Naunyn-Schmiedeberg's

Archivesof Pharmacology

Naunyn-Schmiedeberg's Arch Pharmacol (1984) 327:267-272

9 Springer-Verlag 1984

The transport of (+)-amphetamine by the neuronal noradrenaline carrier* H. B6nisch

Institut ft~rPharmakologie und Toxikologieder Universit&itWtirzburg, Versbacher Strasse 9, D-8700 Wi~rzburg, Federal Republic of Germany Summary. PC-12 cells (a clonal line of rat phaeochromocytoma cells) take up noradrenaline by a transport system which is identical with the neuronal amine transport system ("uptakes"). The uptake of 3H-noradrenaline into reserpine-pretreated PC-12 cells (monoamine oxidase inhibited) was saturable (Km = 0.6 • 0.1 ~tmol/1), dependent on sodium and chloride, and competitively inhibited by ( + )-amphetamine (Ki = 0.18 +_0.04 gmol/1), cocaine (Ki = 0.55 _+0.15 ~tmol/1) and desipramine (Ki = 4.3 + 0.6 nmol/1). The uptake and accumulation of 3H (+)-amphetamine showed characteristics comparable to those of 3H-noradrenaline, since the uptake of 3H(+)-amphetamine (0.1 gmol/1) was reduced by omission of sodium or chloride from the incubation medium. The sodium-sensitive component of uptake and accumulation of 3H(+)-amphetamine was fully inhibited by cocaine and desipramine. The ICs0 of desipramine for inhibition of the sodiumsensitive component of the l-rain uptake of 3H (+)-amphetamine (20 nmol/1) was about 2 nmol/1, i.e., identical with the Ki for inhibition of uptake of 3H-noradrenaline. At concentrations above 1 Nnol/1, desipramine additionally caused an inhibition of the sodium-independent permeation of 3H (+)-amphetamine into PC-12 cells. Hence, by using a homogeneous population of cells endowed with "uptake1", it is possible to demonstrate - besides a pronounced lipophilic entry - a carriermediated uptake of 3H ( + )-amphetamine. Key words: (+)-amphetamine - Neuronal uptake - Sympathomimetic amines

ample evidence that phenolic amines (such as tyramine) are transported by "uptake1" (Ross 1976), it has been impossible to demonstrate a cocaine:sensitive and sodiumdependent uptake by sympathetic neurones of isolated organs of non-phenolic amines like (+)-amphetamine (Ross and Renyi 1966; Thoenen et al. 1968; B6nisch and Rodrigues-Pereira 1983). After Paton (1973) had demonstrated a cocaine-sensitive efflux of noradrenaline from adrenergic nerve terminals, it has been proposed (Paton 1974) that non-phenolic amines enter adrenergic nerve terminals only by passive diffusion and cause a pronounced carrier-mediated efflux of noradrenaline, which is inhibited by cocaine. Thus, the question remains which of the two explanations for the inhibitory effect of cocaine is correct: Evidence for a carrier-mediated neuronal uptake of (+)-amphetamine would give a clear answer to this question. Neuronal uptake of (+)-amphetamine has been studied in isolated organs in which adrenergic nerve terminals represent less than one percent of the tissue. Due to its relatively high lipophilicity (Mack and Btnisch 1979) (+)-amphetamine enters all cells of an isolated organ. The high degree of nonspecific distribution of (+)amphetamine into all cells might have masked a carriermediated uptake of (+)-amphetamine into the small volume of adrenergic nerve terminals within an isolated organ. However, with a system consisting exclusively of adrenergic neurones, it should be possible to determine whether (+)-amphetamine is subject to carrier-mediated uptake. PC-12 ceils (a clonaI cell line of rat phaeochromocytoma cells) possess a noradrenaline transport system which is identical with "uptake1" (Greene and Rein 1977).

Introduction

The non-phenolic amine amphetamine, like the phenolic amine tyramine, exerts its sympathomimetic effect by liberating noradrenaline from sympathetic nerve terminals (for review see Trendelenburg 1972). Since cocaine inhibits the release ofnoradrenaline (by sympathomimetic amines), it has been proposed (Trendelenburg 1972) that this release requires the uptake of the releasing amine by the cocaine-sensitive and sodium-dependent neuronal amine transport system ("uptake1", Iversen 1967). While there is * Some of the results were communicated to the German Pharmacological Society (B6nisch 1981). This study was supported by the Deutsche Forschungsgemeinschaft(Bo 521) Send offprint requests to H. B6nisch at the above address

Methods Cell culture. PC-12 cells were grown on plastic tissue culture dishes in Dulbecco's modified Eagle's medium (Gibco catalog No. H 16; without antibiotics) completed with 10% heat inactivated horse serum and 5% fetal calf serum (both from Gibco); for further details see Greene and Rein (1977). To cause depletion of endogenous catecholamines and inhibition of vesicular amine uptake, PC-12 cells were cultured 18-24 h prior to experiment in the presence of 10 ~tmol/1 reserpine (dissolved in dimethylsulfoxide, final concentration 0.1%). This exposure of PC-12 cells to reserpine causes a depletion of endogenous catecholamines (noradrenaline and dopamine) by more than 90% (Greene and Rein 1977).

268

Uptake of ~H-noradrenaline and 3H( + )-amphetamine. Cell cultures were aspirated free of culture medium and washed once with prewarmed (37~ physiological salt solution (KRH, i.e., Hepes-buffered Krebs-Ringer saline) of the following composition (in retool/l): NaC1 125, KC14.8, Hepes 25, MgSO4 1.2, KH2PO4 1.2, glucose 5.6, ascorbic acid 1.0; the solution was finally adjusted with Tris to pH 7.4. In some experiments this solution was modified in that NaC1 was replaced isosmotically by Tris. HC1 or sodium isethionate. All solutions contained 0.1 mmol/1 pargyline (as hydrochloride) to inhibit monoamine oxidase. The cultures were preincubated for 15 min at 37 ~ C in 2 ml of KRH (or modified KRH). Thereafter this preincubation solution was replaced by the incubation solution which contained a tritiated amine (either (-)-noradrenaline or (+)-amphetamine) and 14C-sorbitol (as extracellular space marker). When the effect of an inhibitor of amine uptake was studied, the inhibitor was already present in the preincubation solution. After incubation for a given time, the incubation solution was aspirated, and the tissue culture dishes (with adhering cells) were washed twice with 4 ml ice-cold preincubation solution. Finally, the washed cells were dissolved in 1.5 ml of 0.1% (v/v) Triton X-100 (dissolved in 5 retool/1 Tris. HC1, pH7.4). After standing for about 1 h at ambient temperature, the radioactivity of the cells (dissolved in the detergent) was determined by scintillation counting (as described earlier, B6nisch and Trendelenburg 1974). The accumulation of tritium was corrected for sorbitol space. The protein content of this solution was determined by the method of Lowry et al. (1951). All data are related to milligram total cell protein, where 1 mg corresponds to about 107 cells and 2.14~1 intracellular water space (Harder and B6nisch 1984). In one series of experiments, the deamination of 3Hnoradrenaline was determined in the presence or absence of sodium and in the presence or absence of desipramine. In these experiments, cells were first pre-incubated for 15 min in a pargyline-free KRH-solution; this solution was identical with the solution used for the subsequent incubation, with the exception of 3H-noradrenaline which was absent in the preincubation solution. After aspiration of the preincubation solution, the cells were incubated for 60 min with 3H-noradrenaline. Thereafter, the incubation fluid above the cell monolayer was removed and acidified with HC104 (final concentration 0.4 tool/l). The cells were washed twice with 2 ml of ice-cold preincubation solution and then exposed to 2 ml HC104 (0.4 tool/l). An aliquot of the perchloric acid cell extract and of the acidified incubation fluid were subjected to column chromatography involving alumina and Dowex 50 WX4 (Graefe et al. 1973, 1977) to separate noradrenaline from its metabolites. For determination of the protein content, the denaturated cells (which strongly adhered to the plastic tissue culture dishes) were dissolved in 0.5 ml NaOH (1 tool/l) and 1.5 ml 0.1% (v/v) Triton X-100. Calculations. Arithmetic means (_+ S.E.) are presented throughout. Student's t-test was used to calculate significances, and P < 0.05 was regarded as significant. Substances used. Chemicals (+)-amphetamine sulfate, pargyline hydrochloride and reserpine (Sigma, Mtinchen, FRG), (-)-noradrenaline hydrochloride (Hoechst, Frank-

furt, FRG), cocaine hydrochloride (Merck, Darmstadt, FRG), desipramine hydrochloride (Ciba-Geigy, Basel, Switzerland); all other chemicals were either from Merck (Darmstadt, FRG) or Sigma (Mt~nchen, FRG). Radiochemicals (all from NEN, Dreieich, FRG): 14C-D-Sorbitol (11.84 GBq/mmol), 3H (+)-amphetamine sulfate (791.8 GBq/mmol) and 3H (-)-noradrenaline (118.4 GBq/mmol, Lot No. 1271-115, nominally labelled in position 7). Results

Characteristics of the uptake of 3H-noradrenaline in PC-12 cells Since during long-term subculture of a cell line some characteristics of the cultured cells may change, the uptake of 3H-noradrenaline in PC-12 cells was reexamined. Reserpine-pretreated PC-12 cells were incubated for 1-10 min with 3H-noradrenaline (0.1 or 8 ~tmol/1), in the absence or presence of sodium. Figure 1 shows that at a low concentration of 3H-noradrenaline (0.1 ~tmol/1) the uptake and accumulation was nearly totally dependent on sodium, whereas at the higher concentration of 3H-noradrenaline (8 ~tmol/1) a sodium-independent component was clearly demonstrable. At both concentrations of noradrenaline, the sodium-dependent accumulation increased linearly with time for at least 5 rain (Fig. 1).

a)

b)

20 -

40.

/ 15

/

/ I

T

30"

.4 9

,o

20-

cu

f

f

...... 0

,-7:--7-'-?-,,9 5

rain

o

5

I0 min

Fig. 1. Time course of uptake and accumulation of a 0.1 9ol/1 and b 8 9mol/1 3H-noradrenaline in PC-12 cells in the presence or absence of sodium. Ordinates: accumulation of 3H-noradrenaline in pmol/mg protein. Abscissae: duration of incubation in min. Reserpine-pretreated PC-12 cells (grown in tissue culture dishes) were incubated at 37~ with 0.1 or 8 ~mol/1 3H-noradrenaline and 14C-sorbitol (as extracellular space marker). When sodium was omitted from the incubation fluid (Krebs-Ringer-Hepes, pH 7.4), it was replaced by Tris. To inhibit monoamine oxidase, pargyline hydrochloride (0.1 mmol/1) was present in the incubation fluid. Shown are means (_+S.E.) of 3-4 tissue culture dishes each. Symbols: v - - v total accumulation, o - - o accumulatioh in the absence of sodium, 9 9 sodium-dependent accumulation

269

b)

a)

I

0.3.

c)

I/v

,,v0.6H1

0.8-

0.2. 0.6o.,

0.~O.i-

0.2p

~_

t~

-

~

t

1

~

J

i

0

0.6

10

1.2

omphetomine (jumol/[)

cocoine (3Jmol/[)

20

desiprnmine (nmol/[)

Fig. 2. Dixon analysis of the inhibitory effect of a (+)-amphetamine, b cocaine and e desipramine on the sodium-dependent uptake of ~H-noradrenaline in PC-12 cells. Ordinates: l/v; v= initial rates (in pmol per mg protein per 4 rain) of sodium-dependent uptake of ~Hnoradrenaline (0.3 ~mol/1 open symbols, or 3 ~tmol/1 closed symbols) in the presence of the inhibitors. Abscissae: concentration of the inhibitor in the incubation medium. For experimental details, see legend to Fig. 1 and Methods. Shown are means (___S.E.) of duplicate determinations from 4 experiments each. According to Dixon (1953) the intersect of the two straight lines left from the ordinate and above the abscissa indicates a competitive type of inhibition 15"

aline (Fig. 2) with Ki values of 0.18• ~mol/1 (n=4), 0.55 • 0.15 ~Lmo1/1 (n = 3) and 4.3 _+ 0.6 nmol/1 (n = 3) for amphetamine, cocaine and desipramine, respectively. From the Dixon-plots of Fig. 2, a mean Km for the uptake of 3Hnoradrenaline of 0.98 --- 0.33 ~tmol/1 (n = 10) was calculated. ~[his value agrees fairly well with that obtained by saturation kinetics.

i g

;/

E

=s-

The uptake and accumulation of 3H ( + )-amphetamine

/ 0

,

,

,

,

,

5 rain

.

.

.

.

,

10

Fig. 3. Time course of uptake and accumulation of 0.1 gmol/1 3H (+)-amphetamine in PC-12 cells in the absence and presence of sodium. Ordinate." accumulation of 3H(+)-amphetamine in pmol/mg protein. Abscissa: duration of incubation in rain. The experimental procedure was identical with that described in the legend to Fig. 1 for 3H-noradrenaline with the exception that pargyline was not present in the incubation fluid. Shown are means (+_S.E.) of 3-4 tissue culture dishes each. Symbols: v - - v total accumulation, o -- o accumulation in the absence of sodium (replaced by Tlis), 9 -- 9 sodium-dependent accumulation

When initial (4 min) rates of uptake were determined for several concentrations of 3H-noradrenaline (0.1-8 ~tmol/1), the sodium-dependent component obeyed saturation kinetics. Half-saturation (Kin) of the transport was observed at a concentration of 0.6+_0.1 ~tmol/1 ~H-noradrenaline (n = 3). The sodium-independent component of uptake, on the other hand, showed no saturation; it increased linearly with the concentration of noradrenaline. In a further series of experiments the apparent affinity of amphetamine, cocaine and desipramine for the noradrenaline transport system of PC-12 cells was determined. All three compounds caused a competitive inhibition of the sodium-dependent component of uptake of 3H-noradren-

The uptake and accumulation of 3H(+)-amphetamine in reserpine-pretreated PC-12 cells was studied under several experimental conditions and compared with that of 3Hnoradrenaline. In contrast to the accumulation of 3H-noradrenaline shown in Fig. 1, the accumulation of 3 H ( + ) amphetamine rapidly approached steady-state, independently of whether sodium was present or not (Fig. 3). The sodium-independent component of uptake of 0.1 ~mol/1 3H-amphetamine (Figs. 3 and 4) was relatively high when compared with that of 0.1 ~mol/1 3H-noradrenaline (Figs. 1 and 4). Nevertheless, after 1 min the sodium-sensitive component of amphetamine uptake (Fig. 3) was identical with that observed after 1 rain fbr noradrenaline uptake (Fig. 1). Omission of chloride from the incubation medium caused an inhibition of the 5-rain uptake of 3 H ( + ) - a m phetamine as well as of ~H-noradrenaline (Fig. 4). The degree of inhibition (by omission of chloride) of the uptake of amphetamine was comparable to that observed after omission of sodium (Fig. 4b). Since 100~tmol/1 pargyline as hydrochloride was present in the experiments with 3H-noradrenaline (to inhibit monoamine oxidase), the degree of inhibition of uptake of 3H-noradrenaline by omission of chloride was smaller than that by omission of sodium. Finally, cocaine (30 ~tmol/1) caused about the same degree of inhibition of the 5-rain uptake of 3 H ( § phetamine and ~H-noradrenaline as did omission of sodium (Fig. 4). When the inhibitory effect of desipramine was studied at several concentrations of desipramine, a

270 biphasic inhibition of the 1-min uptake of 0.02 ~tmol/1 3H(+)-amphetamine was observed (Fig. 5). A concentration of about 2 nmol/1 desipramine caused half-maximal inhibition of the sodium-dependent component of uptake. At concentrations of desipramine higher than 1 ~tmol/1, there was an inhibition of the sodium-independent uptake b)

a)

T

r (x)

BO

l

",'."~:. )

:)>> o

..'.'

6o

./,/ 8 ' '"6 t,.O.

',' " - 'i

of ~H (+)-amphetamine with an IC50 of at least 10 gmol/1 (Fig. 5). To test whether this effect of desipramine was due to a non-specific "membrane-stabilizing" effect of high concentrations of desipramine, the influence of 10 ~tmol/1 desipramine on the sodium-independent deamination of ~H-noradrenaline (8 ~tmol/1) was tested. In the presence of sodium (i.e., when 3H-noradrenaline entered the cells mainly by carrier-mediated transport) 54.4+ 3.8% (n=4) of the amine was deaminated (mainly to dihydroxyphenylglycol) within 60 min (at 37 ~ In the absence of sodium, i.e., in the absence of carrier-mediated transport, only 5.06_+ 0.32% (n =4) of the offered 3H-noradrenaline was deaminated; this percentage decreased to 0.53-t-0.10% (n=4) in the presence of 10gmol/1 desipramine. The intraceHular accumulation of unchanged 3H-noradrenaline was not simultaneously increased when the deamination was inhibited by desipramine.

i>....
t._

~- 20

(X)

(X)

I,'. . . '.' " 9

(X)

>>..i %..-

O.

confn COC -No + - [ [ -

contr. CO[ -No* - C t (~) (17) (6) (1tO 16)

(n)

(25)

(3)

(25)

(11)

Fig. 4. Inhibition by cocaine and by omission of sodium or chloride of the 5-min uptake of a 0.1 ~tmol/1 3H-noradrenaline and b 0.1 ~tmol/1 3H(+)-amphetamine in PC-12 cells. Height of columns: 5-min uptake of 3H-amine in percent of control. The 5rain uptake of ~H-noradrenaline or 3H(+)-amphetamine was determined as described in the legends to Figs. 1 and 3. Abbrevations: contr.=controls; COC=in the presence of 30 ~tmol/1 cocaine; -Na+=in the absence of sodium (sodium replaced by Tris); -Cl-=in the absence of chloride (chloride replaced by isethionate). Shown are means (• S.E.) of n tissue culture dishes. (*) In the experiments with 3H-noradrenaline the incubation medium contained a residual concentration of 0.1 mmol/l chloride (due to the presence of pargyline hydrochloride in the incubation medium); (X) significantly different from controls (p < 0.001).

100. ~~...~.._ {i_..•_,•_ ...........

, ONo+

/

\, 50. *6

o~ r -~..r

0

,b -9 ,b-8

.......

\,

Ib -7 ,b -6 Jb-~

,'0-4

desiprornine (mot/t)

Fig. 5. Inhibition by desipramine of the 1-min uptake of 0.02 ~tmol/1 3H(+)-amphetamine in PC-12 cells. Ordinate." 1-min

uptake of ~H(+)-amphetamine in percent of controls; (controls = in the absence of desipramine). Abscissa: concentration of desipramine. Reserpine-pretreated PC-12 cells were incubated for 1 min with 3H(+)-amphetamine (0.02 ~tmol/1), 14C-sorbitol and different concentrations of desipramine (0.001-100 ~tmol/1). Controls were determined in the presence and absence of sodium. 0 Na += sodium replaced by Tris (open symbol and broken line). The l-rain uptake of ~H(+)-amphetamine in the presence and absence of sodium was 0.69 + 0.03 pmol/mg protein (n = 12) and 0.54_+0.01 pmol/mg protein (n = 9), respectively. Shown are mean values (• S.E.) of at least 4 tissue cfilture dishes

Discussion

The view that (+)-amphetamine is taken up in adrenergic neurones originates from the observation that cocaine and other inhibitors of "uptake1" antagonize the pharmacological action (i.e. the release of noradrenaline) of this indirectly acting amine (Fleckenstein and St6ckle 1955; Trendelenburg 1972). After Paton (1973) had shown that cocaine inhibits carrier-mediated outward transport of noradrenaline, he proposed, that the indirectly acting nonphenolic amines might enter adrenergic neurones by lipophilic diffusion (and not by "uptake1") in order to somehow induce carrier-mediated outward transport of noradrenaline (that is cocaine-sensitive). However, this proposal does not explain how (+)-amphetamine (or other non-phenolic amines) initiates a carrier-mediated efflux ofnoradrenaline. Noradrenaline efflux by "facilitated exchange diffusion", however, might be the simpliest explanation for the mode of action of indirectly acting sympathomimetic amines, if these amines are transported by "uptake1". However, hitherto all attempts to demonstrate that (+)-amphetamine and other nonphenolic phenylethylamine derivatives are actively transported into sympathetic neurones have failed. For (+)-amphetamine only a non-saturable and cocaine-resistant accumulation was demonstrated in slices of the mouse heart (Ross and Renyi 1966) or in the isolated perfused rat heart (Thoenen et al. 1968). Even with a new technique for the measurement of initial rates of amine removal from the extracellular space of an isolated perfused organ, it was not possible to detect a cocaine-sensitive uptake of (+)-amphetamine in the isolated perfused rabbit heart (Btnisch and Rodrigues-Pereira 1983). Similarly negative results were obtained for the neuronal uptake of other nonphenolic phenylethylamines such as ephedrine (Jacquot etal. 1969; Patil etal. 1974; Golko and Paton 1976). However, as discussed by Ross (1976) and Trendelenburg (1978), the failure to demonstrate carrier-mediated uptake of non-phenolic phenylethylamines is not conclusive evidence against carrier-mediated uptake, since the uptake of (+)-amphetamine (and other nonphenolic phenylethylamines) has been measured in tissues in which adrenergic neurones represent less than 1% of the total tissue~ The large non-specific distribution volume might have masked a neuronal uptake of these amines. Because of its relatively high lipophilicity (Mack and Btnisch

271 1979), (+)-amphetamine not only easily penetrates into all kinds of cells but it also rapidly diffuses out of them, with the result of a relatively low net accumulation (also within adrenergic nerve endings) as demonstrated by a rapid approach to steady-state accumulation within PC-12 ceils (see Fig. 3). In order to overcome these experimental obstacles, a homogeneous population of cells (PC-12 cells) was used in the present study. Like cultured chromaffine cells (Kenigsberg and Trifaro 1980; Cena etal. 1984), PC-12 cells possess a noradrenaline transport system which has the characteristics of "uptake1". In the present study it was shown, that the transmembranal transport ofnoradrenaline into PC-12 cells consists of two components: a very small sodium-independent, non-saturable component and a sodium-dependent, saturable component. The Km for the sodium-dependent uptake of noradrenaline was comparable with the Km value reported by Greene and Rein (1977) for PC-12 cells and it was within the range of K m values reported for "uptake1", (Iversen 1967; Graefe et al. 1978). In addition, like "uptake1", the uptake of noradrenaline into PC-12 cells was competitively inhibited by (+)-amphetamine, cocaine and with very high affinity by desipramine. Furthermore, the uptake of noradrenaline into PC-12 cells was inhibited by the omission of chloride from the incubation medium. For "uptake1" an absolute dependency on chloride was shown by Sanchez-Armass and Orrego (1977). The uptake and accumulation of (+)-amphetamine into PC-12 cells shows properties similar to those of the uptake of noradrenaline, since it is inhibited by omission of sodium or chloride from the incubation medium and by the uptake-inhibitors cocaine and desipramine. The ICs0 of desipramine for inhibition of the sodium-dependent component of the 1-min uptake of (+)-amphetamine is comparable with the Ki of desipramine for inhibition of the uptake of noradrenaline. Taken together, these data prove that (+)-amphetamine is transported into PC-12 cells by the noradrenaline transport system of these cells, which obviously corresponds to "uptakes". Support for this conclusion comes from the observation (B6nisch, unpublished observations) that (+)-amphetamine (like tyramine) causes a carrier-mediated effiux of extravesicularly accumulated noradrenaline from PC-12 cells, whereas non-transported inhibitors of uptake (cocaine, desipramine) have no facilitatory, or even a slight inhibitory, effect on noradrenaline efflux. Thus, the sodium-dependent uptake and accumulation does not represent simply the binding of this amine to the transport system without subsequent transport. At 0.1 ~tmol/1 (+)-amphetamine, i.e., at about half of the Ki of (+)-amphetamine, the sodium-independent l-rain uptake of 3H(+)-amphetamine was 4 times higher than the sodium-dependent (carrier-mediated) l-rain uptake (see Fig. 3). This discrepancy means that it was impossible to determine a full saturation curve for the sodium-dependent uptake of (+)-amphetamine. Hence, the kinetic constants Km and Vmaxcannot be determined. If there is deamination of noradrenaline in the absence of sodium, the amine must have entered the cells exclusively by passive diffusion. Any inhibitory effect of desipramine must then be interpreted as a "membranestabilizing" effect that hinders the passive diffusion of noradrenaline. An inhibition of monoamine oxidase by high

concentrations of desipramine can be ruled out, because the 3H-noradrenaline content of the PC-12 cells was n o t increased in the presence of desipramine. Inhibition ~of monoamine oxidase - with no impairment of passive inward diffusion - should have increased the intracellular 3H-noradrenaline concentration. Tricyclic antidepressants are highly lipid soluble d~ugs. They have a pronounced tendency to accumulate in membranes and are known to cause non-specific membrane effects such as uncoupling of oxidative phosphorylation in isolated heart mitochondria (Bachmann and Zbinden 1979) or "stabilization" of the erythrocyte membrane in hypo-osmotic solution (Despopoulos 1970). The latter effect was observed at 10 ~tmol/1 desipramine which, in the present study, caused pronounced inhibition of the sodium-independent accumulation of 3H(+)-amphetamine and of the sodium-independent deamination of 3H-noradrenaline. It should be mentioned that for central adrenergic neurones a carrier-mediated uptake of (+)-amphetamine has been proposed. Azzaro et al. (1974) observed a temperature-, cocaine- and desipramine-sensitive accumulation of (+)-amphetamine in a synaptosomal preparation of rat cerebral cortex. However, since high concentrations of desipramine (100 ~mol/1) had to be used to inhibit this accumulation, it remains questionable whether this observation can be regarded as an evidence for carrier-mediated neuronal uptake of (+)-amphetamine. In a recent study, Rutledge and Vollmer (1979) provided further evidence for a carrier-mediated uptake of (+)-amphetamine in rat cortex synaptosomes. The uptake of tritiated (+)-amphetamine (determined at 25~ for 5 min) was sodiumdependent and saturable, although with a very high Km of 58 ~tmol/1. In addition, from the Lineweaver-Burk plots of inhibition of (+)-amphetamine uptake by desipramine, a very high K i ofdesipramine of 13 ~tmol/1 can be calculated. Thus, these results do not provide conclusive evidence for a transport of (+)-amphetamine by the noradrenaline transport system. The difficulty of demonstrating a carriermediated neuronal uptake of (+)-amphetamine into synaptosomes again might be due to the fact that synaptosomes are by no means a homogeneous population of "pinched off' adrenergic nerve terminals (Morgan 1976). However, with a homogeneous population of cells which possess the noradrenaline transport system of adrenergic neurones (like PC-12 cells) it has been possible to demonstrate a carrier-mediated uptake of the highly lipophilic (+)-amphetamine by the neuronal noradrenaline transport system. Acknowledgements. The author is grateful to Marianne Babl for

her skilful technical assistance and to Professor Dr. U. Trendelenburg for helpful discussions. References

Azzaro AJ, Ziance RJ, Rutledge CO (1974) The importance of neuronal uptake of amines for amphetamine-induced release of 3H-norepinephrine from isolated brain tissue. J Pharmacol Exp Ther 189:110-118 Bachmann E, Zbinden G (1979) Effect of antidepressant and neuroleptic drugs on respiratory function of rat heart mitochondria. Biochem Pharmaco128:3519-3524 B/Snisch H (1981) Evidence for a cocaine-sensitive and sodiumdependent uptake of amphetamine. Naunyn-Schmideberg's Arch Pharmacol (Suppl) 316:R54

272 B6nisch H, Rodrigues-Pereira E (1983) Uptake of 14C-tyramine and release of extravesicular 3H-noradrenaline in isolated perfused rabbit hearts. Naunyn-Schmideberg's Arch Pharmacol 323: 233-244 Brnisch H, Trendelenburg U (1974) Extraneuronal removal, accumulation and O-methylation of isoprenaline in the perfused heart. Naunyn-Schmideberg's Arch Pharmacol 283: 191-218 Cena V, Montiel C, Sanchez-Garcia P, Garcia AG (1984) Uptake of (3H)-nicotine and (3H)-noradrenaline by cultured chromaffin cells. Br J Pharmacol 81:119-125 Despopoulos A (1970) Antihemolytic actions of tricyclic tranquilizers; structural correlations. Biochem Pharmacol 19: 2907-2919 Dixon M (1953) qlae determination of enzyme inhibitor constants. Biochem J 55:170-171 Fleckenstein A, Strckle D (1955) Zum Mechanismus der Wirkungsverstgrkung trod Wirkungsabschw~tchung sympathomimetischer Amine durch Cocain und andere Pharmaka. II. Die Hemmung der Neuro-Sympathomimetika durch Cocain. Naunyn-Schmiedeberg's Arch Pharmaco1224:401-415 Golko DS, Paton DM (1976) Characteristics of accumulation of ephedrine in rabbit atria. Can J Physiol Pharmacol 54:93-100 Graefe K-H, Stefano FJE, Langer SZ (1973) Preferential metabolism of 3H-(-)-norepinephrine through the deaminated glycol in the rat vas deferens. Biochem Pharmacol 22:1147-1160 Graefe K-H, Stefano FJE, Langer SZ (1977) Stereoselectivity in the metabolism of 3H-noradrenaline during uptake into and efflux from the isolated rat vas deferens. Naunyn-Schmiedeberg's Arch Pharmaco1299:225-238 Graefe K-H, B0nisch H, Keller B (1978) Saturation kinetics of the adrenergic neurone uptake system in the perfused rabbit heart. A new method for the determination of initial rates of amine uptake. Naunyn-Schmiedeberg's Arch Pharmacol 302: 263-273 Greene LA, Rein G (1977) Release, storage and uptake of catecholamines by a clonal cell line of nerve growth factor (NGF) responsive pheochromocytoma cells. Brain Res 129:247-263 Harder R, Brnisch H (1984) Large scale preparation of plasma membrane vesicles from PC-12 pheochromocytoma cells and their use in noradrenaline transport studies. Biochim Biophys Acta, in press Iversen LL (1967) The uptake and storage of noradrenaline in sympathetic nerves. Cambridge University Press, Cambridge Jacquot C, Bralet J, Cohen Y, Valette G (1969) Fixation de la dlepinephrine-14C par le cour isole perfuse de rat. Biochem Pharmacol 18:903-914

Kenigsberg RL, Trifaro JM (1980) Presence of a high affinity uptake system for catecholamines in cultured bovine chromaffine cells. Neuroscience 5: 1547-1556 Lowry OH, Rosenbrough NJ, Farr AL, Randall RJ (1951) Protein measurement with the Folin phenol reagent. J Biol Chem 193: 265-275 Mack F, B/3nisch H (1979) Dissociation constants and lipophilicity ofcatecholamines and related compounds. Naunyn-Schmiedeberg's Arch Pharmacol 310:1-9 Morgan IG (1976) Synaptosomes and cell separation. Neuroscience 1:159-165 Patil PN, Shimada K, Feller DR, Malspeis L (1974) Accumulation of (-)-14C-ephedrine by the pigmented and the nonpigmented iris. J Pharmacol Exp Ther 188:342-352 Paton DM (1973) Mechanism of efflux of noradrenaline from adrenergic nerves in rabbit atria. Br J Pharmaco149:614-627 Paton DM (1974) Mechanism of inhibition by cocaine of action of indirectly acting sympathomimetic amines. Am Heart J 88: 128-129 Ross SB (1976) Structural requirements for uptake into catecholamine neurons. In: Paton DM (ed) The mechanism of neuronal and extraneuronal transport of catecholamines. Raven Press, New York, pp 67-93 Ross SB, Renyi AL (1966) Uptake of tritiated tyramine and ( + ) amphetamine by mouse heart slices. J Pharm Pharmacol 18: 756-757 Rutledge CO, Vollmer S (1979) Evidence for carrier-mediated efflux of norepinephrine displayed by amphetamine. In: Usdin E, Kopin IJ, Barchas J (eds) Catecholamines: Basic and clinical frontiers, vol I. Pergamon Press, New York, Oxford, pp 304-306 Sanchez-Armass S, Orrego F (1977) A major role of chloride in 3H-noradrenaline transport by rat heart adrenergic nerves. Life Sci 20:1829-1838 Thoenen H, Htirlimann A, Haefely W (1968) Cation dependence of the noradrenaline releasing action of tyramine. Eur J Pharmacol 6:29-37 Trendelenburg U (1972) Classification of sympathetic amines. In: Blaschko H, Muscholl E (eds) Handbook of experimental pharmacology, vo132. Springer Berlin, Heidelberg, New York, pp 336-362 Trendelenburg U (1978) Release induced by phenethylamines. In: Paton DM (ed) The release of catecholamines from adrenergic neurons. Pergamon Press, New York, Oxford, pp 333-354 Received May 22, 1984/Accepted July 2, 1984