Nigral and Striatal Comparative Study of the Neurotoxic Action of 1-Methyl-4-Phenylpyridinium Ion: Involvement of Dopamine Uptake System

Abstract: Microdialysis was used in a comparative study of the neurotoxic action of MPP⁺ in the absence or presence of nomifensine (20 µM) in the striatum and substantia nigra. Three different concentrations of MPP⁺ (1, 2.5, and 5 mM) were perfused for 15 min at 24 (day 1) and 48 h (day 2) after surgery. The dopamine basal value in the striatum was ～17 fmol/min. Nomifensine (20 µM) stimulated dopamine release to ～170 fmol/min. The increase of dopamine extracellular output in the striatum after MPP⁺ perfusion on day 1 was independent of the concentration of MPP⁺ perfused and of the absence or presence of nomifensine (20 µM), being ～2,500 fmol/min. The dopamine basal value in the substantia nigra was below the detection limit of our HPLC equipment. Nomifensine (20 µM) stimulated dopamine release to ～6.3 fmol/min. The increase of dopamine extracellular output in the substantia nigra was MPP⁺ dose-dependent (1 mM, 75 fmol/min; 2.5 mM, 150 fmol/min; and 5 mM, 250 fmol/min) and independent of the presence or absence of nomifensine. On day 2, the presence of nomifensine on day 1 produced a total protection against MPP⁺ (1 mM) perfusion in the striatum, which was not observed against MPP⁺ (5 mM). MPP⁺ (1 mM) did not produce any neurotoxic action in the substantia in the absence or presence of nomifensine. The MPP⁺ (2.5 mM) effect on dopamine extracellular output in the absence of nomifensine (20 µM) in the substantia nigra on day 2 was similar to that of MPP⁺ (1 mM) in the striatum. The presence of nomifensine (20 µM) partially prevented the neurotoxic effect of MPP⁺ (2.5 mM) on dopaminergic cell bodies/dendrites in the substantia nigra. The MPP⁺ (5 mM) effect on dopamine extracellular output was similar in both structures studied in the absence or presence of nomifensine on day 2. These results suggest that terminals in the striatum are more sensitive to the neurotoxicity of MPP⁺ than cell bodies/dendrites in the substantia nigra.     

Age-Dependent Sensitivity of Cultured Peripheral Sympathetic Neurons to 1-Methyl-4-Phenylpyridinium: Role of Glutathione

Abstract: We demonstrate that 1-methyl-4-phenylpyridinium (MPP⁺) is toxic to chick peripheral sympathetic neurons maintained in culture in the presence of nerve growth factor (NGF). When MPP⁺ was added to the culture medium at the time the neurons were plated, cell loss after 3 days in culture was evident at concentrations as low as 3 nM, and near maximal at 1 µM. Toxicity was blocked by brief preincubation with the norepinephrine (NE)-reuptake blocker desipramine (DMI; 10 µM for 30 min). MPP⁺ blocked the uptake of [³H]NE by sympathetic neurons in a dose-dependent manner with a potency roughly equal to DMI. At concentrations up to 10 µM, MPP⁺ had no neurotoxic effect on the survival of sensory neurons maintained in the presence of NGF. The sensitivity of sympathetic neurons to the toxic effects of MPP⁺ diminished gradually with increasing lengths of time in culture. When MPP⁺ was added to the culture medium 48 h after plating, concentrations up to 100 µM did not cause neuronal death. This increasing resistance of sympathetic neurons to MPP⁺-induced cell death could not be explained by an increasing capacity for sequestration of MPP⁺ within synaptic vesicles. The loss of sensitivity with time in culture was, however, accompanied by a threefold increase in the levels of glutathione (GSH). Furthermore, addition of MPP⁺ (1 µM) to cultures previously maintained for 2 days in the presence of the GSH-synthesis inhibitor l -buthionine-[S,R]-sulfoximine (1 µM) caused the same degree of cell death as when added to freshly plated neurons. These results suggest that the observed toxicity of MPP⁺ in freshly plated chick sympathetic neurons may involve the formation of free radicals and that GSH plays a role in protecting sympathetic neurons in vivo from the toxicity of MPP⁺.     

Nitroindazole compounds inhibit the oxidative activation of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) neurotoxin to neurotoxic pyridinium cations by human monoamine oxidase (MAO)

Monoamine oxidase (MAO) B is a mitochondrial enzyme selectively involved in the oxidative activation of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) neurotoxin to toxic pyridinium cations producing Parkinsonism in animal models. Various synthesized 5-nitroindazoles, 6-nitroindazole and the neuroprotectant 7-nitroindazole were examined as inhibitors of MAO and as antioxidants and radical scavengers. The oxidation of MPTP by human MAO-B and mitochondria was assessed by HPLC. Simple nitroindazoles inhibited MPTP oxidation to 1-methyl-4-phenyl-2,3-dihydropyridinium (MPDP⁺) and 1-methyl-4-phenylpyridinium (MPP⁺) in a competitive and reversible manner. 5-Nitroindazole (IC₅₀=0.99 µM, K_i=0.102 µM) and 6-nitroindazole (IC₅₀=2.5 µM) were better inhibitors of human MAO-B than 7-nitroindazole (IC₅₀=27.8 µM). 6-Nitroindazole also inhibited MAO-A. Nitroindazole isomers were good hydroxyl radical (OH^?) scavengers, with 5-nitro-, 6-nitro- and 7-nitroindazole showing similar activity (k ~10¹⁰ M^?1 s^?1). Neuroprotective actions of nitroindazoles (7-nitroindazole) could be linked to their MAO-inhibitory and antiradical properties besides inhibition on nitric oxide synthase (NOS). 5-Nitro- and 6-nitroindazole, previously reported as weak NOS inhibitors, were better inhibitors of human MAO-B and more active against MPTP neurotoxin oxidation (lower MPDP⁺ and MPP⁺ levels) than 7-nitroindazole and acted as good radical scavengers and could be potential neuroprotective agents in addition to MAO-B inhibitors.     

Inhibition of NADH oxidation by 1-methyl-4-phenylpyridinium analogs as the basis for the prediction of the inhibitory potency of novel compounds

Inhibition of NADH dehydrogenase (Complex I) of the mitochondrial respiratory chain by 1-methyl-4-phenylpyridinium (MPP⁺) and its analogs results in dopaminergic cell death. In the present study, the inhibition of mitochondrial respiration and of NADH oxidation in inverted inner membrane preparations by the oxidation products of N-methyl-stilbazoles (N-methyl-styrylpyridiniums) are characterized. These nonflexible MPP⁺ analogs were found to be considerably more potent inhibitors than the corresponding MPP⁺ derivatives. The IC₅₀ values for these compounds and previously published figures for MPP⁺ analogs were then used to select a computer model based on structural parameters to predict the inhibitory potency of other compounds that react at the “rotenone site” in Complex I. A series of 12 novel inhibitors different in structure from the basic set were used to test the predictive capacity of the models selected. Despite major structural differences between the novel test compounds and the MPP⁺ and styrylpyridinium analogs on which the models were based, substantial agreement was found between the predicted and experimentally determined IC₅₀ values. The value of this technique lies in the potential for the prediction of the inhibitory potency of other drugs and toxins which block mitochondrial respiration by interacting at the rotenone sites. © 1996 John Wiley & Sons, Inc.     

I. The development of amine substituted analogues of MPTP as unique tools for the study of MPTP toxicity and Parkinson's disease

We are currently developing amino-substituted MPTP analogues as useful probes for understanding the mechanism of MPTP toxicity and Parkinson's disease. One analogue, 4′-amino MPTP, induces a loss of striatal dopamine and is thus a suitable substitute for MPTP. This probe will be used as a histologically fixable MPTP which can be used to answer detailed anatomical questions concerning the sites of MPTP, MPP⁺ uptake and storage. In addition, antibodies have been raised against MPTP and MPP⁺ in rabbits using diazo-linked bovine serum albumin conjugates. The antibodies have been characterized with regard to their recognition of relevant structural analogues using an enzymelinked immunoassay (ELISA) procedure. Antibodies to MPTP detected MPTP in mouse brain extracts derived from as little as 5 μg of tissue. The antibodies will be used for immunohistochemical localization of 4′-NH₂-MPTP and 4′-NH₂-MPP⁺ in brain, as well as probes for the screening of parkinsonian brain tissue for any MPTP- or MPP⁺-like materials which might exist.     

Ca2+ - and Nitric Oxide-Dependent Stimulation of Cyclic GMP Synthesis in Neuronal Cell Line Induced by P2-Purinergic/Pyrimidinergic Receptor

Abstract: The mechanism by which cyclic GMP synthesis is activated through a nucleotide receptor was studied in mouse neuroblastoma × rat glioma hybrid cells [108CC15 (NG 108-15)]. The transient increase in cyclic GMP level induced by ATP reached its maximum at 20 s and lasted for ～1 min. The maximal rise in cyclic GMP level achieved was highest for ATP and decreased in the following order: ATP = adenosine 5′-(γ-thio)triphosphate > UTP = 2-methylthio-ATP > ADP ? CTP, AMP, α,β-methylene-ATP, 2′- and 3′-O-(4-benzoylbenzoyl)ATP. The EC₅₀ of 1 ± 0.2 µM for UTP was significantly lower than that for ATP (14 ± 8 µM) and for all the other nucleotides tested. The rank order of potency is consistent with the pharmacology of a P_2u receptor. At submaximal concentrations of the nucleotides ATP and UTP, the rise in cyclic GMP level was inhibited by suramin (IC₅₀ = 40–60 µM) or the pyridoxal phosphate analogue pyridoxal phosphate-6-azophenyl-2′,4′-disulfonic acid (IC₅₀ = 20–30 µM). Pretreatment of cells with the Ca²⁺ ionophore ionomycin or with 2,5-di(tert-butyl)-1,4-benzohydroquinone, an inhibitor of Ca²⁺-ATPase in the endoplasmic reticulum, a maneuver to deplete internal Ca²⁺ stores, suppressed the ATP- or UTP-induced stimulation of cyclic GMP synthesis. Similarly, loading of the cells with the Ca²⁺ chelator 1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid inhibited cyclic GMP formation by ATP. Preincubation with forskolin to raise the cyclic AMP level potentiated the ATP-induced rise in cyclic GMP level by 60%. The cyclic GMP response caused by ATP was suppressed either by arginine analogues (IC₅₀ for nitroarginine = 1 µM) or by hemoglobin (IC₅₀ = 2 µM). This indicates that ATP/UTP via a P₂-receptor causes formation of nitric oxide, which activates guanylate cyclase. The synthesis of nitric oxide depends on a preceding rise in cytosolic Ca²⁺ level, mostly due to release of Ca²⁺ from internal stores. Bradykinin induces a rise in cyclic GMP level with an amplitude and time course comparable to that caused by ATP. Therefore, we studied cross-desensitization between ATP and bradykinin receptors. Pretreatment with bradykinin completely suppressed a subsequent response to ATP. However, stimulation with ATP reduced a following response to bradykinin by ～40% only. This indicates a heterologous cross-desensitization predominantly in one direction (bradykinin ? ATP).     

Sulphur-Containing Amino Acids Modulate Noradrenaline Release from Hippocampal Slices

Abstract: The l - and d -enantiomers of the sulphur-containing amino acids (SAAs)—homocysteate, homocysteine sulphinate, cysteate, cysteine sulphinate, and S-sulphocysteine—stimulated [³H]noradrenaline release from rat hippocampal slices in a concentration-dependent manner. The relative potencies of the l -isomers (EC₅₀ values of 1.05–1.96 mM) were of similar order to that of glutamate (1.56 mM), which was 10-fold lower than that of NMDA (0.15 mM), whereas the d -isomers exhibited a wider range of potencies (0.75 to >5 mM). All stimulatory effects of the SAAs were significantly inhibited by the voltage-sensitive Na⁺ channel blocker tetrodotoxin (55–71%) and completely blocked by addition of Mg²⁺ or Co²⁺ to the incubation medium. All SAA-evoked responses were concentration-dependently antagonized by the selective NMDA receptor antagonist d -(?)-2-amino-5-phosphonopentanoic acid (IC₅₀ values of 3.2–49.5 µM). 6-Cyano-7-nitroquinoxaline-2,3-dione (CNQX), a non-NMDA receptor antagonist, at 100 µM inhibited the [³H]noradrenaline release induced by glutamate and NMDA (65 and 76%, respectively) and by all SAAs studied (65–85%), whereas 10 µM CNQX only inhibited the effects of S-sulpho-l -cysteine and l - and d -homocysteate (33, 32, and 44%, respectively). However, the more selective AMPA/kainic acid receptor antagonist 6-nitro-7-sulphamoylbenzo(f)quinoxaline-2,3-dione (100 µM), which did not antagonize the [³H]noradrenaline release induced by glutamate and NMDA, reduced only the S-sulpho-l -cysteine-evoked response (25%). Thus, the stimulation of Ca²⁺-dependent[³H]noradrenaline release from hippocampal slices elicited by the majority of the SAAs appears to be mediated by the NMDA receptor.     

The internal rotenone‐insensitivae NADPH dehydrogenase contributes to malate oxidation by potato tuber and pea leaf mitochondria

Inside-out submitochondrial particles from both potato (Solanum tuberosum L. cv. Bintje) tubers and pea (Pisum sativum L. cv. Oregon) leaves possess three distinct dehydrogenase activities: Complex I catalyzes the rotenone-sensitive oxidation of deamino-NADH, ND_in(NADPH) catalyzes the rotenone-insensitive and Ca²⁺-dependent oxidation of NADPH and ND_in(NADH) catalyzes the rotenone-insensitive and Ca²⁺-independent oxidation of NADH. Diphenylene iodonium (DPI) inhibits complex I, ND_in(NADPH) and ND_in (NADH) activity with a K_i of 3.7, 0.17 and 63 µM, respectively, and the 400-fold difference in K_i between the two ND_in made possible the use of DPI inhibition to estimate ND_in (NADPH) contribution to malate oxidation by intact mitochondria. The oxidation of malate in the presence of rotenone by intact mitochondria from both species was inhibited by 5 µM DPI. The maximum decrease in rate was 10–20 nmol O₂ mg^?1 min^?1. The reduction level of NAD(P) was manipulated by measuring malate oxidation in state 3 at pH 7.2 and 6.8 and in the presence and absence of an oxaloacetate-removing system. The inhibition by DPI was largest under conditions of high NAD(P) reduction. Control experiments showed that 125 µM DPI had no effect on the activities of malate dehydrogenase (with NADH or NADPH) or malic enzyme (with NAD⁺ or NADP⁺) in a matrix extract from either species. Malate dehydrogenase was unable to use NADP⁺ in the forward reaction. DPI at 125 µM did not have any effect on succinate oxidation by intact mitochondria of either species. We conclude that the inhibition caused by DPI in the presence of rotenone in plant mitochondria oxidizing malate is due to inhibition of ND_in(NADPH) oxidizing NADPH. Thus, NADP turnover contributes to malate oxidation by plant mitochondria.     

Pharmacology of Sodium-Dependent High-Affinity l-[3H]Glutamate Transport in Glial Cultures

Abstract: Pharmacological and molecular biological studies provide evidence for subtypes of sodium-dependent high-affinity glutamate (Glu) transport in the mammalian CNS. At least some of these transporters appear to be selectively expressed in different brain regions or by different cell types. In the present study, the properties of l -[³H]Glu transport were characterized using astrocyte-enriched cultures prepared from cerebellum and cortex. In both brain regions, the kinetic data for sodium-dependent transport were consistent with a single site with K_m values of 91 ± 17 µM in cortical glial cells and 66 ± 23 µM in cerebellar glial cells. The capacities were 6.1 ± 1.6 nmol/mg of protein/min in cortical glial cells and 8.4 ± 0.9 nmol/mg of protein/min in cerebellar glial cells. The potencies of ～40 excitatory amino acid analogues for inhibition of sodium-dependent transport into glial cells prepared from cortex and cerebellum were examined, including compounds that are selective inhibitors of transport in synaptosomes prepared from either cerebellum or cortex. Of the analogues tested, 14 inhibited transport activity by >50% at 1 mM concentrations. Unlike l -[³H]Glu transport in synaptosomes prepared from cerebellum or cortex, there were no large differences between the potencies of compounds for inhibition of transport measured in glial cells prepared from these two brain regions. With the exception of (2S,1′R,2′R)-2-(carboxycyclopropyl)glycine and l -α-aminoadipate, all of the compounds examined were ～10–200-fold less potent as inhibitors of l -[³H]Glu transport measured in glial cells than as inhibitors of transport measured in synaptosomes prepared from their respective brain regions. The pharmacology of transport measured in these glial cells differs from the reported pharmacology of the cloned Glu transporters, suggesting the existence of additional uncloned Glu transporters or Glu transporter subunits.     

Evidence for Hydroxyl Radical Scavenging Action of Nitric Oxide Donors in the Protection Against 1-Methyl-4-phenylpyridinium-induced Neurotoxicity in Rats

In the present study we provide evidence for hydroxyl radical (^•OH) scavenging action of nitric oxide (NO^•), and subsequent dopaminergic neuroprotection in a hemiparkinsonian rat model. Reactive oxygen species are strongly implicated in the nigrostriatal dopaminergic neurotoxicity caused by the parkinsonian neurotoxin, 1-methyl-4-phenylpyridinium (MPP⁺). Since the role of this free radical as a neurotoxicant or neuroprotectant is debatable, we investigated the effects of some of the NO^• donors such as S-nitroso-N-acetylpenicillamine (SNAP), 3-morpholinosydnonimine hydrochloride (SIN-1), sodium nitroprusside (SNP) and nitroglycerin (NG) on in vitro ^•OH generation in a Fenton-like reaction involving ferrous citrate, as well as in MPP⁺-induced ^•OH production in the mitochondria. We also tested whether co-administration of NO^• donor and MPP⁺ could protect against MPP⁺-induced dopaminergic neurotoxicity in rats. While NG, SNAP and SIN-1 attenuated MPP⁺-induced ^•OH generation in the mitochondria, and in a Fenton-like reaction, SNP caused up to 18-fold increase in ^•OH production in the latter reaction. Striatal dopaminergic depletion following intranigral infusion of MPP⁺ in rats was significantly attenuated by NG, SNAP and SIN-1, but not by SNP. Solutions of NG, SNAP and SIN-1, exposed to air for 48 h to remove NO^•, when administered similarly failed to attenuate MPP⁺-induced neurotoxicity in vivo. Conversely, long-time air-exposed SNP solution when administered in rats intranigrally, caused a dose-dependent depletion of the striatal dopamine. These results confirm the involvement of ^•OH in the nigrostriatal degeneration caused by MPP⁺, indicate the ^•OH scavenging ability of NO^•, and demonstrate protection by NO^• donors against MPP⁺-induced dopaminergic neurotoxicity in rats.     

Selective Inhibition of the Expression of Signal Transduction Proteins by Lithium in Nerve Growth Factor-Differentiated PC12 Cells

Abstract: Muscarinic receptor in human neuroblastoma SK-N-BE(2)C cells was identified and characterized. Treatment of the cells with carbachol evoked the generation of inositol 1,4,5-trisphosphate (IP₃) with a peak level reached at 1 min after stimulation. Carbachol increased intracellular Ca²⁺ ([Ca²⁺]_i) with an EC₅₀ value of 35 µM. In addition, carbachol produced a 1.3–3-fold increase in the cyclic AMP (cAMP) level compared with untreated control and elevated synergistically the cAMP level in the treatment with prostaglandin E₂ (PGE₂). The M₃ antagonist p-fluorohexahydrosiladifenidol (IC₅₀ = 0.5–0.8 µM) inhibited the increases in [Ca²⁺]_i, IP₃, and cAMP more effectively than the M₁ antagonist pirenzepine (IC₅₀ = 5–9 µM) and the M₂ antagonist methoctramine (IC₅₀ = 20–30 µM). The involvements of [Ca²⁺]_i elevation and protein kinase C activation induced by phospholipase C activation were tested in the carbachol-induced cAMP production. The calcium chelator BAPTA/AM (75 µM) inhibited significantly the synergistic effects of carbachol and PGE₂ on the production of cAMP, whereas the Ca²⁺ ionophore ionomycin (1 µM) clearly enhanced PGE₂-induced cAMP production. However, phorbol 12-myristate 13-acetate did not enhance PGE₂-stimulated cAMP production. These data suggest that phospholipase C-linked M₃ receptors are present and that stimulation of the receptors activates adenylyl cyclase, at least in part, by the Ca²⁺-dependent system in the neuronal cells.     

Transgenic Murine Dopaminergic Neurons Expressing Human Cu/Zn Superoxide Dismutase Exhibit Increased Density in Culture, but No Resistance to Methylphenylpyridinium-Induced Degeneration

Abstract: Primary dopaminergic neuronal cultures with increased superoxide dismutase (SOD) activity were established for studying the role of superoxide anion (O₂^?) in 1-methyl-4-phenylpyridinium (MPP⁺)-induced degeneration of dopamine (DA) neurons. Mean SOD activity in cultures prepared from transgenic (human) Cu/Zn SOD (hSOD1) mice was 2.46–2.60 times greater than in cultures prepared from nontransgenic control mice. After 1 and 2 weeks in culture, the mean density of DA neurons [number of tyrosine hydroxylase-immunoreactive (TH-ir) cells per visual field] was significantly higher in cultures prepared from transgenic mice compared with those prepared from nontransgenic control mice (4.55–5.63 TH-ir neurons per field in hSOD1 cultures vs. 2.66–2.8 TH-ir neurons per field in control cultures). However, uptake of [³H]DA relative to uptake of [³H]GABA was only slightly greater in hSOD1 cultures than in normal cultures (14.1 nmol of DA/100 nmol of GABA vs. 12.1 nmol of DA/100 nmol of GABA). Resistance to MPP⁺ toxicity was not significantly different from that in normal cultures when based on density of surviving TH-ir cell bodies (EC₅₀ = 0.54 µM in hSOD1 and EC₅₀ = 0.37 µM in normal cultures). A more sensitive measure of DA neuron integrity and function ([³H]DA uptake) also failed to demonstrate increased resistance of hSOD1 cultures to the toxin (EC₅₀ = 73.7 nM in hSOD1 and EC₅₀ = 86.2 nM in controls). These results do not support the hypothesis that neurotoxicity of the active metabolite of MPTP, MPP⁺, is mediated by generation of O₂^? in the cytoplasm. Nevertheless, mesencephalic cultures with increased hSOD1 activity appear to survive better than normal control cultures in the oxidatively stressful environment of cell culture incubators, and such mesencephalic cells may be useful for cell grafting studies in animal models of Parkinson's disease.     

Characterization of Inhibitor-Sensitive and -Resistant Adenosine Transporters in Cultured Human Fetal Astrocytes

Abstract: The kinetic characteristics of [³H]adenosine uptake, the extent to which accumulated [³H]adenosine was metabolized, the effects such metabolism had on measurements of apparent Michaelis-Menten kinetic values of K_T and V_max, and the sensitivities with which nucleoside transport inhibitors blocked [³H]adenosine accumulations were determined in cultured human fetal astrocytes. K_T and V_max values for accumulations of [³H]-labeled purines using 15-s incubations in the absence of the adenosine deaminase inhibitor erythro-9-(2-hydroxy-3-nonyl)adenine (EHNA) and the adenosine kinase inhibitor 5′-iodotubercidin (ITU) were 6.2 µM and 0.15 nmol/min/mg of protein for the high-affinity and 2.6 mM and 21 nmol/min/mg of protein for the low-affinity components respectively. In the presence of EHNA and ITU, where <4% of accumulated [³H]adenosine was metabolized, transport per se was measured, and kinetic values for K_T and V_max were 179 µM and 5.2 nmol/min/mg of protein, respectively. In the absence of EHNA and ITU, accumulated [³H]adenosine was rapidly metabolized to AMP, ADP, and ATP, and caused an appearance of “concentrative” uptake in that the intracellular levels of [³H]-labeled purines (adenosine plus its metabolites) were 1.4-fold higher than in the medium. No apparent concentrative accumulations of [³H]adenosine were found when assays were conducted using short incubation times in the absence or presence of EHNA and ITU. The nucleoside transport inhibitors dipyridamole (DPR), nitrobenzylthioinosine (NBI), and dilazep biphasically inhibited [³H]adenosine transport; for the inhibitor-sensitive components the IC₅₀ values were 0.7 nM for NBI, 1.3 nM for DPR, and 3.3 nM for dilazep, and for the inhibitor-resistant component the IC₅₀ values were 2.5 µM for NBI, 5.1 µM for dilazep, and 39.0 µM for DPR. These findings, in cultured human fetal astrocytes, represent the first demonstration of inhibitor-sensitive and -resistant adenosine transporters in nontransformed human cells.     

DLP1-dependent mitochondrial fragmentation mediates 1-methyl-4-phenylpyridinium toxicity in neurons: implications for Parkinson's disease

Selective degeneration of nigrostriatal dopaminergic neurons in Parkinson’s disease (PD) can be modeled by the administration of the neurotoxin 1‐methyl‐4‐phenylpyridinium (MPP⁺). Because abnormal mitochondrial dynamics are increasingly implicated in the pathogenesis of PD, in this study, we investigated the effect of MPP⁺ on mitochondrial dynamics and assessed temporal and causal relationship with other toxic effects induced by MPP⁺ in neuronal cells. In SH‐SY5Y cells, MPP⁺ causes a rapid increase in mitochondrial fragmentation followed by a second wave of increase in mitochondrial fragmentation, along with increased DLP1 expression and mitochondrial translocation. Genetic inactivation of DLP1 completely blocks MPP⁺‐induced mitochondrial fragmentation. Notably, this approach partially rescues MPP⁺‐induced decline in ATP levels and ATP/ADP ratio and increased [Ca²⁺]_i and almost completely prevents increased reactive oxygen species production, loss of mitochondrial membrane potential, enhanced autophagy and cell death, suggesting that mitochondria fragmentation is an upstream event that mediates MPP⁺‐induced toxicity. On the other hand, thiol antioxidant N‐acetylcysteine or glutamate receptor antagonist D‐AP5 also partially alleviates MPP⁺‐induced mitochondrial fragmentation, suggesting a vicious spiral of events contributes to MPP⁺‐induced toxicity. We further validated our findings in primary rat midbrain dopaminergic neurons that 0.5 μm MPP⁺ induced mitochondrial fragmentation only in tyrosine hydroxylase (TH)‐positive dopaminergic neurons in a similar pattern to that in SH‐SY5Y cells but had no effects on these mitochondrial parameters in TH‐negative neurons. Overall, these findings suggest that DLP1‐dependent mitochondrial fragmentation plays a crucial role in mediating MPP⁺‐induced mitochondria abnormalities and cellular dysfunction and may represent a novel therapeutic target for PD.     

Cloning and Expression of a Betaine/GABA Transporter from Human Brain

Abstract: A cDNA clone encoding a human γ-aminobutyric acid (GABA) transporter has been isolated from a brain cDNA library, and its functional properties have been examined in mammalian cells. The nucleotide sequence predicts a transporter with 614 amino acids and 12 putative transmembrane domains. The highest degree of amino acid identity is with a betaine/GABA transporter originally cloned from the dog termed BGT-1 (91%) and a related transporter from mouse brain (87%). These identities are similar to those for species homologues of other neurotransmitter transporters and suggest that the new clone represents the human homologue of BGT-1. The transporter displays high affinity for GABA (IC₅₀ of 30 µM) and is also sensitive to phloretin, l -2,4-diaminobutyric acid, and hypotaurine (IC₅₀ values of ～150–400 µM). The osmolyte betaine is ～25-fold weaker than GABA, displaying an IC₅₀ of ～1 mM. The relative potencies of these inhibitors at human BGT-1 differ from those of mouse and dog BGT-1. Northern blot analysis reveals that BGT-1 mRNA is widely distributed throughout the human brain. The cloning of the human homologue of BGT-1 will further our understanding of the roles of GABA and betaine in neural function.     

Pharmacological Characterization of the Voltage-Dependent Ca2+ Channels Present in Synaptosomes from Rat and Chicken Central Nervous System

Abstract: The voltage-dependent calcium channels present in mammalian and chicken brain synaptosomes were characterized pharmacologically using specific blockers of L-type channels (1,4-dihydropyridines), N-type channels (ω-conotoxin GVIA), and P-type channels [funnel web toxin (FTX) and ω-agatoxin IVA]. K⁺-induced Ca²⁺ uptake by chicken synaptosomes was blocked by ω-conotoxin GVIA (IC₅₀ = 250 nM). This toxin at 5 µM did not block Ca²⁺ entry into rat frontal cortex synaptosomes. FTX and ω-agatoxin IVA blocked Ca²⁺ uptake by rat synaptosomes (IC₅₀ = 0.17 µl/ml and 40 nM, respectively). Likewise, in chicken synaptosomes, FTX and ω-agatoxin IVA affected Ca²⁺ uptake. FTX (3 µl/ml) exerted a maximal inhibition of 40% with an IC₅₀ similar to the one obtained in rat preparations, whereas with ω-agatoxin IVA saturation was not reached even at 5 µM. In chicken preparations, the combined effect of saturating concentrations of FTX (1 µl/ml) and different concentrations of ω-conotoxin GVIA showed no additive effects. However, the effect of saturating concentrations of FTX and ω-conotoxin GVIA was never greater than the one observed with ω-conotoxin GVIA. We also found that 60% of the Ca²⁺ uptake by rat and chicken synaptosomes was inhibited by ω-conotoxin MVIID (1 µM), a toxin that has a high index of discrimination against N-type channels. Conversely, nitrendipine (10 µM) had no significant effect on Ca²⁺ uptake in either the rat or the chicken. In conclusion, Ca²⁺ uptake by rat synaptosomes is potently inhibited by different P-type Ca²⁺ channel blockers, thus indicating that P-type channels are predominant in this preparation. In contrast, Ca²⁺ uptake by chicken synaptosomes is sensitive to ω-conotoxin GVIA, FTX, ω-agatoxin IVA, and ω-conotoxin MVIID. This suggests that a channel subtype with a mixed pharmacology is present in chicken synaptosomes.     

Endogenous Hydrogen Sulfide is Involved in Asymmetric Dimethylarginine-induced Protection Against Neurotoxicity of 1-Methyl-4-phenyl-pyridinium Ion

Asymmetric dimethylarginine (ADMA), an endogenous nitric oxide synthase (NOS) inhibitor, is profoundly protective against 1-methy-4-phenylpyridinium ion (MPP⁺)-induced neurotoxicity. Reactive oxygen species (ROS) overproduction contributes to the neurotoxicity of MPP⁺; while hydrogen sulfide (H₂S) is a pivotal endogenous antioxidant. This study is to assess the potential role of endogenous H₂S in the neuroprotection of ADMA against MPP⁺-induced toxicity in PC12 cells. We showed that ADMA prevented MPP⁺-induced inhibition of endogenous H₂S generation through inhibiting the down-regulation of cystathionine-β-synthetase (CBS, the major enzyme responsible for endogenous H₂S generation in PC12 cells) expression and activity elicited by MPP⁺. ADMA obviously attenuated MPP⁺-triggered accumulation of intracellular ROS, dissipation of mitochondrial membrane potential (MMP), release of cytochrome c (Cyt-c), and downregulation of Bcl-2 protein expression in PC12 cells. Inhibition of CBS activity by amino-oxyacetate and CBS silencing with a short hairpin RNA vector targeting rat CBS gene reversed the protective action of ADMA against MPP⁺-caused cytotoxicity, ROS overproduction, and MMP loss in PC12 cells. These results indicate that the protection of ADMA against MPP⁺-mediated neurotoxicity involves the melioration of MPP⁺-induced inhibition of endogenous H₂S generation. Our findings suggest that modulation of H₂S production provide new therapeutic targets for the treatment of neurodegenerative disease, such as Parkinson’s disease.     

Anaerobic Glycolysis Protection against 1-Methy-4-Phenylpyridinium (MPP<Superscript>+</Superscript>) Toxicity in C6 Glioma Cells

The neurotoxin 1-methy-4-phenylpyridinium (MPP⁺) is used for its’ capacity to induce Parkinsonism through its inhibitory effects on mitochondrial complex I. This inhibition disrupts cellular energy formation and aerobic glycolysis. The objective of this study was to demonstrate that the toxic effect of mitochondrial aerobic pathway inhibition with MPP⁺ can be reduced by stimulating anaerobic glycolysis using glucose supplementation. In this study, C6 Glioma cell viability was examined in the presence of different concentrations of MPP alone and with the addition of glucose. The results obtained indicate that there was a significant increase (P < 0.001) in cell viability in cells treated with glucose and MPP⁺ verses cells treated with MPP⁺ alone. Fluorometric analysis using 100 uM Rhodamine 123 indicated mitochondrial membrane potential was not restored in MPP⁺ treated cells with glucose; however, normal cell viability was confirmed using 2 ug/ml Fluorescein diacetate. This dual fluorescence indicated mitochondrial damage from MPP⁺ while glucose augmented cell survival. Further confirmation of cell survival upon damage to the mitochondria was evident in TUNEL staining. Positive staining was prominent only in MPP⁺ treatment groups alone, while control and co-treated groups exhibited little to no TUNEL staining. ATP measurements of all MPP⁺ treated groups exhibited a significant (P < 0.001) decrease verses control. Groups co-treated with MPP⁺ and glucose revealed a significant increase (250 μM group: P < 0.001) in ATP. It was concluded from this study that glucose supplementation was able to sustain cellular viability and ATP production through anaerobic glycolysis despite the inhibitory effect of MPP⁺ on aerobic glycolysis.     

β-Adrenergic Modulation of Glial Inwardly Rectifying Potassium Channels

Abstract: Cultured spinal cord astrocytes (2–13 days in vitro) express several different potassium current types, including delayed rectifier, transient A-type, and inward rectifier (K_ir) K⁺ currents. Of these, K_ir is believed to be of critical importance in the modulation of extracellular [K⁺] in the CNS. Using the whole-cell patch-clamp technique, we analyzed modulation of K_ir currents by β-adrenergic receptor activation. The selective β-adrenergic agonist isoproterenol (1–100 µM) and epinephrine (1–100 µM) each reduced peak K_ir current amplitudes to 52.7 ± 12.5 and 63.6 ± 7.0%, respectively, at 100 µM. Forskolin (K_D of ～25 µM), an activator of adenylate cyclase (AC), and dibutyryl-cyclic AMP (1 mM), a membrane-permeable analogue of cyclic AMP (cAMP), were each used to increase [cAMP]_i, the product of AC, and resulted in similar reductions of K_ir currents. By contrast, 1,9-dideoxyforskolin (1–50 µM), a forskolin analogue that does not activate AC, did not affect K_ir currents, indicating that AC activity is a required element for K_ir modulation. Three inhibitors of PKA—Rp-adenosine 3′,5′-cyclic monophosphothioate, H-7, and adenosine 3′,5′-cyclic monophosphate-dependent protein kinase inhibitor—failed to inhibit K_ir current reduction by β-adrenergic agonists. These results indicate that β-adrenergic receptor ligands can modulate K_ir currents and suggest that this modulation involves activation of AC but not protein kinase A. Such modulation may provide a mechanism by which neurons can modulate glial K_ir currents and thereby may affect glial K⁺“spatial buffering” in the CNS.     

Characterization and Distribution of a Cloned Rat μ-Opioid Receptor

Abstract: We have cloned and expressed a rat brain cDNA, TS11, that encodes a μ-opioid receptor based on pharmacological, physiological, and anatomical criteria. Membranes were prepared from COS-7 cells transiently expressing TS11 bound [³H]diprenorphine with high affinity (K_D = 0.23 ± 0.04 nM). The rank order potency of drugs competing with [³H]diprenorphine was as follows: levorphanol (K_i = 0.6 ± 0.2 nM) ≈β-endorphin (K_i = 0.7 ± 0.5 nM) ≈ morphine (K_i = 0.8 ± 0.5 nM) ≈ [d -Ala², N-Me-Phe⁴,Gly-ol⁵]-enkephalin (DAMGO; K_i = 1.6 ± 0.5 nM) ? U50,488 (K_i = 910 ± 0.78 nM) > [d -Pen^2,5]-enkephalin (K_i = 3,170 ± 98 nM) > dextrorphan (K_i = 4,100 ± 68 nM). The rank order potencies of these ligands, the stereospecificity of levorphanol, and morphine's subnanomolar K_i are consistent with a μ-opioid binding site. Two additional experiments provided evidence that this opioid-binding site is functionally coupled to G proteins: (a) In COS-7 cells 50 µM 5′-guanylylimidodiphosphate shifted a fraction of receptors with high affinity for DAMGO (IC₅₀ = 3.4 ± 0.5 nM) to a lower-affinity state (IC₅₀ = 89.0 ± 19.0 nM), and (b) exposure of Chinese hamster ovary cells stably expressing the cloned μ-opioid receptor to DAMGO resulted in a dose-dependent, naloxone-sensitive inhibition of forskolin-stimulated cyclic AMP production. The distribution of mRNA corresponding to the μ-opioid receptor encoded by TS11 was determined by in situ hybridization to brain sections prepared from adult female rats. The highest levels of μ-receptor mRNA were detected in the thalamus, medial habenula, and the caudate putamen; however, significant hybridization was also observed in many other brain regions, including the hypothalamus.