Selective destruction of dopaminergic neurons by low concentrations of 6-OHDA and MPP+: protection by acetylsalicylic acid aspirin

We optimized a mesencephalic cell culture system to employ low concentrations of 6-hydroxydopamine (6-OHDA) and 1-methyl-4 phenylpyrdium (MPP⁺), neurotoxins known to trigger oxidative stress in dopaminergic cells. Both 6-OHDA and MPP⁺ at 5 μM reproducibly reduced the survivial of dopaminergic neurons by 50–70% (p<0.02) without affecting the survival of the non-dopaminergic neuronal population. We found that 1 mM of the non-steroidal anti-inflammatory drug (NSAID), acetylsalicylic acid (ASA), significantly (p<0.05) increased the survival of dopaminergic neurons exposed to either neurotoxin. The mechanisms underlying neuroprotection by ASA may be of therapeutic import in Parkinson's disease.     

Protective effect of fluvastatin, a new inhibitor of 3-hydroxy-3-methylglutaryl coenzyme A reductase, on MPP(+)-induced hydroxyl radical in the rat striatum

We examined whether fluvastatin, an inhibitor of low-density lipoprotein (LDL) oxidation, can resist 1-methyl-4-phenylpyridine (MPP⁺)-induced hydroxyl radical generation (·OH) in the extracellular fluid of rat striatum. Rats were anesthetized and sodium salicylate in Ringer's solution (0.5 nmol/μl/min) was infused through a microdialysis probe to detect the generation of ·OH as reflected by the nonenzymatic formation of 2,3-dihydroxybenzoic acid (DHBA) in the striatum. MPP⁺ (5 mM; total dose 75 nmol) clearly produced an increase in ·OH formation. However, fluvastatin (100 μM) reduced the ·OH formation by the action of MPP⁺. These results indicated that fluvastatin, a potent inhibitor of LDL oxidation, may resist the formation of ·OH products of MPP⁺.     

MPP(+) and glutamate in the degeneration of nigral dopaminergic neurons

Glutamate neurotoxicity can be an experimental oxidative stress, and we investigated glutamate toxicity against cultured rat mesencephalic neurons. Although glutamate showed similar toxicity against dopaminergic and nondopaminergic neurons, nitric oxide (NO) showed neurotoxicity restricted exclusively in nondopaminergic neurons. An inhibitor of NO synthase had no significant effect on the glutamate toxicity against dopaminergic neurons, however, it had a significant antagonistic effect on that against nondopaminergic neurons. These findings indicate the presence of two mechanisms of glutamate neurotoxicity, one being not mediated by NO, found in dopaminergic neurons, and the other being mediated via NO, found in nondopaminergic neurons. In contrast to NO, peroxynitrite (ONOO⁻), an active metabolite of NO, caused significant cytotoxicity against dopaminergic and nondopaminergic neurons, suggesting that conversion of NO to ONOO⁻ is suppressed in dopaminergic neurons. After pretreatment with small doses of methyl-4-phenylpyridium ion (MPP⁺), NO caused significant cytotoxicity against dopaminergic neurons, and glutamate toxicity was enhanced only against dopaminergic neurons. Therefore, sublethal dose of MPP⁺ enhances glutamate toxicity against dopaminergic neurons, probably by the facilitation of suppressed NO conversion to ONOO⁻ in dopaminergic neurons. Finally, to provide basic data for neuroprotective therapy in Parkinson's disease, we investigated neuroprotection against glutamate toxicity by dopamine agonists. Preincubation with the D2 type dopamine agonists provides neuroprotection against glutamate neurotoxicity and the protective effects blocked by a D2 antagonist, indicating that D2 agonists provide protection mediated not only by the inhibition of dopamine turnover, but also via D2 type dopamine receptor.     

Inhibition of the Met-enkephalin-induced K current in B-cluster neurons of Aplysia by nitric oxide donor

The effects of sodium nitroprusside (SNP), a nitric oxide (NO) donor, on a methionine-enkephalin (Met-E)-induced K⁺ current recorded from B-cluster neurons in Aplysia cerebral ganglion were investigated with voltage-clamp and pressure ejection techniques. Bath-applied SNP (10–25 μM) reduced the Met-E-induced K⁺ current in the neurons without affecting the resting membrane conductance and holding current. The inhibitory effects of SNP were reversible. Pretreatment with methylene blue (10 μM), a non-specific inhibitor of guanylate cyclase, and hemoglobin (50 μM), a NO scavenger, decreased the SNP-induced inhibition of the Met-E-induced current. Intracellular injection of 1 mM guanosine 3′,5′-cyclic monophosphate (cGMP) or bath-applied 3-isobutyl-1-methylxanthine (IBMX; 50 μM), a nonspecific phosphodiesterase inhibitor, inhibited the Met-E-induced current. Furthermore, 1H-[1,2,4] oxadiazolo[4,3- a]quinoxalin-1-one (ODQ, 1 μM), a more specific inhibitor of NO-stimulated guanylate cyclase, decreased the SNP-induced inhibition of the Met-E-induced current. These results suggest that SNP induces suppression of the Met-E-induced K⁺ current recorded from B-cluster neurons of Aplysia cerebral ganglion via stimulation of cGMP formation.     

Indole-N-methylated beta-carbolinium ions as potential brain-bioactivated neurotoxins.

N-Methyl-4-phenylpyridinium ion (MPP⁺), a highly toxic metabolite produced in the brain from a street drug contaminant, is selectively taken up by nigrostriatal dopaminergic neurons and accumulated intraneuronally in mitochondria. There it inhibits respiration, causes neuronal death and, in primates, provokes a parkinsonian condition. It has been suggested that endogenously generated or activated agents resembling MPP⁺ may contribute to the development of Parkinson's disease. We report here that simple β-carbolines derived from tryptophan or related open chain indoles, when specifically methyl-substituted on both (2[β] and 9[indole]) available nitrogens, display mitochondrial inhibitory potencies and neurotoxic effects in vitro (PC12 cultures) and in vivo (striatal microdialysis) which approach or even surpass MPP⁺. These results take on physiological significance with our finding that brain enzyme activity catalyzesS-adenosylmethionine-dependent methylations of the β- and indole-nitrogens in β-carbolines that have been detected in vivo. The unusual 9[indole]-N-methyl transfer, previously unrecognized in animals, apparently requires prior methylation of the 2[β]-nitrogen. Sequential di-N-methylation of endogenous or xenobiotic β-carbolines to form unique, neurotoxic 2, 9-N, N′-dimethyl-β-carbolinium ions may serve as a brain bioactivation route in chronic neurodegenerative conditions such as Parkinson's disease.     

Enhanced restoration of striatal dopamine concentrations by combined GM1 ganglioside and neurotrophic factor treatments

Intraperitoneal injection of GM1 ganglioside or intracerebroventricular infusion of basic fibroblast growth factor (FGF-2) or epidermal growth factor (EGF) partially restored dopamine and 3,4-dihydroxyphenylacetic acid (DOPAC) levels in the striatum of young MPTP-treated mice. Combined treatments of GM1 ganglioside with FGF-2 or EGF produced a greater restoration of striatal dopamine levels than treatments with GM1 or either of the neurotrophic factors alone. GM1 treatment, but not trophic factor treatments, caused significant sparing of substantia nigra pars compacta (SNc) tyrosine hydroxylase (TH)-positive neurons. These results confirm previous findings that GM1 provides trophic support for damaged dopamine neurons and suggests that GM1, FGF-2, and EGF may also enhance dopaminergic function in residual neurons. The results also suggest that a potentially fruitful approach to treating degenerative disorders of the dopamine system may be the use of combined trophic factor therapies.     

Tachykinins protect cholinergic neurons from quinolinic acid excitotoxicity in striatal cultures

The neuroprotective effect of tachykinins against excitotoxic death of cholinergic neurons was studied in rat striatal cell cultures. Quinolinic acid (QUIN) and kainic acid (KA) produced a dose dependent decrease in choline acetyttransferase activity, but KA was more potent. Our results show that substance P (SP) totally reversed the toxicity induced by 125 μM QUIN but not by 40 μM KA. This effect was also observed using protease inhibitors or a SP-analog resistant to degradation, [Sar⁹]-Substance P. The survival of neuron specific enolase- and acetylcholinesterase (AChE)-positive cells after treatment with QUIN alone or in the presence of SP was also examined. We observed that, while a decrease in total cell number produced by QUIN was not prevented by SP treatment, AChE-positive cells were rescued from the toxic damage. To characterize the SP protective effect we used more selective agonists of the three classes of neurokinin (NK) receptors. [Sar⁹, Met(O₂)¹¹]-Substance P (NK₁ receptor agonist), [Nle¹⁰]-Neurokinin A (NK₂ receptor agonist) or [Me-Phe⁷]-Neurokinin B (NK₃ receptor agonist) were all able to block the toxic effect of QUIN on cholinergic activity. These results show that tachykinins provide an important protective support for striatal neurons, suggesting a possible therapeutical benefit in neurodegenerative disorders affecting cholinergic neurons.     

Prostacyclin release from the coronary vascular wall by vasoactive substances

Which vasoactive substances that are synthesized in vivo could induce the release of a sufficient amount of prostacyclin (PGI₂) to inhibit platelet aggregation from the vascular wall was investigated in the isolated dog heart perfused by a modified method of Langendorff. Infusion of 5 μM bradykinin or 25 u/ml crude thrombin into the heart for 30 sec resulted in the transient appearance of inhibitory activity of platelet aggregation. The inhibitory activity was stable at alkaline pH but unstable at acidic pH and thermolabile. The appearance of the inhibitory activity was prevented by treatment of the coronary vessel with 30 μM indomethacin or 1 mM tranylcypromine. These results indicated that the inhibitory activity was caused by PGI2. When 25 μM acetylcholine, 25 μM noradrenaline, 25 μM isoproterenol, 10 μM adenosine triphosphate (ATP 5 μM adenosine, 1 μM angiotensin II, 25 μM histamine or 1 μM serotonin was infused for 30 sec, no inhibitory activity of platele aggregation was observed. Bradykinin (5 × 10⁻⁹ 5 × 10⁻⁶ M) and purified thrombin (1 × 10⁻⁹ 1 × 10⁻⁷ M) induced a dose-dependent release of PGI₂ which was assayed using a radioimmunoassay for 6-keto-prostaglandin F₁ (6-keto-PGF₁).     

Chronic depolarization induced by veratridine increases the survival of rat retinal ganglion cells ‘in vitro’

During the last decades it has been shown that trophic molecules released by target, afferent and glial cells play a pivotal role controlling neuronal cell death. Trophic molecules are able to inhibit this regressive event during development as well as during degenerative diseases. One of the mechanisms involved in the control of neuronal survival by afferent cells requires the release of trophic molecules stimulated by electrical activity. It has been demonstrated that veratridine (a depolarizing agent that keeps the Na⁺ channels opened) induces an increase in neuronal survival. In the present work we show that 3 μM veratridine induced a two-fold increase on the survival of retinal ganglion cells after 48 h in culture. The veratridine effect was inhibited by 50 μM amiloride (an inhibitor of Ca²⁺ channels), 25 μM benzamil (an inhibitor of Na⁺ channels), 30 μM dantrolene and 7.5 μM caffeine (both inhibitors of Ca²⁺ release from the endoplasmatic reticulum) and 10 μM BAPTA-AM (an intracellular Ca²⁺ chelator). However, 5 μM nifedipine (a selective inhibitor of voltage-dependent -type Ca²⁺ channels) and 100 μM MK 801 (an inhibitor of NMDA receptors) did not block the veratridine effect. On the other hand, treatment with 10 μM genistein (an inhibitor of tyrosine kinase enzymes), 20 μM fluorodeoxyuridine (an inhibitor of cell proliferation) or 10 μM atropine (an antagonist of muscarinic receptors) completely abolished the effect of veratridine. Taken together, our results indicate that veratridine increases the survival of rat retinal ganglion cells through mechanisms involving Na⁺ influx, intracellular Ca²⁺ release, activation of tyrosine kinase enzymes and cellular proliferation. They also indicate that cholinergic activity plays an important role in the veratridine effect.     

Independent regulation of activation and inactivation phases in non-contractile Ca transients by nicotinic receptor at the mouse neuromuscular junction

Non-contractile Ca²⁺ mobilization (not accompanied by muscle contraction) occurs by the prolonged activation of nicotinic acetylcholine receptor in mouse diaphragm muscles treated with anticholinesterase. To elucidate the regulation properties of non-contractile Ca²⁺ mobilization by nicotinic receptor, the modes of action of competitive and depolarizing neurmuscular blockers were investigated. (+)-Tubocurarine (0.07–0.1 μM), pancuronium (0.05 μM) and -bungarotoxin (0.03–0.06 μM) decreased decay time (T₂, duration of inactivation phase) without changes in rise time (T₁, duration of activation phase) of non-contractile Ca²⁺ transients. These competitive antagonists also suppressed their peak amplitude at higher concentrations than those affectingT₂. Contractile Ca²⁺ transients were not inhibited by these antagonists at the concentrations used. Decamethonium (1 μM), a depolarizing blocker, suppressed the peak amplitude of non-contractile Ca²⁺ transients without affecting their duration. In contrast, succinylcholine (0.3 μM) suppressed both peak amplitude andT₁ without changingT₂, presumably via the receptor desentization. Succinylcholine but not decamthonium inhibited contractile Ca²⁺ transients at the concentrations used. These results demonstrate that the activation and inactivation phase in non-contractile Ca²⁺ transients are independently regulated by nicotinic acetylcholine receptor.     

The involvement of calcium in epinephrine or ADP potentiation of human platelet aggregation

The mechanism by which low doses of epinephrine or ADP potentiate primary platelet aggregation was investigated. Aspirin (lmg/ml)-treated human blood platelets were isolated by albumin density gradient centrifugation. Platelet ⁴⁵Ca uptake associated with epinephrine or ADP addition was determined over a 240 sec time course. Pretreatment of the platelets with ADP (0.5μM) significantly increased aggregation in response to epinephrine (0.1μM). This increased aggregation was associated with a substantially greater ⁴⁵Ca uptake than that which occurred in the presence of epinephrine (0.1μM) alone. The potentiated epinephrine response was inhibited by the Ca²⁺ antagonist verapamil (25μM). This inhibition could in turn be reduced by Ca²⁺ (1mM) addition. Pretreatment of platelets with epinephrine (0.1μM) also increased aggregation in response to ADP (0.5μM). Although this potentiated response was not associated with measurable ⁴⁵Ca uptake, it was nevertheless completely abolished by verapamil (25μM) treatment. These findings suggest that low doses of ADP promote the ability of epinephrine to stimulate an increase in membrane permeability to Ca²⁺.     

Calcium homeostasis in rat septal neurons in tissue culture

Septal neutons from embryonic rats were grown in tissue culture. Microfluorimetric and electrophysiological techniques were used to study Ca²⁺ homeostasis in these neurons. The estimated basal intracellular free ionized calcium concentration ([Ca²⁺]_i) in the neurons was low (50–100 nM). Depolarization of the neurons with 50 mM K⁺ resulted in rapid elevation of [Ca²⁺]_i to 500–1,000 nM showing recovery to baseline [Ca²⁺]_i over several minutes. The increases in [Ca²⁺]_i caused by K⁺ depolarization were completely abolished by the removal of extracellular [Ca²⁺], and were reduced by 80% by the ‘L-type’ Ca²⁺ channel blocker, nimodipine (1 μM). [Ca²⁺]_i was also increased by the excitatory amino andl-glutamate, quisqualate, AMPA and kainate. Responses to AMPA and kainate were blocked by CNOX and DNOX. In the absence of extracellular Mg²⁺, large fluctuations in [Ca²⁺]_i were observed that were blocked by removal of extracellular Ca²⁺, by tetrodotoxin (TTX), or by antagonists ofN-methyld-aspartate (NMDA) such as 2-amino 5-phosphonovalerate (APV). In zero Mg²⁺ and TTX, NMDA caused dose-dependent increases in [Ca²⁺]_i that were blocked by APV. Caffeine (10 mM) caused transient increases in [Ca²⁺]_i in the absence of extracellular Ca²⁺, which were prevented by thapsigargin, suggesting the existence of caffeine-sensitive ATP-dependent intracellular Ca²⁺ stores. Thapsigargin (2 μM) had little effect on [Ca²⁺]_i, or on the recovery from K⁺ depolarization. Removal of extracellular Na⁺ had little effect on basal [Ca²⁺]_i or on responses to high K⁺, suggesting that Na⁺/Ca²⁺ exchange mechanisms do not play a significant role in the short-term control of [Ca²⁺]_i in septal neurons. The mitochondrial uncoupler, CCCP, caused a slowly developing increase in basal [Ca²⁺]_i; however, [Ca²⁺]_i recovered as normal from high K⁺ stimulation in the presence of CCCP, which suggests that the mitochondria are not involved in the rapid buffering of moderate increases in [Ca²⁺]_i. In simultaneous electrophysiological and microfluorimetric recordings, the increase in [Ca²⁺]_i associated with action potential activity was measured. The amplitude of the [Ca²⁺]_i increase induced by a train of action potentials increased with the duration of the train, and with the frequency of firing, over a range of frequencies between 5 and 200 Hz. Recovery of [Ca²⁺]_i from the modest Ca²⁺ loads imposed on the neuron by action potential trains follows a simple exponential decay (τ = 3–5s).     

Regulation of neurotransmitter enzyme by quisqualate subtype glutamate receptors in cultured cerebellar and hippocampal neurons

The possible involvement of ionotropic and metabotropic quisqualate (QA) receptors in neuronal plasticity was studied in cultured glutamtergic cerebellar or hippocampal cells in terms of the specific activity of phosphate-activated glutaminase, an enzyme important in the synthesis of the putative neurotransmitter pool of glutamate. When cerebellar of hippocampal neurons were treated with QA, it elevated the specific activity of glutaminase in a dose-dependent manner. The half-maximal effect was obtained at about 0.1 μM, the maximum increase was at about 1 μM, but levels higher than 10 μM QA produced progressive reduction in glutaminase activity. In contrast, QA had little effects on the activities of lactate dehydrogenase and aspartate aminotransferase and the amount of protein, indicating that the increase in glutaminase was relatively specific. The QA-mediated increase in glutaminase was mimicked by the ionotropic QA receptor agonist -amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA; EC₅₀, about 0.5 μM), but not by the metabotropic QA receptor agonist trans-(±)-1-aino-cyclopentyl-1,3,dicarboxyalte (t-ACPD; up to 0.5 mM). The specific ionotropic QA receptor antagonist 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) inhibited QA- and AMPA-mediated increases in glutaminase activity in a dose-dependent manner, whereas other glutamate receptor antagonists, -2-amino-5-phosphonovalerate, γ- -glutamyl aminomethyl sulphonic acid and γ- -glutamyl diethyl ester were ineffective. The elevation of neurotransmitter enzyme was Ca²⁺-dependent. The increase in Ca²⁺ influx essentially through the activation of L-type voltage-operated Ca²⁺ channels, and not the mobilization of internal Ca²⁺ stores, was responsible for these QA receptor-mediated long-term plastic changes in hippocampal and cerebellar neurons.     

Uptake of leucine and alanine by cultured cerebral capillary endothelial cells

Pure cultures of rat cerebral capillary endothelium have been used to study the A- and L-systems of amino acid transport. Leucine is taken up by a non-concentrative mechanism that can be saturated, and competivively inhibited by phenylalanine. Uptake is rapid, with equilibiration apparent 3–min (al experiments performed at 37 °C). The K_M for transport was 83 μM ± 26(mean ±S.E.M.., n = 3 which is in good agreement with recent vi vivo report using unanesthtissed rats. Alanine was transported by a saturable, concentrative mechanism. Dependence on Na⁺ -ions was demonstrated by lack of specific uptake in Na⁺ -ffree buffer and reduced uptake after preincubation in ouabain — Na⁺, K⁺ -ATPase inhibitor. The K_M was 325 μM ±88 (mean ± S.E.M., n = 3). The finding ac an active A-system transporter in vitro suggests that the cells may have lost the polarity they demonstrate in vivo. The relevance of these findings to transport of nutrients nd drugs across the blood-brain barrier is discussed.     

Presynaptic D2 dopaminergic receptors mediate inhibition of excitatory synaptic transmission in rat neostriatum

The effect of dopamine (DA) on excitatory synaptic transmission was studied in rat neostriatal neurons using intracellular- and whole-cell voltage clamp-recording methods. Depolarizing excitatory postsynaptic potentials (EPSPs) were evoked by cortical stimulation. Superfusion of DA (0.01–10 μM) reversibly decreases EPSP in a concentration-dependent manner and with a estimated IC₅ of 0.3 μM. In addition, the inhibitory effect induced by DA at a low concentratiion (0.1 μM) was antagonized by sulpiride (1–10 nM), a selective D₂ dopaminergic receptor antagonist. However, D₁ dopaminergic receptor antagonist SKF-83566 (1–5 μM) did not affect the blocking effect by DA 0.1 μM. Based on these findings, we conclude that DA at a low concentration ( 0.1 μM) reduced the excitatory response of neostriatal neurons following cortical stimulation via the activation of D₂, but not D₁ dopaminergic receptors, located on the terminals of corticostriatal neurons.     

Mitochondrial permeability in neuronal death: possible relevance to the pathogenesis of Parkinson's disease

1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) causes catecholaminergic nerve cell loss and a syndrome similar to Parkinson's disease (PD). The metabolite of MPTP, MPP⁺ (1-methyl-4-phenylpyridinium), decreases mitochondrial complex I activity similar to that in the PD nigra. Opening of a multi-protein, mitochondrial membrane pore constitutes a critical decisional event in some forms of apoptosis. We review recent findings showing that the permeability transition pore (PTP) opening caused by a decrease in the mitochondrial membrane potential (ΔΨ_M) contributes to MPP⁺-induced apoptosis. The reduction in ΔΨ_M appears to result from decreased proton pumping at complex I and therefore decreased complex I activity may also contribute to apoptosis in PD.     

The co-expression of VR1 and VRL-1 in the rat vagal sensory ganglia

Immunohistochemistry for two nociceptive transducers, the vanilloid receptor 1 (VR1) and vanilloid receptor 1-like receptor (VRL-1), was performed on the vagal sensory ganglia. In the jugular ganglion, VR1-immunoreactive (IR) neurons were small to medium-sized (range 49.7–1125.6 μm², mean±S.D. 407.7±219.7 μm²), whereas VRL-1-IR neurons were medium-sized to large (range 223.6–1341.1 μm², mean±S.D. 584.3±253.5 μm²). In the nodose ganglion, VR1- and VRL-1-IR neurons were mostly small to medium-sized (VR1: range 148.5–1464.4 μm², mean±S.D. 554.3±207.4 μm²; VRL-1: range 161.7–1166.2 μm², mean±S.D. 541.9±186.2 μm²). The double immunofluorescence method revealed that co-expression of VR1-immunoreactivity among VRL-1-IR neurons was more abundant in the nodose ganglion (63%) than in the jugular ganglion (4%). The present study suggests that co-expression of VR1 and VRL-1 may be more common in visceral sensory neurons than in somatic sensory neurons.