Loss of mitochondrial membrane potential is dependent on the apoptotic program activated: prevention by R-2HMP.

Recent evidence suggests that the mitochondrial membrane potential begins to decrease well before the cells commit to apoptotic death. By using cultured cerebellar granule cells, two types of apoptosis can be induced, one by adding cytosine arabinoside (Ara-c; p53-dependent apoptosis) and one by lowering the K(+) concentrations of the medium (p53-independent apoptosis). Cultures show clear signs of increased apoptosis (chromatin condensation as visualized with bis-benzamide) after 12 hr which increases with time up to 24 hr. A fluorescent probe, chloromethyl-tetramethylrhodamine methyl ester (CMTMR), a lipophilic, potentiometric dye, which when introduced into the media accumulates within mitochondria in proportion to the mitochondrial membrane potential, was added at various time points after the induction of apoptosis. In Ara-c-induced apoptosis, there was a shift in the distribution of cell populations towards low-intensity CMTMR fluorescence, whereas in control and low-K(+) cultures, there was no such shift. This effect was observed as early as 6 hr after adding Ara-c. The antiapoptotic drug R-N-2-heptyl-N-methylpropargylamine hydrochloride (R-2HMP) reversed this loss of mitochondrial membrane potential in Ara-c-induced apoptosis; the effect was antagonized by the S-2HMP.

Neuronal sparing and behavioral effects of the antiapoptotic drug, (-)deprenyl, following kainic acid administration.

(-)Deprenyl is an irreversible inhibitor of monoamine oxidase B (MAO-B) frequently used as an adjunct therapy in the treatment of Parkinson's Disease. Recent evidence, however, has found that deprenyl's metabolites are associated with an antiapoptotic action within certain neuronal populations. Interestingly, deprenyl's antiapoptotic actions appear not to depend upon the inhibition of MAO-B. Due to a paucity of information surrounding (-)deprenyl's ability to spare neurons in vivo, a series of studies was conducted to further investigate this phenomenon within an apoptotic neuronal death model: kainic acid induced excitotoxicity. Results indicated that (-)deprenyl increased hippocampal neuronal survival compared to saline-matched controls following kainic acid insult. Furthermore, it was discovered that (-)deprenyl treatment could be stopped 14 days following CNS insult by kainate, with evidence of neuronal sparing still present by day 28. In open-field locomotor activity testing of kainate-treated animals, those given subsequent (-)deprenyl treatment showed habituation curves similar to control subjects, while saline-treated animals did not. Given deprenyl's antiapoptotic actions, it is proposed that (-)deprenyl may be beneficial in the treatment of a variety of neurodegenerative diseases where evidence of apoptosis exists, such as Parkinson's and Alzheimer's Disease, by slowing the disease process itself.

R-deprenyl and R-2-heptyl-N-methylpropargylamine prevent apoptosis in cerebellar granule neurons induced by cytosine arabinoside but not low extracellular potassium.

R-Deprenyl and R-2-heptyl-N-methylpropargylamine (R-2-HMP) are compounds that have been shown to reduce neuronal death in various in vitro and in vivo models involving apoptosis but do not always prevent apoptosis. In the present study we have examined the effects of these compounds and their S enantiomers on cytosine arabinoside (ara C)-induced apoptosis and low K+-induced apoptosis in cerebellar granule cells in primary culture. It was found that R-deprenyl and R-2-HMP could prevent ara C-induced apoptosis with an EC50 around 10(-9) M but could not prevent low K+-induced apoptosis. S-Deprenyl and S-2-HMP did not prevent apoptosis under any conditions but were found to antagonize the antiapoptotic actions of R-deprenyl and R-2-HMP. Using the fluorescent mitochondrial dye chloromethyltetramethylrhodamine methyl ester it was found that there was a loss of mitochondrial function in cerebellar granule cells exposed to ara C but not low K + medium. R-Deprenyl and R-2-HMP prevented the ara C-induced loss of mitochondrial function. It is concluded that R-deprenyl and R-2-HMP prevent apoptosis of cerebellar granule cells by a mechanism that is independent of monoamine oxidase inhibition and that they act on the same site to prevent specifically apoptosis involving a loss of mitochondrial membrane potential, possibly p53-dependent apoptosis.

Qualitative analysis of membrane currents in glial cells from normal and gliotic tissue in situ: down-regulation of Na+ current and lack of P2 purinergic responses.

To date, the electrophysiological properties of glial cells located in reactive scar tissue are unknown. To address this issue two subtypes of hippocampal glial cells, located in thin vital slices of normal or gliotic brain tissue, were analysed for their voltage controlled ion channels using the patch-clamp technique. Reactive gliosis was induced in adult rats by a single peritoneal injection of kainic acid. The intensity of the following seizures was rated ascending from 1 to 6. Rats which exhibited seizures of level 3 or higher showed, within three days, a marked loss of pyramidal cells (60% in CA1 and CA3) and an increase in the density of glial fibrillary acidic protein immunostaining, representing an apparent increase in the number and size of astrocytes in all layers of the hippocampal CA1 subfield. Reactive and normal astrocytes of one subtype, electrophysiologically characterized by time-independent potassium currents, did not significantly differ in membrane potential and potassium conductivity. Glutamine synthetase-positive, but mostly glial fibrillary acidic protein-negative, glial cells (presumably representing immature astrocytes) were also included in this study. This subtype of glial cells showed several voltage- and time-dependent potassium currents and, under control conditions, tetrodotoxin-sensitive voltage-gated Na+ channels, which were almost completely lost after reactive gliosis. Another part of this study focuses on the sensitivity of reactive and control glial cells for extracellular ATP. Several in vitro studies suggest that P2 purinergic receptors on glial cells could trigger the induction of reactive gliosis. In contrast to results described on cultured astrocytes, we found in situ that hippocampal glial cells were not sensitive to ATP or stable P2 receptor agonists in control or in gliotic brain slices. In summary, the presence of at least two different subtypes of hippocampal astrocytes was demonstrated for control as well as for gliotic brain tissue. A dramatic down-regulation of tetrodotoxin-sensitive sodium channels in one subpopulation of reactive astrocytes was shown. This result supports the hypothesis that the presence of active neurons could be required to maintain glial voltage-gated sodium channels. Furthermore, we conclude that there is no longtime expression of P2 purinoceptors on hippocampal astrocytes in situ, and therefore the involvement of astrocytic ATP receptors in the genesis of reactive gliosis is unlikely.

(-)Deprenyl reduces delayed neuronal death of hippocampal pyramidal cells.

Ischemia-induced delayed neuronal death can be mediated by apoptosis, and (-)deprenyl has been shown to block apoptosis in dopaminergic and cholinergic neurons. This study has investigated whether (-)deprenyl can prevent delayed neuronal death of hippocampal pyramidal cells. Rats were subjected to unilateral hypoxia-ischemia and treated with (-)deprenyl (0.25 mg/kg, s.c.) or saline daily. After sacrifice the left and right hippocampi were examined histologically. Unilateral delayed neuronal death was seen in the CA1, CA3 and CA4 fields up to 14 days after the ischemia. After 14 days' treatment with (-)deprenyl there was 66%, 91% and 96% reduction in delayed neuronal death in the CA1, CA3 and CA4 fields, respectively. (-)Deprenyl was effective when given at the onset or after ischemia, but not when given 2 h before ischemia. The reduction in ischemia-induced delayed neuronal death is consistent with an anti-apoptotic mechanism of (-)deprenyl.

Inhibition of MAO-B by (-)-deprenyl alters dopamine metabolism in the macaque (Macaca facicularis) brain.

The present study has examined whether MAO-B has a role in DA metabolism in the primate CNS in situ. Eleven macaques (macaca facicularis) were used in this study to examine the effects of (-)-deprenyl (1 mg/kg, i.v., 2 and 24 hours). (-)-Deprenyl administration completely and selectively blocked MAO-B activity and blocked DA metabolism in the caudate nucleus and frontal cortex. DA metabolism in the substantia nigra was not affected by MAO-B inhibition. Changes in DA metabolism were accompanied by changes in 5-hydroxytryptamine (5HT) turnover: 5-hydroxyindole acetic acid (5HIAA) levels increased in the caudate and decreased in the frontal cortex. Levels of 2-phenylethylamine (PE), a putative modulator of dopaminergic transmission, were increased by MAO-B inhibition in all three brain regions examined. It is concluded that in some regions of the primate brain, in contrast to the rat, MAO-B has an important role in DA metabolism.

An intact glutamatergic trigeminal pathway is essential for the cardiac response to simulated diving.

Nasal water flow plus concomitant expiratory apnea in anesthetized (Innovar-Vet), paralyzed, and artificially ventilated rats produces immediate bradycardia. To investigate the origin of this response, four procedures were used to block the trigeminal pathway. 1) Trigeminal receptors within the nasal passages were anesthetized by infusing local anesthetic through the external nares. 2) Trigeminal nerves that innervate the nasal passages were sectioned bilaterally as they passed through the orbit. 3) The trigeminal neural pathway was blocked within the brain stem by either electrolytically lesioning or infusing local anesthetic into the spinal trigeminal nucleus interpolaris (Sp5I). 4) Synaptic transmission within Sp5I was prevented by infusing glutamate receptor antagonists D-2-amino-7-phosphonoheptanoic acid and 6,7-dinitroquinoxaline-2,3-dione. After each of the procedures was completed, the cardiovascular responses to nasal water flow plus apnea were either attenuated or eliminated. The major conclusion of this study is that an intact glutamatergic trigeminal pathway is required for manifestation of the cardiovascular responses to nasal stimulation. Evidence also suggests that N-methyl-D-aspartate (NMDA) and non-NMDA glutamate receptors are both required for synaptic neurotransmission within Sp5I.

Neurochemical, neuroprotective and neurorescue effects of aliphatic N-methylpropargylamines; new MAO-B inhibitors without amphetamine-like properties.

A series of aliphatic N-methylpropargylamine MAO-B inhibitors have been synthesized and their structural and functional relationships have been investigated. 2-Hexyl-N-methylpropargylamine (2-HxMP), for example, has been found to be a highly potent, irreversible, selective, MAO-B inhibitor both in vitro and in vivo. The R-(-)-enantiomers are much more active than the S-(+)-enantiomers at inhibiting MAO-B activity. Some of these compounds protect mouse nigrostriatal dopamine neurons against the neurotoxin MPTP and the mouse hippocampal noradrenergic system against the neurotoxin N-(2-chloroethyl)-N-ethyl-2-bromobenzylamine (DSP-4). They rescue hippocampal neurons after damage induced by ischemia and kainic acid treatment, as well as motoneurons in young mice following facial nerve axotomy. Such rescue effects are, interestingly, unrelated to inhibition of MAO-B activity. Some of the aliphatic propargylamines enhance the survival of neuroblastoma cells co-cultured with astrocytes following serum depletion. They stimulate the expression of AADC mRNA and inhibit GFAP mRNA expression. They do not possess amphetamine-like properties and exhibit no effect on noradrenaline or dopamine uptake nor do they increase hypertensive effects in the tyramine pressor test. Unlike R(-)-deprenyl, 2-HxMP does not potentiate dopamine toxicity in vitro. These new MAO-B inhibitors may possess significant chemotherapeutic implications for certain psychiatric and neurodegenerative disorders.

The effects of administration of monoamine oxidase-B inhibitors on rat striatal neurone responses to dopamine.

1. (-)-Deprenyl has been shown to potentiate rat striatal neurone responses to dopamine agonists at doses not altering dopamine metabolism. Since there are a number of effects of (-)-deprenyl which could result in this phenomenon, we have investigated the effects of MDL 72,145 and Ro 19-6327, whose only common effect with (-)-deprenyl is an inhibition of monoamine oxidase-B (MAO-B), on rat striatal neurone responses to dopamine and on striatal dopamine metabolism. 2. Using in vivo electrophysiology, i.p. injection of either MDL 72,145 or Ro 19-6327 was found to produce a dose-dependent potentiation of striatal neurone responses to dopamine but not gamma-aminobutyric acid. 3. Neurochemical investigations revealed that this occurred at doses (0.25-1 mg kg-1) which, while not affecting levels of dopamine or its metabolites, 3,4-dihydroxyphenylacetic acid or homovanillic acid, did cause a significant, dose-dependent, elevation in striatal levels of the putative neuromodulator, 2-phenylethylamine (PE). 4. Inhibition of PE synthesis by i.p. injection of the aromatic L-amino acid decarboxylase inhibitor, NSD 1015, produced a reversal of the effects of MDL 72,145 and Ro 19-6327. 5. Neurochemical analysis revealed this to occur at a dose of NSD 1015 (10 mg kg-1) selective for reduction of elevated PE levels. 6. These results suggest that PE can act as a neuromodulator of dopaminergic responses and that MAO-B inhibitors may potentiate neuronal responses to dopamine via the indirect mechanism of elevation of PE following MAO-B inhibition.

Regulation of aromatic L-amino acid decarboxylase in rat striatal synaptosomes: effects of dopamine receptor agonists and antagonists.

1. In this study we investigated the effects of dopamine receptor agonists and antagonists on rat striatal synaptosomal aromatic L-amino acid decarboxylase (AADC) activity. 2. The results show that 10(-5)-10(-7) M cis-flupenthixol increased the striatal synaptosomal AADC activity (by 25% to 57%) in a time-dependent manner. SCH 23390 and remoxipride alone had little or no effect on striatal synaptosomal AADC activity, but in combination they increased AADC activity by 20%, suggesting that the increases in striatal synaptosomal AADC activity occurred only after blockade of both dopamine D1 and D2 receptors. 3. Treatment with (+)-amphetamine and (+/-)-2-(N-phenylethyl-N-propyl)amino-5- hydroxytetralin hydrochloride ((+/-)-PPHT) produced a reduction of striatal synaptosomal AADC activity in a concentration- and time-dependent manner. SKF 38393 and (-)-quinpirole, however, exhibited no effect on striatal synaptosomal AADC activity, suggesting that only the mixed dopamine receptor agonists can reduce the AADC activity. Incubation with apomorphine at a concentration of 10(-4) M inhibited the AADC activity by 74% and this inhibition cannot be antagonized by SCH 23390, remoxipride or cis-flupenthixol, suggesting that apomorphine-induced inhibition of striatal synaptosomal AADC activity was not mediated by dopamine receptors. 4. cis-Flupenthixol can reverse the reduction of AADC activity induced by (+)-amphetamine and (+/-)-PPHT. The inhibition of AADC activity elicited by (+/-)-PPHT also can be reversed by SCH 23390 and remoxipride. 5. The inhibition of striatal synaptosomal AADC activity induced by (+/-)-PPHT is calcium-dependent and protein kinase C may play a role in the regulation of striatal AADC activity. 6. These studies show that striatal synaptosomal AADC activity is regulated by dopamine receptors and indicate that in vitro dopamine DI and D2 receptors have a synergistic effect in this regulation.

Dopamine metabolism in the guinea pig striatum: role of monoamine oxidase A and B.

These studies were carried out to determine whether the greater abundance of monoamine oxidase B in the guinea pig affects the actions of (-)-deprenyl on dopamine metabolism in whole tissue or in extracellular fluid. The administration of (-)-deprenyl in doses that do not affect monoamine oxidase A activity (1-4 mg kg-1, 2 h) increases striatal 2-phenylethylamine and dopamine concentrations and reduces 3,4-dihydroxyphenylacetic acid. No effects were observed on striatal homovanillic acid, 5-HT and 5-hydroxyindole acetic acid. Inhibition of monoamine oxidase A with clorgyline with doses up to 8 mg kg-1 (2 h) does not affect striatal 2-phenylethylamine but increases dopamine and 5-HT concentrations and reduces 3,4-dihydroxyphenyl-acetic acid and 5-hydroxyindole acetic acid. (-)-Deprenyl (2-4 mg kg-1) did not change the extracellular concentrations of dopamine but the higher dose produced a limited reduction in extracellular 3,4-dihydroxyphenylacetic acid. Inhibition of monoamine oxidase A and monoamine oxidase B with pargyline (75 mg kg-1, 2 h) significantly increased the levels of extracellular dopamine and reduced those of their acid metabolites. These results show that in the guinea pig striatum inhibition of monoamine oxidase B by (-)-deprenyl impairs the metabolism of dopamine in the whole tissue but does not produce a marked increase in extracellular dopamine.

The effects of monoamine oxidase B inhibition on dopamine metabolism in rats with nigro-striatal lesions.

The purpose of this study was to examine whether monoamine oxidase type B (MAO-B) has a role in striatal dopamine metabolism in animals with a unilateral lesion of the medial forebrain bundle, and whether 2-phenylethylamine (PE) could have a role in amplification of dopamine (DA) responses in DA depleted striatum. Inhibition of MAO-B did not alter DA metabolism in lesioned striata. PE accumulation decreased with loss of DA as long as there was no DA dysfunction. In lesioned striata with dysfunction of DA transmission at the synaptic level, PE accumulation increased, suggesting a compensatory increase in PE synthesis. This increase in PE levels does not appear to be mediated by an increase in the total striatal aromatic L-amino acid decarboxylase (AADC) activity. We conclude that inhibition of MAO-B has no effect on DA metabolism in the hemi-parkinsonian rat striatum and that PE could be involved in the antiparkinsonian action of MAO-B inhibitors.

The effect of L-deprenyl on behavior, cognitive function, and biogenic amines in the dog.

Behavioral and pharmacological effects of oral administration of L-deprenyl in the dog are described. Spontaneous behavior is unaffected at doses below 3 mg/kg while at higher doses there was stereotypical responding. There was evidence of improved cognitive function in animals chronically treated with a 1 mg/kg dose but the effectiveness varied considerably between subjects. Chronic administration produced a dose dependent inhibition in brain, kidney and liver monoamine oxidase B, and had no effect on monoamine oxidase A. There were also dose dependent increases in brain phenylethylamine and in plasma levels of amphetamine. Dog platelets did not have significant levels of MAO-B. Brain dopamine and serotonin metabolism were unaffected by L-deprenyl at doses up to 1 mg/kg. It appears that for the dog, deamination of catecholamines is controlled by MAO-A. Nevertheless, it is suggested that L-deprenyl serves as a dopaminergic agonist, and there is also evidence that it affects adrenergic transmission. These catecholaminergic actions may account for the effects of L-deprenyl on behavior and cognitive function.

The potentiation of cortical neuron responses to noradrenaline by 2-phenylethylamine is independent of endogenous noradrenaline.

2-Phenylethylamine (PE) is an endogenous brain amine which produces sympathomimetic responses and potentiates cortical neuron responses to noradrenaline (NA). In order to examine further the mechanism of action of PE, extracellular recordings were made of the activity of single neurones in the cerebral cortex in urethane-anesthetized rats. Sympathomimetic responses to PE were blocked by pretreatment with reserpine, reserpine plus alpha-methyl-p-tyrosine and desipramine. It is concluded that the sympathomimetic responses to PE are indirect. 2-Phenylethylamine potentiated cortical neuron responses to electrical stimulation of the locus coeruleus in a dose-dependent manner. This was seen when PE was given systemically (with as little as 1 microgram/kg) and iontophoretically. The effects of PE were not reproduced by its metabolite phenylacetic acid or its putative metabolite phenylethanolamine. Iontophoretic applications of PE (0-6 nA, 2-5 minutes) potentiated cortical neuron responses to iontophoretically applied NA, without affecting the spontaneous firing rate, or the responses to iontophoretically applied GABA or acetylcholine. This effect of PE was not blocked by pretreatment with alpha-methyl-p-tyrosine or desipramine, and was potentiated by pretreatment with reserpine and reserpine plus alpha-methyl-p-tyrosine. It is probable that the ability of PE to modulate neuronal responses to NA does not involve the presynaptic NA terminal or endogenous NA and it is likely that PE acts directly to increase the efficacy of NA. These findings are consistent with the hypothesis that the physiological role of PE is to modulate catecholaminergic transmission within the central nervous system.

Decarboxylation of L-dopa by cultured mouse astrocytes.

Cultured astrocytes contain immunologically specific aromatic L-amino acid decarboxylase (AADC) protein and express the AADC gene. Following incubation with L-Dopa, glial cultures synthesize and metabolize dopamine. The addition of pyridoxal 5-phosphate did not change the rate of dopamine synthesis. The formation of dopamine was blocked by NSD-1015. These experiments show that mouse cultured astrocytes are capable to convert L-Dopa into dopamine in a dose-dependent fashion.