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Rat striatal dopamine tetrahydrobiopterin content following an intrastriatal injection of manganese chloride
Life Sciences, Vol. 38, pp. 2121-2127 Printed in the U.S.A.
Pergamon Press
RAT STRIATAL DOPAMLNE AND TETRAHYDRDBIOPTERINOONTENT FOLLOWING AN INTRASrRIATAL INJECTION OF ~iANGANESE CHLORIDE.
A. Lista, J. Abarca, C. Ramos and A.J. Daniels* Laboratory of Biochemical Pharmacology Department of Cell Biology P. Universidad Cat61ica de Chile (Received in final form March 18, 1986) Summary Injection of manganese into the rat corpus striatum causes a rapid fall in the biopterin and dopamine (DA) content ipsilateral to the lesion. Two weeks after the lesion both biopterin and DA are partially recovered. Controls, injected with saline or magnesium, do not show alterations in their DA or cofactor levels. It is proposed that the fall in DA levels results from a rapid displacement of the amine from its storage sites by manganese followed by a decrease in the rate of DA synthesis causes by the drop in cofactor levels. Manganese poisoning is characterized by classical symptoms of Parkinson's disease accompanied by a substantial decrease in brain dopamine (1,2,3) suggest ing that the mechanisms underlying the neurotoxic effect of the metal ion are in some way related to a functional deficiency of the nigro-striatal dopaminergic pathway. We have shown previously (4) that rat striatal slices incubated in the presence of manganese, accumulate as much metal ion (3 vg/g tissue) as that found in the striatum of monkeys exposed to manganese for 18 months (5). Under these conditions, the accumulated metal ion displaces dopamine from its storage sites and can be released by K + depolarization (4). Dopamine synthesis depends upon the activity of tyrosine hydroxylase (6) a mixed function oxidase that requires molecular oxygen and L-erythro-5,6,7,8tetrahydrobiopterin (BH4) as a cofactor (7,8). It has been shown that cerebrospinal fluid (CSF) from Parkinsonian patients contains half the amount of BH 4 than that of age matched control patients (9,10). To our knowledge, there is no information in the literature on the effects of manganese, either acute or chronically administered, on brain cofactor levels. Since there is good evidence that tyrosine hydroxylase can be finely regulated through the availability of its natural cofactor (11,12) in this work we have studied, as a first approach, the fate of striatal biopterin and dopamine after a single intrastriatal injection of manganese chloride. ~t~ds Male Sprague Dawley rats (250 g) were anesthetized with pentobarbital (35 mg/kg) and injected under stereotaxic control into the right striatum with different concentrations of manganese chloride (in saline) in a total volume of 1 ~I (0.2 ~ft/min) through a i0 ~i Hamilton syringe that was left in place for five minutes after the injection. Coordinates for the placement of the syringe tip were: anterior-posterior 7.4 ram., dorsal-ventral 4.0 mm and lateral 2.5 according to the stereotaxic Atlas of K6nig & Klippel (13). *Address reprint requests to: Dr. A.J. Daniels, The Wellcome Research Labs., Dept. Med. Biochem., 3030 Cornwallis Rd., Research Triangle Park, NC 27709 0024-3205/86 $3.00 + .00 Copyright (c) 1986 Pergamon Press Ltd.
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Animals were killed by decapitation at different times after the lesion and the striata dissected, weighed and quickly frozen. At each time point, age matched non injected controls were also sacrificed. Each striatum was homgenized separately in 1.5 ml of 0.I N HCI for 15 sec with an Ultra Turrax (Ika-Werk) and aliquots were taken for the determination of biopterins and dopamine. Biopterins were measured by the method of Fukushima and Nixon (14). Basically, the method consists of oxidation by iodine of the various forms of biopterin under acidic and alkaline conditions. All forms of biopterin are stable under acidic conditions, whereas only biopterin and dihydrobiopterin are stable under oxidation in alkaline conditions. The difference between biopterin values under acid an alkaline conditions represents cofactor-active forms of biopterin (BH4 plus quinoid dihydrobiopterin). Since we found that 85 to 90 percent of the tissue biopterin was present as BH 4 and this proportion remained unchanged throughout the time course of the experiment, routinely we determined only total biopterins by acidic oxidation. This allowed us to compare the values obtained in the lesioned striatum with those in the contralateral, from the same animal. Otherwise, tissue samples must be pooled from at least two animals to perform acidic and alkaline oxidation. The biopterin initially present and that formed during oxidation is purified by ion-exchange colunm chromatography, followed by high performance liquid chromatography (C18, i0~, Whatman) and detected by its native fluorescence using an LDC Fluoromenitor III with a 340-380 nm excitation source and a 418-700 nm emission filter. Dopamine was determined by precipitating 0.3 ml of homogenate with perchloric acid (0.1 N final concentration) and the clear supernatant analyzed by reversed phase ion-pairing HPLC (C18, 5U, Rainin) and electrochemical detection (BAS). The mobile phase consisted of 70 mM sodium phosphate, 0.2 mM sodium octyl sulphate, 0.01%EDTA and 10% methanol pH 2.6. The oxidation potential was set at 0.7V. T i s s u e manganese c o n t e n t was d e t e r m i n e d tometry ( P e r k i n Elmer 370) i n a s e p a r a t e group and c o n t r a l a t e r a l s t r i a t a from t ~ r a t s (8 r a t s p a r a t e l y and homogenized i n 15% t r i c h l o r o a c e t i c a n a l y z e d f o r metal i o n c o n t e n t .
by atomic a b s o r p t i o n s p e c t r o p h o of l e s i o n e d animals. Lesioned p e r t i m e p o i n t ) were pooled sea c i d and t h e c l e a r s u p e r n a t a n t
+
R e s u l t s a r e e x p r e s s e d as the mean - s t a n d a r d e r r o r of t h e mean. S t a t i s t i c a l d i f f e r e n c e s were t e s t e d by a two t a i l e d p a i r e d t - t e s t .
Results The e f f e c t o f an i n t r a s t r i a t a l i n j e c t i o n o f manganese (10 t o 50 ~g) on the t i s s u e b i o p t e r i n c o n t e n t i s p r e s e n t e d i n T a b l e I . A dose dependent dec r e a s e of the c o f a c t o r l e v e l can be o b s e r v e d i n the l e s i o n e d s i d e w i t h r e s p e c t to t h a t i n the c o n t r a l a t e r a l s t r i a t u m . A small but s i g n i f i c a n t d e c r e a s e i n b i o p t e r i n i n the l e s i o n e d s i d e o c c u r s 24 h r s a f t e r the i n j e c t i o n o f 10 ug o f manganese. The e f f e c t i s much g r e a t e r , and even e x t e n d s to the non l e s i o n e d c o n t r a l a t e r a l s t r i a t u m , when t h e dose i s i n c r e a s e d t o 50 ug. The i n t e r m e d i a t e dose o f 25 ~g of manganese s t i l l p r o d u c e s a s i g n i f i c a n t e f f e c t b u t o n l y i n t h e l e s i o n e d s i d e t h e r e f o r e , i t was chosen f o r the n e x t e x p e r i m e n t s . No d i f f e r e n c e s were found i n the b i o p t e r i n o r dopamine c o n t e n t between the r i g h t and l e f t c o r pus s t r i a t u m i n c o n t r o l s r a t s ( n o t shown).
To study how specific was the effect of manganese and at the same time rule out a simple osmotic effect, we injected rats with an equivalent amount of magnesium or with saline. Table II shows that 24 or 48 hrs after an intrastriatal injection of 25 ~g of magnesium (MgCI2) there is no change in the tissue biopterin content. Biopterin levels also remain unaltered 48 hrs after an injection of saline.
Vol. 38, No. 23, 1986
Manganese and Striatal Biopterln and DA
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TABLE I Effect of an Intrastriatal Injection of M~CI 2 on Biopterin Levels
Total Biopterin (ng/gr. tissue)
Manganese (~g) I0 25 50
(5) (8) (ii)
Lesioned
Contralateral
t 227.8 -+ 3.5 * 170.9 + 4.4 * 100.8 + 2.0
258.9 + 4.0 250.6 + 5.3 188.4 +- 4.4
Animals were sacrificed 24 hrs after injection of different amounts of manganese (MnCI2) into the right striation. In parenthesis the number of animals. Results are expressed as the mean + S.E.M. t 0.05 > p > 0.02; * p < 0.001 by paired t test. TABLE II Effect of an Intrastriatal
Injection of Magnesium Chloride or Saline on Biopterin Levels
Total Biopterin (ng/gr. tissue) Conditions
Saline MgCI 2 MgCl 2
(48 h) (24 h) (48 h)
(3) (5) (5)
Injected
Contralateral
251.9 +- 13.6 242.3 + 11.4 221.2 -+ 9.6
250.6 + 8.0 229.1 + 6.8 227.0 -+ 4.4
In parenthesis the number of animals. Results are expressed as the mean + S.E.M. No statistical differences (paired t-test) were found between the injected and contralateral stricture.
A parallel decrease of both, DA and biopterin is observed when the time course of a single intrastriatal injection of 25 ~g of manganese is followed. Figure 1 shows that 4 hrs after the injection, the lesioned side already presents a significant decrease in DA and biopterin with respect to the contralateral side. The decrease in biopterin content is maximal between 48 and 72 hrs and then slowly recovers however, not totally even after two weeks. Only at the time point of 72 hrs there is a significant decrease in the biopterin content of the contralateral striatum with respect to that in non injected controls (see legend to figure I). The decrease in DA is maximal by 72 hrs and then also starts to recover a l t h ~ g h after two weeks it has not reached the contralateral DA content which, in contrast to the biopterin content, does not vary with respect to that in non injected controls.
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Manganese and Striatal Biopterin and DA
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[ ] confrolofero[ []
tesioned
Tofol biopferin ..T.. T
--T-,T
250"
t
200-
Oopomine 10
1"
T
T -r
*
8
///
150
._~
o~ 100//
~,2
50"
//1
T
t
///
== .4--
T
//
/// /// /// /// ///
4 2L~ L~ 72 168 336 hr
1 /, 2L~/,8 72 168 336 hr
FIG. 1 Time course of the effect of intrastriatally injected n~nganese on DA and biopterin levels, Results are expressed as the mean -+ S.E.M. obtained from 5-8 animals per time point. An equal number of non-injected controls were sacrificed at each ti~e, The striatal biopterin ~und DA values for non-injected controls were: 231 - 22.9 ng/g tissue and 9.3 "- 0.9 ~g/g tissue respectively. * p < 0.001; f p < 0.01; ~ 0.02 > p > 0.01: lesioned vs contralateral (paired t test) ** p < 0.001: contralateral vs non injected control (t-test).
Manganese
S
3
/1t
~
2 ¸
Ill
o
[ ] lesioned
, ~,
E -~-
C~ /// fl] I/I
1
4 24 48 72 168 hr FIG. 2
Manganese clearance curve. Results are shown as the total content of metal ion (~g) per striation and expressed as the mean -+ S.E.M. n=8 animals per time point.
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Manganese and Striatal Biopterin and DA
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The manganese clearance curve is shown in Figure 2. One hour after the lesion, 20 percent of the metal ion injected is still in the striat~ and thereafter slowly diffuses out. It can be observed that 24 hrs after the injection, manganese starts accumulating in the contralateral side. Discussion In this paper we have shown that intrastriatally administered manganese causes a large depletion of DA and biopterin in the striatum ipsilateral to the lesion. The metal ion's effect presents a fast onset however, its action is apparently not long lasting. Eventhough we have not followed the subcellular distribution of manganese, we think that the rapid initial fall in striatal DA could be explained by displacement of the amine from its storage sites by the metal ion, followed by extravesicular enzy~tic oxidation or non enzymatic autooxidation favoured in the presence of manganese (15). Support for this hypothesis derives from our previous findings where manganese readily acc~ulates into rat striatal slices and once incorporated can be co-released with DA by K + depolarization following the same time course (4). In addition, we have found that manganese can induce the release of [3H]DA previously taken up by dopaminergic terminals from rat striatal slices (4) and also, we have shown that chromaffin granules, the storage vesicles for adrenaline in the adrenal medulla, accumulate manganese and therein by binding to ATP displaces the amine from its storage complex (16,17). Biopterin levels also show a decrease shortly after the lesion although not to the extent of that of DA. The fall in cofactor could be due to a direct oxidation by manganese or the products of DA autooxidation (free radicals, H202) enhanced by nmnganese. We have not followed the activity of catalase or peroxidase after the lesion with manganese however, preliminary experiments lesioning rats pretreated with Nafenopin, a fatty acid analog that produces a proliferation of brain peroxisomes (18), do not indicate any protective action of this agent against the effect of the metal ion on striatal DA or biopterin. It is interesting to observe that while manganese diffuses and starts accumulating in the contralateral striatum by 24 hrs, it is only after 72 hrs that we observe an effect over the biopterin content but still no effect on DA levels. This could be interpreted in terms that the metal ion in the contralateral side does not reach the DA storage sites but can induce a slow oxidation of the cofactor present in the extravesicular compartment. There is evidence suggesting that the intraneuronal concentration of BH4 is subsaturating or very close to Km (19,20) and thus limit the activity of tyrosine hydroxylase in dopaminergic neurons. A fall in cofactor levels in the striatum, induced by manganese, would therefore decrease the rate of DA synthesis until new cofactor is synthesized maintaining the low levels of DA. In fact, we have found that striatal slices, previously incubated in the presence of 1 mM manganese, show a ~ r k e d depression on their capacity to synthesize [14C]dopamine from [14C]tyrosine (unpublished results). In addition, experiments in progress in our laboratory show that after intranigral injections of manganese there is a parallel time course of decrease in cofactor and tyrosine hydroxylation capacity in the striatum ipsilateral to the injection. We have not followed the activity of GTP cyclohydrolase (the first enzyme in ~ 4 biosynthesis) after the lesion with manganese but this could be a determinant factor in the recovery of biopterin and DA levels. It has been suggested that manganese could produce its neurotoxic effect
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Vol. 38, No. 23, 1986
in a s i m i l a r way as 6-hydroxydopamine (21, 15). Under our e x p e r i m e n t a l c o n d i t i o n s however, t h e e f f e c t s of manganese a r e of r e l a t i v e s h o r t d u r a t i o n , sugg e s t i n g t h a t the metal ion m u l d n o t cause a d e g e n e r a t i o n of t h e dopaminergic nerve t e r m i n a l s . N e v e r t h e l e s s , we can not r u l e out t h e p o s s i b i l i t y t h a t manganese could indeed produce a p a r t i a l d e g e n e r a t i o n of t h e n e r v e t e r m i n a l s and, t h a t t h e recovery o f b i o p t e r i n and DA l e v e l s could d e r i v e from a compensatory i n c r e a s e d s y n t h e s i s in t h e remaining t e r m i n a l s . On t h e o t h e r hand, most of the s t r i a t a l c o f a c t o r has been s u g g e s t e d to be c o n t a i n e d in dopaminergic t e r minals (22) t h e r e f o r e , one would expect an equal d e c r e a s e of b e t h b i o p t e r i n and DA i f t h e r e i s a d e g e n e r a t i v e p r o c e s s . While the r a t i o of the p e r c e n t d e c r e a s e o f both s u b s t r a t e s can be c a l c u l a t e d to be l i n e a r throughout t h e exp e r i m e n t , q u a n t i t a t i v e l y the p e r c e n t d e c r e a s e of b i o p t e r i n and DA a r e q u i t e different. In conclusion, our results would indicate that after an intrastriatal inj e c t i o n of manganese, the metal ion produces a d e c r e a s e in DA c o n t e n t by two mechanisms: F i r s t , by d i s p l a c e m e n t o f the amine from i t s s t o r a g e s i t e s , and second by a d i r e c t or i n d i r e c t e f f e c t o f manganese (enhancing e x t r a v e s i c u l a r DA a u t o o x i d a t i o n ) o v e r t h e b i o p t e r i n c o f a c t o r d e c r e a s i n g t h e r a t e of DA synt h e s i s . At t h i s p o i n t we do not know whether t h e observed recovery o f DA and b i o p t e r i n l e v e l s corresponds to compensatory mechanisms t h a t c o u l d be followed in the l o n g term by a d e g e n e r a t i o n o f the t e r m i n a l s o r i f t h e e f f e c t of manganese under our c o n d i t i o n s i s t r u l l y r e v e r s i b l e . We a r e c u r r e n t l y i n v e s t i g a t i n g t h i s m a t t e r as w e l l as the e f f e c t of manganese over s t r i a t a l dopamine r g i c t e r m i n a l s when i n j e c t e d i n t o the s u b s t a n t i a n i g r a . #~knpwl edgements We thank Mrs Lucy Chacoff f o r t y p i n g t h e manuscript. This work was supp o r t e d by g r a n t s (to A . J . D . ) 65/84 from t h e U n i v e r s i d a d Cat61ica de C h i l e ; 1192/84 from Conicyt and a donation (TXRC/83/4-233) from t h e Wellcome Research L a b o r a t o r i e s (USA). References i.
O. HORNYKIEWICZ, in Biochemistry and Pharmacology of the Basal Ganglia (Eds. E. Costa, L. CA3te and Nu Yahe), pp 171-185. Raven Press, New York (1966). 2. I. ~ N A , O. MARIN, S. FUENZALIDA and G.C. (1)TZIAS. Neurology, 17; 11231129 (1967). 3. S.J. MDSTAFA and S.V. gAANDRA. J. Neurochem. 18; 931-933 (1971). 4. A.J. DANIELS, K. GYSLING and J. ABARCA. Biochem. Pharmacol. 30; 1833-1837 (1981). S. S.V. C]~ANDRA, R.8. SRIVASTAVAand G.S. SHU]~A. Toxic. L e t t . 4; 189-192 (1979). 6. N. LEVITT, S. SPECTOR, A. SJOERDSMA and S. UDENFRII~qD. J. Pharmac. Exp. Ther. 148; 1-8 (1965). 7. S. KAUFb,P~q and D.B. FISHER in Yolecular i~chanisms of Oxygen Activation (ed. O. Hayashi) pp 285-369 Academic Press, New York (1974). 8. N. WEINER. Psychopharmacol. Bull. 14; 40-44 (1978). 9. W. IDVENBERG, R.A. LEVINE, D.S. ROBIN---~N, M. EBERT, A.C. W I L L I A ~ and D.B. CALNE. Science 204; 624-626 (1979). I0. A.C. WILLIAMS, R.A. LEVINE, T.N. (IIASE, W. LOVI~ERG and D.B. CALNE, J. Neurol. Neurosurg and Psychiat. 43; 735-738 (1980). ii. O.H. VIVEROS, C-L. LEE. M.M. ABOU----DONIA,J.C. NIXON and C.A. NIGIOL. Science 213; 349-350 (198]). 12. M.M. ABOU-DONIA, A.J. DANIELS, S.P. WILSON, C.A. NI(IIOL and O.H. VIVEROS in Chemistry and Biology of Pteridines (ed. J.A. Blair) pp 777-781 Walter
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13. 14. 15. 16. 17.
18. 19. 20. 21. 22.
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de Gruyter, Berlin (1983). J.F.R. KONIG and R.A. KLIPPEL. The Rat Brain - A stereotaxic atlas of the forebrain and lower parts of the brain stem. Krieger Huntingdon. New York (1963). T. FUKUSHIMA and J.C. NIXON, Anal. Biochem. 102; 176-188 (1980). J. DONALDSON, F.S. LABELLA and D. GESSER. Neurotoxicology 2; 53-64 (1981). A.J. DANIELS, R.J.P. WILLI/~S and P.E. WRIGHT. Neuroscienc-e 3; 573-585 (1978). A.J. DANIELS, L.N. JOHNSON and R.J.P. W I L L I ~ . J. Neurochem. 33; 923929 (1979). F. LEIGHTON and O. LAZO. Arch. Biol. Med. Exp. 15; 447-456 (1982). R. KETTLER, G. BARTHOLINI and A. PLETSCHER. Nature (London) 249; 476478 (1974). R.L. PATRICK and J.D. BARCHAS. J. Pharmacol. Exp. Ther. 197; 97-104 (1976). G. (DHEN and R.E. HEIKKILA. J. B i o l . Chem. 249; 2447-2452 (1974). R.A. LEVINE, L.P. MILLER and W. LOVENBERG. Science 214; 919-921 (1981).
Pergamon Press
RAT STRIATAL DOPAMLNE AND TETRAHYDRDBIOPTERINOONTENT FOLLOWING AN INTRASrRIATAL INJECTION OF ~iANGANESE CHLORIDE.
A. Lista, J. Abarca, C. Ramos and A.J. Daniels* Laboratory of Biochemical Pharmacology Department of Cell Biology P. Universidad Cat61ica de Chile (Received in final form March 18, 1986) Summary Injection of manganese into the rat corpus striatum causes a rapid fall in the biopterin and dopamine (DA) content ipsilateral to the lesion. Two weeks after the lesion both biopterin and DA are partially recovered. Controls, injected with saline or magnesium, do not show alterations in their DA or cofactor levels. It is proposed that the fall in DA levels results from a rapid displacement of the amine from its storage sites by manganese followed by a decrease in the rate of DA synthesis causes by the drop in cofactor levels. Manganese poisoning is characterized by classical symptoms of Parkinson's disease accompanied by a substantial decrease in brain dopamine (1,2,3) suggest ing that the mechanisms underlying the neurotoxic effect of the metal ion are in some way related to a functional deficiency of the nigro-striatal dopaminergic pathway. We have shown previously (4) that rat striatal slices incubated in the presence of manganese, accumulate as much metal ion (3 vg/g tissue) as that found in the striatum of monkeys exposed to manganese for 18 months (5). Under these conditions, the accumulated metal ion displaces dopamine from its storage sites and can be released by K + depolarization (4). Dopamine synthesis depends upon the activity of tyrosine hydroxylase (6) a mixed function oxidase that requires molecular oxygen and L-erythro-5,6,7,8tetrahydrobiopterin (BH4) as a cofactor (7,8). It has been shown that cerebrospinal fluid (CSF) from Parkinsonian patients contains half the amount of BH 4 than that of age matched control patients (9,10). To our knowledge, there is no information in the literature on the effects of manganese, either acute or chronically administered, on brain cofactor levels. Since there is good evidence that tyrosine hydroxylase can be finely regulated through the availability of its natural cofactor (11,12) in this work we have studied, as a first approach, the fate of striatal biopterin and dopamine after a single intrastriatal injection of manganese chloride. ~t~ds Male Sprague Dawley rats (250 g) were anesthetized with pentobarbital (35 mg/kg) and injected under stereotaxic control into the right striatum with different concentrations of manganese chloride (in saline) in a total volume of 1 ~I (0.2 ~ft/min) through a i0 ~i Hamilton syringe that was left in place for five minutes after the injection. Coordinates for the placement of the syringe tip were: anterior-posterior 7.4 ram., dorsal-ventral 4.0 mm and lateral 2.5 according to the stereotaxic Atlas of K6nig & Klippel (13). *Address reprint requests to: Dr. A.J. Daniels, The Wellcome Research Labs., Dept. Med. Biochem., 3030 Cornwallis Rd., Research Triangle Park, NC 27709 0024-3205/86 $3.00 + .00 Copyright (c) 1986 Pergamon Press Ltd.
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Animals were killed by decapitation at different times after the lesion and the striata dissected, weighed and quickly frozen. At each time point, age matched non injected controls were also sacrificed. Each striatum was homgenized separately in 1.5 ml of 0.I N HCI for 15 sec with an Ultra Turrax (Ika-Werk) and aliquots were taken for the determination of biopterins and dopamine. Biopterins were measured by the method of Fukushima and Nixon (14). Basically, the method consists of oxidation by iodine of the various forms of biopterin under acidic and alkaline conditions. All forms of biopterin are stable under acidic conditions, whereas only biopterin and dihydrobiopterin are stable under oxidation in alkaline conditions. The difference between biopterin values under acid an alkaline conditions represents cofactor-active forms of biopterin (BH4 plus quinoid dihydrobiopterin). Since we found that 85 to 90 percent of the tissue biopterin was present as BH 4 and this proportion remained unchanged throughout the time course of the experiment, routinely we determined only total biopterins by acidic oxidation. This allowed us to compare the values obtained in the lesioned striatum with those in the contralateral, from the same animal. Otherwise, tissue samples must be pooled from at least two animals to perform acidic and alkaline oxidation. The biopterin initially present and that formed during oxidation is purified by ion-exchange colunm chromatography, followed by high performance liquid chromatography (C18, i0~, Whatman) and detected by its native fluorescence using an LDC Fluoromenitor III with a 340-380 nm excitation source and a 418-700 nm emission filter. Dopamine was determined by precipitating 0.3 ml of homogenate with perchloric acid (0.1 N final concentration) and the clear supernatant analyzed by reversed phase ion-pairing HPLC (C18, 5U, Rainin) and electrochemical detection (BAS). The mobile phase consisted of 70 mM sodium phosphate, 0.2 mM sodium octyl sulphate, 0.01%EDTA and 10% methanol pH 2.6. The oxidation potential was set at 0.7V. T i s s u e manganese c o n t e n t was d e t e r m i n e d tometry ( P e r k i n Elmer 370) i n a s e p a r a t e group and c o n t r a l a t e r a l s t r i a t a from t ~ r a t s (8 r a t s p a r a t e l y and homogenized i n 15% t r i c h l o r o a c e t i c a n a l y z e d f o r metal i o n c o n t e n t .
by atomic a b s o r p t i o n s p e c t r o p h o of l e s i o n e d animals. Lesioned p e r t i m e p o i n t ) were pooled sea c i d and t h e c l e a r s u p e r n a t a n t
+
R e s u l t s a r e e x p r e s s e d as the mean - s t a n d a r d e r r o r of t h e mean. S t a t i s t i c a l d i f f e r e n c e s were t e s t e d by a two t a i l e d p a i r e d t - t e s t .
Results The e f f e c t o f an i n t r a s t r i a t a l i n j e c t i o n o f manganese (10 t o 50 ~g) on the t i s s u e b i o p t e r i n c o n t e n t i s p r e s e n t e d i n T a b l e I . A dose dependent dec r e a s e of the c o f a c t o r l e v e l can be o b s e r v e d i n the l e s i o n e d s i d e w i t h r e s p e c t to t h a t i n the c o n t r a l a t e r a l s t r i a t u m . A small but s i g n i f i c a n t d e c r e a s e i n b i o p t e r i n i n the l e s i o n e d s i d e o c c u r s 24 h r s a f t e r the i n j e c t i o n o f 10 ug o f manganese. The e f f e c t i s much g r e a t e r , and even e x t e n d s to the non l e s i o n e d c o n t r a l a t e r a l s t r i a t u m , when t h e dose i s i n c r e a s e d t o 50 ug. The i n t e r m e d i a t e dose o f 25 ~g of manganese s t i l l p r o d u c e s a s i g n i f i c a n t e f f e c t b u t o n l y i n t h e l e s i o n e d s i d e t h e r e f o r e , i t was chosen f o r the n e x t e x p e r i m e n t s . No d i f f e r e n c e s were found i n the b i o p t e r i n o r dopamine c o n t e n t between the r i g h t and l e f t c o r pus s t r i a t u m i n c o n t r o l s r a t s ( n o t shown).
To study how specific was the effect of manganese and at the same time rule out a simple osmotic effect, we injected rats with an equivalent amount of magnesium or with saline. Table II shows that 24 or 48 hrs after an intrastriatal injection of 25 ~g of magnesium (MgCI2) there is no change in the tissue biopterin content. Biopterin levels also remain unaltered 48 hrs after an injection of saline.
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Manganese and Striatal Biopterln and DA
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TABLE I Effect of an Intrastriatal Injection of M~CI 2 on Biopterin Levels
Total Biopterin (ng/gr. tissue)
Manganese (~g) I0 25 50
(5) (8) (ii)
Lesioned
Contralateral
t 227.8 -+ 3.5 * 170.9 + 4.4 * 100.8 + 2.0
258.9 + 4.0 250.6 + 5.3 188.4 +- 4.4
Animals were sacrificed 24 hrs after injection of different amounts of manganese (MnCI2) into the right striation. In parenthesis the number of animals. Results are expressed as the mean + S.E.M. t 0.05 > p > 0.02; * p < 0.001 by paired t test. TABLE II Effect of an Intrastriatal
Injection of Magnesium Chloride or Saline on Biopterin Levels
Total Biopterin (ng/gr. tissue) Conditions
Saline MgCI 2 MgCl 2
(48 h) (24 h) (48 h)
(3) (5) (5)
Injected
Contralateral
251.9 +- 13.6 242.3 + 11.4 221.2 -+ 9.6
250.6 + 8.0 229.1 + 6.8 227.0 -+ 4.4
In parenthesis the number of animals. Results are expressed as the mean + S.E.M. No statistical differences (paired t-test) were found between the injected and contralateral stricture.
A parallel decrease of both, DA and biopterin is observed when the time course of a single intrastriatal injection of 25 ~g of manganese is followed. Figure 1 shows that 4 hrs after the injection, the lesioned side already presents a significant decrease in DA and biopterin with respect to the contralateral side. The decrease in biopterin content is maximal between 48 and 72 hrs and then slowly recovers however, not totally even after two weeks. Only at the time point of 72 hrs there is a significant decrease in the biopterin content of the contralateral striatum with respect to that in non injected controls (see legend to figure I). The decrease in DA is maximal by 72 hrs and then also starts to recover a l t h ~ g h after two weeks it has not reached the contralateral DA content which, in contrast to the biopterin content, does not vary with respect to that in non injected controls.
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Manganese and Striatal Biopterin and DA
Vol. 38, No. 23, 1986
[ ] confrolofero[ []
tesioned
Tofol biopferin ..T.. T
--T-,T
250"
t
200-
Oopomine 10
1"
T
T -r
*
8
///
150
._~
o~ 100//
~,2
50"
//1
T
t
///
== .4--
T
//
/// /// /// /// ///
4 2L~ L~ 72 168 336 hr
1 /, 2L~/,8 72 168 336 hr
FIG. 1 Time course of the effect of intrastriatally injected n~nganese on DA and biopterin levels, Results are expressed as the mean -+ S.E.M. obtained from 5-8 animals per time point. An equal number of non-injected controls were sacrificed at each ti~e, The striatal biopterin ~und DA values for non-injected controls were: 231 - 22.9 ng/g tissue and 9.3 "- 0.9 ~g/g tissue respectively. * p < 0.001; f p < 0.01; ~ 0.02 > p > 0.01: lesioned vs contralateral (paired t test) ** p < 0.001: contralateral vs non injected control (t-test).
Manganese
S
3
/1t
~
2 ¸
Ill
o
[ ] lesioned
, ~,
E -~-
C~ /// fl] I/I
1
4 24 48 72 168 hr FIG. 2
Manganese clearance curve. Results are shown as the total content of metal ion (~g) per striation and expressed as the mean -+ S.E.M. n=8 animals per time point.
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Manganese and Striatal Biopterin and DA
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The manganese clearance curve is shown in Figure 2. One hour after the lesion, 20 percent of the metal ion injected is still in the striat~ and thereafter slowly diffuses out. It can be observed that 24 hrs after the injection, manganese starts accumulating in the contralateral side. Discussion In this paper we have shown that intrastriatally administered manganese causes a large depletion of DA and biopterin in the striatum ipsilateral to the lesion. The metal ion's effect presents a fast onset however, its action is apparently not long lasting. Eventhough we have not followed the subcellular distribution of manganese, we think that the rapid initial fall in striatal DA could be explained by displacement of the amine from its storage sites by the metal ion, followed by extravesicular enzy~tic oxidation or non enzymatic autooxidation favoured in the presence of manganese (15). Support for this hypothesis derives from our previous findings where manganese readily acc~ulates into rat striatal slices and once incorporated can be co-released with DA by K + depolarization following the same time course (4). In addition, we have found that manganese can induce the release of [3H]DA previously taken up by dopaminergic terminals from rat striatal slices (4) and also, we have shown that chromaffin granules, the storage vesicles for adrenaline in the adrenal medulla, accumulate manganese and therein by binding to ATP displaces the amine from its storage complex (16,17). Biopterin levels also show a decrease shortly after the lesion although not to the extent of that of DA. The fall in cofactor could be due to a direct oxidation by manganese or the products of DA autooxidation (free radicals, H202) enhanced by nmnganese. We have not followed the activity of catalase or peroxidase after the lesion with manganese however, preliminary experiments lesioning rats pretreated with Nafenopin, a fatty acid analog that produces a proliferation of brain peroxisomes (18), do not indicate any protective action of this agent against the effect of the metal ion on striatal DA or biopterin. It is interesting to observe that while manganese diffuses and starts accumulating in the contralateral striatum by 24 hrs, it is only after 72 hrs that we observe an effect over the biopterin content but still no effect on DA levels. This could be interpreted in terms that the metal ion in the contralateral side does not reach the DA storage sites but can induce a slow oxidation of the cofactor present in the extravesicular compartment. There is evidence suggesting that the intraneuronal concentration of BH4 is subsaturating or very close to Km (19,20) and thus limit the activity of tyrosine hydroxylase in dopaminergic neurons. A fall in cofactor levels in the striatum, induced by manganese, would therefore decrease the rate of DA synthesis until new cofactor is synthesized maintaining the low levels of DA. In fact, we have found that striatal slices, previously incubated in the presence of 1 mM manganese, show a ~ r k e d depression on their capacity to synthesize [14C]dopamine from [14C]tyrosine (unpublished results). In addition, experiments in progress in our laboratory show that after intranigral injections of manganese there is a parallel time course of decrease in cofactor and tyrosine hydroxylation capacity in the striatum ipsilateral to the injection. We have not followed the activity of GTP cyclohydrolase (the first enzyme in ~ 4 biosynthesis) after the lesion with manganese but this could be a determinant factor in the recovery of biopterin and DA levels. It has been suggested that manganese could produce its neurotoxic effect
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Vol. 38, No. 23, 1986
in a s i m i l a r way as 6-hydroxydopamine (21, 15). Under our e x p e r i m e n t a l c o n d i t i o n s however, t h e e f f e c t s of manganese a r e of r e l a t i v e s h o r t d u r a t i o n , sugg e s t i n g t h a t the metal ion m u l d n o t cause a d e g e n e r a t i o n of t h e dopaminergic nerve t e r m i n a l s . N e v e r t h e l e s s , we can not r u l e out t h e p o s s i b i l i t y t h a t manganese could indeed produce a p a r t i a l d e g e n e r a t i o n of t h e n e r v e t e r m i n a l s and, t h a t t h e recovery o f b i o p t e r i n and DA l e v e l s could d e r i v e from a compensatory i n c r e a s e d s y n t h e s i s in t h e remaining t e r m i n a l s . On t h e o t h e r hand, most of the s t r i a t a l c o f a c t o r has been s u g g e s t e d to be c o n t a i n e d in dopaminergic t e r minals (22) t h e r e f o r e , one would expect an equal d e c r e a s e of b e t h b i o p t e r i n and DA i f t h e r e i s a d e g e n e r a t i v e p r o c e s s . While the r a t i o of the p e r c e n t d e c r e a s e o f both s u b s t r a t e s can be c a l c u l a t e d to be l i n e a r throughout t h e exp e r i m e n t , q u a n t i t a t i v e l y the p e r c e n t d e c r e a s e of b i o p t e r i n and DA a r e q u i t e different. In conclusion, our results would indicate that after an intrastriatal inj e c t i o n of manganese, the metal ion produces a d e c r e a s e in DA c o n t e n t by two mechanisms: F i r s t , by d i s p l a c e m e n t o f the amine from i t s s t o r a g e s i t e s , and second by a d i r e c t or i n d i r e c t e f f e c t o f manganese (enhancing e x t r a v e s i c u l a r DA a u t o o x i d a t i o n ) o v e r t h e b i o p t e r i n c o f a c t o r d e c r e a s i n g t h e r a t e of DA synt h e s i s . At t h i s p o i n t we do not know whether t h e observed recovery o f DA and b i o p t e r i n l e v e l s corresponds to compensatory mechanisms t h a t c o u l d be followed in the l o n g term by a d e g e n e r a t i o n o f the t e r m i n a l s o r i f t h e e f f e c t of manganese under our c o n d i t i o n s i s t r u l l y r e v e r s i b l e . We a r e c u r r e n t l y i n v e s t i g a t i n g t h i s m a t t e r as w e l l as the e f f e c t of manganese over s t r i a t a l dopamine r g i c t e r m i n a l s when i n j e c t e d i n t o the s u b s t a n t i a n i g r a . #~knpwl edgements We thank Mrs Lucy Chacoff f o r t y p i n g t h e manuscript. This work was supp o r t e d by g r a n t s (to A . J . D . ) 65/84 from t h e U n i v e r s i d a d Cat61ica de C h i l e ; 1192/84 from Conicyt and a donation (TXRC/83/4-233) from t h e Wellcome Research L a b o r a t o r i e s (USA). References i.
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