1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) disrupts social memory/recognition processes in the male mouse

Male mice treated with MPTP or vehicle were tested for their ability to demonstrate a memory-recognition response as evaluated in a habituation-dishabituation task. Treatment with MPTP severely disrupted the male's habituation-dishabituation response profile compared to vehicle treated animals. Administration of L-DOPA at 45 min prior to behavioral testing in MPTP animals restored their performance on the habituation-dishabituation test to levels observed in vehicle treated animals. There was also a tendency for L-DOPA to produce enhanced responsiveness in vehicle treated animals. Mice treated with MPTP had significantly reduced concentrations of norepinephrine within the olfactory bulb and hippocampus. Vehicle treated mice administered L-DOPA had significantly increased dopamine concentrations within the corpus striatum. These results suggest that, in addition to its putative effects upon the nigrostriatal dopaminergic system and motor behavior, MPTP is also exerting substantial effects upon other systems. In particular, the noradrenergic system and its potential involvement with memory/recognition processes in the CD-1 mouse appears to be very sensitive to the neurotoxic effects of MPTP.     

Effects of ageing on 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) neurotoxic effects on striatum and brainstem in the rat

In 3- and 18-month-old male Wistar rats, levels of dopamine (DA), dihydroxyphenylacetic acid (DOPAC), ascorbic acid (AA), dehydroascorbic acid (DHAA), noradrenaline (NA), uric acid, glutathione (GSH) and 1-methyl-4-phenylpyridinium ion (MPP+) were determined by HPLC in the striatum and/or in the brainstem 24 h after single injections of MPTP (12-35 mg/kg i.p.). Aged rats had lower baseline levels of AA and GSH, compared to young rats. In aged rats, MPTP 35 mg/kg induced a 70% death rate and a decrease in striatal DOPAC/DA ratio which was significantly correlated to MPP+ concentrations (r = -0.840, P < 0.005); in addition, MPTP did not increase AA oxidation. In the brainstem, the MPTP-induced decrease in NA levels and increase in uric acid levels were significantly correlated to the MPP+ concentrations (r = -0.709, P < 0.05, and r = +0.888, P < 0.001, respectively). In conclusion, evidence is given of a mechanism of toxicity of MPTP involving oxidative stress produced by xanthine oxidase; in addition, in aged rats the neuronal antioxidant system (levels of AA and GSH) is considerably lower than in young rats and may play an enabling role in the MPTP age-related neurotoxic effects on striatum and brainstem.     

Rat liver microsomal enzyme catalyzed oxidation of 1-cyclopropyl-4-phenyl-1,2,3,6-tetrahydropyridine

NADPH supplemented rat liver microsomal enzyme preparations catalyze the conversion of 1-cyclopropyl4-phenyl-1,2,3,6-tetrahydropyridine to the p-hydroxyphenyl (low yield), descyclopropyl (high yield) and 2,3-dihydropyridinium and, subsequently, pyridinium (intermediary yield) metabolites. When the methine proton of the cyclopropyl group was replaced with a deuteron, a normal deuterium isotope effect (1.4) was observed on the formation of the decyclopropylated metabolite and an inverse isotope effect (0.6) on the dihydropyridinium metabolite. A larger deuterium isotope effect (3.6) was observed on the ring alpha-carbon oxidation pathway with the 2,2,6,6-d4 analogue as substrate. These results and the observation that the ratios of the rates of these two alpha-carbon oxidation pathways are independent of initial substrate concentrations suggest that both pathways are catalyzed by the same active site of one form of P450. These transformations are discussed in terms of metabolic pathways that have been proposed for the cytochrome P450 catalyzed alpha-carbon oxidation of amines.     

Dermal application of lisuride on parkinsonism induced by 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) in the common marmoset and on cases with Parkinson''s disease

BACKGROUND: Follow-up study of patients with surgical repair of aneurysmal subarachnoidal hemorrhage (SAH), looking for clinical outcome predictors. PATIENTS AND METHODS: Sixty two patients consecutively admitted to a teaching hospital, from January 1992 to December 1995 were included in the study. We studied preoperative, intraoperative and postoperative features looking for their relationship with the outcome. The ultimate outcome was evaluated by means of Glasgow Outcome Scale on discharge and 6 months later. RESULTS: Smoking (p = 0.0001) and arterial hypertension (AHT) (p = 0.0186) were more frequent in these patients than in general population, but without relationship to the outcome as with the age of the clinical status on admission. The greatest statistical relationship was found between the level of consciousness on postoperative awakening (measured by the Hunt and Hess scale), and the outcome (p = 2.53 x 10(-8). From our results we made an algorithm that correctly assigned 92% of studied patients to their outcome. CONCLUSIONS: All patients admitted on with aneurysm SAH deserve intensive care treatment besides their clinical grade. The level of consciousness on postoperative awakening was a good outcome predictor.     

Oxidation of 1-amino-4-phenyl-1,2,3,6-tetrahydropyridine, a 1-amino analog of MPTP, by type A and B monoamine oxidase

A 1-amino analog of MPTP, 1(N)-amino-4-phenyl-1,2,3,6-tetrahydropyridine, was synthesized and the oxidation was examined using human synaptosomal mitochondria as sources of type A and B monoamine oxidase. An oxidation product, 1-amino-4-phenylpyridinium ion, was quantified by high-performance liquid chromatography-fluorometric detection. The amino analog was a substrate of both type A and B monoamine oxidase and the oxidation depended linearly on the enzyme amount and the reaction time with an optimal pH around 7.5. After the systemic injection of the amino analog in C57/black mice for one week, 1-amino-4-phenylpyridinium ion was detected in the brain. 1(N)-Amino-4-phenyl-1,2,3,6-tetrahydropyridine was proved to be cytotoxic to pheochromocytoma PC12 cells, and it may be a new neurotoxin bioactivated through the oxidation by type A and B monoamine oxidase.     

1-Methyl-4-phenyl-1,2,3,6-tetrahydropyride neurotoxicity is attenuated in mice overexpressing Bcl-2

The proto-oncogene Bcl-2 rescues cells from a wide variety of insults. Recent evidence suggests that Bcl-2 protects against free radicals and that it increases mitochondrial calcium-buffering capacity. The neurotoxicity of 1-methyl-4-phenyl-1,2,3, 6-tetrahydropyride (MPTP) is thought to involve both mitochondrial dysfunction and free radical generation. We therefore investigated MPTP neurotoxicity in both Bcl-2 overexpressing mice and littermate controls. MPTP-induced depletion of dopamine and loss of [3H]mazindol binding were significantly attenuated in Bcl-2 overexpressing mice. Protection was more profound with an acute dosing regimen than with daily MPTP administration over 5 d. 1-Methyl-4-phenylpyridinium (MPP+) levels after MPTP administration were similar in Bcl-2 overexpressing mice and littermates. Bcl-2 blocked MPP+-induced activation of caspases. MPTP-induced increases in free 3-nitrotyrosine levels were blocked in Bcl-2 overexpressing mice. These results indicate that Bcl-2 overexpression protects against MPTP neurotoxicity by mechanisms that may involve both antioxidant activity and inhibition of apoptotic pathways.     

Dynamic changes in striatal dopamine D2 and D3 receptor protein and mRNA in response to 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) denervation in baboons

Loss of nigrostriatal neurons leads to striatal dopamine deficiency and subsequent development of parkinsonism. The effects of this denervation on D2-like receptors in striatum remain unclear. Most studies have demonstrated increases in striatal dopamine D2-like receptors in response to 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-mediated denervation, but others have found either decreases or no change in binding. To clarify the response to denervation, we have investigated the time-dependent changes in dopamine D2, D3, and D4 receptor protein and mRNA levels in unilaterally MPTP-lesioned baboons. MPTP (0.4 mg/kg) was infused into one internal carotid artery, producing a contralateral hemi-parkinsonian syndrome. After MPTP treatment, the animals were maintained for 17-480 d and then euthanized. MPTP decreased ipsilateral dopamine content by >90%, which did not change with time. Ipsilateral D2-like receptor binding in caudate and putamen initially decreased then increased two- to sevenfold over the first 100 d and returned to near baseline levels by 480 d. Relative levels of D2 mRNA were essentially unchanged over this period. D4 mRNA was not detected. In contrast, D3 mRNA increased sixfold by 2 weeks and then decreased. At the peak period of increase in binding sites, all D2-like receptors were in a micromolar affinity agonist-binding state, implying an increase in uncoupled D2 but not D3 receptor protein. Taken together, these data suggest that MPTP-induced changes in D2-like dopamine receptors are complex and include translational or post-translational mechanisms.     

Nullification of a positive correlation between urinary contents of alpha1-microglobulin and ulinastatin with intracerebroventricularly administered 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) and 1-methyl-4-phenylpyridinium (MPP+) in mice

BACKGROUND: Wolff-Parkinson-White syndrome is thought to be a congenital disease, however, its exact prevalence is not known. This may be because of the intermittent activity of accessory pathways in some cases and fluctuations in autonomic tone. AIMS: To investigate the prevalence of ventricular preexcitation by electrocardiography and reported symptoms in each school age child in Yamanashi prefecture. METHODS: From 1994 to 1996, answers to a questionnaire, results of physical examination, and electrocardiography were obtained from all schoolchildren in Yamanashi prefecture (n = 92,161; total population 880,000) on admission to elementary school (age 6 to 7 years, n = 28,395), junior high school (age 12 to 13 years, n = 31,206), and high school (age 14 to 15 years, n = 32,837). RESULTS: Elementary and junior high school students had a significantly lower prevalence of preexcitation than high school students (0.073% and 0.070% v 0.174%, p < 0.001). The prevalence of left free wall pathway was highest in high school students (n = 27) compared with elementary (n = 6) and junior high school students (n = 5) (p < 0.005). The only symptom noted in the answers to the questionnaire was palpitations. The symptomatic cases were more frequent in high school (n = 13) than in elementary (n = 1) and junior high school (n = 2) children, but not significantly. No student with preexcitation had associated heart disease or family history of Wolff-Parkinson-White syndrome or sudden death. CONCLUSIONS: The prevalence of preexcitation in younger schoolchildren was less frequent than previously reported. The prevalence of preexcitation and left free wall pathways increased with age. The symptoms were few and there was no significant morbidity.     

Elevation of antioxidant potency in the brain of mice by low-dose gamma-ray irradiation and its effect on 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced brain damage

The elevation of endogenous thiol-related antioxidants and free radical scavenging enzymes in the brain of C57BL/6 female mice after low-dose gamma-ray irradiation and its inhibitory effect on 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced brain damage were investigated. The brain level of the reduced form of glutathione (GSH) increased soon after irradiation with 50 cGy of gamma-rays, reached a maximum at 3 h post-treatment, and remained elevated until 12 h. Thioredoxin (TRX) was also transiently increased after irradiation. The activities of free radical scavenging enzymes, including Cu/Zn-superoxide dismutase, catalase and glutathione peroxidase, were significantly induced after irradiation as well. Cerebral malondialdehyde was remarkably elevated by MPTP treatment, and this elevation was suppressed by pre-irradiation (50 cGy). The contents of GSH and TRX were significantly decreased by MPTP treatment in comparison with those of the control group. These reductions both seemed to be attenuated by pre-irradiation with gamma-rays. These results suggest that low-dose gamma-ray irradiation induces endogenous antioxidative potency in the brain of mice and might be effective for the prevention and/or therapy of various reactive oxygen species-related neurodegenerative disorders, such as Parkinson's disease and Alzheimer's disease.     

Functional impairment of nigrostriatal neurons progresses following withdrawal of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine

C57 BL/6 mice were rendered severely parkinsonian by exposure to high doses of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine. The fluorescent retrograde tracer Fast Blue was injected into the neostriatum one (group A) or five weeks (group B) following exposure to 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine. Neurons located in the substantia nigra pars compacta and in the centre median-parafascicular complex were analysed. There was no variation in the number and distribution of Fast Blue-labelled perikarya located in the centre median-parafascicular complex, which are insensitive to 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine. No variation was seen in the number of Nissl-stained perikarya located in the substantia nigra pars compacta, indicating that neurons had not degenerated. The number and the density of Fast Blue retrogradely-labelled neurons located in the same region were decreased in group A by 41% and in group B by 55%. Fast Blue labelling provided a measure of functional impairment in viable neurons. The Fast Blue-to-Nissl cell ratio was 55% in controls and declined to 20% in group A and to 17% in group B mice. The present study shows that (1) functional inactivation of viable neurons can be measured by using a fluorescent retrograde tracer following exposure to 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine and (ii) inactivation of retrograde axonal transport progresses from one to five weeks following withdrawal of the toxin. Fluorescent retrograde probes may be used to measure the anatomical substrate of recovery induced by drugs or by brain grafts in parkinsonian animals.     

Cytotoxicity of 1-amino-4-phenyl-1,2,3,6-tetrahydropyridine and 1-amino-4-phenylpyridinium ion, 1-amino analogues of MPTP and MPP+, to clonal pheochromocytoma PC12 cells

AIMS: To compare the tolerability and efficacy of a fixed combination solution of dorzolamide/timolol (Cosopt), administered twice daily with the concomitant administration of its components, dorzolamide (Trusopt) twice daily and timolol (Timoptic) twice daily. METHODS: After a 2 week timolol run in, patients with open angle glaucoma or ocular hypertension were randomised (1:1) to receive treatment with either the dorzolamide/timolol combination solution twice daily (combination) or the dorzolamide solution twice daily plus timolol maleate solution twice daily (concomitant) for 3 months. RESULTS: 299 patients were entered and 290 patients completed the study. Compared with the timolol baseline, additional IOP lowering of 16% was observed at trough (hour 0) and 22% at peak (hour 2) at month 3 in both the concomitant and combination groups. The IOP lowering effects of the two treatment groups were clinically and statistically equivalent as demonstrated by the extremely small point differences (concomitant--combination) observed in this study--0.01 mm Hg at trough and 0.08 mm Hg at peak. The safety variables of the concomitant and combination groups were very similar. Both combination and concomitant therapy were well tolerated and few patients discontinued due to adverse effects. CONCLUSIONS: The dorzolamide/timolol combination solution administered twice daily is equivalent in efficacy and has a similar safety profile to the concomitant administration of the components administered twice daily.     

Motor response to a dopamine D3 receptor preferring agonist compared to apomorphine in levodopa-primed 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine monkeys

The profile of dopamine receptor subtype activation contributing to the therapeutic efficacy and motor response complications of levodopa (nonselective pro-agonist) in Parkinson's disease remains unclear. Potent, selective, short-acting dopamine D2 receptor subfamily agonists show good antiparkinsonian efficacy but produce dyskinesias comparable to levodopa. Nonetheless, agonists displaying higher affinity for dopamine receptors other than the D2 subtype may have a better therapeutic index. To clarify this issue, we compared the nonselective dopamine D1/D2 receptor subfamilies agonist apomorphine to the dopamine D3 receptor preferring agonist [R-(+)-trans-3,4,4a,10b-tetrahydro-4-propyl-2H,5H-[1]benzopyrano[4 , 3-b]-1,4-oxazin-9-ol] (PD 128,907) in 6 levodopa-primed , 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine-lesioned parkinsonian monkeys with reproducible dyskinesias. Single s.c. dosing with the lowest fully effective dose of apomorphine (averaging 27.9 +/- 4.5 microg/kg) and PD 128,907 (averaging 41.7 +/- 4.4 microg/kg) yielded equivalent antiparkinsonian efficacy on the behavioral scale and portable activity monitoring used. A comparable significant dose-dependent increase in the response magnitude and duration was seen with two higher doses. The severity of dyskinesia was also similar between the two drugs. When the lower dose for each drug was administered six times at a fixed 90-min interval, both drugs remained efficacious with no significant tolerance observed. The D3 receptor preferring antagonist U-99194A significantly reduced the motor effects of both apomorphine and PD 128,907. Thus, increased D3 receptor tone does not acutely ameliorate dyskinesias in levodopa-primed parkinsonian monkeys. Given the reported lack of affinity of PD 128,907 for central D1 receptors, our data support the concept that the pharmacological activation of D1 receptors is not mandatory for relief of parkinsonism and production of dyskinesia.     

1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine as a substrate of cytochrome P450 2D6: allosteric effects of NADPH-cytochrome P450 reductase

1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), a neurotoxin that produces Parkinsonism symptoms in man, has been examined as a substrate of recombinant human cytochrome P450 2D6. When cumene hydroperoxide is used as an oxygen and electron donor, a single product is formed, identified as 4-phenyl-1,2,3,6-tetrahydropyridine. The K(m) for formation of this product (130 microM) is in agreement with the dissociation constants for MPTP binding to the enzyme determined by optical and nuclear magnetic resonance (NMR) spectroscopy. When the reaction is carried out with nicotinamide adenine dinucleotide phosphate (reduced) (NADPH) and recombinant human NADPH-cytochrome P450 reductase, a second product, identified as 1-methyl-4-(4'-hydroxyphenyl)-1,2,3,6-tetrahydropyridine, is formed in addition to 4-phenyl-1,2,3,6-tetrahydropyridine. The K(m) values for formation of these two products are 19 microM and 120 microM, respectively. Paramagnetic relaxation experiments have been used to measure distances between the protons of bound MPTP and the heme iron, and these have been used to construct models for the position and orientation of MPTP in the active site. For the cytochrome alone, a single mode of binding was observed, with the N-methyl close to the heme iron in a position appropriate for the observed N-demethylation reaction. In the presence of the reductase, the data were not consistent with a single mode of binding but could be explained by the existence of two alternative orientations of MPTP in the active site. One of these, characterized by a dissociation constant of 150 microM, is essentially identical to that observed in the absence of the reductase. In the second, which has a K(d) of 25 microM, the MPTP is oriented so that the aromatic ring is close to the heme iron, in a position appropriate for p-hydroxylation leading to the formation of the product seen only in the presence of the reductase. In the case of codeine, another substrate for cytochrome P450 2D6, the addition of reductase had no effect on the nature of the product formed, the dissociation constant, or the orientation in the binding site. These observations show that NADPH-cytochrome P450 reductase has an allosteric effect on the active site of cytochrome P450 2D6 that affects the binding of some substrates but not others.     

Probing the mechanism of bioactivation of MPTP type analogs by monoamine oxidase B: structure-activity studies on substituted 4-phenoxy-, 4-phenyl-, and 4-thiophenoxy-1-cyclopropyl-1,2,3,6-tetrahydropyridines

Previous studies have shown that 4-benzyl-1-cyclopropyl-1,2,3,6-tetrahydropyridine is an excellent monoamine oxidase B (MAO-B) substrate (kappa cat/KM = 1538 min-1 mM-1) although the corresponding 4-phenyl analog displays MAO-B inactivating properties only. This behavior led us to speculate that the pathway for the MAO-B catalyzed oxidation of these tetrahydropyridines may not necessarily proceed via an initial single electron transfer step as proposed by others but rather through an initial alpha-carbon hydrogen atom abstraction step. In the present studies we have examined the interactions of various 4-phenoxy-, 4-phenyl-, and 4-thiophenoxy-1-cyclopropyl-1,2,3,6-tetrahydropyridine derivatives, some of which bear substituents on the phenyl ring. The 4-thiophenoxy- and all of the 4-phenoxytetrahydropyridine derivatives proved to be substrates but not inactivators of MAO-B, while several of the 4-phenyltetrahydropyridine derivatives were inactivators but not substrates. A case of particular interest was 1-cyclopropyl-4-(2-methylphenyl)-1,2,3,6-tetrahydropyridine, which displayed only substrate properties. The results are discussed in terms of two catalytic pathways, one of which involves partitioning of the proposed cyclopropylaminyl radical cation intermediate between cyclopropyl ring opening and proton loss while the second involves partitioning of the parent amine between an initial single electron transfer step, leading to cyclopropylaminyl radical cation formation and enzyme inactivation, and an initial alpha-carbon hydrogen atom abstraction step, leading to an allylic radical and dihydropyridinium product formation.     

Cytokine-induced production of macrophage inflammatory protein-1alpha (MIP-1alpha) in cultured human astrocytes

The human cytotoxic T cell line TALL-104 displays potent anti-tumor effects in animals with spontaneous and induced malignancies. We investigated the biodistribution of TALL-104 cells in tumor bearing and healthy mice. 111In-labeled TALL-104 cells, injected intravenously, localized primarily in the lungs for the first 2 h, and redistributed to liver, spleen, and kidneys in the following 24 h both in immunodeficient and immunocompetent mice. TALL-104 cells consistently accumulated in the tumor and at sites of metastases. In animals injected with free 111In-oxine, most of the radioactivity remained in the blood pool with no significant organ accumulation. These data support the tumor homing properties of TALL-104 cells, information which is crucial to their therapeutic efficacy in forthcoming clinical trials.     

New insights into the compartmentation of glutamate and glutamine in cultured rat brain astrocytes

Studies from several groups have provided evidence that glutamate and glutamine are metabolized in different compartments in astrocytes. In the present study we measured the rates of 14CO2 production from U-[14C]glutamate and U-[14C]glutamine, and utilized both substrate competition experiments and the transaminase inhibitor aminooxyacetic acid (AOAA) to obtain more information about the compartmentation of these substrates in cultured rat brain astrocytes. The rates of oxidation of 1 mM glutamine and glutamate were 26.4 +/- 1.4 and 63.0 +/- 7.4 nmol/h/mg protein, respectively. The addition of 1 mM glutamate decreased the rate of oxidation of glutamine to 26.3% of the control rate, demonstrating that glutamate can effectively compete with the oxidation of glutamine by astrocytes. In contrast, the addition of 1 mM glutamine had little or no effect on the rate of oxidation of glutamate by astrocytes, demonstrating that the glutamate produced intracellularly from exogenous glutamine does not dilute the glutamate taken up from the media. The addition of 5 mM AOAA decreased the rate of 14CO2 production from glutamine to 29.2% of the control rate, consistent with earlier studies by our group. The addition of 5 mM AOAA decreased the rate of oxidation of concentrations of glutamate < or = 0.1 mM by approximately 50%, but decreased the oxidation of 0.5-1 mM glutamate by only approximately 20%, demonstrating that a substantial portion of glutamate enters the tricarboxylic acid (TCA) cycle via glutamate dehydrogenase (GDH) rather than transamination, and that as the concentration of glutamate increases the relative proportion entering the TCA cycle via GDH also increases. To determine if the presence of an amino group acceptor (i.e. a ketoacid) would increase the rate of metabolism of glutamate, pyruvate was added in some experiments. Addition of 1 mM pyruvate increased the rate of oxidation of glutamate, and the increase was inhibited by AOAA, consistent with enhanced entry of glutamate into the TCA cycle via transamination in the presence of pyruvate. Enzymatic studies showed that pyruvate increased the activity of mitochondrial aspartate aminotransferase (AAT). Overall, the data demonstrate that glutamate formed intracellularly from glutamine enters the TCA cycle primarily via transamination, but does not enter the same TCA cycle compartment as glutamate taken up from the extracellular milieu. In contrast, extracellular glutamate enters the TCA cycle in astrocytes via both transamination and GDH, and can compete with, or dilute, the oxidation of glutamate produced intracellularly from glutamine.     

Stimulation of glutamate uptake and Na,K-ATPase activity in rat astrocytes exposed to ischemia-like insults

The postsynaptic actions of glutamate are rapidly terminated by high affinity glutamate uptake into glial cells. In this study we demonstrate the stimulation of both glutamate uptake and Na,K-ATPase activity in rat astrocyte cultures in response to sublethal ischemia-like insults. Primary cultures of neonatal rat cortical astrocytes were subjected to hypoxia, or to serum- and glucose-free medium, or to both conditions (ischemia). Cell death was assessed by propidium iodide staining of cell nuclei. To measure sodium pump activity and glutamate uptake, 3H-glutamate and 86Rb were both simultaneously added to the cell culture in the presence or absence of 2 mM ouabain. Na,K-ATPase activity was defined as ouabain-sensitive 86Rb uptake. Concomitant transient increases (2-3 times above control levels) of both Na,K-ATPase and glutamate transporter activities were observed in astrocytes after 4-24 h of hypoxia, 4 h of glucose deprivation, and 2-4 h of ischemia. A 24 h ischemia caused a profound loss of both activities in parallel with significant cell death. The addition of 5 mM glucose to the cells after 4 h ischemia prevented the loss of both sodium pump activity and glutamate uptake and rescued astrocytes from death observed at the end of 24 h ischemia. Reoxygenation after the 4 h ischemic event caused the selective inhibition of Na,K-ATPase activity. The observed increases in Na,K-ATPase activity and glutamate uptake in cultured astrocytes subjected to sublethal ischemia-like insults may model an important functional response of astrocytes in vivo by which they attempt to maintain ion and glutamate homeostasis under restricted energy and oxygen supply.     

Neuroprotection from focal ischemia by 4-phenyl-1-(4-phenylbutyl) piperidine (PPBP) is dependent on treatment duration in rats

OBJECTIVES: We sought to determine whether treatment with high dose verapamil prevents restenosis in patients at high risk for reoccurrence after successful percutaneous transluminal coronary angioplasty (PTCA). BACKGROUND: Restenosis is the major limitation of PTCA. Calcium antagonists have demonstrated some potential as inhibitors of this process. METHODS: A total of 98 patients with peripheral occlusive arterial disease (POAD), stable angina pectoris, mild hypertension and at least one additional risk factor increasing the likelihood of restenosis after angioplasty were selected for this placebo-controlled, double-blind, randomized trial. Verapamil (240 mg twice daily) or placebo was taken for 6 months. Efficacy variables assessed before and after angioplasty and at 6 weeks and 6 months after PTCA included thickness of the intima/media complex degree of stenosis, interventricular septal thickness, crurobrachial pressure ratios of dorsalis pedis and posterior tibial arteries, distance to claudication and total vessel diameter. RESULTS: No significant intergroup differences emerged before or immediately after PTCA. Six weeks after angioplasty, a significant thickening of the intima/media complex in the treated vascular segment of 14.3% occurred in the placebo group versus 0% among verapamil patients (p < 0.01). At 6 months, the intima/media thickness was 35.7% greater in the placebo group but had decreased by 14.3% in the verapamil group (p < 0.001). At 6 months, a marked reduction in septal thickness was observed in the verapamil group versus that in the placebo group (p < 0.001). The rate of restenosis was also significantly lower in the verapamil group (p < 0.001). Few minor side effects were reported. CONCLUSIONS: In patients with POAD at increased risk for restenosis, the administration of high dose verapamil prevented recurrent stenosis for 6 months after successful peripheral angioplasty and was well tolerated.