Pekka Rauhala

Neuroprotection by S-nitrosoglutathione of brain dopamine neurons from oxidative stress

The proposed anti‐ and pro‐oxidant effects of nitric oxide (NO) derivatives, such as S‐nitrosoglutathione (GSNO) and peroxynitrite, were investigated in the rat nigrostriatal dopaminergic system. Intranigral infusion of freshly prepared GSNO (0–16.8 nmol, i.n.) prevented iron‐induced (4.2 nmol, i.n.) oxidative stress and nigral injury, reflected by a decrease in striatal dopamine levels. This neuroprotective effect of GSNO was verified by ex vivo imaging of brain dopamine uptake sites using 125I‐labeled RTI‐55. In addition, in vitro data indicate that GSNO concentration‐dependently inhibited iron‐evoked hydroxyl radical generation and brain lipid peroxidation. In this iron‐induced oxidant stress model, GSNO was approximately 100‐fold more potent than the antioxidant glutathione (GSH). Light‐exposed, NO‐exhausted GSNO produced neither antioxidative nor neuroprotective effects, which indicates that NO may mediate at least part of GSNOs effects. Moreover, GSNO completely (and GSH only partially) inhibited the weak pro‐oxidant effect of peroxynitrite, which produced little injury to nigral neurons in vivo. This study provides relevant in vivo evidence suggesting that nanomol GSNO can protect brain dopamine neurons from iron‐induced oxidative stress and degeneration. In conclusion, S‐nitrosylation of GSH by NO and oxygen may be part of the antioxidative cellular defense system.—Rauhala, P., Lin, A. M.‐Y., Chiueh, C. C. Neuroprotection by S‐nitrosoglutathione of brain dopamine neurons from oxidative stress. FASEB J. 12, 165–173 (1998)

The redox pathway of S-nitrosoglutathione, glutathione and nitric oxide in cell to neuron communications.

Recent results demonstrated that S-nitrosoglutathione (GSNO) and nitric oxide (*NO) protect brain dopamine neurons from hydroxyl radical (*OH)-induced oxidative stress in vivo because they are potent antioxidants. GSNO and *NO terminate oxidant stress in the brain by (i) inhibiting iron-stimulated hydroxyl radicals formation or the Fenton reaction, (ii) terminating lipid peroxidation, (iii) augmenting the antioxidative potency of glutathione (GSH), (iv) mediating neuroprotective action of brain-derived neurotrophin (BDNF), and (v) inhibiting cysteinyl proteases. In fact, GSNO--S-nitrosylated GSH--is approximately 100 times more potent than the classical antioxidant GSH. In addition, S-nitrosylation of cysteine residues by GSNO inactivates caspase-3 and HIV-1 protease, and prevents apoptosis and neurotoxicity. GSNO-induced antiplatelet aggregation is also mediated by S-nitrosylation of clotting factor XIII. Thus the elucidation of chemical reactions involved in this GSNO pathway (GSH GS* + *NO-->[GSNO]-->GSSG + *NO-->GSH) is necessary for understanding the biology of *NO, especially its beneficial antioxidative and neuroprotective effects in the CNS. GSNO is most likely generated in the endothelial and astroglial cells during oxidative stress because these cells contain mM GSH and nitric oxide synthase. Furthermore, the transfer of GSH and *NO to neurons via this GSNO pathway may facilitate cell to neuron communications, including not only the activation of guanylyl cyclase, but also the nitrosylation of iron complexes, iron containing enzymes, and cysteinyl proteases. GSNO annihilates free radicals and promotes neuroprotection via its c-GMP-independent nitrosylation actions. This putative pathway of GSNO/GSH/*NO may provide new molecular insights for the redox cycling of GSH and GSSG in the CNS.

APPARENT ROLE OF HYDROXYL RADICALS IN OXIDATIVE BRAIN INJURY INDUCED BY SODIUM NITROPRUSSIDE

Sodium nitroprusside (disodium nitroferricyanide) has been suggested to cause cytotoxicity through either the release of cyanide and/or nitric oxide. The present study investigated a possible mechanism that after a brief release of nitric oxide, iron moiety of breakdown products of sodium nitroprusside could cause a long lasting oxidative stress, such as hydroxyl radical generation, lipid peroxidation and cytotoxicity. Intranigral administration of sodium nitroprusside (0-16.8 nmol) to rats induced an acute increase in lipid peroxidation in the substantia nigra and a chronic dopamine depletion in the caudate nucleus. Photodegraded (nitric oxide-exhausted) sodium nitroprusside, however, still produced lipid peroxidation and neurotoxicity in the midbrain. Moreover, non-iron containing nitric oxide-donor compounds, such as S-nitroso-N-acetylpenicillamine, did not cause oxidative brain injury in vivo suggesting that nitric oxide may not mediate neurotoxicity induced by sodium nitroprusside. Additional in vitro studies demonstrated that both freshly prepared (nitric oxide donor) and photodegraded (nitric oxide-exhausted) sodium nitroprusside generated hydroxyl radicals in the presence of ascorbate and also increased lipid peroxidation in brain homogenates. These pro-oxidative effects of sodium nitroprusside were blocked by nitric oxide, S-nitroso-N-acetylpenicillamine, oxyhemoglobin, and deferoxamine (iron chelator). The present results suggest that iron moiety, rather than nitric oxide, may mediate the pro-oxidative properties of sodium nitroprusside. With this new information in mind, the misuse of sodium nitroprusside as a selective nitric oxide donor in both basic and clinical uses should be urgently addressed.

PEROXIDATION OF BRAIN LIPIDS IN VITRO : NITRIC OXIDE VERSUS HYDROXYL RADICALS

The pro-oxidant effects of hydroxyl radical (.OH, ferrous ammonium sulfate/Fe2+) or nitric oxide (NO., S-nitroso-N-acetylpenicillamine/SNAP) generating compounds were studied in rat brain homogenate preparations. Submicromolar concentrations of Fe2+, but not SNAP (up to 100 microM), increased the formation of fluorescent products of malondialdehyde in cortical homogenates. In fact, iron-catalyzed brain lipid peroxidation was inhibited by SNAP (100 microM), but not by light-exposed SNAP or its degradation product penicillamine (100 microM). This study provides relevant evidence to suggest that submicromolar concentrations of Fe2+ can potentiate lipid peroxidation in disrupted brain tissue. NO. released from SNAP did not stimulate, but rather inhibited brain lipid peroxidation. These results support the hypothesis that NO., as opposed to .OH radicals, is not a pro-oxidant but rather an antioxidant.

The COMT inhibitor, entacapone, reduces levodopa-induced elevations in plasma homocysteine in healthy adult rats

Summary.Levodopa treatment has been shown to increase plasma homocysteine levels in Parkinson’s disease (PD) patients and this may lead to an increased risk for coronary arterial diseases. Levodopa is metabolised via O-methylation by catechol-O-methyltransferase (COMT) using S-adenosyl-L-methionine (SAM) as the methyl donor, this leading to the subsequent formation of homocysteine. In this study, the effects of the COMT inhibitor, entacapone, on levodopa-induced hyperhomocysteinaemia were studied in rats. Using a single dose acute treatment paradigm, entacapone (10 or 30 mg/kg) prevented the levodopa (30 or 100 mg/kg) induced rise in plasma homocysteine levels in a dose-dependent manner. Five-day sub-chronic treatment with levodopa (3 × 100 mg/kg per day) resulted in a marked rise in plasma homocysteine levels when measured 2 hours post-treatment on Day 5. These levels fell but remained greater than baseline at 8 hours post-treatment on Day 5. Consistent with findings in the acute treatment test paradigm, the co-administration of entacapone (30 mg/kg) significantly (p<0.001) reduced levodopa-induced hyperhomocysteinaemia for up to 2 hours post-treatment on Day 5 of the sub-chronic study. These results suggest that entacapone may reduce levodopa-induced hyperhomocysteinaemia in PD patients.

Resistance to salt-induced hypertension in catechol-O-methyltransferase-gene-disrupted mice

Background Previous studies have indicated that catechol-O-methyltransferase (COMT) can modulate renal dopaminergic tone. Objective To test the hypothesis that COMT blockade protects from salt-induced hypertension. Methods COMT gene-disrupted (−/−) mice and wild-type controls received a high-sodium diet (NaCl 6%) for 3 weeks. Blood pressure and heart rate were recorded by radiotelemetry. Tissue and urine samples were assessed by light microscopy and high-performance liquid chromatography. The effects of nitecapone treatment were also examined. Systolic blood pressure and heart rate during normal sodium diet were similar in COMT (−/−) and wild-type mice. The high-sodium diet increased night-time systolic and diastolic blood pressures in wild-type mice, whereas blood pressure in COMT (−/−) mice remained unaltered. In wild-type mice, the sodium-induced increase in blood pressure was completely normalized by treatment with the COMT inhibitor, nitecapone. At baseline, 24-h urinary excretion of levodopa (l-DOPA), dopamine and noradrenaline was increased by 145, 85 and 74%, respectively, in COMT (−/−) mice compared with wild-type controls. In COMT (−/−) and wild-type mice, a high-sodium diet increased urinary l-DOPA excretion by 405 and 660% (reflected as 102 and 212% increases in dopamine excretion), respectively. The absolute amounts of urinary l-DOPA and dopamine remained 60 and 20% greater in COMT (−/−) mice. The high-sodium diet did not influence renal cortical COMT activity. Conclusion Our findings suggest that COMT deficiency in mice increases the availability of l-DOPA, leading to enhanced dopaminergic tone, which may be associated with resistance to salt-induced hypertension. The findings of the present study also underline the importance of COMT in the regulation of blood pressure, sodium excretion and renal dopaminergic tone.

Effects of atypical antioxidative agents, S-nitrosoglutathione and manganese, on brain lipid peroxidation induced by iron leaking from tissue disruption.

Abstract: A fluorescent assay of brain lipid peroxidation was used for screening new antioxidants for the prevention of neurodegeneration caused by free radicals. Incubation of rat brain homogenates led to a temperature‐dependent increase in production of fluorescent adducts of peroxidized poly‐unsaturated fatty acids; it was inhibited completely by lowering the incubation temperature to 4°C. This tissue disruption‐induced brain lipid peroxidation at 37°C was blocked by deferoxamine (IC50= 0.3 μM) and EDTA; it was augmented by adding submicromolar iron and hemoglobin. Ferrous ions pro‐oxidative activities were five times more potent than ferric ion. Micromolar manganese completely inhibited lipid peroxidation, confirming earlier unexpected in vivo reports. Trolox and vitamin C suppressed brain lipid peroxidation with IC50 values of 20 and 500 μM, respectively. U‐78517F was approximately 20 times more potent than Trolox. 17β‐Estradiol, hydralazine, S‐nitrosoglutathione and 3‐hydroxybenzylhydrazine were as potent as Trolox. Melatonin, glutathione, α‐lipoic acid and l‐deprenyl were about 20 times less potent than Trolox. Surprisingly, N‐tert‐butyl‐α‐phenylnitrone was a weak antioxidant. Furthermore, this procedure can also detect pro‐oxidative side effects of vitamin C, oxidized glutathione, penicillamine and Angelis salt. The present results obtained from this selective fluorescent assay are consistent with earlier reports that iron complexes promote while manganese inhibits brain lipid peroxidation caused by cell disruption. S‐Nitrosoglutathione, melatonin, 17β‐estradiol, and manganese have been successfully tested in cell/animal models for their potential neuroprotective effects. In conclusion, monitoring fluorescent adducts of peroxidizing polyunsaturated fatty acids in brain homogenates is a simple, quantitative method for studying iron‐dependent brain lipid peroxidation and for screening of potential neuroprotective antioxidants in both in vitro and in vivo preparations.

Angeli's Salt Induces Neurotoxicity in Dopaminergic Neurons In Vivo and In Vitro

In this study, we investigated the hypothesis that the pro-oxidative properties of Angelis salt (AS), a nitroxyl anion (HNO/NO m ) releasing compound, cause neurotoxicity in dopaminergic neurons. The pro-oxidative properties were demonstrated in vitro by measuring hydroxylation products of salicylate and peroxidation of lipids under various redox conditions. AS (0-1000 w M) released high amounts of hydroxylating species in a concentration dependent manner. AS also increased lipid peroxidation in brain homogenates at concentrations below 100 w M, while inhibiting it at 1000 w M concentration. The AS induced pro-oxidative effects were completely suppressed by copper (II), which converts nitroxyl anion to nitric oxide, as well as by a potent nitroxyl anion scavenger glutathione. Neurotoxicity towards dopaminergic neurons was tested in rat nigrostriatal dopaminergic system in vivo and by using primary mesencephalic dopaminergic neuronal cultures in vitro. Intranigral infusion of AS (0-400 nmol) caused neurotoxicity reflected as a dose dependent decrease of striatal dopamine seven days after treatment. The effect of the 100 nmol dose was more pronounced when measured 50 days after the infusion. Neurotoxicity was also confirmed as a decrease of tyrosine hydroxylase positive neurons in the substantia nigra. Neither sulphononoate, a close structural analog of AS, nor sodiumnitrite caused changes in striatal dopamine, thus reflecting lack of neurotoxicity. In primary dopaminergic neuronal cultures AS reduced [ 3 H] dopamine uptake with concentrations over 200 w M confirming neurotoxicity. In line with the quite low efficacy to increase lipid peroxidation in vitro, infusion of AS into substantia nigra did not cause increased formation of fluorescent products of lipid peroxidation. These results support the hypothesis that AS derived species oxidize critical thiol groups, rather than membrane lipids, potentially leading to protein oxidation/dysfunction and demonstrated neurotoxicity. These findings may have pathophysiological relevance in case of excess formation of nitroxyl anion.

FREE RADICALS AND MPTP-INDUCED SELECTIVE DESTRUCTION OF SUBSTANTIA NIGRA COMPACTA NEURONS

Publisher Summary This chapter summarizes several recent studies that investigate the possible role of free radicals in 1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine( MPTP)-induced selective dopaminergic neurotoxicity for supporting the oxidant stress hypothesis in Parkinsons disease. Among brain dopamine neurons, iron-containing substantia nigra compacta neurons are subjected to a continuous oxidant stress. Hydroxyl radicals and semiquinone radicals are generated during iron-catalyzed dopamine auto-oxidation. Furthermore, dopamine-derived oxidants propagate lipid peroxidation that leads to generation of lipid radicals and accumulation of malondialdehyde. Therefore, the accumulation of age pigments such as dopamine melanin in the midbrain substantia nigra compacta neurons may be a reliable biological marker for oxidative stress. The inhibition of complex I may donate unpaired electron to oxygen, leading to the generation of reactive oxygen species. It has been shown that high levels of 1-methyl-4-phenylpyridinium ion (MPP+) inhibit complex I activities in mitochondria1 preparations. MPP+-induced oxidative injury in the cultured midbrain dopamine neurons is suppressed by antioxidants, including lazaroid, trolox, selegiline, and melatonin. Additional in vivo studies further suggest that hydroxyl radicals and associated oxidative stress mediate MPTP-induced neurotoxicity. Based on the peroxynitrite hypothesis, attempts have been made to protect nigral neurons from injury mediated by toxic nitric oxide derivatives through inhibiting neuronal nitric oxide synthase. Neuronal nitric oxide synthase is blocked by both 7-nitroindazole and L-NG-nitro-arginine methyl ester. However, only 7-nitroindazole protects nigral neurons from MPTP-induced dopamine depletion.

Beneficial effects of co-administration of catechol-O-methyltransferase inhibitors and L-dihydroxyphenylalanine in rat models of depression.

The administration of catechol-O-methyltransferase inhibitors alone changed neither the behavior of the rats in two animal models of depression, the forced swimming test (entacapone and tolcapone) or in the learned helplessness paradigm (tolcapone), nor the locomotor activity. L-Dihydroxyphenylalanine (L-DOPA) and carbidopa treatment as such decreased motility but did not improve the behavior in the antidepressant tests. Co-administration of catechol-O-methyltransferase inhibitors and L-DOPA/carbidopa increased the performance of rats in both tests without increasing locomotor activity. Catechol-O-methyltransferase inhibitors could be beneficial as adjunct drugs of L-DOPA not only in Parkinsons disease but also in the coincident depressive illness.