Biochemical and spectroscopic characterization of Escherichia coli aconitases (AcnA and AcnB).

Escherichia coli contains two major aconitases (Acns), AcnA and AcnB. They are distantly related monomeric Fe-S proteins that contain different arrangements of four structural domains. On the basis of the differential expression of the acnA and acnB genes, AcnA has been designated as an aerobic-stationary-phase enzyme that is specifically induced by iron and oxidative stress, whereas AcnB functions as the major citric-acid-cycle enzyme during exponential growth. The biochemical and kinetic properties of the purified enzymes have now shown that AcnA is more stable than AcnB, has a higher affinity for citrate, and operates optimally over a wider pH range, consistent with its role as a maintenance or survival enzyme during nutritional or oxidative stress. In contrast, the better performance at high substrate concentrations and greater instability of AcnB indicate that AcnB is specifically adapted to function as the main catabolic enzyme and, by inactivation, to rapidly modulate energy metabolism in response to oxidative or pH stress, either directly or indirectly by regulating post-transcriptional gene expression. EPR and magnetic-CD spectroscopy showed that the iron-sulphur clusters of the bacterial Acns (and their binding sites) strongly resemble those of the mammalian enzymes. The EPR and MCD spectra of the oxidized inactive form of AcnB confirmed the presence of a [3Fe-4S](1+) (S=1/2) cluster. Comparisons showed that the EPR spectrum of AcnB more closely resembled that of mammalian mitochondrial Acn (m-Acn), whereas the spectrum of AcnA more closely resembled that of the cytoplasmic enzyme (c-Acn). The MCD spectra revealed spectroscopic signatures similar to that of m-Acn. Reconstitution of the active [4Fe-4S](2+) forms followed by one-electron reduction gave rise to EPR spectra that are almost identical with those reported for the mammalian enzymes.

Behavioural and biochemical consequences following activation of 5HT1-like and GABA receptors in the dorsal raphé nucleus of the rat.

The behavioural and biochemical response to the 5-HT1-like receptor compounds, 5-carboxamidotryptamine (5-CT), 8-hydroxy-2-(di-n-propylamino) tetralin (8-OH-DPAT), buspirone and ipsapirone and the GABA agonist, muscimol, injected into the dorsal raphé nucleus (DRN) are reported. All compounds increased social interaction under high light, unfamiliar conditions and increased punished responding in a Vogel conflict test. At doses ranging from 5-25 times greater than those which were effective in these anxiety models, muscimol, 5-CT and 8-OH-DPAT induced a marked hypothermia and a flattening of body posture. Buspirone, on the other hand, failed to induce a significant reduction in core temperature or produce a marked flattening of posture in all animals, even at doses 100 times those effective in anxiety models. Following injection of muscimol, 5-CT, 8-OH-DPAT and buspirone into the dorsal raphé nucleus, all tended to reduce the 5-HIAA:5-HT ratios in the frontal cortex, hippocampus and hypothalamus. These findings, together with available electrophysiological data suggest that these behavioural responses are a consequence of a depression of the firing of cells in the dorsal raphé nucleus, with a corresponding decrease in functional activity of 5-HT in the forebrain.

5-Hydroxytryptamine involvement in the locomotor activity suppressant effects of amphetamine in the mouse.

d-Amphetamine, in doses lower than required to increase motor activity, reduced mouse spontaneous locomotor activity when this was assessed using cages equipped with photocell units, using treadwheels, or the measurement of spontaneous climbing behaviour. Acute treatments with the serotonergic agonists quipazine and 5-hydroxy-DL-tryptophan also reduced wheel running activity, spontaneous locomotor activity assessed using photocell cages, and spontaneous climbing behaviour; fenfluramine caused a similar effect. Pretreatment with 5-hydroxy-DL-tryptophan enhanced the inhibitory effects of d-amphetamine. A 3-day treatment with fenfluramine, or lesions of the median raphe nucleus (but not the dorsal raphe nucleus) abolished the ability of d-amphetamine to reduce motor activity in the three test systems. It is concluded that low doses of d-amphetamine can reduce locomotor activity and that the effects may be mediated via an enhancement of the release of 5-hydroxytryptamine from the system arising in the median raphe nucleus.

MPP+ can disrupt the nigrostriatal dopamine system by acting in the terminal area.

Acute intracerebral injections of MPP+ in the mouse can cause behavioural and biochemical correlates of nigrostriatal dopamine dysfunction, but these are most marked and consistent when injections are directed at the dopamine nerve terminal area (CP) rather than the cell body area (SN). This adds further support to the hypotheses that a locus of neurotoxic action of MPTP/MPP+ may reside in the dopamine cell terminal regions.

The effect of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) on striatal and limbic catecholamine neurones in white and black mice. Antagonism by monoamine oxidase inhibitors.

Albino mice and pigmented mice were treated for 6 days with 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) at the maximum tolerated doses (2 days at 30 mg/kg i.p., 2 days at 40 mg/kg i.p. and 2 days at 50 mg/kg i.p. in white mice, 6 days at 30 mg/kg i.p. in pigmented mice) and the effects of simultaneous treatment with the monoamine oxidase inhibitors, deprenyl (1 mg/kg, i.p.), MDL 72145 (0.5 mg/kg, i.p.) and clorgyline (5 mg/kg, i.p.), determined behaviourally (daily for 6 days and for 4 days after withdrawal) and biochemically (92 hr after withdrawal of drug). In albino mice MPTP caused depletions of dopamine (90%), dihydroxyphenylacetic acid (DOPAC; 82%) and homovanillic acid (HVA; 65%) in the striatum and in dopamine (54%), DOPAC (51%) and HVA (53%) in the nigra. However, MPTP was not selective in its action since the levels of dopamine and its metabolites were also reduced in limbic tissue. Further, MPTP affected the function of noradrenaline, with reduced levels in tissues of the striatum (74%) and nigra (46%). Pigmented mice were as susceptible as albino mice to the actions of MPTP to reduce the levels of dopamine and metabolites in the striatum. However, the limbic areas and substantia nigra of the pigmented mouse were more resistant to the actions of MPTP. Treatment with deprenyl and MDL 72145 (but not clorgyline) could be shown to reduce the biochemical and behavioural consequences of the action of MPTP (although behavioural changes, development of severe motor incapacitation and prostrate appearance, appeared to be non-specific).(ABSTRACT TRUNCATED AT 250 WORDS)

Biochemical changes caused by the infusion into the substantia nigra of the rat of MPTP and related compounds which antagonise dihydropteridine reductase.

1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), 1-methyl-4-phenylpyridinium bromide (MPP+), 1-methyl-4-(3', 4'-dihydroxyphenyl)pyridinium bromide, 4-(3',4'-dihydroxyphenyl)pyridine, 4-phenyl-1,2,3,6-tetrahydropyridine and 4-(3',4'-dimethoxyphenyl)1,2,3,6-tetrahydropyridine were infused bilaterally into the substantia nigra of the rat (10 micrograms/24 hr for 4 days). The ability to inhibit spontaneous locomotor activity and to reduce levels of neurotransmitters and metabolites in the nigrostriatal system (striatum, substantia nigra) was compared with activity to inhibit dihydropteridine reductase (DHPR) in vitro. The compound MPP+ was most effective to reduce motor responding and to decrease levels of dopamine, DOPAC and HVA (50-56%) in the striatum in addition to reducing levels of dopamine, DOPAC, noradrenaline, serotonin and 5-HIAA (42-86%) in the substantia nigra, yet MPP+ has been shown to have very weak ability to inhibit DHPR. In contrast, 4-(3',4'-dihydroxyphenyl)pyridine and 1-methyl-4-(3',4'-dihydroxyphenyl)pyridinium bromide were in the order of 10(4) and 2 X 10(5) times, respectively, more potent than MPP+ to inhibit DHPR in vitro, but these compounds failed to modify dopamine neuronal function when assessed in vivo. Therefore, there would not appear to be any correlation between the ability to modify dopamine neuronal function, as assessed behaviourally or biochemically, and ability to inhibit DHPR in synaptosomes from the striatum of the rat in vitro.

The neurotoxic actions of 6-hydroxydopamine infused into the rat substantia nigra.

6-Hydroxydopamine (6-OHDA) 10 and 40 micrograms/24 h infused bilaterally for 4 days into the rat substantia nigra (SN) caused a 'freezing' akinetic response which was apparent within 24 h and which persisted throughout the period of infusion. The infusion of 10 or 40 micrograms/24 h 6-OHDA into the SN failed to cause any change in motor coordination, or induce limb and body rigidity. The infusion of 40 micrograms/24 h 6-OHDA led to significant reductions in the striatal levels of dopamine, 3,4-dihydroxyphenylacetic acid, homovanillic acid and noradrenaline; no changes were observed following the 4-day infusion of 10 micrograms 6-OHDA. The consequences of infusing 6-OHDA into the SN are discussed in relation to those induced by 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) and 1-methyl-4-phenylpyridine (MPP+) where the spectrum of behavioural and biochemical change caused by 6-OHDA is shown not to mimic the actions of either MPTP or MPP+.

Evidence that 5-hydroxytryptamine may exert both facilitatory and inhibitory control of electrical field stimulation-evoked contractions in longitudinal muscle taken from the body of guinea-pig stomach.

Electrical field stimulation (FS) of guinea-pig stomach body longitudinal muscle strips caused frequency-related contractions mediated via cholinergic mechanisms. Metoclopramide (10(-8)-10(-5) M), MDL 72222 (10(-9)-10(-7) M) and 5-hydroxytryptophan (5-HTP) (3 X 10(-7)-2.4 X 10(-4) M) enhanced these contractions in all tissues, whereas 5-hydroxytryptamine (5-HT) (3 X 10(-8)-3 X 10(-5) M) enhanced the contractions, but only in approximately 15% of the tissues tested. FS-induced contractions were also enhanced in tissues treated in-vitro with p-chlorophenylalanine (2.5 X 10(-7)-2.5 X 10(-5) M) or monofluoromethyldopa (6 X 10(-7)-10(-4) M) or in tissues taken from animals having received p-chlorophenylalanine or monofluoromethyldopa. It is concluded that cholinergic-mediated contractions of stomach strips are subject to 5-HT modulation in two ways. The predominant action of endogenous 5-HT is to exert an inhibitory tone mediated via a metoclopramide and MDL 72222-sensitive 5-HT neuronal receptor. Exogenously applied 5-HT has little overt action to increase the essentially maximal inhibitory action of endogenous 5-HT, but acts on a 5-HT facilitatory receptor system to enhance contractions. Therefore, the actions of 5-HT agonists and antagonists to modify contractions in stomach strips will reflect the balance between 5-HT inhibitory and facilitatory influences, and the specificity of action of the compounds for the two 5-HT receptor systems.

1-methyl-4-phenylpyridine is neurotoxic to the nigrostriatal dopamine pathway.

Systemic administration of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) is neurotoxic to cerebral dopaminergic neurones in several animal species, and can cause parkinsonism in man. The mechanism of this action may be indirect. MPTP is oxidized in the brain to a pyridinium species, 1-methyl-4-phenylpyridine (MPP+)6. This oxidation is greatly decreased by inhibition of monoamine oxidase B6, as are the biochemical effects of MPTP in the mouse and its neurotoxicity in the monkey. We now show that MPP+ exerts a powerful neurotoxic action on the nigrostriatal dopamine system of the rodent.

Neuropharmacological manipulations with MPTP.

Studies have been carried out in the rodent and marmoset to assess (i) the selectivity of MPTP action to the nigrostriatal system, (ii) the possibility that melanin pigmentation may influence the actions of MPTP, (iii) whether the metabolites of MPTP may contribute to its actions on dopamine cells, (iv) the site(s) of action of MPTP in the brain, (v) the mechanism(s) of action of MPTP and whether it is possible to prevent the damaging effects of MPTP/metabolites on the brain dopamine systems. The peripheral administration of MPTP in the mouse causes depletions of dopamine and its metabolites in both the striatal and limbic systems: this action is similar in both white and pigmented mice. The MAO-B inhibitor deprenyl was shown to antagonize the actions of MPTP, and its metabolite formed via oxidation through MAO, MPP+, was shown to disrupt striatal dopamine function as determined behaviorally (as motor impairment) or biochemically (as loss of dopamine and its metabolites) when injected into the cerebral ventricles of mouse or infused into the substantia nigra of rat brain. MPTP and MPP+ act in the midbrain to interfere with dopamine cell functioning, and studies in both the rodent and marmoset are presently analyzing possible additional actions in forebrain regions. It is therefore proposed that the neurotoxic action of MPTP, effected via its metabolite MPP+, may not be selective for the nigrostriatal system. Nevertheless, the nature and profile of neurotoxicity effected by MPTP and, in particular, MPP+ should ensure continuing analyses of the relevance of such action to an understanding of the etiology of, treatment of, and even the possible prevention of Parkinson's disease in man.

Laterality of dopamine function and neuroleptic action in the amygdala in the rat.

Rats selected according to turn preference in an open field were categorised as showing left or right hemispheric dominance (turning to right or left respectively). Dopamine was persistently infused into the central area of the left or right amygdala of animals showing left or right hemispheric dominance. Infusion was effected for 9 days by Alzet osmotic minipumps, implanted subcutaneously, which delivered 25 micrograms/24 hr of dopamine via chronically-indwelling, stereotaxically-located injection units. Dopamine caused marked and consistent hyperactivity only when infused into the left amygdala when the right hemisphere was dominant. This hyperactivity developed during the first day of infusion and persisted throughout the 9-day infusion period. Both (-)sulpiride and fluphenazine, injected unilaterally into the amygdala, antagonised the hyperactivity caused by infusion of dopamine into the left amygdala in animals with right hemispheric dominance. This antagonism could be effected both from the infused amygdala [5-50 pg (-)sulpiride, 25-100 pg fluphenazine] and from the contralateral amygdala [100-250 pg (-)sulpiride, 25-100 pg fluphenazine]. Thus, a laterality was shown for the action of dopamine in the amygdala of the rat. In contrast, neuroleptic agents failed to show an exclusive laterality of action, but were able to act in either hemisphere to antagonise the effects of dopamine injected into the left amygdala. Interhemispheric biochemical differences "within-animals" could not be shown, although differences were seen between rats having right or left hemispheric dominance.(ABSTRACT TRUNCATED AT 250 WORDS)

Biochemical correlates of motor changes caused by the manipulation of dopamine function in the substantia nigra of the mouse.

2-Di-n-propylamino-5,6-dihydroxytetralin, injected bilaterally into the substantia nigra of the mouse, caused dose-dependent motor inhibition which was associated with decreased levels of DOPAC and increased levels of dopamine in the striatum. (-)Sulpiride, injected into the substantia nigra, antagonised the locomotor depression although the partial antagonism of the elevation in the level of dopamine in the striatum and of the reduction in levels of DOPAC did not achieve significance. The specificity of the action of tetralin on dopamine receptors was shown by the failure of prazosin and yohimbine to antagonise the locomotor depression induced by tetralin and the reduction in levels of DOPAC. The selectivity of the action of tetralin for the dopamine system was shown by its failure to affect levels of noradrenaline, serotonin and 5-hydroxyindoleacetic acid in the striatum. The injection of tetralin into the substantia nigra also caused biochemical changes in limbic areas (nucleus accumbens and tuberculum olfactorium), where the levels of dopamine and DOPAC were elevated, and in the frontal cortex where the levels of DOPAC were reduced. These changes were antagonised by a concomitant injection of (-)sulpiride into the substantia nigra. It is concluded that the action of dopamine agonists in the midbrain can decrease the functional activity in the ascending dopaminergic pathways.

The toxic actions of MPTP and its metabolite MPP+ are not mimicked by analogues of MPTP lacking an N-methyl moiety.

1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), its metabolite 1-methyl-4-phenylpyridine (MPP+) and three analogues of MPTP, lacking an N-methyl moiety, namely, 4-phenylpiperidine (I), 4-phenyl-1,2,3,6-tetrahydropyridine (II) and 4-phenylpyridine (III), were infused continuously for a period of 4 days into the rat substantia nigra. Within 12 h of commencing the bilateral infusion of MPTP or MPP+, rats showed marked motor deficits with reduction in locomotor activity, loss of ability to move the forelimbs and grip with forepaws and, following MPP+ infusions, similar loss of movement in the hindlimbs associated with the development of limb and body rigidity. These motor deficits were not induced by the 3 analogues of MPTP on infusion into the substantia nigra. After 4 days of infusion, the motor deficits caused by MPTP and, in particular, MPP+, were still marked, and for MPP+ these correlated with marked loss of striatal dopamine (DA), 3,4-dihydroxyphenylacetic acid (DOPAC) and homovanillic acid. 4-Phenyl-1,2,3,6-tetrahydropyridine caused a small loss in striatal DA and DOPAC, but the other analogues failed to modify the striatal content of DA or its metabolites. Small alterations of chemical structures related to MPTP and its metabolite can critically alter ability to induce behavioural and neurochemical changes reflecting toxicity on the nigrostriatal DA system.

The neurotoxic actions of 1-methyl-4-phenylpyridine (MPP+) are not prevented by deprenyl treatment.

1-Methyl-4-phenylpyridine (MPP+) injected into the cerebral ventricles (ICV) of mouse caused depletions of striatal dopamine (DA)(-42%), 3,4-dihydroxyphenylacetic acid (DOPAC) (-34%) and homovanillic acid (HVA) (-16%) content without significant reductions in levels of noradrenaline (NA), serotonin (5-HT) or 5-hydroxyindoleacetic acid (5-HIAA). When deprenyl was administered before MPP+, striatal DA and its metabolites were further depleted, and striatal NA and 5-HT levels also were reduced. Further, whilst ICV MPP+ alone failed to influence the biochemistry of the limbic areas (nucleus accumbens plus tuberculum olfactorium), in the presence of deprenyl MPP+ caused 20-40% reductions in levels of limbic NA, DA, DOPAC, HVA, 5-HT and 5-HIAA. Therefore, deprenyl treatment does not prevent the neurotoxic actions of MPP+; indeed, a more extensive neurotoxicity for MPP+ is revealed in the presence of this monoamine oxidase inhibitor.

Inhibition of serum melatonin concentration and synthesis of brain indolealkylamines by monofluoromethyldopa in the rat.

The simultaneous effect of alpha-monofluoromethyldopa (MFMD) on the synthesis of rat brain indolealkylamines has been investigated both in the daytime and in the dark phase. The effect on serum melatonin concentration has also been determined in the dark period. MFMD inhibits 5-hydroxytryptophan decarboxylase in the pineal gland, hypothalamus, and the cerebral cortex. Simultaneous measurement of 5-hydroxytryptophan (5-HTP), 5-hydroxytryptamine (5-HT), and 5-hydroxyindoleacetic acid (5-HIAA) levels in the three brain areas revealed that MFMD caused large increases in pineal 5-HTP concentrations and substantial decreases in pineal 5-HT and 5-HIAA both in the light and dark phases; a significant decrease in daytime hypothalamic 5-HT content after 7-h pretreatment; and a large increase in dark-phase cortical 5-HT concentration after 4-h pretreatment. Serum melatonin levels were also reduced by the action of MFMD in the dark period. The evidence suggests that differences occur in the rate-limiting steps in the indolealkylamine biosynthesis in the three brain areas.