THR-18, a 18-mer peptide derived from PAI-1, is neuroprotective and improves thrombolysis by tPA in rat stroke models.

OBJECTIVE: The thrombolytic treatment of stroke is limited by a narrow therapeutic time window and is associated with significant adverse side effects. To improve this situation, the modulation of tissue-type plasminogen activator (tPA) activity by a synthetic plasminogen activator inhibitor-1-derived 18-mer peptide (THR-18) was examined in two models of stroke in rats. METHODS: In the first model (thromboembolic), stroke was induced by intra-carotid injection of micro-clots to rats, and tPA (6 mg/kg) was intravenously infused for 30 minutes with or without THR-18 (1 mg/kg) at 4 hours post-induction. In the second model [transient middle cerebral artery occlusion (tMCAO)], stroke was induced for 2 hours by a transient mechanical occlusion. tPA and/or THR-18 (0.02, 0.1, and 1 mg/kg) were intravenously infused for 60 minutes at the time of reperfusion. RESULTS: In the thromboembolic model, cerebral blood flow, measured before and up to 5.5 hours post-induction, revealed that tPA administration caused reperfusion of flow at 30 minutes post-infusion. Later on, an additional increase in reperfusion was seen in the tPA+THR-18 group, and not with tPA alone. In both models, the frequency of intracranial hemorrhage in the tPA-treated group was found to be significantly higher than the control, and this tPA effect was attenuated by THR-18. In the thromboembolic study, infarct size and brain edema were similar in the control and tPA-treated rats. However, the combination of tPA and THR-18 caused a statistically significant reduction in both parameters (infarct size 17.8 versus 25.0%, brain edema 5 versus 8%, tPA+THR-18 versus control, respectively). In the tMCAO mechanical model, infarct size and brain edema were both increased by tPA treatment as compared to the control group, and this increase was markedly diminished by THR-18 co-administration. Neurobehavioral assessment of the tMCAO animals performed at 72 hours post-stroke induction revealed significant improvements (P<0.05-0.01) in neuroscores in all groups of animals treated with peptide-tPA, as compared to the tPA monotherapy group. A significant (P<0.05) improvement in the neurological outcome was also seen in the THR-18 monoterapy group, as compared to the control animals, thus demonstrating a clear neuroprotective effect by the peptide on its own. DISCUSSION: The results support the use of THR-18 together with tPA in the thrombolytic therapy of stroke, in order to achieve better patency, less tPA-induced damage, and possibly a widening of tPA therapeutic time window.

Attenuation of methamphetamine induced dopaminergic neurotoxicity by flupirtine: microdialysis study on dopamine release and free radical generation.

Flupirtine is a triaminopyridine derived centrally acting analgetic, which has been found to display neuroprotective effects in models of excitotoxic cell damage, global, and focal ischemia, but no direct interaction with any component of the N-methyl-D-aspartate (NMDA) and glutamate triggered Ca(2+)-channel. Additionally flupirtine shows potent antioxidant effects in isolated mitochondria and cell culture. Work in models of monoamine depletion and neuroleptic induced catalepsy in rats suggests a interaction of flupirtine with the dopaminergic neurotransmitter system as well. This prompted us to examine the effect of flupirtine on methamphetamine toxicity in mice and to investigate the influence on dopamine release and free radical formation in the rat striatum by microdialysis that may explain methamphetamine neurotoxicity. Pretreatment of C57-BL mice with flupirtine (4 x 10 mg/kg) significantly attenuated the striatal dopamine loss after methamphetamine application (4 x 5 mg/kg). In rats, a single injection of 10 mg/kg flupirtine reduced the methamphetamine induced striatal dopamine release by almost 50%, as measured by in vivo microdialysis. Flupirtine, however, did not influence the increase of free radical formation after methamphetamine infusion, which was assayed after infusion of salicylic acid by quantification of 2,3- and 2,5-dihydroxybenzoic acid. This suggests that other mechanisms rather than dopamine metabolism and autoxidation, may contribute to methamphetamine neurotoxicity.

The nitroxide antioxidant tempol is cerebroprotective against focal cerebral ischemia in spontaneously hypertensive rats.

Free radicals appear to participate in the final common pathway of neuronal death in ischemia and may therefore be an adequate target for therapy. Tempol is a nitroxide antioxidant with proven protective efficacy in several animal models, including myocardial ischemia, that has not been previously tested in models of permanent cerebral ischemia. Spontaneously hypertensive rats underwent permanent middle cerebral artery occlusion (PMCAO). Following dose-response and time-window-finding experiments rats were given vehicle or tempol (50 mg/kg) subcutaneously 1 h after PMCAO (n = 10/group). Five animals in each group were evaluated with a motor scale 24 h after the infarct and were then sacrificed and the injury volume was measured. The remaining animals were examined daily with the motor scale and also with a Morris water maze test on days 26-30 after PMCAO and sacrificed on day 30. Motor scores at all time points examined were significantly better in the tempol-treated animals (P < 0.05 for all). Significantly better performance in the water maze test for performance on days 26-30 was noted in the tempol group compared with the vehicle-treated group (P < 0.05). Injury volumes at days 1 and 30 were significantly reduced in the tempol group (9.83 +/- 1.05 vs 19.94 +/- 1.43% hemispheric volume, P = 0.0009, and 13.2 +/- 2.97 vs 24.4 +/- 2.38% hemispheric volume, P = 0.02, respectively). In conclusion, treatment with tempol led to significant motor and behavioral improvement and reduced injured tissue volumes both in the short and in the long term after stroke.

Bax and Bak coalesce into novel mitochondria-associated clusters during apoptosis.

Bax is a member of the Bcl-2 family of proteins known to regulate mitochondria-dependent programmed cell death. Early in apoptosis, Bax translocates from the cytosol to the mitochondrial membrane. We have identified by confocal and electron microscopy a novel step in the Bax proapoptotic mechanism immediately subsequent to mitochondrial translocation. Bax leaves the mitochondrial membranes and coalesces into large clusters containing thousands of Bax molecules that remain adjacent to mitochondria. Bak, a close homologue of Bax, colocalizes in these apoptotic clusters in contrast to other family members, Bid and Bad, which circumscribe the outer mitochondrial membrane throughout cell death progression. We found the formation of Bax and Bak apoptotic clusters to be caspase independent and inhibited completely and specifically by Bcl-X(L), correlating cluster formation with cytotoxic activity. Our results reveal the importance of a novel structure formed by certain Bcl-2 family members during the process of cell death.

Modification of dopamine release by selective inhibitors of MAO-B.

Chronic low dose deprenyl treatment in rats causes an increase in striatal extracellular dopamine level, without significant reduction in deaminated metabolite formation. This effect could be the result of increased endogenous levels of the MAO-B substrate beta-phenylethylamine, which is both a releaser of dopamine as well as an inhibitor of the neuronal membrane active dopamine uptake. In guinea pigs, however, striatal extracellular dopamine was not increased either by deprenyl or by clorgyline. Local infusion of the dopamine uptake inhibitor GBR-12909 caused a greater increase in striatal dopamine in microdialysate in rats than in guinea pigs. Intra-species differences in synaptic architecture or in density of dopamine transporter expression may account for these differences.

Selective monoamine oxidase subtype inhibition and striatal extracellular dopamine in the guinea-pig.

Striatal microdialysate levels of dopamine (DA) in conscious guinea-pigs were measured following acute (1 day) and chronic (21 days) treatment with deprenyl (2 and 0.25 mg kg(-1) s.c., respectively) or clorgyline (4 and 1 mg kg(-1) s.c., respectively), as well as by combination treatment using the same doses of the two inhibitors. These treatments caused selective inhibition of monoamine oxidase type B (MAO-B) or monoamine oxidase type A (MAO-A) respectively. Neither acute nor chronic treatments with deprenyl or clorgyline increased basal or KCl-induced DA levels. Acute and chronic clorgyline treatments were accompanied by significant reductions in striatal microdialysate 3,4-dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA). On the other hand, both acute and chronic deprenyl treatments were accompanied by significant increases in microdialysate HVA with no effect on DOPAC levels. Acute or chronic combined treatment with clorgyline and deprenyl increased tissue but not microdialysate DA levels. The combination treatment given chronically also reduced KCl-induced DA release but enhanced amphetamine-induced DA release. Microdialysate DA levels increased to a smaller extent in guinea-pig than in rat following local striatal infusion of GBR-12909 (100 microM). The difference between guinea-pigs and rats in the response to GBR-12909, could be the result of a lower dopaminergic innervation and/or density of DA transporter. This difference may explain why striatal microdialysate DA levels increased following chronic deprenyl treatment in the rat but not in the guinea-pig.

Acidic dopamine metabolites are actively extruded from PC12 cells by a novel sulfonylurea-sensitive transporter.

Incubation of PC 12 cells with the sulfonylurea drug, glipizide (1-100 microM), increased intracellular levels of the acidic metabolites of dopamine, 3,4-dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA). The levels of these acids in the medium were decreased, indicating the presence of a sulfonylurea-sensitive organic anion transporter. In the present study, we demonstrate that the sulfonylurea-sensitive transport of acidic dopamine metabolites is unidirectional, ATP dependent, unaffected by ouabain or by tetrodotoxin and blocked by drugs that interact with the multidrug-resistance protein-1 (MRP1). However, over-expression of MRP1 did not affect transport of the acid metabolites. The pharmacological profile and ion dependence of the transporter also differs from that of known ATP-binding cassette (ABC) family members. Using microdialysis, we also demonstrated a sulfonylurea-sensitive transport process in the striatum of freely moving rats. These results show that acidic dopamine metabolites are actively secreted from dopaminergic cells into surrounding extracellular fluid by a previously undescribed transporter.

3,4-Dihydroxyphenylacetaldehyde potentiates the toxic effects of metabolic stress in PC12 cells.

3,4-Dihydroxyphenylacetaldehyde (DOPAL) is a toxic metabolite formed by the oxidative deamination of dopamine. This aldehyde is mainly oxidized to 3,4-dihydroxyphenylacetic acid (DOPAC) by aldehyde dehydrogenase (ALDH), but is also partly reduced to 3, 4-dihydroxyphenylethanol (DOPET) by aldehyde or aldose reductase (ARs). In a previous study, we found that rotenone, a complex I inhibitor, induced a rapid accumulation of DOPAL and DOPET in the medium of cultured PC12 cells. Here, we examined the potential role of DOPAL in the toxicity induced by complex I inhibition in PC12 cells and compared the effects of rotenone on concentrations of DOPAL and DOPET to those of MPP(+). DOPAL and DOPET levels were increased by rotenone but decreased by MPP(+). Inhibition of ALDH by daidzein reduced the formation of DOPAC and increased the accumulation of DOPAL. Inhibition of ARs (with AL1576) diminished DOPET formation and elevated DOPAL concentrations. Combined inhibition of ALDH and ARs markedly elevated DOPAL concentrations while diminishing DOPET and DOPAC levels. The elevation of DOPAL levels induced by combined inhibition of ALDH and ARs had no effect on cell viability. However, combined inhibition of ALDH and ARs potentiated rotenone-induced toxicity. Both the potentiation of toxicity and the increase in DOPAL levels were blocked by inhibition of monoamine oxidase with clorgyline indicating that accumulation of DOPAL was responsible for the potentiated rotenone-induced toxicity following combined inhibition of ALDH and ARs. Since complex I dysfunction is reported to be involved in the pathogenesis of Parkinson's disease, DOPAL potentiation of the deleterious effects of complex I inhibition may contribute to the specific vulnerability of dopaminergic neurons to injury.

Metabolic stress in PC12 cells induces the formation of the endogenous dopaminergic neurotoxin, 3,4-dihydroxyphenylacetaldehyde.

3,4-Dihydroxyphenylacetaldehyde (DOPAL) has been reported to be a toxic metabolite formed by the oxidative-deamination of dopamine (DA) catalyzed by monoamine oxidase. This aldehyde is either oxidized to 3,4-dihydroxyphenylacetic acid (DOPAC) by aldehyde dehydrogenase, an NAD-dependent enzyme or reduced to 3, 4-dihydroxyphenylethanol (DOPET) by aldehyde or aldose reductase. In the present study we examined whether levels of DOPAL are elevated by inhibition of the mitochondrial respiratory chain. Using inhibitors of mitochondrial complexes I, II, III and IV we found that inhibition of complex I and III increased levels of DOPAL and DOPET. Nerve growth factor-induced differentiation of PC12 cells markedly potentiated DOPAL and DOPET accumulation in response to metabolic stress. DOPAL was toxic to differentiated PC12 as well as to SK-N-SH cell lines. Because complex I dysfunction has been implicated in the pathogenesis of Parkinson's disease, the accumulation of DOPAL may explain the vulnerability of the dopaminergic system to complex I inhibition. The rapid appearance of DOPAL and DOPET after inhibition of complex I may be a useful early index of oxidative stress in DA-forming neurons.

Effect of glipizide on dopamine synthesis, release and metabolism in PC12 cells.

Sulfonylureas block ATP-dependent K(+) channels (K/ATP channels) in pancreatic beta cells and brain gamma-aminobutyric acid (GABA) containing neurons causing depolarization-evoked insulin or GABA release. In high concentrations, sulfonylureas also inhibit catecholamine release from bovine adrenal chromaffin cells and isolated guinea pig aorta. In this study, we examined the effect of glipizide, a sulfonylurea, on dopamine release from PC12 cells and found that neither basal nor K(+)-stimulated dopamine release was affected. Although PC12 cells expressed mRNA for the K/ATP channel, functional K/ATP channels could not be demonstrated electrophysiologically, consistent with the lack of effect of glipizide on dopamine release. Glipizide did, however, increase cytoplasmic retention of the acidic dopamine metabolites, 3, 4-dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA), indicating blockade of their outward transport. The cellular accumulation of DOPAC was accompanied by reduced tyrosine hydroxylase activity and reduced formation of dopamine and its metabolites presumably by a negative feedback effect of the increased cytoplasmic concentrations of DOPAC.

Sources and physiological significance of plasma dopamine sulfate.

Dopamine in the circulation occurs mainly as dopamine sulfate, the sources and physiological significance of which have been obscure. In this study, plasma concentrations of dopamine sulfate were measured after a meal, after fasting for 4 days, and during i.v. L-DOPA, nitroprusside, or trimethaphan infusion in volunteers; after dopamine infusion in patients with L-aromatic-amino-acid decarboxylase deficiency; in arterial and portal venous plasma of gastrointestinal surgery patients; and in patients with sympathetic neurocirculatory failure. Meal ingestion increased plasma dopamine sulfate by more than 50-fold; however, prolonged fasting decreased plasma dopamine sulfate only slightly. L-DOPA infusion produced much larger increments in dopamine sulfate than in dopamine; the other drugs were without effect. Patients with L-aromatic amino acid decarboxylase deficiency had decreased dopamine sulfate levels, and patients with sympathetic neurocirculatory failure had normal levels. Decarboxylase-deficient patients undergoing dopamine infusion had a dopamine sulfate/dopamine ratio about 25 times less than that at baseline in volunteers. Surgery patients had large arterial-portal venous increments in plasma concentrations of dopamine sulfate, so that mesenteric dopamine sulfate production accounted for most of urinary dopamine sulfate excretion, a finding consistent with the localization of the dopamine sulfoconjugating enzyme to gastrointestinal tissues. The results indicate that plasma dopamine sulfate derives mainly from sulfoconjugation of dopamine synthesized from L-DOPA in the gastrointestinal tract. Both dietary and endogenous determinants affect plasma dopamine sulfate. The findings suggest an enzymatic gut-blood barrier for detoxifying exogenous dopamine and delimiting autocrine/paracrine effects of endogenous dopamine generated in a "third catecholamine system."

Effect of low-dose treatment with selegiline on dopamine transporter (DAT) expression and amphetamine-induced dopamine release in vivo.

1. Chronic treatment with low doses of the selective monoamine oxidase (MAO) type B inhibitors selegiline [(-)-deprenyl] and rasagiline, causes elevation in extracellular level of 3,4-dihydroxyphenylethylamine (dopamine) in the rat striatum in vivo (Lamensdorf et al., 1996). The present study was carried out to determine whether this effect of selegiline could be the result of an inhibition of the high-affinity dopamine neuronal transport process. 2. Changes in activity of the dopamine transporter (DAT) in vivo following selegiline treatment were evaluated indirectly by microdialysis technique in the rat, from the change in striatal dopamine extracellular concentration following systemic amphetamine administration (4 mg kg(-1), i.p.). Striatal levels of the DAT molecule were determined by immunoblotting. Uptake of [3H]-dopamine was determined in synaptosomes from selegiline-treated animals. 3. Amphetamine-induced increase in striatal extracellular dopamine level was attenuated by one day and by chronic (21 days) treatment with selegiline (0.25 mg kg(-1), s.c.). 4. Striatal levels of DAT were elevated after 1 and 21 days treatment with selegiline, but were not affected by clorgyline, rasagiline, nomifensine or amphetamine. 5. The increase in DAT expression, and attenuation of amphetamine-induced dopamine release, were not accompanied by a change in [3H]-dopamine uptake in synaptosomes of selegiline-treated animals. 6. The results suggest that a reversible inhibition of dopamine uptake occurs following chronic low dose selegiline treatment in vivo which may be mediated by an increase in endogenous MAO-B substrates such as 2-phenylethylamine, rather than by the inhibitor molecule or its metabolites. Increased DAT expression appears to be a special property of the selegiline molecule, since it occurs after one low dose of selegiline, and is not seen with other inhibitors of MAO-A or MAO-B. The new DAT molecules formed following selegiline treatment appear not to be functionally active.

Kir6.2 oligoantisense administered into the globus pallidus reduces apomorphine-induced turning in 6-OHDA hemiparkinsonian rats.

ATP-sensitive inwardly rectifying potassium channels (KATPs) couple cell metabolism with its membrane potential. The best characterized KATP is the pancreatic KATP which is an heteromultimer of Kir6.2 and SUR1 protein subunits. KATPs are found in a variety of excitable cells, including neurons of the central nervous system. Basal ganglia (BG), especially in the substantia nigra (SN) reticulata and the globus pallidus (GP), have a high density of KATPs. Pharmacological modulation of the KATPs within the BG alters GABAergic activity and produces behavioural changes. However, the relatively high concentrations of drugs used might not have been entirely selective for the KATPs and may have acted at presynaptic nerve terminals as well as on the post-synaptic neurons. As an alternative means of examining the role of KATPs in regulating motor behavior, we used oligoantisense technology to diminish selectively Kir6.2 formation in the GP neurons. We then examined the effect of reduction in Kir6.2 expression on apomorphine-induced turning behavior in rats with unilateral 6-hydroxydopamine (6-OHDA) lesions of the SN. Two weeks after injection of 6-OHDA, contralateral circling in response to apomorphine (0.25 mg/kg sc) was recorded. Kir6.2 antisense oligodeoxyribonucleotide (ODN) was then administered daily for 6 days into the GP ipsilateral to the 6-OHDA injection. Responses to apomorphine were then tested again and the animals killed to determine the effect of the antisense ODN on Kir6. 2 mRNA. Administration of Kir6.2 antisense ODN significantly attenuated apomorphine-induced contralateral turning and specifically reduced Kir6.2 mRNA in the injected GP. These results are consistent with pharmacological experiments which suggest that KATP channels in the GP are involved in motor responses to apomorphine in 6-OHDA lesioned rats, localizing the effects to the GP neurons, probably through modulation of the GABAergic system.

Reduced striatal tyrosine hydroxylase activity is not accompanied by change in responsiveness of dopaminergic receptors following chronic treatment with deprenyl.

Deprenyl is the only selective monoamine oxidase B (MAO-B) inhibitor that is in clinical use for the treatment of Parkinson's disease. Our previous studies showed that chronic treatment of rats with low (MAO-B selective) doses of deprenyl inhibited dopamine (DA) re-uptake and enhanced DA release in the striatum. These changes could affect DA synthesis rate by activation of negative feedback loops. Chronic deprenyl treatment has also been suggested to cause down-regulation of release-modulating DA receptors. The effects of chronic and acute treatment with deprenyl on ex vivo striatal tyrosine hydroxylase activity were therefore studied, by determination of steady-state tissue level of DOPA following administration of NSD-1015 (100 mg/kg i.p.). In addition, we assessed changes in the in vivo sensitivity of dopaminergic receptors from the reduction in DOPA extracellular level after systemic apomorphine administration (2.5 mg/kg s.c.), following elevation of microdialysate DOPA by systemic or local aromatic amino acid decarboxylase inhibition with NSD-1015. Chronic treatment with deprenyl (0.25 mg/kg s.c. daily for 21 days) caused a significant reduction in tyrosine hydroxylase activity to 60% of control, with no change in the apomorphine-induced reduction of microdialysate DOPA and DOPAC. The reduction in tyrosine hydroxylase activity is compatible with our previous results showing an increase in striatal DA extracellular level following chronic treatment with deprenyl. The increased extracellular striatal DA level could reduce tyrosine hydroxylase activity through activation of a negative feedback loop, by activation of either presynaptic or postsynaptic DA receptors.

Effect of long-term treatment with selective monoamine oxidase A and B inhibitors on dopamine release from rat striatum in vivo.

Acute inhibition of monoamine oxidase B (MAO-B) in the rat does not affect striatal dopamine (DA) metabolism, but chronic MAO-B inhibition with deprenyl has been reported to increase the release of striatal DA, as shown using in vitro techniques. To see whether chronic MAO-B inhibition also causes an increase in DA release in vivo, rats were treated for 21 days with either deprenyl (0.25 mg/kg), TVP-1012 [R(+)-N-propargyl-1-aminoindan mesylate; 0.05 mg/kg], an irreversible inhibitor of MAO-B that is not metabolized to amphetamines, clorgyline (0.2 mg/kg), or saline (all doses once daily by subcutaneous injection). Concentric 4-mm-long microdialysis probes were implanted in the left striatum under pentobarbital/chloral hydrate anesthesia on day 21, and microdialysate DA, 3,4, dihydroxyacetic acid (DOPAC), and 4-hydroxy-3-methoxyphenyl acetic acid (HVA) were determined in the conscious animals on day 22. Baseline levels of DA were as follows: control, 0.34 +/- 0.04 (n = 13); deprenyl, 0.88 +/- 0.10 (n = 8, p < 0.01); TVP-1012, 0.94 +/- 0.20 (n = 7, p < 0.01); clorgyline, 0.90 +/- 0.12 (n = 7, p < 0.01) pmol/20 min. Levels of DOPAC and HVA were reduced only in the clorgyline-treated group. The incremental release of DA induced by depolarizing concentration of K+ (100 mM bolus of KCl in perfusate) was significantly greater in clorgyline- and deprenyl-treated rats and elevated (nonsignificantly) in TVP-1012-treated rats. Chronic treatment with the MAO-B inhibitors reduced striatal MAO-B activity by 90%, with 15% (TVP-1012) or 40% (deprenyl) inhibition of MAO-A. Clorgyline inhibited MAO-A by 95%, with 30% inhibition of MAO-B. A single dose of deprenyl (0.25 mg/kg, 24 h before microdialysis) had no significant effect on striatal efflux of DA. The results show that DA metabolism was reduced only by clorgyline, whereas neuronal release of DA was enhanced by both MAO-A and MAO-B inhibitors on chronic administration. The enhanced DA release by chronic MAO-B inhibition does not appear to be dependent on production of amphetamine-like metabolites of the inhibitor. Possible mechanisms for the release-enhancing effect of the MAO-B inhibitors include elevation in levels of endogenous beta-phenylethylamine, or an inhibition of DA reuptake, which develops only on chronic administration, because both deprenyl and TVP-1012 have only very weak effects on amine uptake in acute experiments.

Pharmacology and neuroprotective properties of rasagiline.

Rasagiline [R(+)-N-propargyl-1-aminoindane] is a selective irreversible inhibitor of MAO-B which is not metabolised to amphetamine-like derivatives. Like deprenyl, when given to rats in a dose selective for inhibition of MAO-B, it does not affect striatal extracellular fluid dopamine levels, but when administered chronically (21 days) it increased striatal microdialysate dopamine without reduction in deaminated metabolites. Similarly to deprenyl, rasagiline (10(-6)M) increased the percentage of tyrosine hydroxylase positive cells in a primary culture of rat fetal mesencephalic cells (6 days in culture). Rasagiline, but not deprenyl, also increased the number of neurons per field in this organotypic culture.