1997 Elsevier Science Ltd. Printed in Great Britain. ... quisqualate (QA) and their corresponding antagonists, Dizocilpine maleate (MK-801), D-7-glutamyl-.
~ Pergamon
Neurochem. Int. Vol. 31, No. 4, pp. 607-616, 1997
PII: S0197-0186(96)00141-6
© 1997ElsevierScienceLtd Printedin Great Britain.All rightsreserved 0197-0186/97$17.00+0.00
NITRIC OXIDE INVOLVEMENT IN R E G U L A T I N G THE DOPAMINE TRANSPORT IN THE STRIATAL REGION OF RAT BRAIN VERONICA CHAPARRO-HUERTA, t CARLOS BEAS-ZARATE, MONICA URElqA G U E R R E R O I and ALFREDO FERIA-VELASCO2 ~Lab. de Neuroquimica, Div. de Patol. Expl., C.I.B.O., I.M.S.S., Depto. de Biol. Cel. y Molec., Div. de Cs. Biol. y Ambs., C.U.C.B.A., Universidad de Guadalajara, Guadalajara, Mexico 2Biotechnologyand Experimental Pathology Group, CIATEJ, Guadalajara, Jalisco, Mexico (Received 13 June 1996; accepted 28 November 1996)
Abstract--Spontaneous [3H]dopamine([3H]DA)overflow was measured from striatal slices in the presence of different glutamate (Glu) receptor agonists such as N-methyl-D-aspartate(NMDA), kainate (KA) and quisqualate (QA) and their corresponding antagonists, Dizocilpine maleate (MK-801), D-7-glutamylaminomethanesulfonic acid (GAMS) and 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX), respectively. [3H]DA uptake and release in the presence of L-Arginine (L-Arg) and N6-nitro-arginine (L-N-Arg), an inhibitor of nitric oxide (NO) synthesis were also evaluated. L-N-Arg alone or combined with L-Arg significantlyreduced [3H]DA uptake at 10 and 100#M from 33% to 44% from striatal slices. Whereas, in brain synaptosomal fractions L-Arg induced a biphasic effect on that [3H]DA uptake in a dose dependent manner, and L-N-Argshowed an absolute inhibition in 80°90% of this [3H]DA uptake at 1-500#M. The amino acids, lysine, valine and histidine (100#M) had a little effect inhibitory on [3H]DA uptake from synaptosomal fractions. Glu agonists, NMDA (10/~M) and KA (10 #M) importantly increased the spontaneous [3H]DA overflow, which was blocked by MK-801 (10 #M) and GAMS (10/~M), respectively.QA had no effect on [3H]DA release. L-Arg (100200#M) potentiated the spontaneous [3H]DA overflow in a dose dependent fashion from striatal slices, being reverted by 10/~M L-N-Argalone or in combination with all other compounds; whereas, lysine, histidine and valine did not modify that spontaneous [3H]DA overflow. Results support the hypothesis related to the participation of NO on DA transport possibly synthesized at the dopaminergic (DAergic) terminals in the striatum; also that L-Arg concentration may determine alternative mechanisms to regulate the DAergic activity at the striatum. © 1997 Elsevier Science Ltd
The selective vulnerability of the neuronal system is a remarkable characteristic of brain degenerative disorders such as Parkinson's disease, Huntington's disease and Alzheimer's disease, among others (Coyle and Puttfarcken, 1993). Although the mechanism of this neuronal destruction is unknown, it has been suggested that excitotoxicity by activating the glutamate
(Glu) N M D A receptor (NMDA-R) is involved (Hyman et al., 1992). This neurotoxicity is dependent upon extracellular calcium (Ca 2+) influx and events following it resulting in irreversible damage and cell death (Regan et al., 1993). NMDA-Rs have been broadly studied for their involvement in pathological and physiological processes such as memory and learning (Manzoni and Bockaert, 1993). *To whom all correspondence should be addressed at the In vitro and in vivo studies have shown that gluM. Sci. Carlos BEPS Lab. de Neurobiologia Div. de tamatergic fibers act as stimuli on the dopaminergic Patol. Expl., C.I.B.O., IMSS Apartado Postal #4-160 terminal in the striatum (Carlsson and Carlsson, 1990; Guadalajara, Jalisco, 44421 Mexico. Krebs et al., 1991) and regulating the dopamine (DA) Abbreviations: [3H]DA, [3H]dopamine; Glu, Glutamate; NMDA, N-methyl-Ig-aspartate;KA, kainate; QA, quis- transport in this region appears to be N M D A - R qualate; MK-801, Dizocilpine maleate; GAMS, D-7-glu- mediated (Leviel et al., 1990). Thus, the presence of tamylaminomethanesulfonic acid; CNQX, 6-cyano-7- Glu receptors in this region might be part of the mechnitroquinoxaline-2,3-dione; L-Arg, L-arginine; L-N-Arg, N6-nitro-arginine; NO, nitric oxide; NOS, nitric oxide anism involved in some of the processes triggering synthase; NMDA-R, NMDA receptor; Glu-Rs, Glu neuronal degeneration (Fuller et al., 1992; Dawson receptors; D~-R, dopamine receptor D~. et al., 1993). 607
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O n the other h a n d , nitric oxide (NO) p r o d u c t i o n might play a m a j o r role in some central nervous system (CNS) pathologies ( M o n c a d a et al., 1989). Ca 2+ influx a c c o m p a n y i n g sustained N M D A - R activation appears to be associated to n e u r o n a l d a m a g e ( R o t h m a n a n d Olney, 1987; G a r t h w a i t e et al., 1989a) and neurotoxicity, via an increased N O p r o d u c t i o n ( D a w s o n et al., 1991). In 1991, D a w s o n et al. showed a protection from excitotoxicity t h r o u g h nitric oxide synthase (NOS) inhibitors such as monomethyl-L-arginine a n d N Gnitro-L-Arginine (L-N-Arg). These produce a dose dependent decreased N M D A induced cell d e a t h (Dawson et al., 1991). These c o m p u n d s are also involved in n e u r o n a l degeneration induced in primary cortical cultures (Puttfarcken et al., 1992). L-N-Arg has also been reported as the m o s t powerful N O S inhibitor b o t h in vitro a n d in vivo (Dwyer et al., 1991). However, even if the N O produced by G l u receptor activation has been involved in some neurodegenerative p h e n o m e n a , the m e c h a n i s m s triggering these processes have yet to be clarified. Thus, the purpose o f this p a p e r was to evaluate D A uptake in the presence a n d absence o f N O p r o d u c t i o n inhibitors a n d the s p o n t a n e o u s D A release induced by G l u receptor agonists a n d antagonists in the presence a n d absence o f N O p r o d u c t i o n inhibitors by using striatal slices a n d synaptosomes.
MATERIAL AND METHODS
[3H]DA uptake and release in the presence and absence of different drugs. In some experiments, raw synapstosome fractions were used. These were prepared from tissue homogenized in 10 ml 0.32 M sucrose and then centrifuged at 1500g in a Beckman JA-20 rotor for 20 min. The supernatant was centrifuged again at 40,000g for 20 min. The resulting pellet was resuspended in 0.32 M sucrose. [~H]DA uptake experiments Uptake experiments were conducted following the scheme previously reported by Beas-Zfirate et al. (1989). Striatal slices (with almost 25 mg of tissue) or crude synaptosomal fractions (0.5mg protein) were preincubated in l ml of Krebs-bicarbonate medium made with 110 mM NaCI, 3 mM KCI, 1.2 mM MgCI2, 1.2 mM NaHzPO4, 25 mM NaHCO3, 1.5 mM CaCI2, 10 mM glucose, 0.01% ascorbic acid and 0.002% pargyline adjusted at a pH of 7.4 with a CO2-O2 mixture (545%). The samples were pre-incubated in a stir bath at 3 7 C for 10min and 20#1 of [3H]DA were added (for a final concentration of 0.1 #M and 0.05 1.0mM for the kinetics studies). In some experiments, DA uptake was measured in the presence of L-arginine (L-Arg) (0.001-1 mM) or with L-NArg (I-500/~M) as a nitric oxide synthase (NOS) inhibitor and in the presence of different aminoacids such as lysine, valine and histidine (0.2 mM). These were added at the same time as the [3H]DA. The incubation was suspended after 20 min by filtration through fiberglass GF/B (Whatman) filters and washed in 10ml of medium. The radioactivity retained in the filters was read with a model LS6000 Beckman scintiscan. Blanks were derived by incubating at O~'~C in every case. Every sample was measured twice. Data are presented as the quantity of labeled dopamine ([3H]DA) accumulated (total uptake minus the blank) for 20 min per mg of tissue or mg of protein, as required.
Materials
[~H]DA release experiments
The different drugs used in this research such as L-arginine (L-Arg) (a NO synthesis precursor), pargyline, L-lysine, Lvaline, L-histidine, and L-N~-nitroarginine (L-N-Arg) (a NOS inhibitor) were purchased from the Sigma Chemical Co. (St Louis, MO, U.S.A.) while N-methyl-D-aspartate (NMDA), kainate (KA) and quisqualate (QA) (Glu agonists); Dizocilpine maleate (MK-801), D-gamma-glutamylaminomethanesulfonic acid (GAMS) and 6-eyano-7nitroquinoxaline-2,3-dione (CNQX) (Glu antagonists) were supplied by RBI (Natick, MA, U.S.A). Other reagents were commercially available with a high degree of purity. 3,4-[7-3H(N)]Dihydroxyphenylethylamine or [3H]dopamine ([3H]DA) (46Ci/mmol) was purchased from New England Nuclear, Wilmington, DE, U.S.A.
Release experiments were conducted following the BeasZ~rate et al. (1989) scheme. To measure the [3H]DA baseline flow, a Krebs-bicarbonate medium made with 115 mM NaCl, 3 mM KCI, 1.2 mM MgSO4, 1.2 mM NaH2PO4, 25 mM NaHCO3, 1.5 mM CaCI2, 10 mM glucose, 0.01% ascorbic acid and 0.002% pargyline, was used, adjusted at a pH of 7.4 with a CO2~O2 mixture (5-95%). Samples were incubated for 20 min in 1 ml of incubation medium at 37°C together with [3H]DA. The samples were then transferred to a filter perfusion Whatman GF/B system. Both the filters and tissue were washed in a Brandel Perfusion model SP-06 at a 0.5ml flow for 15min. The three consecutive 5 min fractions were then collected and taken as the baseline flow. Spontaneous release was conducted with a solution containing Glu agonists for the following four fractions (20 min) with a 10 mM concentration in all cases except for L-Arg (10-200#M). Finally, three additional fractions were collected with a baseline medium. Every fraction was collected in vials with radioactive fluid and radioactivity was measured with a model LS6000 Beckman scintiscan. Spontaneous [3H]DA release was calculated as the amount of DA released in the presence of drugs over the baseline flow (read as 0%). Results are expressed as the overflow percentage of total radioactivity present in the samples with fraction number 6 being the one with the greatest release.
Methods Male Wistar adult (24(I-280 g) rats were used. They were kept under standard conditions with 12 x 12 h light~larkness cycles and free access to water and food. The animals were decapitated and the brain was removed. The brain was immediately washed with a 0.32 M sucrose solution in temperatures ranging from 0 to 4°C. The striatum was dissected out. From the slices, 25 mg of tissue was taken for each study. The slices were subjected to spontaneous
Dopamine release and nitric oxide at striatum
Data analysis
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Also, the effect of some aminoacids such as valine, lysine and histidine were tested to find the L-Arg specificity on [3H]DA uptake. The results showed a slightly decreased uptake induced by different aminoacids (His > Lys > Val) (Fig. 3).
Statistical significancebetween group means was found by an ANOVA analysis using the SPSS computer program. RESULTS
[3H]DA uptake
[3H]DA release
The results show that L-Arg at 10 and 100 aM concentrations did not significantly change the [3H]DA uptake in striatal slices when compared to the control (Fig. 1). L-N-Arg, however, when applied at the same concentrations, reduced the [3H]DA uptake by 33% and 41%, respectively (Fig. 1). The combination of L-Arg and L-N-Arg at 10 and 100 aM concentrations induced inhibition by 23% and 44% of [3H]DA uptake, respectively, when compared to the control (Fig. 1). Synaptosome [3H]DA uptake was significantly increased by 1-10aM of L-Arg [Fig. 2(A)] while high concentrations (0.1-1 mM) were inhibited depending on the concentration [Fig. 2(A)]. L-N-Arg 0 - 5 0 0 aM), however, significantly decreased [3H]DA uptake by 80-90% [Fig. 2(B)]. In order to determine the possible inhibition mechanism by L-N-Arg, [3H]DA uptake kinetics studies were conducted in the presence of the inhibitor L-NArg at 1 and 100aM concentrations. The results did not show typical inhibition although a definite 8090% inhibition was seen (Table 1).
Release results showed that Glu-R agonists induce a differential increase in spontaneous [3H]DA release in striatal slices. Thus NMDA, KA and QA application at a 10 #M concentration increased the release by 97, 77 and 15%, respectively, compared to the transmitter baseline release [Fig. 4(A, B and C)]. Nonetheless, the independent presence of MK-801, GAMS and CNQX decreased [3H]DA release over the baseline value [Fig. 4(A, B and C)]. Like Glu agonists, L-Arg (10 aM) had a stimulating effect on this release by more than 220% [Fig. 4(A, B and C)]. NMDA, combined with L-Arg, produced no major changes on the [3H]DA release over those seen in the presence of N M D A alone [Fig. 4(A)]. In all cases, however, L-N-Arg, 10aM, alone or combined with other compounds, decreased spontaneous [3H]DA release [Fig. 4(A, B and C)]. On the other hand, L-Arg alone induced a concentration dependent stimulating effect [Fig. 5(A)]. Thus, at 100 and 200 aM concentrations, L-Arg combined with N M D A or Kainate, 10#M, increased [3H]DA release proportionately to that seen with L-
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Fig. 1. Effect of L-Arg ( 10 and 100/~M), L-N-Arg ( 10 and 100tzM) and L-Arg+ L-N-Arg (10 and 100 #M) on [3H]DAuptake from striatal slices. Data represent the means +__SD of 8-10 experiments; determinations were done by duplicate. Statistically different from control at: [] P < 0.05; *P < 0.001.
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