Rona R. Ramsay

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

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 inhibition by 4-phenylpyridine unaffected but causes efflux of MPP+ from the mitochondria and a reversal of its inhibitory action. In isolated inner membranes the inhibition 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.

Uptake of the neurotoxin 1-methyl-4-phenylpyridine (MPP+) by mitochondria and its relation to the inhibition of the mitochondrial oxidation of NAD+-linked substrates by MPP+

1-methyl-4-phenylpyridine (MPP+), a major product of the oxidation of the neurotoxic amine 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) has been postulated to be the compound responsible for destruction of nigrostriatal neurons in man and primates and for inhibition of mitochondrial NADH oxidation which leads to cell death. We have confirmed that 0.5 mM MPP+ inhibits extensively the oxidation of NAD+-linked substrates in intact liver mitochondria in State 3 and after uncoupling, while succinate oxidation is unaffected. However, in inverted mitochondria, inner membrane preparations, and Complex I NADH oxidation is not significantly affected at this concentration of MPP+, nor are malate and glutamate dehydrogenases or the carriers of these substrates inhibited. We report here the discovery of an uptake system for MPP+ in mitochondria which is greatly potentiated by the presence of malate plus glutamate and inhibited by respiratory inhibitors, suggesting an energy-dependent carrier. A 40-fold concentration of MPP+ in the mitochondria occurs in ten minutes. This might account for the inhibition of malate and glutamate oxidation in intact mitochondria.

Evidence that the blockade of mitochondrial respiration by the neurotoxin 1-methyl-4-phenylpyridinium (MPP+) involves binding at the same site as the respiratory inhibitor, rotenone

It has been postulated that 1-methyl-4-phenylpyridinium (MPP+) blocks mitochondrial respiration by combining at the same site as rotenone, a potent inhibitor of NADH oxidation in mitochondria, known to act at the junction of NADH dehydrogenase and coenzyme Q (CoQ). The present experiments show that MPP+ and two of its analogs indeed act in a concentration dependent manner to prevent the binding of [14C]-rotenone to submitochondrial particles (ETP) and significantly decrease the inhibition of electron transport caused by rotenone. It therefore appears that MPP+ binds at the same site as rotenone or an adjacent site, supporting the hypothesis that its neurotoxic action is due to the inhibition of mitochondrial respiration.

Mechanism of the neurotoxicity of 1-methyl-4-phenylpyridinium (MPP)+, the toxic bioactivation product of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)☆

It is widely believed that the nigrostriatal toxicity of MPTP is due to its oxidation by brain monoamine oxidase first to MPDP+, and eventually to MPP+. Following uptake by the synaptic dopamine reuptake system, it is concentrated in the matrix of striatal mitochondria by an energy-dependent carrier, energized by the electrical gradient of the membrane. At the very high intramitochondrial concentrations thus reached, MPP+ combines with NADH dehydrogenase at a point distal to its iron-sulfur clusters but prior to the Q10 combining site. This leads to cessation of oxidative phosphorylation, ATP depletion, and cell death. Other pyridine derivatives act similarly on NADH dehydrogenase but they are not acutely toxic unless concentrated by the MPP+ carrier.

Inhibition of NADH oxidation by pyridine derivatives

The neurotoxicity of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine, an impurity in an illicit drug, is expressed after its oxidation to 1-methyl-4-phenylpyridinium by monoamine oxidase. The pyridinium is concentrated by carrier-mediated transport into the mitochondria where it inhibits NADH dehydrogenase and, hence, ATP synthesis. Some structurally related compounds have been tested for their effect on the oxidation of NAD+-linked substrates in intact mitochondria, and for the inhibition of the accumulation of the pyridinium into mitochondria and of NADH dehydrogenase activity in a membrane preparation. Some pyridine derivatives are more inhibitory to NADH dehydrogenase than is 1-methyl-4-phenylpyridinium but these are not concentrated into mitochondria by the uptake system. 4-Phenylpyridine, one of the most effective inhibitors, both occurs naturally and is an environmental pollutant.

Enhancement by tetraphenylboron of the interaction of the 1-methyl-4-phenylpyridinium ion (MPP+) with mitochondria

Inhibition of mitochondrial energy production by MPP+ may be the key step in chemically-induced Parkinsons disease. Tetraphenylboron (TPB-) markedly enhances the effect of MPP+. Inhibition of respiration and uptake of MPP+ are accelerated, the former by up to two orders of magnitude. TPB increases the final concentration of MPP+ in the matrix by 2-3 fold, insufficient to explain the rapid inhibition of respiration. TPB- lowers the membrane surface potential by only about 20%, but increases the partitioning of MPP+ into organic solvent by one order of magnitude. TPB- also enhances the effect of MPP+ on inverted membranes, reducing the I50 by an order of magnitude. We suggest that TPB- acts by ion pairing with MPP+ to facilitate penetration into mitochondria as well as access to a hydrophobic inhibition site on NADH dehydrogenase.

Processing of MPTP by monoamine oxidases: implications for molecular toxicology

MPTP (1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine), a selective nigrostriatal neurotoxin, is bioactivated by MAO-B (and less effectively by MAO-A) to 2,3-MPDP+ and this intermediate undergoes further oxidation to MPP+, partly through the activity of MAO forms. MPTP and its two primary metabolites are competitive inhibitors of both A and B forms of MAO. MPTP and 2,3-MPDP+ are also mechanism-based inactivators of both forms of the enzyme. A catalytic mechanism, involving the formation of radical intermediates, is proposed for the MAO-mediated oxidation of MPTP. Post-oxidation biochemical sequelae, possibly involved in the expression of neurotoxicity, include the active accumulation of MPP+ via dopamine reuptake systems, the energy-driven uptake of MPP+ by mitochondria and the inhibition of NADH dehydrogenase by pyridine derivatives. A scheme linking these events as steps in the molecular mechanism of action of MPTP is proposed and discussed in terms of the selective toxicity of the neurotoxin towards nigrostriatal cells.

PALMITOYL-L-CARNITINE, A METABOLIC INTERMEDIATE OF THE FATTY ACID INCORPORATION PATHWAY IN ERYTHROCYTE MEMBRANE PHOSPHOLIPIDS

In this paper we report that palmitoyl-L-carnitine can be a metabolic intermediate of the fatty acid incorporation pathway into erythrocyte membrane phosphatidylcholine, and phosphatidylethanolamine. Phospholipid acylation was evaluated by measuring the incorporation of radioactive [1-14C]-palmitoyl-L-carnitine in membrane erythrocyte ghost phospholipids in the presence or absence of CoA. CoA highly stimulated the incorporation of [1-14C]-palmitic acid into both the phospholipids examined, although the incorporation was also evident in the absence of added CoA. Incorporation of [1-14C]-palmitic acid into phosphatidylcholine was greater than into phosphatidylethanolamine. 2-Bromo-palmitoyl-CoA, an irreversible inhibitor of the erythrocyte carnitine palmitoyltransferase, inhibited the acylation process.

A case of carnitine palmitoyltransferase II deficiency in human skeletal muscle

A 20‐year‐old man was shown to have a deficiency of carnitine palmitoyltransferase (CPT) II in skeletal muscle. The evidence was: (i) there was no significant oxidation of [9,10‐3H]palmitate or of [1‐14C]palmitate in mitochondrial fractions from fresh skeletal muscle from the patient; (ii) all the CPT activity in a homogenate of fresh muscle from the patient was overt (CPT I) with no increase in activity after the inner membrane was disrupted; (iii) all the CPT activity in the patients muscle was inhibited by malonyl‐CoA; and (iv) an immunoreactive peptide of 67 kDa corresponding to CPT II, present in mitochondria from controls, was absent in those from the patient.

Kinetic properties of cloned human liver monoamine oxidase A

Monoamine oxidases deaminate many amines, including neurotransmitters, by oxidation followed by spontaneous breakdown of the imine product. The reduced enzyme is reoxidized slowly by oxygen, but in the presence of amines, the rate of reoxidation is markedly enhanced. The extent of enhancement depends on the amine substrate, kynuramine enhancing the rate 125-fold, but 5-hydroxytryptamine only 6-fold. Here we describe the properties of human liver monoamine oxidase A which has been cloned into and overexpressed in yeast. The purified enzyme has a higher Km for oxygen than does the placental enzyme, but the steady-state parameters for the endogenous amines are the same. Tertiary amines are oxidized at slightly different rates by the two enzymes. The consequences of the branched pathway mechanism with substrate-dependent enhancement of reoxidation for the steady-state levels of the various enzyme species is discussed.