Yumiko Komori

Role of nitric oxide in learning and memory and in monoamine metabolism in the rat brain.

1 We investigated the effects of NG‐nitro‐L‐arginine methyl ester (L‐NAME), an inhibitor of nitric oxide (NO) synthase, on the performance of rats in a radial arm maze and in habituation tasks, and on monoamine metabolism in the brain. 2 Daily administration of L‐NAME (10–60 mg kg−1) resulted in a dose‐dependent impairment of performance during the acquisition of the radial arm maze task, while it failed to affect performance in those rats that had previously acquired the task. 3 The rate of decrease in locomotor activity in the habituation task in the L‐NAME‐treated rats was significantly less than that in control rats. 4 NG‐nitro‐D‐arginine methyl ester (D‐NAME, a less active inhibitor of NO synthase) showed no effects in the above behavioural tasks. 5 NO synthase activity was significantly decreased in both the L‐NAME and D‐NAME‐treated rats, with the magnitude of inhibition being greater in the L‐NAME‐treated animals. 6 The content of 5‐hydroxyindoleacetic acid (5‐HIAA) in the hippocampus and the 5‐HIAA/5‐hydroxytryptamine ratio in the hippocampus and cortex were significantly decreased in the L‐NAME (60 mg kg)‐treated rats compared with these values in the controls. 7 Striatal 3,4‐dihydroxyphenylacetic acid (DOPAC) content was significantly increased in the L‐NAME (60 mg kg_1)‐treated rats compared with the values in the controls, while the DOPAC/dopamine ratio was not changed. 8 These results suggest that: (i) NO may play an important role in performance during the acquisition, but not retention, of the radial arm maze task, and (ii) that endogenous NO may be involved in the regulation of monoamine metabolism.

Role of nitric oxide and cyclic GMP in the dizocilpine-induced impairment of spontaneous alternation behavior in mice.

The activation of N-methyl-D-aspartate receptors induces the synthesis of nitric oxide, which activates soluble guanylate cyclase and leads to the formation of cyclic GMP in the brain. The inhibition of nitric oxide production, as well as the blockade of N-methyl-D-aspartate receptors, has been reported to prevent the induction of hippocampal long-term potentiation and learning and memory formation in vivo, although the effects of inhibitors of nitric oxide synthase are still controversial. We investigated the putative role of nitric oxide and cyclic GMP in dizocilpine-induced memory impairment in mice. The nitric oxide synthase inhibitors, NG-nitro-L-arginine methyl ester and 7-nitro indazole, as well as dizocilpine, a non-competitive N-methyl-D-aspartate receptor antagonist, dose-dependently impaired spatial working memory in mice, assessed by their spontaneous alternation behavior in a Y-maze. The inhibitory effects of both NG-nitro-L-arginine methyl ester and dizocilpine on their behavior were completely reversed by 8-bromo-cyclic GMP. Cyclic GMP levels in the cerebellum were reduced by treatment with dizocilpine. NG-Nitro-L-arginine methyl ester and 7-nitro indazole reduced cyclic GMP levels in the cerebral cortex/hippocampus and cerebellum, and the suppressive effect of NG-nitro-L-arginine methyl ester on cyclic GMP levels in the cerebral cortex/hippocampus was reversed by co-treatment with L-arginine. Cyclic AMP levels in the brain were not affected by treatment with either dizocilpine, NG-nitro-L-arginine methyl ester, or 7-nitro indazole. Neither NG-nitro-L-arginine methyl ester nor L-arginine had any effect on monoamine and acetylcholine metabolism in the brain. These results suggest that the reduction in nitric oxide/cyclic GMP production in the brain may be responsible for dizocilpine-induced impairment of spontaneous alternation behavior in a Y-maze.

Reduction in the number of NADPH-diaphorase-positive cells in the cerebral cortex and striatum in aged rats.

Nitric oxide (NO) plays an important role as a diffusible messenger in learning and memory. To determine whether changes in NO production in the brain may be involved in aging-associated brain dysfunction, we measured the performance of aged rats in a radial arm maze task, and carried out histochemical examination of the changes in NADPH diaphorase (NADPH-d)-containing neurons in the brains of aged rats. The performance of aged rats (30 months old) in a radial arm maze task was significantly impaired compared to the performance of young rats (3 months old). The number of neurons containing NADPH-d reactivity in the cerebral cortex and striatum of aged rats was significantly reduced, by approximately 50 and 30 percent, respectively, compared to that in young rats. NO synthase activity in discrete brain regions of aged rats, i.e., in the cerebral cortex, striatum and hippocampus was not different from that in young rats, although the activity in the cerebellum of aged rats was significantly lower than that in young rats. These results suggest that the reduction in the number of NADPH-d-positive cells in the brains of aged rats may be involved in aging-associated learning impairment in rats.

Brain dysfunction associated with an induction of nitric oxide synthase following an intracerebral injection of lipopolysaccharide in rats

We investigated the pathophysiological role of nitric oxide synthesized by inducible nitric oxide synthase in the brain, by injecting lipopolysaccharide directly into the rat cerebral cortex/hippocampus. The levels of nitric oxide metabolites, nitrite and nitrate, began to increase in a dose-dependent manner with a 3-h lag, and reached approximately seven-fold the basal levels 8 h after the direct injection of lipopolysaccharide (5 microg). The lipopolysaccharide-induced increase in nitrite and nitrate levels was inhibited by treatment with the specific inducible nitric oxide synthase inhibitor aminoguanidine. The protein synthesis inhibitor cycloheximide delayed the onset of the increase in nitric oxide metabolite levels, and reduced the peak levels. Lipopolysaccharide increased Ca2+-independent, but not Ca2+-dependent, nitric oxide synthase activity in the brain. Intense nicotinamide adenine dinucleotide phosphate-diaphorase activity was observed in round cells in the vicinity of the site of injection of lipopolysaccharide 8 h after the injection. Neuronal death was observed seven days after the injection of lipopolysaccharide. Spatial memory, as assessed by performance in a water maze task and spontaneous alternation behavior in a Y-maze, was significantly impaired in rats which had had previous bilateral injections of lipopolysaccharide into the hippocampus. The lipopolysaccharide-induced neuronal death and spatial memory impairments were prevented by aminoguanidine. These results suggest that direct injection of lipopolysaccharide into the brain causes an induction of inducible nitric oxide synthase in vivo. Furthermore, it is suggested that nitric oxide produced by inducible nitric oxide synthase is responsible for the lipopolysaccharide-induced brain dysfunction.

Role of nitric oxide in the development of tolerance and sensitization to behavioural effects of phencyclidine in mice

1 To determine whether nitric oxide (NO) was involved in tolerance and sensitization to the effects of phencyclidine (PCP), we examined NO synthase activity and the number of NADPH‐diaphorase (NADPH‐d)‐positive cells in discrete brain regions of saline‐, acute PCP‐ and repeated PCP‐treated mice. We also investigated the effects of a NO synthase inhibitor, NG‐nitro‐L‐arginine methyl ester (L‐NAME), on the behavioural changes induced by repeated PCP treatment in mice. 2 Acute PCP (1, 3, and 10 mg kg−1, s.c.) treatment induced dose‐dependent hyperlocomotion, motor incoordination and stereotyped behaviours, consisting of sniffing, head movement and ataxia in mice. 3 In mice treated repeatedly with PCP (1, 3, and 10 mg kg−1 day−1), s.c., once a day for 14 days), the sniffing, head movement, ataxia and motor incoordination induced by PCP were attenuated (indicating the development of tolerance to these behaviours), whereas the hyperlocomotion induced by PCP was potentiated (indicating the development of sensitization to hyperlocomotion). The development of tolerance and sensitization to PCP‐induced behaviours in the repeated PCP‐treated mice was more marked at the dose of 10 mg kg−1 day−1) than at other doses. 4 NO synthase activity in the cerebral cortex and cerebellum, but not in the striatum and hippocampus, was significantly decreased by acute PCP (10 mg kg−1) treatment in comparison with saline treatment, and such changes in activity in the cerebral cortex and cerebellum were reversed by repeated PCP treatment (10 mg kg−1 day−1). 5 The number of neurones containing NADPH‐d reactivity in the cerebral cortex, nucleus accumbens, and striatum of acute and repeated PCP‐treated mice showed no change in comparison with saline‐treated mice. 6 Tolerance to PCP (10 mg kg−1 day−1)‐induced ataxia and motor incoordination was significantly attenuated by combined treatment with L‐NAME (50 mg kg−1 day−1 i.p.). 7 Sensitization to PCP‐induced hyperlocomotion was further enhanced by combined treatment with L‐NAME (50 mg kg−1 day−1). However, NG‐nitro‐D‐arginine methyl ester (d‐NAME, 50 mg kg−1 day−1, i.p.), a less active enantiomer of L‐NAME, had no effect, suggesting a stereospecific mechanism. 8 The PCP‐induced behaviours in animals that had exhibited tolerance and sensitization to PCP (10 mg kg−1 day−1) were not influenced by acute L‐NAME (5 and 50 mg kg−1, i.p.) or D‐NAME (50 mg kg−1, i.p.) treatment. 9 These results suggest that NO may play an important role in the development, but not in the maintenance, of tolerance and sensitization to the effects of PCP in mice.

Hemorrhagic activity and muscle damaging effect of Pseudomonas aeruginosa metalloproteinase (elastase).

Pseudomonas aeruginosa elastase, a Bacillus subtilis thermolysin-like zinc-proteinase was examined for hemorrhagic activity and its effect on muscle and endothelial cells. Subcutaneous and intramuscular injections of elastase into mice caused severe hemorrhage with an acute increase of creatine phosphokinase activity in serum. The elastase also possessed fibrinogenolytic and fibrinolytic activities. The Aalpha and Bbeta chains of fibrinogen were completely hydrolyzed as demonstrated by their electrophoretic disappearance on SDS polyacrylamide gels. The pathological study indicates that elastase induces changes in the structure of the vascular wall and causes leakage of the plasma component and red and white blood cells into the extravascular tissue. This is further supported by results showing injury to cultured endothelial cells and macrophages. These data indicate that P. aeruginosa elastase directly affects endothelial cells and destroys the basement membrane of blood vessels to cause hemorrhage. Since fibrinogenolytic activity is an additional component of this elastase and this activity induces the hemorrhagic tendency, the damage in tissues could become increasingly severe.

Biochemical and physiological studies on a kallikrein-like enzyme from the venom of Crotalus viridis viridis (Prairie rattlesnake)

A kallikrein-like enzyme was isolated from Crotalus viridis viridis (Prairie rattlesnake) venom by Sephadex G-50, DEAE-Sephacel and heparin-Sepharose CL-6B column chromatography. The purified enzyme has a molecular mass of 32 kDa and an isoelectric point of 5.4. The enzyme catalyzed the hydrolysis of arginine esters, kallikrein substrates Pro-Phe-Arg-MCA and Z-Phe-Arg-MCA. The specificity of the enzymes substrate requirement is demonstrated by the fact that no proteolytic activity was detected against either dimethyl casein or fibrinogen. The enzyme also cleaves kininogen analogs to release bradykinin. Although the enzyme induced contraction of the isolated rat uterus directly at high concentrations, more forceful contractions resulted when the reaction mixture of the enzyme and bovine plasma was applied to the uterus. The reaction mixture of 5.10(-11) M of the enzyme and plasma caused contractions equal to that of 10(-9) M of bradykinin. Additionally the enzyme demonstrated capillary permeability-increasing activity and hypotensive activity on the anesthetized rat, suggesting that the enzyme releases the dilator of the wall of capillaries from plasma. Uterine contraction, capillary permeability-increasing activity and arginine esterolytic activity were inhibited by diisopropyl fluorophosphate, indicating that the serine hydroxyl group is essential for enzymatic and biological activities. It was demonstrated that the NH2-terminal region of the enzyme has significant similarities in sequence with kallikrein-like enzymes from other snake venoms and porcine pancreatic kallikrein.

Primary structure and biological activity of snake venom lectin (APL) from Agkistrodon p. piscivorus (Eastern cottonmouth)

A lectin (APL) was purified from the venom of Agkistrodon piscivorus piscivorus (Eastern cottonmouth moccasin). APL is a disulfide-linked, homodimeric protein consisting of identical monomers of molecular weight 16,200. Native rabbit and human erythrocytes were agglutinated by APL and the activity was found to be calcium-dependent. Galactose, lactose, rhamnose and EGTA strongly inhibited the hemagglutination activity of APL. The complete amino acid sequence determined by Edman sequencing of the S-pyridylethylated derivative and its peptides derived from enzymatic digestion indicate the structure of APL to be highly homologous with lectins and the platelet glycoprotein Ib (GPIb)-binding proteins isolated from other snake venoms. These results suggest that APL belongs to the C-type beta-galactoside binding lectin family which possess structural similarities with the C-terminal carbohydrate-recognition domain (CRD) of animal membrane lectins.

Effect of bilineobin, a thrombin-like proteinase from the venom of common cantil (Agkistrodon bilineatus)

A thrombin-like proteinase, named bilineobin, was isolated from Agkistrodon bilineatus venom by Sephadex G-75, DEAE-Sephacel and Heparin-Sepharose CL-6B column chromatography. The purified enzyme has a mol. wt of 57,000 and catalysed the hydrolysis of arginine esters and thrombin substrates Boc-Val-Pro-Arg-MCA and Boc-Asp(OBz)-Pro-Arg-MCA. Although bilineobin converted fibrinogen into fibrin resulting in the production of fibrinopeptides, the activity was relatively low (0.65 NIH units/mg). Fibrinopeptides released upon hydrolysis by this proteinase were identified as fibrinopeptide A (FpA) and fibrinopeptide B (FpB) by measuring fast atom bombardment (FAB) mass spectra and amino acid sequence. This indicates that bilineobin hydrolyses the Arg(19)-Gly(20) bond in the A alpha chain and the Arg(21)-Gly(22) bond in the B beta chain of the bovine fibrinogen molecule. Kinetic study of FpA and FpB release reveals that bilineobin has a preference for cleaving the B beta chain. In addition, bilineobin is resistant to thrombin inhibitors such as hirudin. These suggest that the mechanism of action of bilineobin is similar but not identical to that of thrombin. It was demonstrated that the NH2-terminal region of bilineobin has significant similarities in sequence with thrombin-like proteinases from other snake venoms; however, only three residues were common with thrombin up to residue number 24.

Proof of proteolytic activity of hemorrhagic toxins, HR-2a and HR-2b, from Trimeresurus flavoviridis venom

Two hemorrhagic toxins, HR-2a and HR-2b were originally isolated from Trimeresurus flavoviridis (Habu) venom by Takahashi and Ohsaka (1970). It was reported by the original investigators that no proteolytic activity was detected when casein was used as the substrate. HR-2a and HR-2b were isolated in this laboratory and their proteolytic activities were tested using a variety of different substrates and assay methods. HR-2a and HR-2b were found to contain, respectively, 200 and 219 amino acid residues. Toxicological and biochemical properties of HR-2a and HR-2b were further investigated and are reported in this paper.