphetamine] versus percent mean alkalinization was linear (R = 0.99). Similar results were obtained for each amine tested (see Table 1). Data points were averaged from three experimental values.

of protein per min. Thus, pharmacologically relevant concentrations of amphetamine appear to have no effect on the ATPase activity of isolated chromaffin granules. As previously shown, the weak bases ammonium and chloroquine caused dose-dependent alkalinization, as did relatively high concentrations of serotonin and dopamine (Table 1; see Johnson et al., 1978). We found that in addition to amphetamine, cocaine and phencyclidine resulted in dose-dependent alkalini-

Psychostimulants 801

Table

Reduce

1. Alkalinization

Intracellular

of Isolated

Compound Neurotransmitters Dopamine Serotonin Commonly-used weak bases Ammonium (as NH&II) Chloroquine Psychoactive drugs o-Amphetamine sulfate L-Amphetamine sulfate L-Amphetamine free base Cocaine Fenfluramine lmipramine Phencyclidine Tyramine Controls DOPAC Tyrosine

pH Gradients

Chromaffin (COOH)

Granules pK

by Psychoactive PK

G

9.2 4.919.8

2249 692

2185 15

9.2 8.5110.1

106 3

6 40

9.9

51 46 108 137 64 28 28 433

<4 <4 <4 <4 <4 <4 67 68

8.4 9.9 8.6 9.4 9.2 4.5 2.3

Amines

9.4

WV

No alkalinization No alkalinization

Tc (9

(10,000 PM) (500 PM)

pK values of the compounds were taken from the literature (see below). Some compounds have two proton-accepting sites and thus dual pK values. The pK values in the second column are generally understood to reflect amine pK values (Mack and Bonisch, 1979). We measured the I& at the point halfway between maximum acidification after addition of ATP and valinomycin and that after FCCP incubation, which abolishes the pH gradient (see Figure 4A). For all the effective compounds, a minimum of five concentrations were tested in triplicate (R > 0.98; see Figure 4B). The T, was measured as the time constant for the pH gradient to fall to l/e at the I&. Observations were in duplicate; In(time) versus percent mean alkalinization was plotted, and the slope was calculated (R > 0.96). Citations for pK values are as follows: ammonium, Roos and Boron, 1981; amphetamine, DOPAC, dopamine, tyramine, and tyrosine, Mack and Bonisch, 1979; chloroquine, DeDuve et al., 1974; cocaine, Dean, 1985; fenfluramine, Beckett and Moffat, 1969; imipramine, Schmalzing, 1989; phencyclidine, Weinstein et al., 1983; and serotonin, the Merck index.

zation with a characteristic exponential time course (Table 1). Other clinically important amines, including the antidepressants fenfluramine and imipramine and the sympathomimetic tyramine, showed similar results. However, the times and concentrations required for inhibition of the proton gradient differed considerably. The dose-response curve for each of the compounds tested fit a first-order exponential (R > 0.98 for all drugs tested). Concentrations in the low micromolar range caused granular alkalinization. Amphetamine and cocaine had kinetic profiles resembling NH&I; L-amphetamine sulfate and o-amphetamine sulfate showed quantitatively similar results and lower ICSO values than the free base form. Tyramine fell between the other phenylethylamine derivatives, dopamine and amphetamine. Phencyclidine, imipramine, and chloroquine had relatively long T, values. Fenfluramine, imipramine, and phencyclidine had low ICSO values, although none of the drugs tested were as effective as chloroquine. The profile of alkalinization by weak base drugs differed significantly from that due to protonophores, since the pH gradient reestablished itself after addition of weak bases but not after FCCP (Figure 4A, Amph versus FCCP). Since our ICSO corresponds to a 50% unquenching of fluorescence, which represents a change of about 0.3 pH units (see Discussion), these figures are not comparable to previous reports of ammonium concentrations that collapse the ApH by one-half, i.e., about 0.9 pH units (see Pollard et al., 1979). If storagevesiclealkalinization isduetoaweak base

mechanism, amine-derived compounds that either do not accept protons or are always charged should not affect proton gradients. We found that 3,4dihydroxyphenylacetic acid (DOPAC), the deaminated monoamine oxidase product of dopamine, caused no alkalinization in isolated chromaffin granules. Tyramine, formed by tyrosine decarboxylation, had an I& of 433 PM, whereas tyrosine, which is overwhelmingly charged at pH 7.4, caused no alkalinization at concentrations up to 500 PM. Effects of Amphetamine on Serotonin Accumulation in Chromaffin Ghosts To observe the direct effect of amphetamine-induced alkalinization on transmitter accumulation, we measured the uptake and release of [3H]serotonin in chromaffin ghosts. Typical synaptic vesicles of midbrain dopamine neurons, like chromaffin ghosts, are thought not to contain the dense cores of whole chromaffin granules (Phillips, 1974;Van Eden et al., 1987; although presence of dense cores may be dependent on fixation; see Bloom, 1970); the dense core is thought to contribute to precipitation of neurotransmitter (Johnson, 1988). Moreover, the ghosfs as prepared contained MOPS rather than the more complex endogenous pH buffering of whole chromaffin granules. Therefore, ghosts may more closely model dopaminergic vesicles than whole granules. It has been previously shown that alkalinization of wholechromaffin granules also leads to loss of transmitter (Johnson and Scarpa, 1979), as we confirmed (data not shown). We found that amphetamine-induced alkaliniza-

NellrOn 802

15

Figure 5. Amphetamine, NH&II, and FCCP Cause Both Alkalinization of Chromaffin Granule Ghosts and Inhibition of [‘H]Serotonin Accumulation

0

a

Two parallel sets of experiments, differing only in that A0 was used to detect the proton gradient and [3H]serotonin was used to 10 f measure transmitter uptake, were run. \ F (A) Addition of ATP induced acidification $ (as measured by quenching of A0 fluoresI 0 5 cence) and serotonin uptake. NHICl and CL amphetamine caused concurrent inhibi5 tion of proton accumulation and serotonin % uptake. 0 (B)Amphetamine, NHKI,and FCCPcaused dose-dependent release of serotonin and in the proton gradient (as measured by unquenching of fluorescence). Background was measured in ATP-free medium and from the data points. Experiments were performed in triplicate. In (B), the scale is adjusted so that 100% relative fluorescence to nonreleasable serotonin, probably bound to charged membrane sites (see Discussion). 2 9 5

reduction subtracted corresponds

tion of the chromaffin ghost preparation was similar tothat intheintactgranulesandthatthealkalinization resulted in reduced neurotransmitter accumulation (Figure 5). To observe neurotransmitter uptake, we used [3H]serotonin, since the vesicular monoamine transporter takes up both catecholamines and serotonin, but shows the highest affinity for serotonin. Uptake of J3H]serotonin was initiated by the addition of ATP (T, of uptake = 30 min). NHdCI (2 mM) and 400 PM amphetamine both greatly reduced the [3H]serotonin uptake and, at comparable levels, the transmembrane pH gradient (Figure 5A). Similarly, 2 mM NH&I, 180 and 720 ).rM amphetamine, and 20 PM FCCP each caused rapid release of previously accumulated [3H]serotonin from ghosts as well as alkalinization of the pH gradient (Figure 5B). Addition of the granule monoamine transport blocker reserpine (I ).rM) did not cause release of [3H]serotonin. Proton and serotonin uptake could be uncoupled by pretreatment with reserpine (1 PM for 30 min). This inhibited [3H]serotonin uptake by84% f 16% without affecting the pH gradient. However, 1 PM reserpine did not inhibit alkalinization by amphetamine or by the higher serotonin concentrations necessary to detect changes in transmembrane pH gradient (see Table 1). This strongly suggests that amphetamine and serotonin at higher concentrations enter the granule primarily by lipophilic partitioning rather than via the monoamine transporter. Discussion

The impetus for this work stemmed from the observation that many psychostimulants are lipophilic weak bases that might be expected to reduce proton gradients. Amphetamine, ammonia, chloroquine, theneurotransmitters, and the psychoactive drugs examined have amine groups with pK values between 8 and 11 (Table I), existing in both neutral and protonated forms at physiological pH. Previous work has suggested thatweak basedrugs, including amphetamine, atropine, desipramine, haloperidol, imipramine, metar-

aminol, morphine, njcotine, procaine, and tyramine, accumulate at acid’ic intracellular sites in cells and in isolated granules or synaptosomes (Batzri et al., 1988; Brown and Garthwaite, 1979; Daniels et al., 1980; Honegger et al., 1983; Johnson et al., 1982; Moe et al., 1988; Schmalzing, 1989; Strobe1 and Bianchi, 1970; Weiss 1968). Other groups have reported that alkalinization of synaptosomes, cells, and isolated vesicles leads to reduced uptake or increased neurotransmitter release(Erecinskaet al., 1987; Holtz, 1978; Johnson, 1988; Maron et al., 1979; Phillips, 1978; Schuldiner et al., 1978; Van der Kloot, 1987). The importance of the weak base mechanism for drug action was first suggested by Johnson and collaborators (1981), who showed a similar mechanism for the action of the anti-parkinsonian drug, amantadine. For the first time, we have shown that several prominent drugs of abuse are weak bases that inhibit intracellular acidification and reduce the vesicular transmembrane pH gradient resulting in a reduced uptake and increased release of aminergic neurotransmitter. Weak

Base

Model

of Psychostimulant

Action

Lipophilic weak bases equilibrate pH gradients across intracellular compartments (DeDuve et al., 1974; Johnson, 1988; Roos and Boron, 1981; Schuldiner et al., 1972). According to Schuldiner et al. (1972), for a weak base with a single proton-accepting group with a pK >>physiological pH, membrane-permeable in its neutral form, and membrane-impermeable in its cationic form, at equilibrium,

[baseldbasel,,

= [H+l,,t/[H+l,,

(1)

When synaptic vesicles reach an internal pH of 5.6 in an external medium of pH 7.4, weak bases such as amphetamine should accumulate over 60-fold. After the vesicle’s buffering capacity is exceeded, these compounds will induce alkalinization of the intravesicular space, reducingthedrivingforce necessaryfor monoamine uptake (Johnson, 1988). Weak base drugs would also compete for protons with neurotransmit-

Psychostimulants 803

Reduce

Intracellular

pH Gradients

ter already present within the granules. The resulting uncharged neurotransmitter would then diffuse out of the granules down its concentration gradient. As discussed above, assuming a granule pH of 5.6 and that AO, which has one proton-accepting amine group (pK = 10.5; Cools and Janssen, 1986), is distributed according to Equation 1, the granule pH represented by the IGO values is about 5.9. If weak base neurotransmitters such as dopamine (pK = 9.17) are distributed in vesicles as reported (Johnson, 1988),

~og(LWL41,,,) = AwLZ + 2Apt-i

(2)

where [A] = the concentration of amine neurotransmitter, Aw = the transmembrane potential, and Z = RTlF = 59 mV, and assuming Aw = +80 mV as reported for chromaffin granules (Johnson, 1988), alkalinization of the vesicular pH gradient from pH 5.6 to 5.9 (which corresponds to our lCsO measurements) should lead to a loss of 75% of the releasable pool of monoamine neurotransmitter. Therefore, a relatively small change in ApH can be expected to affect neurotransmitter accumulation profoundly. We confirmed this model by showing a correlation between drug-induced alterations in the pH gradient and serotonin accumulation (Figure 5) in chromaffin ghosts. From Equations 1 and 2 it can be shown that the weak base mechanism is sufficient to explain the observed inhibition of vesicular serotonin accumulation. In our protocol,weabolished the Av component using valinomycin to be certain that we were examining effects due to ApH. The correspondence between alkalinization and accumulation is somewhat better for uptake than release experiments; this is probably due in part to binding of neurotransmitter by charged vesicle membrane constituents, trapping transmitter that would otherwise be released (see Sulzer et al., 1987). This binding should not be as important in the uptake experiments, in which inhibition of ApH by drugs would reduce the initial entry of neurotransmitter. Different kinetics of the proton and serotonin uptake systems may also be involved; we found that the T, for serotonin uptake was 30 min, whereas that for acidification was 8 min. Additionally, the differences between uptake and release may be due to the dual pK values of serotonin, which has an indole ring amine (pK 4.9) and would therefore increase the buffering capacity in the vicinity of the granule pH. Along these lines, it has been noted that ammonium more readily releases dopamine than serotonin from synaptosomal fractions (Erecinska et al., 1987). Reserpine, although itself a weak base, did not cause detectable release of [3H]serotonin or vesicular alkalinization when used at levels sufficient to block transmitter uptake (1 PM), nor did it block alkalinization by amphetamine. Therefore, the effects of amphetamineon neurotransmitter accumulation are not explained by a reserpine-like effect, i.e., inhibition of the reserpine-sensitive transporter. These results also demonstrate that amphetamine entry into storage

vesicles does not depend primarily on the reserpinesensitive transporter. Our results indicate that pharmacologically relevant concentrations of amphetamine do not block granule ATPase activity. In fact, a small increase in H+-ATPase activity would be predicted if amphetamine is acting to decrease the intravesicular proton concentration. It has been noted that the net efflux of previously accumulated serotonin does not occur until at least 1 hr after ATP has been depleted from the granule incubation medium (Maron et al., 1983). Moreover, it does not appear likely that inhibition of the H+-ATPase would result in both rapid alkalinization (Table 1) and subsequent fast initiation of recoveryof the proton gradient, as seen with amphetamine (Figure 4A). Thus, it appears that amphetamine acts primarily on the pH gradient, and its modulation of transmitter accumulation is due to its weak base action and not to inhibition of the proton pump or the granule monoamine transporter. Alkalinization of Intracellular Sites in VTA Cells Theobservationson VTAdopaminergic neurons stronglysuggestthatthesedrugsactaslipophilicweak bases in cells and are capable of reducing pH gradients in synaptic vesicles. Although the weak base vital dyes A0 and NR stain several populations of intracellular organelles, it is apparent that all detectable acidified sites are affected by exposure to the drugs. Presumably, some of these sites include synaptic vesicles and associated structures, since the subcellular regions where staining is attenuated include areas where vesicles are highly concentrated, i.e., in axonal varicosities and closely underlying the plasma membrane of the cell body. Moreover, we have recently demonstrated that these procedures stain presynaptic sites in retina and varicosities of cultured hippocampal neurons, sites where synaptic vesicles predominate (Augenbraun et al., 1990; Sulzer and Holtzman, 1989). Stained sites in somecells wereaffected byamphetamine more slowly and required higher doses than in other cells; in particular, reduction in staining of glial cell compartments appeared to be slower (see Figure 3). Preliminary results suggest that dopaminergic cells (identified by antigenicity to tyrosine hydroxylase) may be affected by lower levels of amphetamine than otherVTAcells. Furtherworkwill be required toquantifythedifferences in weak base effectsof thesedrugs, including o-and L-amphetamine isomers, on different organelles and between different types of neurons. Kinetic Measurements of Alkalinizdion in Chromaffin Granule Preparations We used isolated chromaffin granules as model synaptic vesicles to compare the weak base actions of psychoactive amines. In the chromaffin granules, I& and T, values of psychoactive amines vary widely (Table 1). Contributing factors may include the amine pK, binding to intragranularconstituents (e.g., Moe et al., 1988; Sulzer et al., 1987, 1990; Zacek et al-., 1990),

Neuron 804

permeability of the protonated form (see Njus et al., 1986), lipophilic interactions with the granule membrane (Mack and Bonisch, 1979), and active uptake mechanisms, including uptake driven by the transmembrane gradient (Ayr; see Johnson et al., 1982). It is striking that the compounds which have two proton-accepting groups had very low lCs0 values; serotonin, which has a much lower I& than dopamine, and chloroquine, which has the lowest I& of all the compounds measured, each have two proton-accepting amines. It is also noteworthy that of the three phenylethylamines we tested (amphetamine, tyramine, and dopamine), the effectiveness of alkalinization increases (i.e., the T, and I& values decrease) in parallel with increasing lipophilicityand pK(seeMack and Bonisch, 1979, for pK and lipophilicity data). In the chromaffin granule preparations, psychoactiveaminesonlytemporarilycollapsed the proton gradient, which reestablished itself over time. By contrast, protonophores like FCCP permanently abolished the proton gradient. The recovery from stimulantinduced alkalinization may be significant, since longterm collapse of ApH should be cytotoxic. However, since the rate of proton pumping appears to be significantly faster than that of monoamine uptake (T, of acidification = 8 min versus T, of serotonin uptake = 30 min in our preparations), there is probably a lag time beforecatecholamines are reaccumulated. In the chromaffin granule preparations, it is likely that the extragranular amphetamine concentration is diminished by binding, uptake, and other mechanisms, so that it becomes less effective at alkalinizing thevesicular interior. In the cultured VTA preparations, on the other hand, amphetamine appears not to be so effectively sequestered, so that recovery is seen only after exchanging the incubation medium to remove amphetamine. Our results demonstrate that weak base effects at the storage vesicle are dose dependent. Presumably, the amount of passive or active entry of the drugs across the plasma membrane is also important. For several reasons, intracellular concentrations of drugs that vesicles are exposed to are, in situ, likely to be far higher than serum levels. Some lipophilic drugs such as cocaine are far more concentrated in cerebral spinal fluid than in serum (Hurd et al., 1988). Moreover, since extracellular pH is about 7.4 while the cytoplasmic pH of neurons is about 7.0 (Roes and Boron, 1981), weak bases should be 2.5-fold concentrated in the cell (see Equation 1). Concentrations may be increased further by specific uptake or binding to cellular constituents. For example, amphetamine is concentrated 45-fold in PC12 cells (Bonisch, 19842, and imipramine and haloperidol are concentrated 500fold in synaptosomes (Schmalzing, 1989), perhaps in part as a result of ionic binding to synaptic vesicle synaptosomal constituents (see Sulzer and Holtzman, 1986; Sulzer et al., 1987; Zaczek et al., 1990) and active uptake at the plasma membrane. In support of the role of ionic binding, we found that 32.5% f 2.5% of

ATP-dependent [3H]serotonin accumulated in chromaffin ghosts was not releasable with FCCP and valinomycin (Figure 5B). For the above reasons, ICsvalues for chromaffin preparations should not be compared with drug serum concentrations, but ratherwith intraneuronal concentrations of the drugs. Cellular Effects Weak base effects in VTA cells are also dose dependent (Figures 2 and 3). To our knowJedge, the level of extracellular amphetamine in the brain after self-administration has not been determined; however, rats will self-administer 43 PM amphetamine locally into the nucleus accumbens 40-70 times per hr (Hoebel et al., 1983). Since we have found clear weak base effects at 1 PM amphetamine, it appears that these effects are important at pharmacological concentrations of amphetamine. For cocaine, it is known that self-administering rats reach 20 PM brain levels (Petit et al., 1989, Sot. Neurosci., abstract). Since 1 uM cocaine inhibited and 100 uM cocaine rapidly abolished staining of acidic sites in our VTAcultures, cocaine in pharmacologically relevant concentrations can overwhelm the buffering capacity of intracellular organelles and induce alkalinization. Our results in dopamine cells and chrqmaffin granules, taken together with reports quantifying effects of extracellular amphetamine (e.g., 100 uM amphetamine induces release of 9% of the cellular pool of dopamine from midbrain/striatal coaggregate cultures[Shalabyetal.,1983])andonthecelIularaccumulation of amphetamine (Bonisch, 1984), indicate that the weak base effects of amphetamine are sufficient to explain its modulation of dopamine uptake and release. In addition to elucidating the primary mechanism of action of amphetamine and related sympathomimetics that have only low affinity for the dopamine transporter, weak baseeffects may explain fundamental aspects of the pharmacological profiles of psychostimulants. For example,this mechanism appears to explain how cocaine and other compounds, understood to operate primarily at the dopamine transporter, reduce accumulation of transmitter in isolated synaptic vesicle preparations lacking the plasma membrane transporter (Carlsson et al., 1963); how nicotine promotes release of dopamine from isolated synaptic vesicles (Kramer et al., 1989); how compounds, including cocaine and imipramine, can apparently elevate transmitter release, not simply reduce transmitter reuptake (Bagchi and Reilly, 1983; Daniels et al., 1980); how amphetamine promotes apparent discharge of neurotransmitter from cytosolic stores in cell bodies or dendrites as well as varicosities, as a result of the interior-acidic pH gradient between the cytosol and surrounding fluid (see above); the mechanism of false transmitters, such as tyramine or 5,6-dihydroxytryptamine; why drugs such as amphetamine cause greater release of dopamine than either serotonin (see above) or acidic amino acid transmitters, for which accumulation depends more

Psychostimulants 805

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Intracellular

pH Gradients

on the transmembrane gradient than on ApH (Maycox et al., 1990); and a potential molecular basis for amphetamine “acceptor” sites (Andersen, 1987). It is important to determine what factors contribute to the diverse behavioral effects of weak base drugs. As mentioned, cocaine, fenfluramine, and imipramine appear to work primarily by blocking plasma membrane neurotransmitter-uptake sites. Cocaine, which in cells causes little neurotransmitter release in comparison to amphetamine, may interfere with its own weak base effects by blocking the plasma membrane monoaminetransporter, impeding extrusion of neurotransmitter. Tyramine may behave differently from amphetamine because it is membrane permeable even in its protonated form (Njus et al., 1986). Phencyclidine may have a particularly complex set of actions, as a result of its affinity for NMDA, acetylcholine, and opiate receptors, as well as its effects on dopamine accumulation (Albuquerque et al., 1983; Johnson, 1983; Sonders et al., 1988). Chloroquine may not detectably affect catecholamine accumulation in vivo because little of the compound crosses the blood-brain barrier (Webster, 1985), although there are recognized psychostimulant side effects of antimalarial doses. We suggest that other psychoactive drugs that are weak bases may regulate neurotransmitter accumulation through vesicle alkalinization under some conditions, including caffeine, chlorpromazine, desipramine, methamphetamine, methylphenidate, I-methyl4phenylpyridinium (MPP+), morphine, nicotine, nomifensine, reserpine, and tetrabenazine. It is also important to recognize that since the weak base mechanism of psychostimulant action is not receptor-mediated, discovery of directly acting amphetamine antagonists is unlikely. The weak base mechanism suggests two important implications: First psychoactive amines should be concentrated in acidic synaptic vesicles and released during exocytosis along with native neurotransmitter. This corelease would result in a spatial and temporal concentration of the drugs at postsynaptic receptors. Second, weak base action should dissociate release of monoamine neurotransmitter from release of neuropeptide cotransmitters. Combined with their effects on transmitter uptake and release, these actions may help to explain their individual pharmacological profiles. Experimental

Procedures

Cell Culture Methods Cultures were prepared from I- to 7-day-old rats. Pups were anesthetized with ketamine (5 mg intraperitoneally), and the VTA was isolated. Tissue was dissociated using a gently stirred oxygenated protease solution (Huettner and Baughman, 1988; Kay and Wong, 1986) and plated onto polyornithine-coated glass coverslips (for experiments using CCD optics) or onto confluent glial monolayers. Serum-free media (di Porzio et al., 1980; Rosenberg and Aizenman, 1989) were modified by addition of superoxide dismutase and catalase (see Rosenberg, 1988). The cultures were maintained at 37OC in 5% CO? and used after 3-12 days in vitro.

Electron Microscoov For electron micrb&pic observation, cultures were incubated in 50 PM Ihydroxy dopamine for 18 hr at 37OC and then fixed in 1.5% glutaraldehyde in 0.1 M phosphate buffer (pH 7.3), with 0.4 mM CaClz for 15 min at room temperature. Postfixation, dehydration, embedding, and staining steps were as reported previously (Sulzer and Holtzman, 1989). lmmunocytochemistry For immunocytochemical demonstration of tyrosine hydroxylase, cultures were fixed in 4% paraformaldehyde in PBS at room temperatureand incubatedforl-3daysat4°Cwith1:200EugeneTech anti-tyrosine hydroxylase antibody (Allendale, NJ) with 0.1% bovine serum albumin and 0.1% Triton X-100 in PBS. Subse quent secondary antibody and amplification steps used the Vectastain Elite Kit (Vector Laboratories). Pharmacological Reagents Reagents were obtained from Sigma unless otherwise noted. L-Amphetamine free base, o-amphetamine sulfate (RBI, Natick, MA), L-amphetamine sulfate (RBI), cocaine hydrochloride, fenfluramine hydrochloride, imipramine hydrochloride, phencyclidine hydrochloride, and tyramine hydrochloride were used. L-Amphetamine free base was used for the data reported in the figures. c-Amphetaminesulfatewasalso used in identical experiments with VTA cultures; results were indistinguishable from those of L-amphetamine free base. Cocaine free base was used in some chromaffin granule experiments, but did not result in a kinetic profile different from that of cocaine hydrochloride. Observation of Intracellular Acidification in Cultured Dopamine Cells For direct microscopic observation, intracellular sites in cultured cells were stained with 0.1% NR or 10 PM A0 in the media. AO-treated cultures were observed with a fluorescein filter set. Cells did not appear to be damaged by NR incubation under these conditions, although we occasionally observed signs of toxicity afterA incubation at higher concentrations (but not at 100 nM; see below). Experiments were performed in triplicate; cultures were scanned after addition of amines, and effects on previously unobserved neurons were similar. In several trials 10 mM quinacrine, another fluorescent weak base vital dye, was used instead of A0 or NR, with similar results. Some cultures were processed after the experiments to detect antigenicity to tyrosine hydroxylase, in order to examine the dopaminergic status of the cells. For instance, the culture used for Figure 3 contained 37.5% * 1.5% dopaminergic neurons. For low-light level imaging, we used a chilled CCD (PhotometTics, Tucson, AZ) on a Zeiss IM35. The excitation beam was attenuated to 10% with a neutral density filter. Cells were incubated for IO-60 min in 100 nM AO. At this concentration nearly all observable A0 fluorescence appeared in the green wavelength under fluorescein optics. There was no bleaching of A0 fluorescence after seven consecutive 5 s exposures taken at 5 min intervals. The cell shown in Figure 3 was subsequently shown to be dopaminergic. Observation of ApH Shifts in Chromaffin Granules Whole chromaffin granule and ghost preparations were isolated following the protocol of Nelson and colleagues (1988). For observation of pH shifts in granules, whole granules (62 pg of protein) were suspended in 20 mM MOPS (p 7.4; buffered with Tris base), 200 mM sucrose, 100 mM KCI, 1 ii M MgCI, at 25’-‘C; this stabilized the pH with all drugs used. ATP-dependent accumulation of A0 into isolated granules could be observed directly by light microscopy. For spectrofluorometric quantification (Varian, Sunnyvale, CA), A0 was added to a final concentration of 670 nM, and fluorescencewas measured after a 6 min preincubation (ex = 496 nm, em = 526 nm). After a 15 min incubation in AO, 1 mM ATP was added to activate the granule proton pump. This activation led to granular acidification observed by fluorescence quenching (Al-Awqati, 1986; Nelson et al., 1988). The measurements reported in Table 1 were performed in the presence

Neuron 806

of 1 uglml valinomycin (Fluka, Ronkonkoma, NY); withoutvalinomycin amphetamine caused similar alkalinization; however, valinomycin increased the total fluorescence signal. All drugs were added a second time after FCCP, and dilutional effects (minor) were subtracted from the measurements. At the concentrations and wavelengths used, there were no aspecific changes in fluorescence caused by psychoactive drugs, FCCP, or ethanol. Quantification of Chromaffin Granule pH The fluorescent weak base quinacrine was used to estimate pH of the granule preparation after addition of ATP according Salama et al. (1980):

ApH

= 0.5 log(QV/l

- Q,

the to

(3)

where Q = the proportion of total fluorescence quenched (ex = 430, em = 500) and V = the total granule and extragranule volumedivided bythegranulevolume(takentobetheexchangeable water volume). For volume measurements, granules were incubated for 12 min at room temperature in medium identical to that used to measure ApH but without AO, ATP, or valinomytin and with the addition of 75 nM [‘4C]sucrose (1 uCi/ml) as a marker of the extragranular medium and 1.3 uCi/ml pH]H*O (Amersham).Aftercentrifugation, the supernatantwas removed, and the pellet was suspended in 200 ul of water, frozen, and thawed to aid in dispersion of the granule contents. Exchangeable granule water volume was estimated by subtracting the pellet [‘4C]sucroseexcludingvolumefrom the pellet [3H]HZOvolume (Pollard et al., 1976) and was determined to be 4.4 f 1.4 ul per mg of protein (n = 4), in close agreement with other reports (Pollard et al., 1976; Johnson, 1988). For A0 measurements, relative fluorescence measurements were used, since A0 has been reported to give spuriously low absolute pH values (Lee and Forte, 1978; Cools and janssen, 1986). Neurotransmitter Accumulation in Chromaffin Ghosts To examine accumulation of serotonin in chromaffin ghosts, 10 ug of chromaffin granule ghosts was incubated in 100 ~rl of 40 mM KCI, 300 mM sucrose, 5 mM MgSO+ 20 mM MOPS (pH 7.7; titrated with NaOH), 1 ug/ml valinomycin, 170 nM [‘Hlserotonin. ATP (5 mM) was added, and the incubation was continued for 30 min at 25OC. For experiments in which serotonin uptake was observed (Figure 5A), drugs were added prior to ATP. In experiments in which release of-serotonin was observed (Figure 5B), drugs were added after the 30 min ATP incubation and incubations continued for 1 min. FCCP (20 PM) was used to abolish the proton gradient; complete inhibition of the gradient was observed with as little as 1.33 )rM FCCP. Incubations were stopped with 900 ul of ice-cold medium without serotonin or valinomycin. Nine hundred microliters of the resulting 1 ml was applied to Whatman GF/B filters and washed with 10 ml of medium. Background was measured in ATP-free medium and subtracted from the data points. Experiments were performed in triplicate. For comparison purposes, proton gradients in ghost preparations were detected in the same media, except that 0.66 uM A0 was substituted for [3H]serotonin. ATPase Activity in Chromaffin Granules A calorimetric assay for inorganic phosphorus adapted from the procedure of Taussky and Shorr (1953) was used to measure granule ATPase activity. Isolated chromaffin granules were preincubated at the same dilution and in the same media as described above with 1 pglml valinomycin for 12 min at room temperature. NaATP (1 mM), with or without 100 uM o-amphetamine sulfate, was then added and the incubation was continued for I2 min at room temperature. The reaction was stopped by addition of 12% trichloroacetic acid. Two hundred microliters of 1% ammonium molybdate with 5% ferrous sulfate was added to 800 ul of the granule-trichloroacetic acid preparation, and the signal at 660 nm was measured after 15 min. The concentration of inorganic phosphorus was read from a calibration curve (R > 0.99) made from known concentrations of potassium phosphate in the media used above, without ATP or chromaffin granules.

Kinetic Measurements For all the amines tested, a minimum of five different concentrations were tested in triplicate; log(concentration) versus percent mean alkalinization was plotted, and a first-order regression line was fitted. The ICw, was measured at the concentration halfway between maximum acidification after addition of valinomycinl ATP and that after FCCP incubation. For all drugs, R 2 0.98. The T, was the time required for the pH gradient to fall to l/e at the I&. Observations were in duplicate; In(time) versus percent mean alkalinization was plotted, a first-order regression line was fitted, and the slope was calculated. For each drug tested, R > 0.96. Acknowledgments Thanks to Leslie Vosshall for invaluable advice and assistance, to Drs. Qais Al-Awqati, Eliene Augenbraun, Shulamit Cidon, Michael Gershon, Eric Holtzman, and Edward Nunes for critical discussion, to Dr. Nicholas Willson for the use of the fluorescence spectrophotometer, and to Mr. Jevons Liu for assistance with electron microscopy. This work was supported by an NARSADYoung Investigator Award (D. S.), Biomedical Research Sup port grant RR05650 (D.‘S.), the Dana Foundation (S. R.) and NIMH grant MHO0705 (S. R.). The costs of publicaton of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 USC Section 1734 solely to indicate this fact. Received

June

1.5, 1990; revised

August

17,199O.

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