Inhibition of mitochondrial NADH dehydrogenase by pyridine derivatives and its possible relation to experimental and idiopathic parkinsonism

Vol. 135,No.

1, 1986

BIOCHEMICALAND

8lOPHYSlCALRESEARCHCOMMUNlCATlONS

February 26, 1986

Pages 269-275

INHIBITION OF MITOCHONDRIAL NADH DEHYDROGENASE BY PYRIDINE DERIVATIVES AND ITS POSSIBLE RELATION TO EXPERIMENTAL AND IDIOPATHIC PARKINSONISM Rona R. Ramsay *§,

Departments

James I.

Salach',

of Pharmaceutical

University Molecular

Jahan

Chemistry+

of California,

Biology

Division',

January

and Thomas P. Singer'*"

and Biochemistry-Biophysics*

San Francisco, Veterans

San Francisco, Received

Dadgar*',

California

Administration

California

94143 Medical

Center

94121

9, 1986

SUMMARY. 4-Phenyl-N-methylpyridinium (MPP+), the oxidation product of the neurotoxic amine MPTP, is considerably more inhibitory to the oxidation of NAD+linked substrates in intact mitochondria in State 3 than is 4-phenylpyridine. On adding uncouplers, the inhibition by MPP+ progressively diminishes, while the effect of 4-phenylpyridine remains. This is in accord with the fact that MPP+ is rapidly concentrated in the mitochondria by an energy-dependent process, while 4-phenylpyridine seems to enter passively with the concentration gradient. Collapse of the electrical gradient after addition of uncouplers thus leaves the inhibiton by 4-phenylpyridine unaffected but causes efflux of MPP+ from the mitochondria In isolated inner membranes the inhibition and a reversal of its inhibitory action. of NADH oxidation via the respiratory chain by 4-phenylpyridine is much greater than by MPP+. MPTP and 4-phenyl-N-methylpyridinone also inhibit more than MPP+, whereas N-methylpyridinium has relatively little effect. The block is not at the point of entry of electrons into the flavoprotein since the NADH-ferricyanide activity is not inhibited by MPP+ at Vmax. @ 1986 Academic Press., hc.

INTRODUCTION. destruction mitter usually

of nigrostriatal

dopamine.

dopamine (1).

It

hydropyridine), elicits causes cessing

neurons

symptoms clinical

level

occurs

as a result

has been demonstrated

symptoms

destruction enzymes

in the (4).

disease

and consequent

the first

the neurotoxic

by brain

a degenerative

that

parkinsonian cell

is

The neurological

estimated

striatal cells

Parkinsonism

that

component

depletion

develop signs

by extensive of the

progressively

appear

neurotrans-

and it

has been

when an 80% decline

of comparable

loss

in

of nigrostriatal

MPTP (1-methyl-4-phenyl-1,2,3,6-tetraof certain

batches

in man and primates substantia

caused

nigra,

The two types

(2,3) but only

of monoamine

of illicit

and that after oxidase,

drugs,

this

compound

metabolic

pro-

primarily 0006-291X/86

269

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Copyright 0 1986 rights qf‘ reproduction

the $1.50

by Academic Press, Inc. in any form reserved.

Vol. 135, No. 1, 1986

the B type, oxidized

have been

to the

implicated

responsible

neurons

for

of NAD+-linked

by a moderate

dation

and thus

presented cells

substrates

the

cell

NADH oxidation Second,

is -not

charged

demonstrating

the

We felt molecular

possible

ago called

end result called contain latter

it

substituted compound

cultures,

implying

disease.

We report

on NADH oxidation

inhibition

to the fact

that,

here

this

found

in State

3 that

NADH oxi-

death.

This

idea

nigrostriatal

PM, respectively), or

in Complex the

has resolved

membrane

problems

uptake

MPP+ inside

I (11).

inner

these

energy-dependent

two closely

by

system

for

the mitochondria

at

Parkinsonism. that

besides

related

Langston's

idiopathic

industrial

depletion

of compound

and intact

study

problems,

the

NADH dehydrogenase

of xenobiotics.

dopamine

type

oxi-

postulated

in the

to cross

of mitochondrial

a comparative

in membranes

in cell

Snyder wastes,

such as 4-phenylpyridine,

moderate

(8)

of the

mitochondrial

preparations

possibility

effect

inhibition

--et al.

(30 and-50

(11)

4-phenyl-

in dopaminergic

mitochondria

blocks

not

to MPP+, the

NADH oxidation.

to idiopathic to the

that

paper

to approach

cumulative

causes

(10)

thought

to block

pyridines,

on the

at the MPP+ levels

of concentrating

needed

of the

it

initially

to be the compound

resulting

of a very active

attention

attention

and brain

Our recent

relevance

of the

liver

generally

was timely

mechanism

and its long

that

than

in rat

in membrane

capable

higher

Based

and monkeys

are

presence

MPP+ in mitochondria, far

(9)

MPTP is

is concentrated

(7).

conservation,

inhibited

freely.

and it

system

First,

molecules

of mitochondria

has been suggested

mM) of MPP+, Nicklas

energy

mice

(5,6).

more slowly

body of the neuron,

two difficulties.

of MPTP-treated

levels

(0.5

prevents

and then

destruction

reuptake

concentration

when MPP+ reaches

bioactivation

The latter neuronal

by the dopamine

dation

form

species.

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

in this

dihydropyridinium

N-methylpyridinium directly

BIOCHEMICAL

might of the

group

(1,Z)

Parkinsonism

is

and D'Amato

(12)

certain

foodstuffs

and reported in mice and also be a causative effect

by MPP+

that in

the

the

Cell

agent

of substituted

of the pyridines

mitochondria.

was from the Aldrich Chemical MATERIALS AND METHODS. 4-Phenylpyridine N-methylpyridine and 1-methyl-4-phenyl-2-pyridinone were kind gifts from Castagnoli. Other materials and methods were as before (5,6,11). 270

Co., Dr. N.

Vol. 135, No. 1, 1986

BIOCHEMICAL

RESULTS AND DISCUSSION. dine

on the

oxidation

mitochondria. MPP+ is

into

of about

concentrated system

inhibitors

taken

a concentration into

for

in which

the

glutamate

with

uptake

oxidation membranes

It

explain

of uncoupler, (Fig.

1).

into

the

in contrast In experiments

mitochondria

are

preloaded

latter

that

and brain

than

may explain

being

to be published

elsewhere

MPP+ in the

presence

actively

malate

inhibition

and

although (Figs.

by MPP+ after

(13)

being

why in mitochondria

by 4-phenylpyridine,

of inhibition of the

on ex-

by the presence

is much more inhibitory

decline

the

4-phenylpyridine,

rather

by MPP+ than

to the persistence

with

seem to be

of MPP+ and 4-phenylpyridine

compound

progressive

that

reaching

was based

was unaffected

This

whereas

does not

of MPP+ in liver

this

liver

(ll),

as a substrate

from

process.

but,

The conclusion

the mitochondria,

concentrations

the

uptake

rat

conditions

mechanism.

appears

is more inhibited

in isolated may also

It

coupled

4-phenylpyridine

of 0.5 mM C3~]-~pp+

passively

equal

these

4-phenylpyridine

by an energy-requiring

pre-incubated

in tightly

under

by this

(13).

may diffuse

concentrated

the mitochondria

recognize

of MPP+ and 4-phenylpyri-

at 0.5 mM concentration

20 mM in minutes,

of 1 mM 4-phenylpyridine neutral,

added

the energy-dependent

does not

periments

were

RESEARCH COMMUNICATIONS

the effects

and L-malate

the mitochondria

responsible

mitochondria

1 compares

of L-glutamate

Both

rapidly

Fig.

AND BIOPHYSICAL

2 and 3). addition

by 4-phenylpyridine

we found

of an energy

that source

when the and an

Oxidation of Glutamate 8 Malate 0.5mM

MPP’

0.5mM

0.5mM

MPP*

0.5mi

4-PP

4-PP

Fi . 1. Polarograp&ic measurement of the respiration of rat liver mitochondria . mg/ml) at 25 . Malate, glutamate, ADP, and 2,4-dinitrophenol (DHP) were added to give 2.5, 5, 0.25, and 0.019 mM concentrations, respectively. Preincubation with MPP+ or 4-phenylpyridine (4-PP) was for 5 min., in the presence of substrate.

-+J

271

Vol. 135,No.1,1986

BIOCHEMICAL

AND BIOPHYSICALRESEARCH

COMMUNICATIONS

100 t 80 .

s E 60 g _C 40 8

+

20

4 6 Inhibitor (mM) 0 O 03 O Inhibitor (mM) 2. Aliquots of 25 ~1 of a beef heart ETP suspension (2 pg/ml) were diluted to 90. 5 ml with 0.25 M sucrose - 0.05 M potassium phosphate, pH 7.6, cogtaining the indicated concentFation of inhibitor and incubated for 5 min. at 25 . The l!Iliz

2

2

8

16

tubes were rapidly chilled and aliquots of 25 ~1 were immediately assayed in a 1 ml reaction volume containing 0.25 M sucrose - 0.05 M phosphate, pH 7.6, 0.28 mM NADH, and the indicated amount of inhibitor,by following the initial rate of disappearance of the 340 nm band of NADH at 25 . Symbols: X-X, 4 phenylpyridine; O-O, MPTP;O--O, N-methyl-4-phenylpyridinone. X-X,

Fig. 3. Conditions N-methylpyridine.

uncoupler the

is

then

added,

an efflux

intramitochondrial

thus

uncouplers Figs.

atory

have no effect

beef

heart.

rate

of NADH oxidation,

inhibitor

for

Note

slight

that,

This

cytoplasmic itself,

It

in accord

substituted

concentrations

is

not at the

suggested

into (8),

with

that

prevailing

is

via membrane

of the

particles

pyridinium 2).

of entry

in the ferricyanide

reached

inside

compound,

also

the site

of pyridine-nucleotide

272

from

of the

initial

the appropriate potent

inhi-

mM MPP+ produces

(i.e.,

only

at 10 mM or higher the mitochondria

MPP+ (11,13).

assay

preparation

with

0.5

occurs

chain

respir-

was the most

(ll),

inhibition

However,

remains.

MPP+, and N-methylpyridin-

data

mM or less

theelectrontransport since

earlier

and

the complete

measurements

4-phenylpyridine

rapidly

of 0.5

(Fig. point

our

outside

of NADH dehydrogenase

in ETP, an inner

that

NAD+;

case of 4-phenylpyridine

of NADH oxidation

complete

concentration

high

equivalents

exceed

preincubation

is seen

MPP+;O-0,

In the

on spectrophotometric

but nearly

concentrations another

dation

after

O-O,

by MPTP, 1-methyl-4-phenyl-2-pyridinone,

inhibition,

trations.

based

Symbols:

inhibition

inhibition

pyridines

are

5 min.

followed

ium.

very

The data

cannot

and the

the

by substituted

2.

of MPP+ occurs.

concentration

2 and 3 compare

chain

bitor,

were as in Fig.

at

Interestingly,

inhibits

concen-

NAD+

NADH oxidation

of inhibition

of NADH oxi-

generated the flavin),

MPP+ does not

reducing

as has been inhibit

at

at Vmax

Vol.

135,

No.

BIOCHEMICAL

1, 1986

AND

BIOPHYSICAL

RESEARCH

COMMUNICATIONS

IFeCNj'ml“ Fig. 4. Double reciprocal plot of NADH-ferricyanide activity. The ETP prepagation used in the experiments of Figs. 2 and 3 was incubated for 5 min. at 30 in 0.04 M triethanolamine buffer, pH 7.8, the indicated amounts of 10-2 M ferricyanide, and inhibitors as follows: O-O, none;@-@, 1 mM 4-phenylpyridine; O-0, 5 mM MPP+; X-X, 10 mM methylpyridine. Assay was initiated by adding NADH to 1.5 X 10-4 concentration. Abscissa, reciprocal concentration of 10-2 M ferricyanide; ordinate, reciprocal absorbance change at 420 nm per min. at 30'.

(Fig.

4).

from

It

the

low

inhibition

potential

site

junction

must

A (16).

in the

decreasing but

it

for

hydrophobic

KM for

apparent

To what death

part

extent

do these

of this

chain enzyme

destruction. inhibit

lack

-linked

The data

interaction

presented

the dehydrogenase

NADH oxida-

of this

is

part the

effect

not

known affinity

of MPP+, exposing ferricyanide

and thus

clear

the

anion,

a lowering

posi-

resulting

of the

of 4-phenylpyridine

(Fig.

4) which

are

support

in accord

the notion is

(8)

triggered ?

with

this

that

nigrostriatal

by inhibition

Obviously,

cell

of the

extensive

inhibition

and,

hence,

at sufficiently

high

concentrations

completely. 273

The uptake

the hy.

speculation.

of ATP synthesis

show that almost

and

lacks

experiments

cessation

amytal,

which

NADH dehydrogenase

to virtual

at the

used by

of its

the

by N-methylpyridinium,

of effect

animals

possibly

concentrations

by virtue

attract

stimulation

studies

MPTP-treated

leads

then

Thus,

of MPP' stimulate

the aromatic

might

at the pH of these

in susceptible

respiratory

dehydrogenase,

electrons

(14,151.

case of rotenone,

ferricyanide

of successful

and the

accepts

Fe-S cluster,

The mechanism

may bind

The much lesser

drophobicbinding uncharged

the

ferricyanide

concentrations

at all

that

which

potential

and Q, as in the

oxidant.

frequency

KM.

low

this

nitrogen,

that

1 of NADH dehydrogenase

high

assay

molecules,

charged

in increased

does

the

Curiously,

seems possible

tively

is

be past

ferricyanide the

many years

Fe-S cluster

of NADH dehydrogenase

piericidin tion

has been known for

mechanism

neuronal

which

MPP+ we

Vol. 135, No. 1, 1986

have described these

(11)

levels,

it

responsible that is

for

as the

neurons.

moment they

remain

To the extent of MPTP, they pyridines,

that

concentrated chondria

pyridine,

data

Other

would

present.

to accumulate also

with

MPTP and that several

oxidation

because

dopamine system

the

hypothesis

NADH dehydrogenase

unresolved

products

questions,

of MPTP for

such

certain

can be provided,

to the ultimate

readily

If,

for

the

the

inner

this

decline

may be a sufficient

hydrophobic

membrane,

be cumulative.

over

Depending

decrease

number

lead

symptoms. 274

is

much

less

inhiis

not

in mito-

pH, like then

diffuse levels

of the

nature,

of time

with

the

of the

com-

NADH dehydrogenase

its

effect

of exposure

to significant

production

4-phenyl-

4-phenylpyridine

frequency

of nigrostriatal

in dopamine

(12),

4-phenylpyridine

in low

a period

on the

may eventually in the

barrier,

of a fraction

of its

of idiopathic

intracellular

resulting

inhibition

substituted

may be even more inhibitory

at

blood-brain

that

MPP+ accumulation

xenobiotics

the mitochondria,

by virtue

for

neurotoxicity

is MPP+, though

is so because

neutral,

cross

(12)

spices

than

responsible

related

and the

in the

This

electrically

of the

etiology

of certain

in membranes

mechanism

into

to the

a constituent

1).

cause

the suggestions

contribute

(Fig.

If

xenobiotic,

to the natural

neurological

MPP+,

extent,

synaptic

compatible

leave

to support

structurally

in the mitochondria

to the

Although from

than

by the mitochondrial

explanations

point

be taken

pumping

gradient

enzyme would

overt

plausible

mitochondria

they

molecules

result

Although

also

by the

concentration

Added

of the

these

to NADH dehydrogenase.

person

toxicity

to NADH dehydrogenase

(13).

up by the

concentrated

observations

4-Phenylpyridine,

in intact

to a significant

not taken

arising

such as 4-phenylpyridine,

more inhibitory

were

agent

to NADH dehydrogenase

conjectural.

might

Parkinsonism.

pound

is

MPP+ (13).

these

selective

dopaminergic

cells,

to reach

of MPP+ in the cytoplasm.

neurons

not actively

neurotoxic target,

highly

bitory

and is

the mitochondria

concentrations

the dopaminergic

concentrating

ultimate

MPP+ into

more inhibitory

by glia

(7),

MPP+ is the its

considerably

oxidized

system

sufficient

steady-state

to reach

rapidly

reuptake

at low

though

expected

is

accumulates

even

MPTP itself, is not

BlOCHEMlCALANDBlOPHYSlCALRESEARCHCOMMUNlCATlONS

cells for

on the of the

neuronal with

age,

the appearance

loss. the of

Vol.

135,

No.

1, 1986

BIOCHEMICAL

AND

BIOPHYSICAL

RESEARCH

COMMUNICATIONS

ACKNOWLEDGMENTS. This work was supported by Program Project HL 16251 from the National Instrtutes of Health, by Grant No. DMB 8416967 from the National Science Foundation, and by the Veterans Administration.

REFERENCES

:: 3. 4. 5. 6. 7. 8. 9. 10. 11.

Calne, D.B., and Langston, J.W. (1983) Lancet 2, 1457-1459. Langston, J.W., Ballard, P., Tetrud, J.W., and Irwin, I. (1983) Science, 219, 979-980. Burns, R.S., Chiueh, C.C., Markey, S.P., Ebert, M.H.E., Jacobowitz, D.M., and Kopin, I.J. (1983) Proc. Nat. Acad. Sci. U.S. 80, 4546-4550. Chiba, K., Trevor, A., and Castagnoli, N., Jr., (1984) Biochem. Biophys. Res. Communs. 120, 574-578. Salach, J.I., Singer, T.P., Castagnoli, N., Jr., and Trevor, A. (1984) Biochem. Biophys. Res. Communs. 125, 831-835. Singer, T.P., Salach, J.I., and Crabtree, D. (1985) Biochem. Biophys. Res. Cornnuns. 127, 707-712. Javitch, J.A., D'Amato, R.J., Strittmatter, S.M., and Snyder, S.H. (1984) Proc. Nat. Acad. Sci. U.S. 82, 2173-2177. Nicklas, W.J., Vyas, I., and Heikkila, R.E. (1985) Life Sci. 36, 2503-2508. Shinka, T., and Castagnoli, N., Jr., to be published. Langston, J.W., Irwin, I., Langston, E.B., and Forno, L.S. (1984) Neurosci. Letts. 48, 87-92. Ramsay, R.R., Salach, J.I., and Singer, T.P. Biochem. Biophys. Res. Comnun.,

in press.

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Snyder, S.H. and D'Amato, R.J. (1985) Nature (London) 317, 198-199. Ramsay, R.R., and Singer, T.P., to be published. Beinert, H., Palmer, G., Cremona, T., and Singer, T.P. (1965) J. Biol. 240, 475-480. Singer, T.P., Gutman, M., and Massey V. (1973) in Iron-Sulfur Proteins, Lowenberg, W. (ed.), Academic Press, New York, p. 225-300. Gutman, M., Singer, T.P., and Casida, J.E. (1970) J. Biol. Chem. 245,

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