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Tissue concentrations of MPTP and MPP+ in relation to catecholamine depletion after the oral or subcutaneous administration of MPTP to mice
Life Sciences, Vol. 45, pp. 2077-2083 Printed in the U.S.A.
Pergamon Press
TISSUE CONCENTRATIONS OF MPTP AND MPP+ IN RELATION TO CATECHOLAMINE DEPLETION AFTER THE ORAL OR SUBCUTANEOUS ADMINISTRATION OF MPTP TO MICE Ray W. Fuller, Susan K. Hemrick-Luecke and Kenneth W. Perry Lilly Research Laboratories, Eli Lilly and Company, Lilly Corporate Center, Indianapolis, IN 46285 (Received in final form September 19, 1989) Summary One hour after MPTP was given to mice at a dose of 30 mg/kg s.c., its concentration in tissues varied in the order kidney>liver> lung>brain>heart. When the same dose of MPTP was given orally, concentrations in most tissues were much lower at 1 hr than after s.c. administration, although the MPTP concentration in liver was only slightly lower. The concentrations of MPP+ (a metabolite of MPTP) at I hr were as high or higher than those of MPTP in all tissues except kidney, and MPP+ disappeared from the various tissues with half-lives from 3-20 hrs. The highest concentrations of MPP+, both absolute and relative to MPTP, were in heart. After oral administration of MPTP, no MPP+ was found in brain, and MPP+ concentrations in other tissues were lower than those after s.c. dosing. The depletion of heart norepinephrine was similar after MPTP administration by either route of administration even though MPTP and MPP+ concentrations in heart were lower after oral administration, suggesting that other metabolites of MPTP might also contribute to heart norepinephrine depletion. When injected subcutaneously into mice, 1-methyl-4-phenyl-l,2,3,6-tetrahydropyridine (MPTP) depletes heart norepinephrine after single or multiple doses (1,2) and depletes brain catecholamines (especially striatal dopamine and cortical norepinephrine) after multiple doses (3,4). After oral administration, MPTP depletes heart norepinephrine as much as after s.c. administration but does not deplete brain catecholamines (5). The experiments described here were done to compare the influence of route of administration on tissue concentrations of MPTP and its pharmacologically active metabolite, l-methyl-4phenylpyridinium (MPP+) (6,7). methods Male CRL/CFW mice weighing 20-30 g (Charles River Breeding Laboratories, Portage, MI) were given MPTP hydrochloride (synthesized in the Lilly Research Laboratories by Dr. David W. Robertson and associates) by s.c. injection or by oral gavage. Tissues were removed from decapitated mice, frozen on dry ice, and stored at -60°C prior to analysis. MPTP and MPP+ concentrations were determined by liquid chromatography with spectrophotometric detection in a slight modification of our earlier method (2). Tissues were homogenized in 10 volumes of 0.I N trichloroacetic acid, and the homogenates were centrifuged. An aliquot (i00 pl) of the supernatant fluid was adjusted to a pH of 5 to 7 with IM sodium acetate and mixed with 500 ~i water. The sample was adsorbed onto a CBA (carboxylic acid) cassette and eluted directly onto a Zorbax ODS C18 column. The mobile phase was 0 . 1 M sulfuric acid + 0.05 M triethylamine + 10% acetonitrile, pH 2.3; flow rate was 1.5 ml/min, and MPP+ and MPTP were detected 0024-3205/89 $3.00 + .00 Copyright (c) 1989 Pergamon Press plc
2078
MPTP C o n c e n t r a t i o n
in Tissues
Vol.
45, No.
22, 1989
by ultraviolet absorbance at 260 nm. Standards were added to tissue homogenates from untreated mice. All results are shown as mean values ± standard errors for 5 mice per group. Statistical comparisons were made by Tukey's test. The oxidation of MPTP in vitro was measured by a Hydrogen peroxide formation MPTP at a concentration of added per reaction vessel,
by mitochondrial preparations from various tissues m o d i f i c a t i o n of the method of Szutowicz et al. (8). was measured colorimetrically following addition of 5 mM. Homogenate equivalent to 5 mg of tissue was and incubation was at 37°C for 60 min.
(a/ 20
ugm MPTP/grn
15
10
_I
5
0
[--'
BRAIN
LIVER
HEART
LUNG
KIDNEY
(b) 2O
ugm MPP+/gm
15
10
I 5
0
........ BRAIN
J
HEART
_i I
LIVER
LUNG
KIDNEY
FIG. I. Tissue d i s t r i b u t i o n of (a) MPTP and (B) MPP+ at 1 hr after subcutaneous (open bars) or oral (shaded bars) a d m i n i s t r a t i o n of MPTP (30 mg/kg) to mice.
Results Fig. la shows the concentrations of MPTP in brain, heart and other tissues from mice treated 1 hr earlier with MPTP. When MPTP was given by s.c. injection (open bars), highest concentrations of MPTP were found in kidney, lower concentrations in liver and lung, a lower concentration in brain, with heart
Vol.
45, No.
22,
1989
MPTP
Concentration
in Tissues
2079
having the lowest concentration among the tissues examined. When MPTP was given by oral gavage (shaded bars), MPTP concentrations were almost undetectable in brain and heart and were very low in lung and kidney relative to those after s.c. injection. In contrast, MPTP c o n c e n t r a t i o n in liver was almost as high when the drug was given p.o. as when it was given s.c. At 2 hrs, MPTP concentrations were one-fourth to o n e - s i x t h those at I hr in all tissues, and at 4 hrs and later MPTP concentrations were essentially undetectable (data not shown). Fig. Ib shows the concentrations of MPP+ in various tissues 1 hr after MPTP injection. When MPTP was injected s.c. (open bars), MPP+ concentration was highest in the heart, lower in liver and lung, and lowest in kidney and brain. After oral a d m i n i s t r a t i o n of MPTP, MPP+ levels were almost undetectable in brain and were slightly lower in other tissues than after s.c. administration of MPTP. The concentrations of MPP+ at various times after MPTP a d m i n i s t r a t i o n s.c. or p.o. are shown in Fig. 2 for all the tissues examined. The concentrations of MPP+ at early times were as above, highest in heart, intermediate in liver and lung, and lowest in kidney and brain after s.c. administration. MPP+ concentrations disappeared most rapidly from brain, heart and liver, less rapidly from lung and kidney after s.c. administration. MPP+ concentrations were almost undetectable in brain after p.o. a d m i n i s t r a t i o n of MPTP and were slightly lower in heart than after s.c. administration. In liver, lung and kidney, MPP+ concentrations were much the same for the two routes of administration. BRAIN
HEART
0.'6
61 i :
LIVER
,o •
KIDNEY
,o ~
Hours after MPTP
FIG. 2. MPP+ c o n c e n t r a t i o n in tissues at various times after subcutaneous (o) or oral (a) a d m i n i s t r a t i o n of MPTP (30 mg/kg) to mice. Table I shows the ability of tissue homogenates from each of the organs studied to oxidize MPTP in vitro. The liver had more than lO times the capacity of any other tissue to oxidize MPTP, and the kidney had the lowest capacity for MPTP oxidation. The ratio of MPP+ c o n c e n t r a t i o n to MPTP c o n c e n t r a t i o n is also shown, revealing a lack of correlation with MPTP oxidation capacity. The heart has by far the highest M P P + / M P T P concentration, but the oxidative capacity of the heart m e a s u r e d in vitro is less than one-tenth that of liver.
2080
MPTP Concentration
in Tissues
Table I. MPTP oxidation by homogenates vitro and MPP+/MPTP ratio in vivo
Tissue
Liver Heart Brain Lung Kidney
Vol. 45, No. 22, 1989
of mouse tissue in
MPTP oxidation nmoles/g tissue/min.
256.4 17.4 8.9 4.9 1.0
± 23.2 ± 1.6 ± 0.5 ± 0.4 ± 0.6
MPP+/MPTP Ratio
1.49 8.45 1.32 ].83 1.00
MPTP was added as substrate at 5 mM for mitochondrial MAO. For MPP+/MPTP ratio, MPTP was injected s.c. at 30 mg/kg, and mice were sacrificed at 1 hour. The depletion of heart norepinephrine by HPTP given s.c. or p.o. is shown in Fig. 3. After either route of administration, there was rapid and persistent depletion of norepinephrine, the depletion being perhaps slightly faster after s.c. administration.
Hours after MPTP
F i g . 3. Time course o f h e a r t n o r e p i n e p h r i n e d e p l e t i o n a f t e r subcutaneous (e) or oral (m) administration of MPTP (30 mg/kg) in mice. Asterisks indicate significant difference from control group. Fig. 4 shows the concentrations of MPP+ in tissues after s.c. administration of MPTP to mice pretreated with 6-hydroxydopamine to destroy noradrenergic nerves or with EXP 561, an inhibitor of the uptake carrier on noradrenergic nerves. The concentration of MPP+ was not altered significantly by either pretreatment in any tissues. In a parallel experiment, 6-hydroxydopamine hydrobromide (I0 mg/kg s.c.) was shown to deplete heart norepinephrine by 86~ in CFW mice. Heart norepinephrine concentration was 4.96 ± 0.21 nmoles/g in control mice and 0.69 ± 0.05 nmoles/g at 4 days in 6-hydroxydopamine-treated mice (mean values ± standard errors for 5 mice per group). Fig. 5 shows the disappearance of MPP+ from heart after administration of MPTP to control mice or to mice pretreated with 6-hydroxydopamine or with EXP 561. No significant differences in the rate of MPP+ disappearance were observed among the three groups.
Vol. 45, No. 22, 1989
16-
MPTP Concentration
in Tissues
2081
ugm/gm
14 12 10 8
Nf/J
6 4
INNV/ INN//
Ng4
2 0
BRAIN HEART LIVER LUNG KIDNEY Fig. 4. MPP+ concentrations in tissues ] hr after the s.c. injection of MPTP (20 mg/kg) to control mice (open bars), mice pretreated with 6-hydroxydopamine hydrobromide (10 mg/kg s.c., 3 days earlier) (crosshatched bars), or mice pretreated with EXP 561 (5 mg/kg i.p., l hr earlier) (slashed bars). 60-
10-
5
;
1
I
f
I
2
4
8
Hours affer MPTP
Fig. 5. MPP+ disappearance from heart after the s.c. injection of MPTP (20 mg/kg) to control mice (open circles), mice pretreated with 6-hydroxydopamine hydrobromide (I0 mg/kg s.c.) 3 days earlier (open) squares), or mice pretreated with EXP 561 (5 mg/kg i.p., I hr earlier) (filled circles). Fig. 6 shows the tissue distribution of MPP+ at I hr after the s.c. injection of MPTP or MPP+. In brain, essentially no MPP+ was present after injection of MPP+. In liver, lung and kidney, MPP+ concentrations were much higher after MPP+ injection than after MPTP injection. In heart, MPP+ concentrations after MPTP injection were higher than in any other tissue studied and were as high as those found after injection of MPP+ itself.
2082
MPTP C o n c e n t r a t i o n
in Tissues
Vol.
45, No.
22, 1989
70605040302010o~
~
BRAIN HEART LIVER LUNG KIDNEY Fig. 6. Tissue d i s t r i b u t i o n of MPP+ at ] hr after subcutaneous administration of MPTP (shaded bars) or MPP+ (slanted bars), both at doses of 30 mg/kg.
Discussion The present experiments show that s.c. administration of MPTP is a more effective route than oral administration for delivering MPTP to tissues, especially the brain, in the mouse. Probably the MPTP is rapidly and extensively metabolized during its first pass through the liver after oral administration. One of the metabolites formed, MPP+, is present in several tissues at reasonably high concentrations after oral administration of MPTP. However, MPP+ apparently does not cross the b l o o d - b r a i n barrier since its levels in brain were very low after oral administration of MPTP or after s.c. administration of MPP+ itself. After oral administration of MPTP, brain concentrations of MPTP and of MPP+ were very low relative to those after s.c. injection of MPTP. At I hr after oral administration of MPTP, MPTP and MPP+ concentrations were about onetwentieth those found in brain after s.c. injection of MPTP. We had earlier reported that orally administered MPTP did not lead to depletion of striatal dopamine (5). That lack of efficacy no doubt relates to the low concentration of MPP+ in brain. The depletion of heart norepinephrine, in contrast, was very similar whether MPTP was given by oral gavage or injected s.c. MPTP disappeared rapidly from heart after I hr regardless of route of administration, yet norepinephrine levels continued to fall and remained low at 24 hrs. MPTP concentrations were very low in heart after the oral administration of MPTP. After oral administration of MPTP, MPP+ concentrations in heart were substantial but distinctly lower than those after s.c. injection of MPTP, although the depletion of heart norepinephrine was nearly the same for the two different routes of administration (5; Fig. 3). One possible explanation is that other metabolites of MPTP besides MPP+ are capable of depleting heart norepinephrine. Alternatively, the concentration of MPP+ attained even after oral administration of MPTP may have produced maximal depletion of heart norepinephrine. The heart was the only tissue in which MPP+ concentrations far exceeded MPTP concentrations at 1 hr after s.c. injection of MPTP. In the heart, MPP+ concentrations were more than seven times those of MPTP at this time point. In contrast, brain, liver and lung had about equal amounts of MPP+ and MPTP, and kidney had only about one-third as much MPP+ as MPTP. Since the heart does not
Vol. 45, No. 22, 1989
MPTP Concentration
in Tissues
2083
oxidize MPTP as rapidly as does liver (Table I), we thought the high concentrations of HPP+ in heart more likely represented accumulation of MPP+ formed elsewhere. The possibility that the high concentrations of MPP+ in heart were due to its accumulation by noradrenergic nerves was tested by comparing MPP+ levels in rats pretreated with 6-hydroxydopamine to destroy noradrenergic nerves or with EXP 561 to inhibit the uptake carrier on noradrenergic nerves. Neither pretreatment affected MPP+ levels in heart or in other tissues, nor did they affect the rate of MPP+ disappearance from heart. Thus, the high concentrations of MPP+ in heart apparently do not result from localization in noradrenergic nerves. Perhaps the accumulation is in other cells in the heart, or perhaps MPP+ is formed in heart in vivo at faster rates than the in vitro measurements would suggest. References I. 2. 3. 4. 5.
R. W. FULLER, R. A. HAHN, H. D. SNODDY and J. H. WIKEL, Biochem. Pharmacol. 33, 2957-2960 (1984). R. W. FULLER, S. K. KEMRICK-LUECKE and D. W. ROBERTSON, Biochem. Pharmacol. 37, 3343-3347 (1988). R. E. HEIKKILA, A. HESS and R. C. DUVOISIN, Science 224, 1451-1453 (1984). H. HALLMAN, L. OLSON and G. JONSSON, Eur. J. Pharmacol. 97, 133-136 (1984). R. W. FULLER and S. K. HEM!RICK-LUECKE, Biochem. Pharmacol. 36, 789-792
(1987). 6. 7. 8.
K. CHIBA, A. TREVOR and N. CASTAGNOLI, JR., Biochem. Biophys. Res. Commun. 120, 574-578 (1984). S. P. MARKEY, J. N. JOHANNESSEN, C. C. CHIUEH, R. S. BURNS and M. A. HERKENHAM, Nature 311, 464-467 (1984). A. SZUTOWICZ, R. D. KOBES and P. J. ORSULAK, Anal. Biochem. 138, 86-94 (1984).
Pergamon Press
TISSUE CONCENTRATIONS OF MPTP AND MPP+ IN RELATION TO CATECHOLAMINE DEPLETION AFTER THE ORAL OR SUBCUTANEOUS ADMINISTRATION OF MPTP TO MICE Ray W. Fuller, Susan K. Hemrick-Luecke and Kenneth W. Perry Lilly Research Laboratories, Eli Lilly and Company, Lilly Corporate Center, Indianapolis, IN 46285 (Received in final form September 19, 1989) Summary One hour after MPTP was given to mice at a dose of 30 mg/kg s.c., its concentration in tissues varied in the order kidney>liver> lung>brain>heart. When the same dose of MPTP was given orally, concentrations in most tissues were much lower at 1 hr than after s.c. administration, although the MPTP concentration in liver was only slightly lower. The concentrations of MPP+ (a metabolite of MPTP) at I hr were as high or higher than those of MPTP in all tissues except kidney, and MPP+ disappeared from the various tissues with half-lives from 3-20 hrs. The highest concentrations of MPP+, both absolute and relative to MPTP, were in heart. After oral administration of MPTP, no MPP+ was found in brain, and MPP+ concentrations in other tissues were lower than those after s.c. dosing. The depletion of heart norepinephrine was similar after MPTP administration by either route of administration even though MPTP and MPP+ concentrations in heart were lower after oral administration, suggesting that other metabolites of MPTP might also contribute to heart norepinephrine depletion. When injected subcutaneously into mice, 1-methyl-4-phenyl-l,2,3,6-tetrahydropyridine (MPTP) depletes heart norepinephrine after single or multiple doses (1,2) and depletes brain catecholamines (especially striatal dopamine and cortical norepinephrine) after multiple doses (3,4). After oral administration, MPTP depletes heart norepinephrine as much as after s.c. administration but does not deplete brain catecholamines (5). The experiments described here were done to compare the influence of route of administration on tissue concentrations of MPTP and its pharmacologically active metabolite, l-methyl-4phenylpyridinium (MPP+) (6,7). methods Male CRL/CFW mice weighing 20-30 g (Charles River Breeding Laboratories, Portage, MI) were given MPTP hydrochloride (synthesized in the Lilly Research Laboratories by Dr. David W. Robertson and associates) by s.c. injection or by oral gavage. Tissues were removed from decapitated mice, frozen on dry ice, and stored at -60°C prior to analysis. MPTP and MPP+ concentrations were determined by liquid chromatography with spectrophotometric detection in a slight modification of our earlier method (2). Tissues were homogenized in 10 volumes of 0.I N trichloroacetic acid, and the homogenates were centrifuged. An aliquot (i00 pl) of the supernatant fluid was adjusted to a pH of 5 to 7 with IM sodium acetate and mixed with 500 ~i water. The sample was adsorbed onto a CBA (carboxylic acid) cassette and eluted directly onto a Zorbax ODS C18 column. The mobile phase was 0 . 1 M sulfuric acid + 0.05 M triethylamine + 10% acetonitrile, pH 2.3; flow rate was 1.5 ml/min, and MPP+ and MPTP were detected 0024-3205/89 $3.00 + .00 Copyright (c) 1989 Pergamon Press plc
2078
MPTP C o n c e n t r a t i o n
in Tissues
Vol.
45, No.
22, 1989
by ultraviolet absorbance at 260 nm. Standards were added to tissue homogenates from untreated mice. All results are shown as mean values ± standard errors for 5 mice per group. Statistical comparisons were made by Tukey's test. The oxidation of MPTP in vitro was measured by a Hydrogen peroxide formation MPTP at a concentration of added per reaction vessel,
by mitochondrial preparations from various tissues m o d i f i c a t i o n of the method of Szutowicz et al. (8). was measured colorimetrically following addition of 5 mM. Homogenate equivalent to 5 mg of tissue was and incubation was at 37°C for 60 min.
(a/ 20
ugm MPTP/grn
15
10
_I
5
0
[--'
BRAIN
LIVER
HEART
LUNG
KIDNEY
(b) 2O
ugm MPP+/gm
15
10
I 5
0
........ BRAIN
J
HEART
_i I
LIVER
LUNG
KIDNEY
FIG. I. Tissue d i s t r i b u t i o n of (a) MPTP and (B) MPP+ at 1 hr after subcutaneous (open bars) or oral (shaded bars) a d m i n i s t r a t i o n of MPTP (30 mg/kg) to mice.
Results Fig. la shows the concentrations of MPTP in brain, heart and other tissues from mice treated 1 hr earlier with MPTP. When MPTP was given by s.c. injection (open bars), highest concentrations of MPTP were found in kidney, lower concentrations in liver and lung, a lower concentration in brain, with heart
Vol.
45, No.
22,
1989
MPTP
Concentration
in Tissues
2079
having the lowest concentration among the tissues examined. When MPTP was given by oral gavage (shaded bars), MPTP concentrations were almost undetectable in brain and heart and were very low in lung and kidney relative to those after s.c. injection. In contrast, MPTP c o n c e n t r a t i o n in liver was almost as high when the drug was given p.o. as when it was given s.c. At 2 hrs, MPTP concentrations were one-fourth to o n e - s i x t h those at I hr in all tissues, and at 4 hrs and later MPTP concentrations were essentially undetectable (data not shown). Fig. Ib shows the concentrations of MPP+ in various tissues 1 hr after MPTP injection. When MPTP was injected s.c. (open bars), MPP+ concentration was highest in the heart, lower in liver and lung, and lowest in kidney and brain. After oral a d m i n i s t r a t i o n of MPTP, MPP+ levels were almost undetectable in brain and were slightly lower in other tissues than after s.c. administration of MPTP. The concentrations of MPP+ at various times after MPTP a d m i n i s t r a t i o n s.c. or p.o. are shown in Fig. 2 for all the tissues examined. The concentrations of MPP+ at early times were as above, highest in heart, intermediate in liver and lung, and lowest in kidney and brain after s.c. administration. MPP+ concentrations disappeared most rapidly from brain, heart and liver, less rapidly from lung and kidney after s.c. administration. MPP+ concentrations were almost undetectable in brain after p.o. a d m i n i s t r a t i o n of MPTP and were slightly lower in heart than after s.c. administration. In liver, lung and kidney, MPP+ concentrations were much the same for the two routes of administration. BRAIN
HEART
0.'6
61 i :
LIVER
,o •
KIDNEY
,o ~
Hours after MPTP
FIG. 2. MPP+ c o n c e n t r a t i o n in tissues at various times after subcutaneous (o) or oral (a) a d m i n i s t r a t i o n of MPTP (30 mg/kg) to mice. Table I shows the ability of tissue homogenates from each of the organs studied to oxidize MPTP in vitro. The liver had more than lO times the capacity of any other tissue to oxidize MPTP, and the kidney had the lowest capacity for MPTP oxidation. The ratio of MPP+ c o n c e n t r a t i o n to MPTP c o n c e n t r a t i o n is also shown, revealing a lack of correlation with MPTP oxidation capacity. The heart has by far the highest M P P + / M P T P concentration, but the oxidative capacity of the heart m e a s u r e d in vitro is less than one-tenth that of liver.
2080
MPTP Concentration
in Tissues
Table I. MPTP oxidation by homogenates vitro and MPP+/MPTP ratio in vivo
Tissue
Liver Heart Brain Lung Kidney
Vol. 45, No. 22, 1989
of mouse tissue in
MPTP oxidation nmoles/g tissue/min.
256.4 17.4 8.9 4.9 1.0
± 23.2 ± 1.6 ± 0.5 ± 0.4 ± 0.6
MPP+/MPTP Ratio
1.49 8.45 1.32 ].83 1.00
MPTP was added as substrate at 5 mM for mitochondrial MAO. For MPP+/MPTP ratio, MPTP was injected s.c. at 30 mg/kg, and mice were sacrificed at 1 hour. The depletion of heart norepinephrine by HPTP given s.c. or p.o. is shown in Fig. 3. After either route of administration, there was rapid and persistent depletion of norepinephrine, the depletion being perhaps slightly faster after s.c. administration.
Hours after MPTP
F i g . 3. Time course o f h e a r t n o r e p i n e p h r i n e d e p l e t i o n a f t e r subcutaneous (e) or oral (m) administration of MPTP (30 mg/kg) in mice. Asterisks indicate significant difference from control group. Fig. 4 shows the concentrations of MPP+ in tissues after s.c. administration of MPTP to mice pretreated with 6-hydroxydopamine to destroy noradrenergic nerves or with EXP 561, an inhibitor of the uptake carrier on noradrenergic nerves. The concentration of MPP+ was not altered significantly by either pretreatment in any tissues. In a parallel experiment, 6-hydroxydopamine hydrobromide (I0 mg/kg s.c.) was shown to deplete heart norepinephrine by 86~ in CFW mice. Heart norepinephrine concentration was 4.96 ± 0.21 nmoles/g in control mice and 0.69 ± 0.05 nmoles/g at 4 days in 6-hydroxydopamine-treated mice (mean values ± standard errors for 5 mice per group). Fig. 5 shows the disappearance of MPP+ from heart after administration of MPTP to control mice or to mice pretreated with 6-hydroxydopamine or with EXP 561. No significant differences in the rate of MPP+ disappearance were observed among the three groups.
Vol. 45, No. 22, 1989
16-
MPTP Concentration
in Tissues
2081
ugm/gm
14 12 10 8
Nf/J
6 4
INNV/ INN//
Ng4
2 0
BRAIN HEART LIVER LUNG KIDNEY Fig. 4. MPP+ concentrations in tissues ] hr after the s.c. injection of MPTP (20 mg/kg) to control mice (open bars), mice pretreated with 6-hydroxydopamine hydrobromide (10 mg/kg s.c., 3 days earlier) (crosshatched bars), or mice pretreated with EXP 561 (5 mg/kg i.p., l hr earlier) (slashed bars). 60-
10-
5
;
1
I
f
I
2
4
8
Hours affer MPTP
Fig. 5. MPP+ disappearance from heart after the s.c. injection of MPTP (20 mg/kg) to control mice (open circles), mice pretreated with 6-hydroxydopamine hydrobromide (I0 mg/kg s.c.) 3 days earlier (open) squares), or mice pretreated with EXP 561 (5 mg/kg i.p., I hr earlier) (filled circles). Fig. 6 shows the tissue distribution of MPP+ at I hr after the s.c. injection of MPTP or MPP+. In brain, essentially no MPP+ was present after injection of MPP+. In liver, lung and kidney, MPP+ concentrations were much higher after MPP+ injection than after MPTP injection. In heart, MPP+ concentrations after MPTP injection were higher than in any other tissue studied and were as high as those found after injection of MPP+ itself.
2082
MPTP C o n c e n t r a t i o n
in Tissues
Vol.
45, No.
22, 1989
70605040302010o~
~
BRAIN HEART LIVER LUNG KIDNEY Fig. 6. Tissue d i s t r i b u t i o n of MPP+ at ] hr after subcutaneous administration of MPTP (shaded bars) or MPP+ (slanted bars), both at doses of 30 mg/kg.
Discussion The present experiments show that s.c. administration of MPTP is a more effective route than oral administration for delivering MPTP to tissues, especially the brain, in the mouse. Probably the MPTP is rapidly and extensively metabolized during its first pass through the liver after oral administration. One of the metabolites formed, MPP+, is present in several tissues at reasonably high concentrations after oral administration of MPTP. However, MPP+ apparently does not cross the b l o o d - b r a i n barrier since its levels in brain were very low after oral administration of MPTP or after s.c. administration of MPP+ itself. After oral administration of MPTP, brain concentrations of MPTP and of MPP+ were very low relative to those after s.c. injection of MPTP. At I hr after oral administration of MPTP, MPTP and MPP+ concentrations were about onetwentieth those found in brain after s.c. injection of MPTP. We had earlier reported that orally administered MPTP did not lead to depletion of striatal dopamine (5). That lack of efficacy no doubt relates to the low concentration of MPP+ in brain. The depletion of heart norepinephrine, in contrast, was very similar whether MPTP was given by oral gavage or injected s.c. MPTP disappeared rapidly from heart after I hr regardless of route of administration, yet norepinephrine levels continued to fall and remained low at 24 hrs. MPTP concentrations were very low in heart after the oral administration of MPTP. After oral administration of MPTP, MPP+ concentrations in heart were substantial but distinctly lower than those after s.c. injection of MPTP, although the depletion of heart norepinephrine was nearly the same for the two different routes of administration (5; Fig. 3). One possible explanation is that other metabolites of MPTP besides MPP+ are capable of depleting heart norepinephrine. Alternatively, the concentration of MPP+ attained even after oral administration of MPTP may have produced maximal depletion of heart norepinephrine. The heart was the only tissue in which MPP+ concentrations far exceeded MPTP concentrations at 1 hr after s.c. injection of MPTP. In the heart, MPP+ concentrations were more than seven times those of MPTP at this time point. In contrast, brain, liver and lung had about equal amounts of MPP+ and MPTP, and kidney had only about one-third as much MPP+ as MPTP. Since the heart does not
Vol. 45, No. 22, 1989
MPTP Concentration
in Tissues
2083
oxidize MPTP as rapidly as does liver (Table I), we thought the high concentrations of HPP+ in heart more likely represented accumulation of MPP+ formed elsewhere. The possibility that the high concentrations of MPP+ in heart were due to its accumulation by noradrenergic nerves was tested by comparing MPP+ levels in rats pretreated with 6-hydroxydopamine to destroy noradrenergic nerves or with EXP 561 to inhibit the uptake carrier on noradrenergic nerves. Neither pretreatment affected MPP+ levels in heart or in other tissues, nor did they affect the rate of MPP+ disappearance from heart. Thus, the high concentrations of MPP+ in heart apparently do not result from localization in noradrenergic nerves. Perhaps the accumulation is in other cells in the heart, or perhaps MPP+ is formed in heart in vivo at faster rates than the in vitro measurements would suggest. References I. 2. 3. 4. 5.
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