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ATP INDUCES RELEASE OF NEWLY SYNTHESIZED DOPAMINE IN THE RAT STRIATUM
Pergamon
0197-0186(95)00105–0
Neurochem.Int. Vol.28,No. 4, pp. 395400, 1996 CopyrightG 1996Els&ierScienceLtd Printedin Great Britain.All rightsreserved 01974186/96$15.00+0.00
ATP INDUCES RELEASE OF NEWLY SYNTHESIZED DOPAMINE IN THE RAT STRIATUM YU-XIANG ZHANG, HIROSHI YAMASHITA, TOMOHIRO OHSHITA, NOBUKATSU SAWAMOTO and SHIGENOBU NAKAMURA* Third Department of Internal Medicine,Hiroshima UniversitySchoolof Medicine,Hiroshima, Japan (Received 31 March 1995; accepted 1 August 1995) Abstract-The effect of ATP on release of dopamine (DA) and its metabolism in the rat striatum was investigated by use of in uiuo microdialysis. DA release was increased and the level of 3,4-dihydroxyphenylacetic acid (DOPAC) was decreased by treatment with 1 mM ATP or ADP for 20 rein, while such treatment failed to change the extracellular level of homovanillic acid (HVA). The ATP-induced increase in DA and decrease in DOPAC were inhibited by suramin, a nonselective Pz purinoceptor antagonist, and by reactive blue 2, a P2~purinoceptor antagonist, but not by xanthine amine congener, an adenosine receptor antagonist, indicating that PZ~purinoceptors were involved in the present observation. The effects of ATP were extracellular Ca2+-dependent and sensitive to cwconotoxin and tetrodotoxin, which indicates that the opening of voltage-sensitive calcium channels and sodium channels to depolarize the DA neuron is required for both ATP-induced DA release and DOPAC decrease in the rat striatum. Pretreatment with ce-methyl-p-tyrosine, but not with reserpine, suppressed the ATP-induced release of DA. These findings suggest that the newly synthesized pool, but not the vesicular pool, of DA is involved in the ATP-induced release of DA in the rat striatum. Copyright ~ 1996 Elsevier Science Ltd.
ATP can serve as a neurotransmitter. Upon binding to its specific receptors, ATP evokes a secretory function in various cells (Boeynems and Pearson, 1990; Dahlquist and Diamant, 1974; Bertrand et al., 1987). ATP is present in the synaptic vesicles, which contain either acetylcholine or noradrenaline, and is released concomitantly with these chemicals upon stimulation (White, 1988). Receptors for ATP are designated P, purinoceptors. Two major classes of receptors, Pzx and PZYpurinoceptors, were identified, through analysis of the nature of the responses to ATP and related compounds in different biological systems, and additional subtypes have recently been reported (Fredholm et al., 1994).Suramin and reactive blue 2 (RB2) inhibits Pj and P2Ypurinoceptor-mediated excitatory responses (Dunn and Blakeley, 1988; Hourani et al., 1992),respectively, whereas the xanthine amine congener (XAC) blocks PI purinoceptor-mediated excitatory responses (Fredholm et al., 1994). Extracelltrlar ATP stimulates the influx of Ca’+ in *To whom all correspondence should be addressed at Third Department of Internal Medicine, Hiroshima University School of Medicine, 1-2-3 Kasumi, Minami-ku, Hirosbima, Japan.
PC12 cells and secretion of catecholamine by approx. five-fold over baseline levels (Inoue et al., 1989). These findings indicated that ATP might affect the Ca2+ influx and dopaminergic transmission in the central nervous system (CNS). There are two types of dopamine (DA) pools, newly synthesized and storage pools (McMillan et al., 1983), but little is known about whether ATP-stimulated DA release may equally involve both pools. We evaluated the effect of ATP on striatal levels of DA and its metabolizes, 3,4-dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA), by an invivomicrodialysis method, with particular reference to the type of DA pool and the involvement of the receptor and ion channel. EXPERIMENTALPROCEDURES Chemicals Suramin was obtained from Funakoshi Co. (Tokyo, Japan). Other chemicals used were purchased from Sigma Chemical Co. (St Louis, MO, U.S.A.).
Surgery and microdialysis procedure Male Wistar rats, 250–300 g, were maintained under conditions of controlled temperature and humidity, and were allowed free access to food and water. Experiments were 395
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performed in strict compliance with the Guidelinesfor Animal Experimentation (Japanese Association for Laboratory Animal Science, 1987). Rats were anesthetized with chloral hydrate (360 mg/kg, i.p.) then placed in a stereotaxic apparatus for the implantation of a microdialysis probe. The skull was exposed and the lambda and bregma points were positioned within 0.1 mm of the same horizontal plane. Stereotaxic coordinates for the striatum, with respect to the bregma, were A, +0,7, L, 2.7, and V, 6.0 mm, according to the atlas of Paxinos and Watson (1982). Using a sharp needle, we made a small slit in the dura to permit the easy lowering of the microdialysis probe into the striatum from the dural surface. A small screw was fixed to the skull to anchor the probe, which was then secured with dental cement. The animals were given free access to food and water for 1 week after surgery to allow them to recover. For perfusion, we used a sterile salt solution with the following composition: NaCl 145 mM, KC1 2.7 mM, CaC12 1.2 mM, MgCl, 1.0 mM, ascorbic acid 0.2 mM, pH 7.2. A Hamilton gas-tight syringe (1 ml) was filled with the solution, placed into a perfusion pump (EP-60, Eicom, Kyoto, Japan) and connected to the microdialysis probe (BDP-1-8-03, Eicom, Kyoto, Japan), whose dialysis membrane was made of regenerated cellulose. The perfusion rate was 2.0 pl/min. Dialysates were collected every 20 min (40 Al) and injected directly into an HPLC–ECD system. Measurement of DA, DOPAC and HVA We determined the concentration of DA, DOPAC and HVA by use of an HPLC–ECD. An HPLC pump system (EP-1O, Eicom, Kyoto, Japan) was connected to an ODS column (CA-50DS, Eicom) that was maintained at 25”C. The detector was a“glassy-carbon electrode set at +0.70 V vs a Ag+/AgCl reference electrode, The mobile phase consisted of citric acid–sodium acetate 0.1 M each (87°/0 of volume), methanol (130/. of volume), sodium octane sulfonate (160 mg/1), and EDTA (5 mg/1), pH 3.9. The flow rate was 1.0 ml/min. Peak areas on the chromatograms were measured with a data processor Chromatocorder 12 (Eicom, Kyoto, Japan). Drug administration Drugs were added to the solution that perfused the dialysis probe for 20 min. Tetrodotoxin (TTX; 2 PM), a calciumfree sohrtion, co-conotoxin GVIA (co-CgTX, 100 nM), XAC (1 yM), summin (1 mM), or RB2 (20 MM) was also infused continuously through the dialysis probe. Nifedipine (10 PM) was dissolved in a perfusate containing 0.01O\O acetone, which
had no apparent effecton the basal levelof DA. The pH of all drug solutionswas adjusted to 7.2. Rate of dffusion of ATP To estimate the rate of diffusion of ATP through the membrane, dialysis probes were perfused at a flow rate of 2.0 pl/min and placed in the perfusing solution in uitro. ATP was dissolved in the perfusate. The amount of ATP diffused through the dialysis tube into the extramembrane solution for 20 min was determined by HPLC with a UV spectrophotometer (Model Uvidec-100-V, Jasco, Tokyo, Japan). Peak areas on the chromatograms were measured with a data processor (Chromatopac C-R2A, Shimadzu, Tokyo, Japan). Statistical analyses Data are reported as means and SEM of independent experiments. Statistical analyses utilized one-way repeated
measurements for ANOVA or the unpaired Student’s t-test, as appropriate. The basal level of DA, DOPAC and HVA were calculated from the mean of three pooled samples prior to the administration of drugs. The effect was calculated as the difference between the average basal level and the levels obtained after the injection of each drug. The difference with a P value <0.05 was considered statistically significant. RESULTS
Effect of ATP on releaseandmetabolismof DA The ATP amount diffused from the dialysis tube represented 6.88 ~ 0.60°A (mean ~ SEM) of the amount of ATP that was perfused for 20 min. No nucleotide or nucleoside other than ATP was detected in the extramembrane solution. The basal level of DA in the dialysates was 0.87 +0.2 pmol/40 pl (n = 20). At the end of the perfusion with 1 mM ATP, an increase in DA level by 72 ~ 7.1 0/0 (P < 0.01) over basal level in the rat striatum was observed (Fig. 1). DA level was gradually reduced and returned to basal leve 120 min after the ATP treatment. ATP, 1 mM, also significantly decreased the DOPAC concentration. This effect of ATP on the DOPAC level was a function of time. DOPAC was reduced by 38+ 30/o of the basal level 120 min after ATP administration (P< 0.01). In contrast, the HVA level was not influ-
200 180 160 5 g 140 n ~ IZO o a 100 % #
*
80
** *
60
40~
-60-30
** ** I
0
30 60 90 120150180 Time (rein)
Fig. 1. Time course of ATP-induced changes in DA, DOPAC, and HVA concentrations. The duration of ATP treatment was 20 min. The perfusion rate was 2 jd/min and the dialysate was collected at 20-min intervals. Values are expressed as average percentage of the basal level which was the concentration of DA, DOPAC or HVA measured before ATP treatment. Plotted values and vertical bars are means and SEM of five independent experiments, respectively. *P <0.05, **P <0.01, significantly different from basal level (one-way ANOVA).
397
ATP induces release of newly synthesized dopamine
‘--1
centration (time course not shown). The maximal response was 148~ IOOAof the basal level (Fig. 2). The administration of 10-3 M ADP or adenosine caused a prolonged decrease in DOPAC (P < 0.05), but had no effect on HVA (Fig. 2). AMP (10-3 M) increased the DOPAC concentration to 450 f Lt5°/0 of the basal level (P <0.01 ; Fig. 2). The HVA level rapidly increased by 14~ 5°Aof basal value after AMP administration, and then returned to basal level (P< 0.05; Fig. 2).
**
■ dopamino
DOPAC
500
❑ HVA
h
al & 400 7 : 3oo%
Effects of XAC, suraminand RB2 on ATP-induced changeinDA metabolize
1oo-
XAC (1 KM), a xanthine antagonist of the adenosine receptor, was infused into the striatum via the Fig. 2. Effect of ADP, AMP, or adenosine on DA, DOPAC, dialysis tube. ATP (1 mM) was administered for 20 and HVA levels in striatal dialysate. All drugs (1 mM) were rein, 60 min after the start of XAC administration. added to the perfusate for 20 min. The perfusion rate was 2 The XAC and ATP combination induced the increase pl/min and dialysate was collected during the 20-min periods. in extracellular DA by 102~ 7.4°/0 (P < 0.01), which The results are expressed as a percentage of the basal values. exceeded increase produced by ATP alone, 72 f 7°/0 Each dialysate sample (40 pl) was injected into HPLC–ECD. The data are the means + SEM (bars) of five seParate experof the basal level (P < 0.05; Table 1). Pretreatment iments. Any vahres that were significantly different from the with XAC did not influence the ATP-induced basal levelsare indicated. *P <0.05, **P <0.01 (Student’s DOPAC decrease: 70t6Y0 vs 62t3Y0 of the basal t-test). level. We administered suramin, a P, purinoceptor antagenced by a 20-min treatment with 1 mM ATP (Fig. onist, 60 min before the administration of ATP to determine whether suramin-sensitive purinoceptors 1). may be involved in the ATP-induced increase in DA. Effect of ADP, AMP or adenosineon release and Suramin given alone had no effect on the extracellular metabolismof DA DA level. ATP (1 mM) was administered 60 min after start of suramin administration. The ATP-induced The administration of adenosine (10-3 M) decreased DA release by 35 +7Y0 of the basal level increase in extracellular DA was inhibited by admin72 ~ 7.1O/. vs (P< 0.05; Fig. 2). AMP (10-3 M) increased DA istering suramin as pretreatment: release by 20 i 80/0 of the basal level (P < 0.05; Fig. 37~5.3Y0 (P < 0.05; Table 1). The ATP-induced decrease in DOPAC was also reduced by the addition 2). The effect of AMP on DA release was less than that of either ATP or ADP. The administration of of suramin: 38 ~ 3°/0 vs 14* 30/o of the basal level 10-3 M ADP led to a significant (P< 0.05; Fig. 2) (P< 0.05; Table 1). RB2, an antagonist of the P2Ypurinoceptor (20 PM) and prolonged increase in the striatal DA conn
ATP
ADP
AMP adenosine
Table 1. Effectsof P, or P2purinoceptorantagonistson ATP-inducedalterationsin DA and relatedmetabolizes
ATP (1mM) ATP+XAC ATP+suramin
ATP+RB2
(pm~jO PI)
DOPAC (pmOl/40LL1)
HVA (pmOl/40PI)
1.9+0.20 2.23+0.10 1.51+0.10”
31.72+3.06 27.42~3.07 48.62+ 1.54”
16.15+1.56 17.16t0.50
1.22f.O.05**
53.72&1.27””
17.00+0,70 14.83+1.00
Extracellular DA, DOPAC and HVA in striatal dialysates were quantified by HPLC with ECD. XAC (1 jIM), suramin (1 #M), and RB2 (20 PM) were perfused for 3 h and, 60 min later, 1 mM ATP wasaddedto the perfusatefor 20 min. Data are the means f SEM of 4–6 separateexperiments. *P <0.05, **P <0.01, as compared with the value obtained when the striatum was treated withATP alone (Student’st-test).
Yu-Xiang Zhang et al,
398
was infused into the striatum through the dialysis tube for 3 h. ATP (1 mM) was administered for 20 rein, 60 min after start of RB2 administration. No increase in DA level was induced by 1mM ATP in rats pretreated with RB2 (Table 1). The ATP-induced decrease in DOPAC was lessened by the addition of RB2: 38+ 3% vs 5 + 3Y0 of the basal level (P< 0.01 ; Table 1). Calcium-dependency and TTX-sensitivityof the ATPinducedchangesinDA andDOPAC Ca’+ was omitted from the perfusate, and 0.5 mM EGTA was added to determine any dependence on extracellular Ca2+. The ATP-related increase in DA was significantly suppressed by 93& 3.5°10when Ca2+ was removed from the perfusate (P <0.01 ; Fig. 3). To investigate whether release of DA was mediated through a voltage-dependent calcium channel (VDCC), 100 nM co-CgTX or 10 ~M nifedipine was infused into the striatum. m-CgTX inhibited the ATPinduced increase in DA from 72+ 7.10/0of basal level to 23+ 3.2°A (P < 0.05). The ATP-related increase in DA was not significantly suppressed by the addition
■ Dopamlno ❑ DOPAC
of nifedipine (data not shown). Removal of Ca2+ from the perfusate significantly diminished the ATPinduced decrease in DOPAC from 5 + 3°/0 to 38 ~ 3°/0 of the basal level (P < 0.01). The ATP-induced decrease in DOPAC was reduced by the addition of o-CgTX from 7 t 5°/0 to 38~ So/o of the basal level (P< 0.05). TTX blocks the release of DA by inhibiting the voltage-dependent Na+ channel (VDNC). When 2 mM TTX was perfused into the striatum through the dialysis probe, 1 mM ATP neither increased the DA level nor decreased the DOPAC level (Fig. 3). Effects of u-MPT andreserpineon the actionof ATP The inhibition of DA synthesis by u-MPT treatment for 20 or 120 min significantly reduced the ATPinduced increase in DA from 72°/0 of the basal level to 20+4% (P < 0.01) or 10~4% (P <0.01 ; Fig. 4), respectively. The vesicular stores of DA were depleted by injection of reserpine (5 mg/kg, i.p.) 180min before ATP administration. The administration of reserpine did not alter the ATP-induced increase in DA: 62+8% vs 72-+7% (P> 0.05; Fig. 4)
❑ HVA
2oo-
3
~ 1800 c .5180 a s - 1401
Rlngu’s
c~um(-)
‘Qm
—
**
m 0 120-
ATP (1 mM) Fig. 3. Effects of Ca’+ depletion, co-CgTX or TTX on ATPin&ced alterations of DA, DOPAC, ‘or HVA. A Ca2+-free perfusing solution was perfused and 0.5 mM EGTA was added for 4 h. ATP was given for 20 rein, 2 h after the start of Ca2+-free condition, co-CgTX (100 nM) was perfused directly into the striatum through the dialysis probe for 4 h and, 2 h later, ATP was given for 20 min. TTX (2 PM) was applied for 200 rein, and 1 mM ATP was administered for 20 rein, 60 min after the start of TTX. The horizontal line indicates the infusion period of TTX. Increases in DA are expressed as a percentage of the basal level. Data are the means ~ SEM (bars) of 4–6 experiments. *P <0.05, **P <0.01, significantly different from ATP-induced increase in DA level (Student’s t-test).
1oo-
●m ** -
*
Rlngor’s
Rooorplno a-MPT
20 mhs
a-MPT
120 mhr
ATP (1 mM) Fig. 4. Effect of reserpine or ct-MPT on the ATP (1 mM, 20 rein) induced increase in release of DA in the rat striatum. Reserpine (RES, 5 mg/kg, i.p.) was administered 3 h before treatment with ATP. u-MPT (250 mg/kg, i.p.) was administered either 20 or 120 min before ATP administration. Values areexpressed as percentage of the basal level. Data are the means ~ SEM of 4–6 separate experiments. *P <0.05, **p <0,01, ascompared with the value obtained when the striatum was treated with ATP alone (Student’st-test).
ATP induces release of newly synthesized dopamine DISCUSSION The present study indicated that ATP is involved in the striatal transmission of DA by the finding that the release of DA induced by ATP may be mediated by P,Y purinoceptors, and that ATP may also affect DA metabolism, as indicated by the ATP-induced change in DOPAC, a metabolize of DA. Inhibition of ATPinduced changes in DA and DOPAC concentrations by suramin, a nonselective Pj purinoceptor antagonist (Dunn and Blakeley, 1988; Hourani et al., 1992), suggests that P2 purinoceptors may be involved in ATP-induced changes in DA and metabolism. Moreover, the effect of RB2 indicated that the ATP receptor may be a PZYsubtype. The cytoplasmic level of ATP in most mammalian cells exceeds 5 mM, so that the extracellular level of ATP could easily rise to a higher micromolar range (Gordon, 1986). The previous in oitro study indicates that ATP stimulates a dose-dependent [3H]DA secretion in PC-12 cells within a range of 20–1000 PM (Sela et al., 1991). In this study, although 0.1 or 1 mM ATP significantly increased the level of DA, the effective concentration of ATP could be in the range 7–7o .aM, as judged from the diffusion rate experiment. Ectonucleotidase is one of the enzymes responsible for removing ATP from the extracelhdar environment, which also exists in the CNS. Nucleotides, including ATP, are reportedly metabolized rapidly in perfused organs (Gordon, 1986). The possibility that the ATP-induced increase of DA and the decrease in DOPAC may be mediated by ATP metabolizes was excluded, since XAC, an adenosine receptor antagonist, did not inhibit the ATP-induced changes in DA and DOPAC. Adenosine is thought to be mainly an inhibitory neurotransmitter in the central nervous system (Phillis and Wu, 1981; Stone, 1981), since it inhibits the release of various neurotransmitters (Fredholm and Dunwiddie, 1988). Our results are consistent with that suggestion. In contrast to adenosine, AMP raised the level of DA slightly and the level of DOPAC significantly. HVA is formed by catechol-O-methyl transferase and, to a lesser extent, by the deaminination of 3-methoxytyramine, and is not a true indicator of nerve terminal DA metabolism (Wood and Altar, 1988). AMP-induced increase in HVA levels may simply follow those of DOPAC. These results indicate that AMP may play a role differing from adenosine or ATP in the rat striatum, although the exact mechanism requires investigation. TTX inhibits action potentials, then blocks the VDNC opening. The complete inhibition of ATP-
399
induced changes in DA and DOPAC levels by TTX observed in the present study suggests that opening of the VDNC to depolarize the presynapse of DA neurons or the generation of action potentials of interneurons is required for ATP-induced release of DA, as well as for the ATP-induced decrease of DOPAC in the rat striatum. ATP-induced changes in DA and DOPAC depend on extracellular Ca2+. The ATP-induced release of DA was inhibited by coCgTX, but not by nifedipine. co-CgTX almost irreversibly blocks the Ca2+ current through the N-type or L-type VDCC (McGraw et al., 1980; Volpe et a[., 1988). Nifedipine, which is a specific blocker of Ltype VDCC (Lee, 1984) and also inhibits the L-type VDCC-mediated release of DA from adrenal chromaffin cells (Nassar and Luxoro, 1992). These findings seem to support the idea that the Ca2+ current is involved in the effect of ATP in inducing extracellular changes in DA and DOPAC, probably via N-type, not L-type, VDCC. Several agents are thought to release brain DA preferentially from different intraneuronal pools, e.g. amphetamine releases DA from the newly synthesized pool of DA (Chiueh and Moore, 1975), whereas methylphenidate and tyramine selectively release DA from the storage pools (Lentzen and Philippu, 1981). Short-term treatment with a-MPT (250 mg/kg, i.p.) leads to an inhibition of DA synthesis without depletion of its storage, prolonged (120 rein) administration of this agent leads to a depletion of both the newly synthesized and the storage pools of DA (Widerlov and Lewander, 1978). The ATP-induced release of DA was reduced by treatment with a-MPT, but not with reserpine. Results indicate that the DA release by ATP treatment may be derived from the newly synthesized DA pool. It has been postulated that the DA recently released and taken up again is the main source of DOPAC (Roth et al., 1976). However, the hypothesis that DOPAC is derived from an intraneuronal pool of newly synthesized DA has a[so been suggested (Zetterstrom et al., 1986). Our results support the notion that decline in the level of DOPAC is closely associated with depletion of the newly synthesized DA pool. In conclusion, the ATP-induced changes in DA and DOPAC levels may be mediated via P2Y purinoceptors. Opening of the N-type VDCC and VDNC to depolarize the DA neuron is required for both the ATP-induced release of DA and the ATP-induced decreased level of DOPAC in the rat striatum. The newly synthesized pool, but not the storage pool, of DA, is involved in the ATP-induced release of DA in the rat striatum.
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Yu-Xiang Zhang et al.
Acknowledgements—This work was supported by a GrantIn-Aid from the Research Committee on Degenerative Disease of the Central Nervous System, The Ministry of Health and Welfare of Japan.
REFERENCES Bertrand G., Chapal J., Loubatieres M. M. and Roye M. (1987) Evidence for two different P,-purinoceptors on beta cell and pancreatic vascular bed. Br. J. Pharmac. 91, 783– 787. Boeynems J. M. and Pearson J. D. (1990) Pz purinoceptors on vascular endothelial cells: physiological significance and transduction mechanisms. Trends Pharmac. Sci. 11, 34-37. Chiueh C. C, and Moore K, E. (1975) D-amphetamineinduced release of’’newly synthesized” and “stored” dopamine from the caudate nucleus in uiuo. J. Pharmac. exp. Ther. 192, 642–653. Dahlquist R. and Diamant B. (1974) Interaction of ATP and calcium on the rat mast cell: effect on histamine release. Acta, Pharmac. Toxic. Copenh. 34, 368–384. Dunn P. M. and Blakeley A. G, H. (1988) Suramin: a reversible P2-purinoceptor antagonist in the mouse vas deferens. Br, J. Pharmac. 93,243-245. Fredholm B. B. and Dunwiddie T. V. (1988) How does adenosine inhibit transmitter release? Trends Pharmac. Sci. 9, 130–134. Fredholm B. B., Fredholm B. B., Abbracchio M. P., Burnstock G., Daly J. W., Harden T. K., Jacobson K. A., Leff P. and Williams M. (1994) Nomenclature and classification of purinoceptors. Pharmac. Reu. 46, 143– 156. Gordon J. L. (1986) Extracelhdar ATP: effects, source and fate. Biochem. J. 233,309-319. Hourani S. M. O., Hall D. A. and Nieman C. J. (1992) Effects of the P2-purinoceptor antagonist, suramin, on human platelet aggregation induced by adenosine 5’-triphosphate. Br. J. Pharmac. 105, 453–457. Inoue K,, Nakazawa K., Fujimori K. and Takanaka A. (1989) Extracellular adenosine 5’-triphosphate-evoked norepinephrine secretion not relating to voltage-gated Ca Lett.106, channels in pheochromocytoma cells. Neurosci.
294-299. Lee K. and Tsien R. W. (1984) Mechanism of calcium channel blockade by verapamil, D600, diltiazem and nitrendipine in single dialysed heart cells. Nature 302, 538-544.
Lentzen H. and Philippu A. (1981) Physico-chemical properties of phenethylamines and their uptake into synaptic vesicles of the caudate nucleus. Biochem. Pharmac. 30, 1759–1764. McMillan B. A. (1983) CNS stimulants: two distinct mechanisms of action for amphetamine-like drugs. Trends Pharmac. Sci. 4, 429–432. McGraw C. F., Somlyo A. V. and Blaustein M. P. (1980) Localization of calcium in presynaptic nerve terminals: an ultrastructural and electron microprobe analysis. J. Ceil Biol. 85, 228–241. Nassar G. V. and Luxoro M. (1992) Catecholamine secretion from adrenal chromaffin cells of the toad (Caudiverbera caudiuerbera): effect of monensin. Comp. Biochem. Physiol. comp. Pharmac. 101,219-225. Paxinos G. and Watson C. (1982) The Rat Brain in Stereolaxic Coordinates. Academic Press, London. Phillis J. W. and Wu P, H. (1981) The role of adenosine and its nucleotides in central synaptic transmission. Neuroscience 16, 187–199. Roth R. H., Murrin L. C. and Walters J, R. (1976) Central dopaminergic neurons: effects of alteration of impulse flow in the accumulation of dihydroxyphenylacetic acid. Eur. J. Pharmac. 36, 163–168. Sela D., Ram E. and Atlas D. (1991) ATP receptor, a putative receptor-operated channel in PC-12 cells. J. biol. Chem. 266, 17,990–17,994. Stone T. W. (1981) Physiological roles for adenosine and adenosine 5’-triphosphate in the nervous system. Neuroscience 6, 523–555. Volpe D,, Krause K, H., Hashimoto S., Zorzato F., Pozzan T., Meldolesi J. and Lew D. P. (1988) “Calciosome”, a cytoplasmic organelle: the inositol l,4,5-triphosphate-sensitive Caz+ store Ofnonmuscle cells? proc.natn.Acad.Sci. U.S.A.85, 1091-1095. Widerlov E. and Lewander T. (1978) The relationship between amphetamine antagonism and depletion of brain catecholamines by alpha-methyl-p-tyrosine in rats. Naunyn. Schmiedebergs Arch. Plrarmac. 304, 125–134. White T. D. (1988) Role ofadeninecompounds in autonomic neurotransmission. Pharmac. Ther. 38, 129–168. Wood P. L, and Altar C. A. (1988) Dopamine release in uiuo from nigrostriatal, mesolimbic, and mesocortical neurons: utility of 3-methoxytyramine measurements. Pharmac. Reu. 40(3), 163–187. Zetterstrom T., Sharp T. and Ungerstedt U. (1986) Further evaluation of the mechanism by which amphetamine reduces striatal dopamine metabolism: an in oiuo brain dialysis study. Eur. J. Pharmac. 132, 1-9.
0197-0186(95)00105–0
Neurochem.Int. Vol.28,No. 4, pp. 395400, 1996 CopyrightG 1996Els&ierScienceLtd Printedin Great Britain.All rightsreserved 01974186/96$15.00+0.00
ATP INDUCES RELEASE OF NEWLY SYNTHESIZED DOPAMINE IN THE RAT STRIATUM YU-XIANG ZHANG, HIROSHI YAMASHITA, TOMOHIRO OHSHITA, NOBUKATSU SAWAMOTO and SHIGENOBU NAKAMURA* Third Department of Internal Medicine,Hiroshima UniversitySchoolof Medicine,Hiroshima, Japan (Received 31 March 1995; accepted 1 August 1995) Abstract-The effect of ATP on release of dopamine (DA) and its metabolism in the rat striatum was investigated by use of in uiuo microdialysis. DA release was increased and the level of 3,4-dihydroxyphenylacetic acid (DOPAC) was decreased by treatment with 1 mM ATP or ADP for 20 rein, while such treatment failed to change the extracellular level of homovanillic acid (HVA). The ATP-induced increase in DA and decrease in DOPAC were inhibited by suramin, a nonselective Pz purinoceptor antagonist, and by reactive blue 2, a P2~purinoceptor antagonist, but not by xanthine amine congener, an adenosine receptor antagonist, indicating that PZ~purinoceptors were involved in the present observation. The effects of ATP were extracellular Ca2+-dependent and sensitive to cwconotoxin and tetrodotoxin, which indicates that the opening of voltage-sensitive calcium channels and sodium channels to depolarize the DA neuron is required for both ATP-induced DA release and DOPAC decrease in the rat striatum. Pretreatment with ce-methyl-p-tyrosine, but not with reserpine, suppressed the ATP-induced release of DA. These findings suggest that the newly synthesized pool, but not the vesicular pool, of DA is involved in the ATP-induced release of DA in the rat striatum. Copyright ~ 1996 Elsevier Science Ltd.
ATP can serve as a neurotransmitter. Upon binding to its specific receptors, ATP evokes a secretory function in various cells (Boeynems and Pearson, 1990; Dahlquist and Diamant, 1974; Bertrand et al., 1987). ATP is present in the synaptic vesicles, which contain either acetylcholine or noradrenaline, and is released concomitantly with these chemicals upon stimulation (White, 1988). Receptors for ATP are designated P, purinoceptors. Two major classes of receptors, Pzx and PZYpurinoceptors, were identified, through analysis of the nature of the responses to ATP and related compounds in different biological systems, and additional subtypes have recently been reported (Fredholm et al., 1994).Suramin and reactive blue 2 (RB2) inhibits Pj and P2Ypurinoceptor-mediated excitatory responses (Dunn and Blakeley, 1988; Hourani et al., 1992),respectively, whereas the xanthine amine congener (XAC) blocks PI purinoceptor-mediated excitatory responses (Fredholm et al., 1994). Extracelltrlar ATP stimulates the influx of Ca’+ in *To whom all correspondence should be addressed at Third Department of Internal Medicine, Hiroshima University School of Medicine, 1-2-3 Kasumi, Minami-ku, Hirosbima, Japan.
PC12 cells and secretion of catecholamine by approx. five-fold over baseline levels (Inoue et al., 1989). These findings indicated that ATP might affect the Ca2+ influx and dopaminergic transmission in the central nervous system (CNS). There are two types of dopamine (DA) pools, newly synthesized and storage pools (McMillan et al., 1983), but little is known about whether ATP-stimulated DA release may equally involve both pools. We evaluated the effect of ATP on striatal levels of DA and its metabolizes, 3,4-dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA), by an invivomicrodialysis method, with particular reference to the type of DA pool and the involvement of the receptor and ion channel. EXPERIMENTALPROCEDURES Chemicals Suramin was obtained from Funakoshi Co. (Tokyo, Japan). Other chemicals used were purchased from Sigma Chemical Co. (St Louis, MO, U.S.A.).
Surgery and microdialysis procedure Male Wistar rats, 250–300 g, were maintained under conditions of controlled temperature and humidity, and were allowed free access to food and water. Experiments were 395
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Yu-Xiang Zhang et al,
performed in strict compliance with the Guidelinesfor Animal Experimentation (Japanese Association for Laboratory Animal Science, 1987). Rats were anesthetized with chloral hydrate (360 mg/kg, i.p.) then placed in a stereotaxic apparatus for the implantation of a microdialysis probe. The skull was exposed and the lambda and bregma points were positioned within 0.1 mm of the same horizontal plane. Stereotaxic coordinates for the striatum, with respect to the bregma, were A, +0,7, L, 2.7, and V, 6.0 mm, according to the atlas of Paxinos and Watson (1982). Using a sharp needle, we made a small slit in the dura to permit the easy lowering of the microdialysis probe into the striatum from the dural surface. A small screw was fixed to the skull to anchor the probe, which was then secured with dental cement. The animals were given free access to food and water for 1 week after surgery to allow them to recover. For perfusion, we used a sterile salt solution with the following composition: NaCl 145 mM, KC1 2.7 mM, CaC12 1.2 mM, MgCl, 1.0 mM, ascorbic acid 0.2 mM, pH 7.2. A Hamilton gas-tight syringe (1 ml) was filled with the solution, placed into a perfusion pump (EP-60, Eicom, Kyoto, Japan) and connected to the microdialysis probe (BDP-1-8-03, Eicom, Kyoto, Japan), whose dialysis membrane was made of regenerated cellulose. The perfusion rate was 2.0 pl/min. Dialysates were collected every 20 min (40 Al) and injected directly into an HPLC–ECD system. Measurement of DA, DOPAC and HVA We determined the concentration of DA, DOPAC and HVA by use of an HPLC–ECD. An HPLC pump system (EP-1O, Eicom, Kyoto, Japan) was connected to an ODS column (CA-50DS, Eicom) that was maintained at 25”C. The detector was a“glassy-carbon electrode set at +0.70 V vs a Ag+/AgCl reference electrode, The mobile phase consisted of citric acid–sodium acetate 0.1 M each (87°/0 of volume), methanol (130/. of volume), sodium octane sulfonate (160 mg/1), and EDTA (5 mg/1), pH 3.9. The flow rate was 1.0 ml/min. Peak areas on the chromatograms were measured with a data processor Chromatocorder 12 (Eicom, Kyoto, Japan). Drug administration Drugs were added to the solution that perfused the dialysis probe for 20 min. Tetrodotoxin (TTX; 2 PM), a calciumfree sohrtion, co-conotoxin GVIA (co-CgTX, 100 nM), XAC (1 yM), summin (1 mM), or RB2 (20 MM) was also infused continuously through the dialysis probe. Nifedipine (10 PM) was dissolved in a perfusate containing 0.01O\O acetone, which
had no apparent effecton the basal levelof DA. The pH of all drug solutionswas adjusted to 7.2. Rate of dffusion of ATP To estimate the rate of diffusion of ATP through the membrane, dialysis probes were perfused at a flow rate of 2.0 pl/min and placed in the perfusing solution in uitro. ATP was dissolved in the perfusate. The amount of ATP diffused through the dialysis tube into the extramembrane solution for 20 min was determined by HPLC with a UV spectrophotometer (Model Uvidec-100-V, Jasco, Tokyo, Japan). Peak areas on the chromatograms were measured with a data processor (Chromatopac C-R2A, Shimadzu, Tokyo, Japan). Statistical analyses Data are reported as means and SEM of independent experiments. Statistical analyses utilized one-way repeated
measurements for ANOVA or the unpaired Student’s t-test, as appropriate. The basal level of DA, DOPAC and HVA were calculated from the mean of three pooled samples prior to the administration of drugs. The effect was calculated as the difference between the average basal level and the levels obtained after the injection of each drug. The difference with a P value <0.05 was considered statistically significant. RESULTS
Effect of ATP on releaseandmetabolismof DA The ATP amount diffused from the dialysis tube represented 6.88 ~ 0.60°A (mean ~ SEM) of the amount of ATP that was perfused for 20 min. No nucleotide or nucleoside other than ATP was detected in the extramembrane solution. The basal level of DA in the dialysates was 0.87 +0.2 pmol/40 pl (n = 20). At the end of the perfusion with 1 mM ATP, an increase in DA level by 72 ~ 7.1 0/0 (P < 0.01) over basal level in the rat striatum was observed (Fig. 1). DA level was gradually reduced and returned to basal leve 120 min after the ATP treatment. ATP, 1 mM, also significantly decreased the DOPAC concentration. This effect of ATP on the DOPAC level was a function of time. DOPAC was reduced by 38+ 30/o of the basal level 120 min after ATP administration (P< 0.01). In contrast, the HVA level was not influ-
200 180 160 5 g 140 n ~ IZO o a 100 % #
*
80
** *
60
40~
-60-30
** ** I
0
30 60 90 120150180 Time (rein)
Fig. 1. Time course of ATP-induced changes in DA, DOPAC, and HVA concentrations. The duration of ATP treatment was 20 min. The perfusion rate was 2 jd/min and the dialysate was collected at 20-min intervals. Values are expressed as average percentage of the basal level which was the concentration of DA, DOPAC or HVA measured before ATP treatment. Plotted values and vertical bars are means and SEM of five independent experiments, respectively. *P <0.05, **P <0.01, significantly different from basal level (one-way ANOVA).
397
ATP induces release of newly synthesized dopamine
‘--1
centration (time course not shown). The maximal response was 148~ IOOAof the basal level (Fig. 2). The administration of 10-3 M ADP or adenosine caused a prolonged decrease in DOPAC (P < 0.05), but had no effect on HVA (Fig. 2). AMP (10-3 M) increased the DOPAC concentration to 450 f Lt5°/0 of the basal level (P <0.01 ; Fig. 2). The HVA level rapidly increased by 14~ 5°Aof basal value after AMP administration, and then returned to basal level (P< 0.05; Fig. 2).
**
■ dopamino
DOPAC
500
❑ HVA
h
al & 400 7 : 3oo%
Effects of XAC, suraminand RB2 on ATP-induced changeinDA metabolize
1oo-
XAC (1 KM), a xanthine antagonist of the adenosine receptor, was infused into the striatum via the Fig. 2. Effect of ADP, AMP, or adenosine on DA, DOPAC, dialysis tube. ATP (1 mM) was administered for 20 and HVA levels in striatal dialysate. All drugs (1 mM) were rein, 60 min after the start of XAC administration. added to the perfusate for 20 min. The perfusion rate was 2 The XAC and ATP combination induced the increase pl/min and dialysate was collected during the 20-min periods. in extracellular DA by 102~ 7.4°/0 (P < 0.01), which The results are expressed as a percentage of the basal values. exceeded increase produced by ATP alone, 72 f 7°/0 Each dialysate sample (40 pl) was injected into HPLC–ECD. The data are the means + SEM (bars) of five seParate experof the basal level (P < 0.05; Table 1). Pretreatment iments. Any vahres that were significantly different from the with XAC did not influence the ATP-induced basal levelsare indicated. *P <0.05, **P <0.01 (Student’s DOPAC decrease: 70t6Y0 vs 62t3Y0 of the basal t-test). level. We administered suramin, a P, purinoceptor antagenced by a 20-min treatment with 1 mM ATP (Fig. onist, 60 min before the administration of ATP to determine whether suramin-sensitive purinoceptors 1). may be involved in the ATP-induced increase in DA. Effect of ADP, AMP or adenosineon release and Suramin given alone had no effect on the extracellular metabolismof DA DA level. ATP (1 mM) was administered 60 min after start of suramin administration. The ATP-induced The administration of adenosine (10-3 M) decreased DA release by 35 +7Y0 of the basal level increase in extracellular DA was inhibited by admin72 ~ 7.1O/. vs (P< 0.05; Fig. 2). AMP (10-3 M) increased DA istering suramin as pretreatment: release by 20 i 80/0 of the basal level (P < 0.05; Fig. 37~5.3Y0 (P < 0.05; Table 1). The ATP-induced decrease in DOPAC was also reduced by the addition 2). The effect of AMP on DA release was less than that of either ATP or ADP. The administration of of suramin: 38 ~ 3°/0 vs 14* 30/o of the basal level 10-3 M ADP led to a significant (P< 0.05; Fig. 2) (P< 0.05; Table 1). RB2, an antagonist of the P2Ypurinoceptor (20 PM) and prolonged increase in the striatal DA conn
ATP
ADP
AMP adenosine
Table 1. Effectsof P, or P2purinoceptorantagonistson ATP-inducedalterationsin DA and relatedmetabolizes
ATP (1mM) ATP+XAC ATP+suramin
ATP+RB2
(pm~jO PI)
DOPAC (pmOl/40LL1)
HVA (pmOl/40PI)
1.9+0.20 2.23+0.10 1.51+0.10”
31.72+3.06 27.42~3.07 48.62+ 1.54”
16.15+1.56 17.16t0.50
1.22f.O.05**
53.72&1.27””
17.00+0,70 14.83+1.00
Extracellular DA, DOPAC and HVA in striatal dialysates were quantified by HPLC with ECD. XAC (1 jIM), suramin (1 #M), and RB2 (20 PM) were perfused for 3 h and, 60 min later, 1 mM ATP wasaddedto the perfusatefor 20 min. Data are the means f SEM of 4–6 separateexperiments. *P <0.05, **P <0.01, as compared with the value obtained when the striatum was treated withATP alone (Student’st-test).
Yu-Xiang Zhang et al,
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was infused into the striatum through the dialysis tube for 3 h. ATP (1 mM) was administered for 20 rein, 60 min after start of RB2 administration. No increase in DA level was induced by 1mM ATP in rats pretreated with RB2 (Table 1). The ATP-induced decrease in DOPAC was lessened by the addition of RB2: 38+ 3% vs 5 + 3Y0 of the basal level (P< 0.01 ; Table 1). Calcium-dependency and TTX-sensitivityof the ATPinducedchangesinDA andDOPAC Ca’+ was omitted from the perfusate, and 0.5 mM EGTA was added to determine any dependence on extracellular Ca2+. The ATP-related increase in DA was significantly suppressed by 93& 3.5°10when Ca2+ was removed from the perfusate (P <0.01 ; Fig. 3). To investigate whether release of DA was mediated through a voltage-dependent calcium channel (VDCC), 100 nM co-CgTX or 10 ~M nifedipine was infused into the striatum. m-CgTX inhibited the ATPinduced increase in DA from 72+ 7.10/0of basal level to 23+ 3.2°A (P < 0.05). The ATP-related increase in DA was not significantly suppressed by the addition
■ Dopamlno ❑ DOPAC
of nifedipine (data not shown). Removal of Ca2+ from the perfusate significantly diminished the ATPinduced decrease in DOPAC from 5 + 3°/0 to 38 ~ 3°/0 of the basal level (P < 0.01). The ATP-induced decrease in DOPAC was reduced by the addition of o-CgTX from 7 t 5°/0 to 38~ So/o of the basal level (P< 0.05). TTX blocks the release of DA by inhibiting the voltage-dependent Na+ channel (VDNC). When 2 mM TTX was perfused into the striatum through the dialysis probe, 1 mM ATP neither increased the DA level nor decreased the DOPAC level (Fig. 3). Effects of u-MPT andreserpineon the actionof ATP The inhibition of DA synthesis by u-MPT treatment for 20 or 120 min significantly reduced the ATPinduced increase in DA from 72°/0 of the basal level to 20+4% (P < 0.01) or 10~4% (P <0.01 ; Fig. 4), respectively. The vesicular stores of DA were depleted by injection of reserpine (5 mg/kg, i.p.) 180min before ATP administration. The administration of reserpine did not alter the ATP-induced increase in DA: 62+8% vs 72-+7% (P> 0.05; Fig. 4)
❑ HVA
2oo-
3
~ 1800 c .5180 a s - 1401
Rlngu’s
c~um(-)
‘Qm
—
**
m 0 120-
ATP (1 mM) Fig. 3. Effects of Ca’+ depletion, co-CgTX or TTX on ATPin&ced alterations of DA, DOPAC, ‘or HVA. A Ca2+-free perfusing solution was perfused and 0.5 mM EGTA was added for 4 h. ATP was given for 20 rein, 2 h after the start of Ca2+-free condition, co-CgTX (100 nM) was perfused directly into the striatum through the dialysis probe for 4 h and, 2 h later, ATP was given for 20 min. TTX (2 PM) was applied for 200 rein, and 1 mM ATP was administered for 20 rein, 60 min after the start of TTX. The horizontal line indicates the infusion period of TTX. Increases in DA are expressed as a percentage of the basal level. Data are the means ~ SEM (bars) of 4–6 experiments. *P <0.05, **P <0.01, significantly different from ATP-induced increase in DA level (Student’s t-test).
1oo-
●m ** -
*
Rlngor’s
Rooorplno a-MPT
20 mhs
a-MPT
120 mhr
ATP (1 mM) Fig. 4. Effect of reserpine or ct-MPT on the ATP (1 mM, 20 rein) induced increase in release of DA in the rat striatum. Reserpine (RES, 5 mg/kg, i.p.) was administered 3 h before treatment with ATP. u-MPT (250 mg/kg, i.p.) was administered either 20 or 120 min before ATP administration. Values areexpressed as percentage of the basal level. Data are the means ~ SEM of 4–6 separate experiments. *P <0.05, **p <0,01, ascompared with the value obtained when the striatum was treated with ATP alone (Student’st-test).
ATP induces release of newly synthesized dopamine DISCUSSION The present study indicated that ATP is involved in the striatal transmission of DA by the finding that the release of DA induced by ATP may be mediated by P,Y purinoceptors, and that ATP may also affect DA metabolism, as indicated by the ATP-induced change in DOPAC, a metabolize of DA. Inhibition of ATPinduced changes in DA and DOPAC concentrations by suramin, a nonselective Pj purinoceptor antagonist (Dunn and Blakeley, 1988; Hourani et al., 1992), suggests that P2 purinoceptors may be involved in ATP-induced changes in DA and metabolism. Moreover, the effect of RB2 indicated that the ATP receptor may be a PZYsubtype. The cytoplasmic level of ATP in most mammalian cells exceeds 5 mM, so that the extracellular level of ATP could easily rise to a higher micromolar range (Gordon, 1986). The previous in oitro study indicates that ATP stimulates a dose-dependent [3H]DA secretion in PC-12 cells within a range of 20–1000 PM (Sela et al., 1991). In this study, although 0.1 or 1 mM ATP significantly increased the level of DA, the effective concentration of ATP could be in the range 7–7o .aM, as judged from the diffusion rate experiment. Ectonucleotidase is one of the enzymes responsible for removing ATP from the extracelhdar environment, which also exists in the CNS. Nucleotides, including ATP, are reportedly metabolized rapidly in perfused organs (Gordon, 1986). The possibility that the ATP-induced increase of DA and the decrease in DOPAC may be mediated by ATP metabolizes was excluded, since XAC, an adenosine receptor antagonist, did not inhibit the ATP-induced changes in DA and DOPAC. Adenosine is thought to be mainly an inhibitory neurotransmitter in the central nervous system (Phillis and Wu, 1981; Stone, 1981), since it inhibits the release of various neurotransmitters (Fredholm and Dunwiddie, 1988). Our results are consistent with that suggestion. In contrast to adenosine, AMP raised the level of DA slightly and the level of DOPAC significantly. HVA is formed by catechol-O-methyl transferase and, to a lesser extent, by the deaminination of 3-methoxytyramine, and is not a true indicator of nerve terminal DA metabolism (Wood and Altar, 1988). AMP-induced increase in HVA levels may simply follow those of DOPAC. These results indicate that AMP may play a role differing from adenosine or ATP in the rat striatum, although the exact mechanism requires investigation. TTX inhibits action potentials, then blocks the VDNC opening. The complete inhibition of ATP-
399
induced changes in DA and DOPAC levels by TTX observed in the present study suggests that opening of the VDNC to depolarize the presynapse of DA neurons or the generation of action potentials of interneurons is required for ATP-induced release of DA, as well as for the ATP-induced decrease of DOPAC in the rat striatum. ATP-induced changes in DA and DOPAC depend on extracellular Ca2+. The ATP-induced release of DA was inhibited by coCgTX, but not by nifedipine. co-CgTX almost irreversibly blocks the Ca2+ current through the N-type or L-type VDCC (McGraw et al., 1980; Volpe et a[., 1988). Nifedipine, which is a specific blocker of Ltype VDCC (Lee, 1984) and also inhibits the L-type VDCC-mediated release of DA from adrenal chromaffin cells (Nassar and Luxoro, 1992). These findings seem to support the idea that the Ca2+ current is involved in the effect of ATP in inducing extracellular changes in DA and DOPAC, probably via N-type, not L-type, VDCC. Several agents are thought to release brain DA preferentially from different intraneuronal pools, e.g. amphetamine releases DA from the newly synthesized pool of DA (Chiueh and Moore, 1975), whereas methylphenidate and tyramine selectively release DA from the storage pools (Lentzen and Philippu, 1981). Short-term treatment with a-MPT (250 mg/kg, i.p.) leads to an inhibition of DA synthesis without depletion of its storage, prolonged (120 rein) administration of this agent leads to a depletion of both the newly synthesized and the storage pools of DA (Widerlov and Lewander, 1978). The ATP-induced release of DA was reduced by treatment with a-MPT, but not with reserpine. Results indicate that the DA release by ATP treatment may be derived from the newly synthesized DA pool. It has been postulated that the DA recently released and taken up again is the main source of DOPAC (Roth et al., 1976). However, the hypothesis that DOPAC is derived from an intraneuronal pool of newly synthesized DA has a[so been suggested (Zetterstrom et al., 1986). Our results support the notion that decline in the level of DOPAC is closely associated with depletion of the newly synthesized DA pool. In conclusion, the ATP-induced changes in DA and DOPAC levels may be mediated via P2Y purinoceptors. Opening of the N-type VDCC and VDNC to depolarize the DA neuron is required for both the ATP-induced release of DA and the ATP-induced decreased level of DOPAC in the rat striatum. The newly synthesized pool, but not the storage pool, of DA, is involved in the ATP-induced release of DA in the rat striatum.
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Acknowledgements—This work was supported by a GrantIn-Aid from the Research Committee on Degenerative Disease of the Central Nervous System, The Ministry of Health and Welfare of Japan.
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