Decarboxylation of exogenous L-DOPA in rat striatum after lesions of the dopaminergic nigrostriatal neurons: the role of striatal capillaries

244

Brain Research, 198 (1980) 244-248 ~i~ Elsevier/North-Holland Biomedical Press

Decarboxylation of exogenous L-DOPA in rat striatum after lesions of the dopaminergic nigrostriatal neurons: the role of striatal capillaries

ELDAD MELAMED, FRANZ HEFTI and RICHARD J. WURTMAN* Laboratory of Neuroendocrine Regulation, Department of Nutrition and Food Science, Massachusetts Institute of Technology, Cambridge, Mass. 02139 (U.S.A.)

(Accepted June 5th, 1980) Key words: L-DOPA - - decarboxylation - - dopamine - - striatum - - capillaries - - Parkinsonism

In rats with unilateral nigrostdatal lesions, L-DOPA-induced dopamine increases in ipsilateral striata were further enhanced after inhibition of DOPA decarboxylase in cerebral microvessels by carbidopa. DOPA levels were similar, but dopamine increases in lesioned striata were smaller, after carbidopa and DOPA (100 mg/kg) than after DOPA alone (500 mg/kg). These findings suggest that after degeneration of dopaminergic terminals, striatal decarboxylation of exogenous DOPA occurs partly, but not exclusively, in the capillaries.

In the intact corpus striatum, the enzyme D O P A decarboxylase (aromatic Lamino acid decarboxylase, D D C ) is localized predominantly in dopaminergic (DA) nerve terminals la. The therapeutic efficacy o f L - D O P A in Parkinsonism is attributed to its enzymatic conversion to D A in the striatum 6. However, it is still undetermined where exogenous L - D O P A is decarboxylated after loss o f nigrostriatal neurons and their D D C content 10. Endothelial cells o f cerebral microvessels contain D D C 2 and it has been proposed lz,as that striatal capillaries may represent an important locus for decarboxylation o f administered D O P A in Parkinsonian patients and in animals with nigrostriatal lesions. However, since cerebrovascular D D C is localized outside the b l o o d - b r a i n barrier 2 and systemically administered D A cannot enter brain parenc h y m a 15, it is questionable if D A formed from exogenous D O P A in capillaries can reach striatal D A receptors and become functionally effective. A recent behavioral study 3 supports this view and suggests that striatal microvessels are probably not the only site o f D O P A decarboxylation after degeneration of D A terminals; in rats with unilateral nigrostriatal lesions, D O P A - i n d u c e d circling behavior (which is apparently mediated by D A formation in the lesioned striatum 17) was not attenuated when D D C in cerebral capillaries was blocked by pretreatment with a specific inhibitor of peripheral D D C . We have examined the biochemical characteristics of a similar experimental situation. Rats with unilateral nigrostriatal lesions were given L - D O P A after *To whom correspondence should be addressed at: room 56-245, Massachusetts Institute of Technology, Cambridge, Mass. 02139, U.S.A.

245 pretreatment with a large dose of a-methyldopa hydrazine (carbidopa), a peripheral DDC inhibitor. Formation of DA and its metabolites, 3,4-dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA) was measured in the lesioned striatum and also in the cerebellum, a brain region lacking DA neuronal input. Male Sprague-Dawley rats (150-200 g, Charles River Breeding Laboratories) were anesthetized with sodium pentobarbital and placed in a Kopf stereotaxic device. 6-Hydroxydopamine (6-OHDA, 8/~g dissolved in 4/A saline containing 0.2 mg/ml ascorbic acid) was injected into the right anteromedial substantia nigra (level A 2420, --2.6 mm dorsoventral, 1.6 mm mediolateral according to K6nig and Klippel7). This was followed immediately by an additional injection of 4 #g 6-OHDA (in 2 #1 saline) into the right medial forebrain bundle (level A 4000, --2.7 mm dorsoventral, 1.8 mm mediolateral) to assure a severe lesion of the nigrostriatal system. Two weeks after lesioning, animals were injected with L-DOPA (100 or 500 mg/kg, i.p.). One group was pretreated with carbidopa (100 mg/kg, i.p.) 60 min before injection with L-DOPA (100 mg/kg). Rats were decapitated 45 min after DOPA administration; brains were removed, and striata and cerebellae were dissected and frozen on dry-ice. Tissues were homogenized in ice-cold distilled water. Aliquots, deproteinized with perchloric acid, were assayed for DOPA, DA, DOPAC, and HVA, using high-performance liquid chromatography with electrochemical detection 4,5. Measurements of tyrosine hydroxylase (TH) activity in buffered aliquots from striatal homogenates 2° were used as indices for severity of nigrostriatal lesions. In lesioned striata, mean reductions in TH activities ranged from 88 to 94 ~, indicating severe destruction of the nigrostriatal system in all groups of animals (Table I). In each group of DOPA-treated rats, DOPA concentrations were almost identical in the cerebellum and in striata ipsilateral to the nigrostriatal lesion, and were significantly higher in both regions than DOPA levels in unlesioned striata (Table I). Since L-DOPA transport through cerebral capillaries probably does not vary significantly among brain regions 19, the lower DOPA concentrations in intact striata may reflect utilization of exogenous DOPA in DA terminals. In lesioned striata, DOPA administration at 100 and 500 mg/kg increased DA concentrations 5- and 16-fold, respectively, compared to those observed in untreated rats (Table I). These increases indicate striatal decarboxylation of exogenous DOPA despite marked reductions in the number of DA terminals, and confirm earlier findings in cats with electrolytic midbrain lesions 9 and in patients with Parkinson's disease 1°. DA was virtually undetectable in cerebellar tissues from untreated rats but increased significantly after DOPA (I 00 and 500 mg/kg; Table I), indicating that exogenous DOPA can be decarboxylated even in a brain region that lacks DA neuronal projections. If these DA increases in lesioned striata originate mainly from DOPA decarboxylation in microvessels, they should be prevented by pharmacological blockade of capillary DDC. We pretreated lesioned rats with a large carbidopa dose (100 mg/kg) that would effectively inhibit DDC in peripheral tissues and cerebral capillaries, without affecting DDC in brain parenchyma 1,a. Combined carbidopa and DOPA (100 mg/kg) administration increased DA levels in lesioned striata (two-fold) and in the cerebellum, compared with those seen in rats receiving DOPA alone (100 mg/kg; Table I). These findings indicate that in

246 the relative, or total, absence of D A terminals, decarboxylation of exogenous D O P A does n o t occur exclusively in cerebral microvessels but also in striatal or cerebellar p a r e n c h y m a t o u s sites outside the capillary system. The higher D A c o n c e n t r a t i o n s observed in lesioned striata (and in the cerebellum) after c o m b i n e d t r e a t m e n t with c a r b i d o p a a n d D O P A are caused by e n h a n c e d tissue D O P A levels due to i n h i b i t i o n of peripheral a n d cerebrovascular D D C (Table I). T o estimate the capillaries' relative c o n t r i b u t i o n to D O P A - i n d u c e d D A increases in lesioned striata, we c o m p a r e d striatal D A concentrations in rats treated with c a r b i d o p a a n d D O P A (100 mg/kg) to those in rats given a large dose of D O P A alone (500 mg/kg), since both treatments produced similar D O P A c o n c e n t r a t i o n s in lesioned striata a n d cerebellar tissues (Table I). D A concentrations in lesioned striata a n d in the cerebellum were significantly lower in rats pretreated with carbidopa t h a n in those given 500 mg/kg D O P A (Table I). This finding indicates that i n h i b i t i o n of striatal and

TABLE I Effect o f combined treatment with DOPA and carbidopa on striatal and cerebellar concentrations o f DOPA, DA, DOPAC and H V A in rats with unilateral nigrostriatal lesions

Rats were injected unilaterally with 6-OHDA, 8 ~g into the substantia nigra and 4 #g into the medial forebrain bundle. After two weeks, they were injected with L-DOPA and decapitated after 45 min ; carbidopa (100 mg/kg) was given 60 min before DOPA. Results are given as means ± S.E.M. (n.d. = not detectable). Differences between groups were analyzed statistically with one-way analysis of variance followed by Scheffe's test; differences between the two striata of the same group, with t-test using related samples. Number of rats in each group is given in parentheses. T H activity

DOPA

DA

(nmol/mg/h)

(ng/mg wet weight)

DOPAC

HVA

Untreated (6) 0.49i0.14 c 0.14±0.05 b 0.11±0.03 ~ lesionedstriatum 0.004±0.001~ n.d. 7.85i0.59 0.59±0.15 0.93±0.24 controlstriatum 0.071±0.002 n.d. 0.03 ±0.02 0.01 ±0.01 0.024-0.01 cerebellum n.d. DOPA (100 mg/kg) (6) 2.37 ±0.40ee 9.20±2.99 e 4.04±0.56 e lesioned striatum 0.006±0.002° 1.71 ±0.19 b 9.14±0.67 11.62±2.84 e 5.33±1.30e control striatum 0.068±0.004 0.74:k0.06 0.57±0.04e 9.31 ±l.40r 4.45±0.70~ cerebellum 1.27 ± 0.16 DOPA + carbidopa (100 mg/kg each) (8) 4.74±0.63eegI 4.73±l.21aeg k 1.81±0.38aegk lesionedstriatum 0.004i0.001c 20.04~1.95eh controlstriatum 0.051±0.004a 8.124-0.94~a 19.13±1.86rg 12.37±2.03 ek 3.74±1.10ek 0.72±0.17eg k 4.84~2.35rg k 1.46±0.64tg k cerebellum 21.88 ± 2.45h DOPA (500 mg/kg) (6) lesioned striatum 0.004±0.001~ 13.83±3.79 bh 7.76±l.48erg 31.20±8.16 eg 6.53:~1.14e control striatum 0.034z~0.004d 7.74±2.29h 20.71±3.25 rg 34.50±5.66 eg 6.36±0.91 ~ 2 . 6 1 ± 0 . 5 2 eg 36.30±4.40fg 5.76±0.38 E cerebellum 14.27 ± 3.74h a,b,e, different from contralateral side, by P < 0.05, P < 0.01, P < 0.001, respectively. a,e,f, different from corresponding side or cerebellum of untreated animals, by P < 0.05, P < 0.01, P < 0.001, respectively. ~.~, different from corresponding side or cerebellum of animals injected with 100 mg/kg L-DOPA, by P < 0.01 ; P < 0.001, respectively. ~,k, different from corresponding side or cerebellum of animals injected with 500 mg/kg L-DOPA, by P < 0.05, P < 0.001, respectively.

247 cerebellar capillary DDC partially blocked decarboxylation of exogenous DOPA in these tissues. Mean absolute increases in striatal DA concentrations (i.e. increases over levels measured in lesioned striata of untreated rats) were 4.25 ng/mg in rats treated with carbidopa and DOPA and 7.27 ng/mg in rats given 500 mg/kg DOPA (Table I), suggesting that approximately 40 ~ of the DA increases that occurred in animals not receiving a DDC inhibitor derived from DOPA decarboxylation in striatal capillaries. Similar calculations suggest that DDC in cerebellar capillaries is responsible for a higher percentage (approximately 75 ~o) of DOPA-induced increases in DA levels in the cerebellum. It has been postulated that cerebrovascular DDC acts as an enzymatic barrier mechanism preventing circulating DOPA from entering the brain by its conversion to DA. The formed DA is then metabolized in the capillaries by monoamine degradative enzymes2. Both monoamine oxidase (MAO) and catechol-O-methyltransferase (COMT) are present in cerebral microvessels s. Our data indicate that DA formed from DOPA decarboxylation in the capillaries is metabolized intravascularly by these enzymes. In untreated rats, DA metabolites DOPAC and HVA were reduced significantly in lesioned striata, compared to contralateral sides, and were almost undetectable in the cerebellum (Table I). L-DOPA administration (100 and 500 mg/kg) produced marked, dose-dependent increases in striatal and cerebellar DOPAC and HVA levels (Table I). After each dose of DOPA, metabolite concentrations in lesioned striata and in the cerebellum were closely similar to those in unlesioned striata, suggesting that their formation was not fully dependent on presence of DA neurons. DDC inhibition and prevention of DA synthesis from exogenous DOPA in the capillaries had marked effects on DA metabolite levels. In rats treated with carbidopa and DOPA, DOPAC and HVA concentrations were significantly lower than in animals given DOPA alone (500 mg/kg), despite similar striatal DOPA levels. Reductions were more pronounced in lesioned striata and cerebellar tissues (Table I). These findings indicate that local degradation by MAO and COMT of DA formed from exogenous DOPA in the capillaries contributes an important fraction to the increases in brain DOPAC and HVA levels after DOPA administration. In conclusion, our study shows that, in a striatum largely deprived of DA terminals, some of the DOPA-induced increases in DA levels originate from DOPA decarboxylation within striatal capillaries. However, DA formation from exogenous DOPA is not abolished after combined destruction of DA neurons and blockade of the vascular DDC. Potentiation by carbidopa of DOPA's effects in Parkinsonian patients 11 and on rotational behavior in lesioned rats a, indicates that DA synthesis outside striatal capillaries is functionally effective. Besides microvessels, proposed striatal sites for decarboxylation of exogenous DOPA after degeneration of nigrostriatal neurons include surviving DA terminals ~, serotoninergic neurons 14 (we have recently shown that this system may not have an important role12), noradrenergic terminals, or non-aminergic striatal interneurons or efferentsla. In the cerebellum, extracapillary decarboxylation of administered DOPA may occur, in part, in noradrenergic nerve terminals 16.

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