Toshihiko Murayama

Changes in expressions of proinflammatory cytokines IL-1β, TNF-α and IL-6 in the brain of senescence accelerated mouse (SAM) P8

The senescence-accelerated mouse (SAM) is known to be a murine model for accelerated aging. The SAMP8 strain shows age-related deterioration of learning and memory at an earlier age than control mice (SAMR1). In the present study, we investigated the changes in expressions of interleukin-1beta (IL-1beta), tumor necrosis factor-alpha (TNF-alpha) and interleukin-6 (IL-6) in the brain of SAMP8. In the hippocampus of 10 months old SAMP8, the expression of IL-1 mRNA was significantly elevated in comparison with that of SAMR1. In both strains of SAMs, increases in IL-1beta protein in the brain were observed at 10 months of age compared with 2 and 5 months. The only differences found between the strain in protein levels were at 10 months and were elevations in IL-1beta in the hippocampus and hypothalamus, and in TNF-alpha and IL-6 in the cerebral cortex and the hippocampus in SAMP8 as compared with SAMR1. However, lipopolysaccharide-induced increases in the expression of these cytokines in brain did not differ between SAMP8 and SAMR1. Increases in expression of proinflammatory cytokines in the brain may be involved in the age-related neural dysfunction and/or learning deficiency in SAMP8.

Guanine nucleotide activation and inhibition of adenylate cyclase as modified by islet-activating protein, pertussis toxin, in mouse 3T3 fibroblasts

Guanine nucleotide regulation of membrane adenylate cyclase activity was uniquely modified after exposure of 3T3 mouse fibroblasts to low concentrations of islet-activating protein (IAP), pertussis toxin. The action of IAP, which occurred after a lag time, was durable and irreversible, and was associated with ADP-ribosylation of a membrane Mr = 41,000 protein. GTP, but not Gpp(NH)p, was more efficient and persistent in activating adenylate cyclase in membranes from IAP-treated cells than membranes from control cells. GTP and Gpp(NH)p caused marked inhibition of adenylate cyclase when the enzyme system was converted to its highly activated state by cholera toxin treatment or fluoride addition, presumably as a result of their interaction with the specific binding protein which is responsible for inhibition of adenylate cyclase. This inhibition was totally abolished by IAP treatment of cells, making it very likely that IAP preferentially modulates GTP inhibitory responses, thereby increasing GTP-dependent activation and negating GTP-mediated inhibition of adenylate cyclase.

Involvement of L-Type-Like Amino Acid Transporters in S-Nitrosocysteine-Stimulated Noradrenaline Release in the Rat Hippocampus

Abstract: Nitrogen oxides, such as nitric oxide, have been shown to regulate neuronal functions, including neurotransmitter release. We investigated the effect of S‐nitroso‐l‐cysteine (SNC) on noradrenaline (NA) release in the rat hippocampus in vivo and in vitro. SNC stimulated [3H]NA release from prelabeled hippocampal slices in a dose‐dependent manner. SNC stimulated endogenous NA release within 30 min to almost five times the basal level in vivo (microdialysis in freely moving rats). In a Na+‐containing Tyrodes buffer, SNC‐stimulated [3H]NA release was inhibited 30% by the coaddition of l‐leucine. In the Na+‐free, choline‐containing buffer, SNC‐stimulated [3H]NA release, which was similar to that in the Na+‐containing buffer, was inhibited markedly by l‐leucine, l‐alanine, l‐methionine, l‐phenylalanine, and l‐tyrosine. The effects of the other amino acids examined were smaller or very limited. The effect of l‐leucine was stronger than that of d‐leucine. A specific inhibitor of the L‐type amino acid transporter, 2‐aminobicyclo[2.2.1]‐heptane‐2‐carboxylate (BCH), inhibited the effects of SNC on [3H]NA release in the Na+‐free buffer. Uptake of l‐[3H]leucine into the slices in the Na+‐free buffer was inhibited by SNC, BCH, and l‐phenylalanine, but not by l‐lysine. The effect of SNC on cyclic GMP accumulation was not inhibited by l‐leucine, although SNC stimulated cyclic GMP accumulation at concentrations up to 25 µM, much less than the concentration that stimulates NA release. These findings suggest that SNC is incorporated into rat hippocampus via the L‐type‐like amino acid transporter, at least in Na+‐free conditions, and that SNC stimulates NA release in vivo and in vitro in a cyclic GMP‐independent manner.

NO donors stimulate noradrenaline release from rat hippocampus in a calmodulin-dependent manner in the presence of L-cysteine

Nitrogen oxides (NO) such as nitric oxide have been suggested to potentiate neurotransmitter release in a variety of neuronal cells. In this study, we showed that NO donors stimulate the release of noradrenaline (NA) from rat hippocampus both in vivo and in vitro. Co‐addition of NO donors (sodium nitroprusside [SNP] or S‐nitroso‐N‐acetylpenicillamine [SNAP]) and thiol compounds (dithiothreitol [DTT] or L‐cysteine) stimulated [3H]NA release from prelabeled hippocampal slices. Microdialysis in freely moving rats was used to ascertain the role of NO in control of NA release from the hippocampus in vivo. Co‐addition of SNAP and L‐cysteine stimulated endogenous NA release within 30 min. The concentration of NA peaked between 30–60 min to almost 3 times basal level. Another thiol compound, glutathione, had no effect on [3H]NA release in the presence of SNP or SNAP. In the presence of SNAP, the effect of L‐cysteine was much higher than that of the D‐isomer, although SNAP did not show stereospecificity. The effect of SNAP/L‐cysteine was rapid and the maximal increase in [3H]NA release was attained 0–1 min after application, which was similar in time course to the effect of KCl. Unlike the release by KCl, SNAP/L‐cysteine‐stimulated NA release was independent of extracellular CaCl2. However, pretreatment with the calmodulin antagonists W‐7 or trifluoperazine significantly reduced the SNAP/L‐cysteine‐stimulated [3H]NA release. Formation of nitric oxide and activation of guanylate cyclase by nitric oxide were not responsible for SNAP/L‐cysteine‐stimulated NA release. These findings suggest that NO donors stimulate NA release from the hippocampus in the presence of thiol compounds such as L‐cysteine in vivo and in vitro in a calmodulin‐dependent, Ca2+‐ and cyclic GMP‐independent manner. The physiological roles of thiol compounds such as L‐cysteine or glutathione as intermediates of NO are discussed.

Inhibition of hydrogen peroxide-induced apoptosis but not arachidonic acid release in GH3 cell by EGF.

Reactive oxygen species (ROS) and arachidonic acid (AA) can both function as extra- and intra-cellular messengers to regulate various cell functions including cell death. The effect of ROS on phospholipase A2 (PLA2) activity and/or AA release has not been extensively studied in neuronal cells. In this study, we investigated the effects of H2O2 on AA release and apoptosis in GH3 cells, a clonal strain from rat anterior pituitary. Incubation with H2O2 for 1 h stimulated [3H]AA release in a concentration-dependent manner from prelabeled GH3 cells. [3H]AA release was inhibited by arachidonyl trifluoromethyl ketone, a specific inhibitor of cytosolic PLA2, and cytosolic PLA2 protein with a molecular mass of 100 kDa was detected by immunoblotting. Culture with 0.2 mM H2O2 and 30 microM AA for 24 h induced lactate dehydrogenase (LDH) leakage, DNA laddering and DNA fragmentation in GH3 cells. In GH3 cells pretreated with EGF (50 ng/ml) for 24 h, LDH leakage and DNA fragmentation by H2O2 and AA were inhibited, although H2O2-induced [3H]AA release was not modified. Mastoparan, a wasp venom peptide, induced [3H]AA release and cell death in GH3 cells. Neither effect of mastoparan was inhibited by EGF treatment. These findings suggest that (1) H2O2 stimulates AA release via activation of cytosolic PLA2, (2) H2O2 and AA induce apoptotic death of GH3 cells and (3) treatment with EGF protects H2O2- and AA-, but not mastoparan-, induced GH3 cell death.

Inhibition of Inducible Nitric Oxide Synthase Expression by Endothelin in Rat Glial Cells Prepared from the Neonatal Rat Brain

Abstract: In primary cultured rat glial cells, a combination of inflammatory cytokines such as tumor necrosis factor‐α (TNF‐α) and interleukin‐1β (IL‐1β) stimulates production of nitrite via expression of the inducible form of nitric oxide synthase (iNOS). In these cells, simultaneous addition of endothelin (ET) decreased iNOS expression and nitrite accumulation induced by TNF‐α/IL‐1β. The inhibitory effect of ET on TNF‐α/IL‐1β‐stimulated iNOS expression appears to be mediated by ETB receptors, because (1) both ET‐1 and ET‐3 inhibited the effects of TNF‐α/IL‐1β on iNOS expression and nitrite accumulation, (2) a selective ETB receptor agonist, Suc‐[Glu9,Ala11,15]‐ET‐1 (8–21) (IRL1620), decreased the effects of TNF‐α/IL‐1β, and (3) a selective ETB receptor antagonist, N‐cis‐2,6‐dimethylpiperidinocarbonyl‐l‐γ‐methylleucyl‐d‐1‐methoxycarbonyltryptophanyl‐d‐norleucine, abolished the inhibitory effects of ETs and IRL1620. Incubation of glial cells with lipopolysaccharide (LPS) caused an increase in iNOS expression. Simultaneous addition of ET‐3 decreased the effects of LPS (10 and 100 ng/ml) on iNOS expression. Furthermore, cyclic AMP‐elevating agents (dibutyryl cyclic AMP and forskolin) inhibited TNF‐α/IL‐1β‐induced and LPS‐induced iNOS expression and nitrite accumulation. These findings suggest that ETs can decrease TNF‐α/IL‐1β‐induced and LPS‐induced iNOS expression via ETB receptors and that cyclic AMP may be involved in this process.

Sodium nitroprusside stimulates noradrenaline release from rat hippocampal slices in the presence of dithiothreitol.

It is becoming apparent that nitrogen monoxide (NO) such as nitric oxide has regulatory roles for neuronal cell functions. We examined the role of NO using NO donors on [3H]noradrenaline (NA) release from prelabeled rat hippocampal slices. Sodium nitroprusside (SNP), which had no effect by itself, stimulated [3H]NA release in a dose-dependent manner (ED50 = 0.5 mM) in the presence of dithiothreitol (DTT). The stimulatory effect of SNP with DTT, but not high K+, was observed in an extracellular Ca(2+)-free buffer. The maximal effect of SNP was obtained after incubation for 1-2 h with DTT in buffer at physiological pH (7.4). The simultaneous addition of SNP and DTT to the slices induced a small effect, and the effect of SNP declined after 3.5 h. SNP stimulated cyclic GMP accumulation in the slices without DTT. NaNO2 and 1-hydroxy-2-oxo-3,3-bis(2-aminoethyl)-1-triazene (a generator of nitric oxide), which stimulated cyclic GMP accumulation by themselves, did not stimulate [3H]NA release in the presence and absence of DTT. 3-Morpholinosydnonimine HC1 (a generator of peroxynitrite) had no effect on the release. The stimulatory effect of SNP and DTT on NA release was inhibited 40% by nitric oxide scavengers such as oxyhemoglobin and 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxy-3-oxide, although cyclic GMP accumulation induced by NO donors was completely inhibited. These findings suggest that SNP reacts with DTT to produce unknown active species, and that cyclic GMP is not a mediator for SNP-stimulated NA release.

Cell death by 1-chloro-2,4-dinitrobenzene, an inhibitor of thioredoxin reductase and its dual regulation by nitric oxide in rats

Thioredoxin (Trx), the oxidized form of which is converted to a reduced form by Trx reductase, regulates cell proliferation and survival. We investigated the effect of 1-chloro-2,4-dinitrobenzene (DNCB), an inhibitor of Trx reductase, on cell death in neuronal PC12 cells and rat glial cells. In both types of cells, culture with DNCB for 4 h stimulated lactate dehydrogenase (LDH) leakage. LDH leakage by DNCB was inhibited by an inhibitor of caspases. Addition of 2,2-(hydroxynitrosohydrazino)bis-ethanamine, of which 50% decays and releases nitric oxide (NO) in 21 h, inhibited DNCB-induced LDH leakage. Addition of 10 microM N-ethyl-2-(1-ethyl-2-hydroxy-2-nitrosohydrazino)-ethanamine (NOC-12), of which 50% decays and releases NO in 100 min, inhibited DNCB-induced LDH leakage, although 0.2 mM NOC-12 enhanced the leakage in PC12 cells. These findings suggest that DNCB induces cell death of neuronal and glial cells accompanied by caspase(s) activation. NO inhibits DNCB-induced cell death in both types of cells, although excess NO showed a toxic effect.

Possible involvement of cytosolic phospholipase A2 in cell death induced by 1-methyl-4-phenylpyridinium ion, a dopaminergic neurotoxin, in GH3 cells

Previously we reported that 1-methyl-4-phenylpyridinium ion (MPP(+)), a dopaminergic neurotoxin, induced apoptosis of GH3 cells established from rat anterior pituitary. In the present study, the role of MPP(+) along with that of other apoptotic factors such as Ca(2+) and H(2)O(2) in cell death was examined. Ionomycin induced DNA fragmentation and lactate dehydrogenase (LDH) leakage in GH3 cells. H(2)O(2) also induced LDH leakage. Co-addition of MPP(+), in conditions where MPP(+) had no effect by itself, enhanced ionomycin- and H(2)O(2)-induced cell death. Because the stimulation of phospholipase A(2) (PLA(2)) causing arachidonic acid (AA) release has been proposed to be involved in neuronal cell death, the effect of MPP(+) on AA release in GH3 cells was investigated. MPP(+) treatment for 8 h enhanced ionomycin- and H(2)O(2)-stimulated AA release mediated by activation of cytosolic PLA(2) in a concentration-dependent manner, although MPP(+) by itself had no effect on AA release. An inhibitor of cytosolic PLA(2) inhibited MPP(+)-induced cell death. These findings suggest a synergistic effect of MPP(+) on Ca(2+)- and H(2)O(2)-induced cell death, and the involvement of cytosolic PLA(2) activation in MPP(+)-induced cell death in GH3 cells. Pretreatment with a caspase inhibitor or EGF did not modify the ionomycin- or H(2)O(2)-induced AA release, or enhancement by MPP(+), but the pretreatment inhibited the cell death in the presence and absence of MPP(+). The involvement of caspase(s) on activation of PLA(2) by MPP(+) was excluded, and EGF inhibited MPP(+)-induced cell death downstream of the AA release.

Endothelin enhances lipopolysaccharide-induced expression of inducible nitric oxide synthase in rat glial cells

Lipopolysaccharide is known to stimulate production of nitrite via expression of inducible nitric oxide (NO) synthase in not only macrophages but also glial cells. We found that in glial cell cultures lipopolysaccharide-stimulated inducible NO synthase expression and nitrite accumulation were synergistically enhanced by pretreatment with endothelin, whereas endothelin itself did not induce these responses. Pretreatment with endothelin-1, endothelin-3, and the selective endothelin type B (ETB) receptor agonist IRL 1620 caused the same effect with similar potencies, suggesting that the synergism was mediated via the endothelin ETB receptor. A protein kinase C inhibitor, calphostin C, suppressed endothelin-3-enhanced inducible NO synthase expression. Pretreatment with either endothelin-3 or phorbol ester enhanced lipopolysaccharide-induced production of tumor necrosis factor-alpha (TNF-alpha). Simultaneous addition of TNF-alpha increased lipopolysaccharide-stimulated inducible NO synthase expression. These results suggest that the increase in inducible NO synthase expression by endothelin was due to the elevated TNF-alpha production via protein kinase C. Our findings present the possibility that endothelin is implicated in neurotoxicity via enhancement of inducible NO synthase expression.