7-Nitroindazole (NI) is a widely used inhibitor of neuronal nitric-oxide synthase (nNOS) used to ... more 7-Nitroindazole (NI) is a widely used inhibitor of neuronal nitric-oxide synthase (nNOS) used to study the role of the neuronal NO pathway in the nervous system. 7-NI prevents convulsions, including 2-amino-4-methylphosphinobutyric acid (glufosinate)-induced convulsions, in experimental models. Herein, we examined nNOS involvement in glufosinate-induced convulsions and the specificity of 7-NI for nNOS. Another nNOS inhibitor, 1-[2-(trifluoromethyl)phenyl]imidazole (TRIM), inhibited NOS activity in vivo, and it prevented glufosinate-induced convulsions. In contrast, an endothelial NOS inhibitor, N 5-(1-iminoethyl)-L-ornithine, inhibited NOS activity in vivo, but it did not prevent the convulsions. These results suggest the involvement of nNOS in glufosinate-induced convulsions. However, a nonspe-cific NOS inhibitor, N-nitro-L-arginine methyl ester, inhibited NOS activity in vivo, but it failed to prevent glufosinate-induced convulsions. 6-NI and indazole, which did not inhibit NOS activity in vivo, suppressed glufosinate-induced convulsions. Moreover, glufosinate elicited convulsions in nNOS-deficient mice. These results suggest the anticonvulsant effects of 7-NI and TRIM on glufosinate-induced convulsions do not involve nNOS inhibition, instead possibly being related to an undefined property of nitrogen-containing chemical structures.
The authors previously demonstrated that 5HT recognition sites in rat aorta are coupled to a phos... more The authors previously demonstrated that 5HT recognition sites in rat aorta are coupled to a phosphoinositide (PI)-specific phospholipase C. Further, they showed that the mechanism of contraction elicited by 5HT is a complicated scenario involving receptor-mediated activation of calcium channels and a phospholipase C. They now report that in rat aorta, the 5HT-induced contraction and PI turnover are modulated by biologically active phorbol esters. In rat aorta the 5HT-induced contraction and PI hydrolysis (ECââ = 10 +/- 3 ..mu..M) were highly correlated (r = 0.95; p < 0.01). Also, the inhibitory potency of a variety of 5HTâ antagonists was correlated with binding to the brain 5HTâ receptor (r = 0.90; p < 0.05). Further, the tumor-promoting phorbol ester, phorbol dibutyrate (PDB) inhibited 5HT-induced PI turnover at low nM concentrations (ICââ approx. = to 30 nM), while the biologically inactive substance 4-..cap alpha..-phorbol was ineffective. Pretreatment of rat aortic rings with PDB at concentrations which desensitized 5HT-induced PI turnover also attenuated the aortic contraction induced by 5HT in the presence of a calcium channel blocker nitrendipine. Tge results suggest that phorbol esters desensitize 5HT receptor-mediated PI turnover and contraction, probably by activation of protein kinase C.
A peptide was partially purified from bovine forebrain which specifically inhibits the binding of... more A peptide was partially purified from bovine forebrain which specifically inhibits the binding of [3H]-ketanserin to the 5HT2 recognition site. The peptide has a MW of approximately 6,000 daltons and is partially destroyed by limited proteolysis. We suggest that the peptide is a candidate for an endacoid for the 5HT2 recognition site.
Journal of Pharmacology and Experimental Therapeutics
The mechanisms of 5-hydroxytryptamine (5-HT)-induced contraction of rat aorta were investigated i... more The mechanisms of 5-hydroxytryptamine (5-HT)-induced contraction of rat aorta were investigated in vitro. The 5-HT-induced contraction could be analyzed into two distinct components (phasic and tonic) by the use of appropriate inhibitors; nifedipine, an inhibitor of voltage-dependent Ca++ channels, inhibited only the phasic component of 5-HT-induced contraction while totally blocking the KCl-induced contraction. 2-Nitro-4-carboxyphenyl-N,N-diphenylcarbamate, an inhibitor of phospholipase C, inhibited the tonic components of 5-HT-induced contraction as well as the 5-HT-induced stimulation of phosphoinositide hydrolysis in rat aorta. This component of contraction was mimicked by a protein kinase C activator 12-O-tetradecanoylphorbol-13-acetate. These results suggest that 5-HT2 receptors differentially regulate a voltage-dependent Ca++ channel and phospholipase C activity; the voltage-dependent Ca++ channel is involved in the phasic component of contraction whereas the phosphoinositide hydrolysis that results in the activation of protein kinase C and calcium mobilization by inositol triphosphate plays a physiologically important role in the tonic component of the aortic contraction.
The effects of interleukin-1 alpha, tumor necrosis factor-alpha and dexamethasone on the inductio... more The effects of interleukin-1 alpha, tumor necrosis factor-alpha and dexamethasone on the induction of nitric oxide synthase mRNA in rat aortic smooth muscle cells were studied. Neither interleukin-1 alpha (up to 100 U/ml) nor tumor necrosis factor-alpha (up to 5000 U/ml) was capable of inducing nitrite/nitrate production and nitric oxide synthase mRNA in smooth muscle cells. In contrast, treatment for 12 hr or longer with a combination of the two synergistically induced nitrite/nitrate and cyclic GMP production in cell culture media and nitric oxide synthase mRNA, both of which were prevented by dexamethasone. Contamination with bacterial lipopolysaccharide, which may affect the induction of nitric oxide synthase, was below 30 pg/ml in all experiments. Our findings show that dexamethasone and these cytokines regulate the induction of nitric oxide synthase at the mRNA level in vascular smooth muscle cells.
Journal of Pharmacology and Experimental Therapeutics
Effects of various alpha adrenergic agents on insulin release and cyclic 3&#39;:5&#39;-ad... more Effects of various alpha adrenergic agents on insulin release and cyclic 3&#39;:5&#39;-adenosine monophosphate (cAMP) accumulation in pancreatic islets were investigated. Clonidine, epinephrine, alpha-methylnorepinephrine and norepinephrine were most potent and methoxamine and phenylephrine least potent in inhibiting the glucose-stimulated insulin release. Yohimbine and phentolamine were the most effective and prazosin was the least effective in antagonizing the epinephrine-inhibited insulin release. Clonidine markedly inhibited the glucagon-stimulated cAMP accumulation, whereas methoxamine showed weak inhibition. Yohimbine markedly increased cAMP accumulation in the presence of epinephrine, whereas prazosin showed little effect. The effects of alpha adrenergic agents on rabbit aorta contraction were also examined for comparison with alpha adrenergic receptors in pancreatic islets. In the aorta, the order of agonist potency was norepinephrine greater than phenylephrine greater than epinephrine greater than methoxamine greater than alpha-methylnorepinephrine and that of antagonist potency was prazosin greater than WB-4101 greater than phentolamine greater than dihydroergotamine greater than phenoxybenzamine greater than yohimbine. These orders of potencies were markedly different from those in pancreatic islets. These results clearly demonstrate that the alpha adrenergic receptors in rat pancreatic islets are different from those on rabbit aorta (alpha-1) and are typical postsynaptic alpha-2 adrenergic receptors.
Using a new method which combines a brain microdialysis technique and measurement of nitrite/nitr... more Using a new method which combines a brain microdialysis technique and measurement of nitrite/nitrate levels by the Griess reaction, it has been proven that activation of N-methyl-D-aspartate (NMDA) receptors in the cerebelli of rats which had been under non-anesthetic and freely moving conditions induces the release of nitric oxide (NO). Since L-NG-monomethylarginine (L-NMMA), which competitively blocks NO synthesis from L-arginine, significantly inhibited the release of nitrite/nitrate from the rat cerebellum, these results indicate that the new method is capable of measuring NO formation from L-arginine following the stimulation of NMDA receptors. This method should prove useful for investigating the relation between brain functions such as behavior, learning and memory and NO in the central nervous system.
Interleukin-1 augments release of norepinephrine, dopamine, and serotonin in the rat anterior hy... more Interleukin-1 augments release of norepinephrine, dopamine, and serotonin in the rat anterior hypothalamus. J Neurosci 13: 3574 -3581 36. Linthorst AC, Flachskamm C, Muller-Preuss P, Holsboer F, Reul JM 1995 Effect of bacterial endotoxin and interleukin-1 on hippocampal serotonergic neurotransmission, behavioral activity, and free corticosterone levels: an in vivo microdialysis study. J Neurosci 15:2920 -2934 37. Weidenfeld J, Abramsky O, Ovadia H 1989 Evidence for the involvement of the central nervous adrenergic system in interleukin-1-induced adrenocortical response.
The Journal of Neuroscience : The Official Journal of the Society for Neuroscience
We investigated the effects of interleukin-1 beta (IL-1 beta), administered directly into the rat... more We investigated the effects of interleukin-1 beta (IL-1 beta), administered directly into the rat anterior hypothalamus (AHY), on monoamine release in the same region by using a brain microdialysis technique and an HPLC-electrochemical detection system. First, to study the local effects of IL-1 beta, we used a microdialysis probe equipped with a microinjection tube for administering IL-1 beta in the same region into which the probe had been inserted. IL-1 beta (1 ng) injected directly into the AHY elicited release of norepinephrine (NE), dopamine (DA), and 5-HT, as well as increases in their metabolites, 4-hydroxy-3-methoxyphenylglycol, 3,4-dihydroxyphenylacetic acid, 4-hydroxy-3-methoxyphenylacetic acid, and 5-hydroxyindole-3-acetic acid, in the AHY. Vehicle alone exerted no effect on monoamine release. Although the elevated levels of NE and DA persisted for more than 6 hr after injection of IL-1 beta, the elevated levels of 5-HT were transient. Second, in order to investigate whet...
To investigate the interaction between endothelin (ET) and the nitric oxide system, we examined t... more To investigate the interaction between endothelin (ET) and the nitric oxide system, we examined the effects of ET-1 and ET-3 on the induction of inducible nitric oxide synthase (iNOS) and guanosine triphosphate cyclohydrolase I (GTP:CHI), the rate-limiting enzyme of de novo synthesis of the cofactor tetrahydrobiopterin (BH4), in rat mesangial cells. ET-1 inhibited the nitrite accumulation induced by a combination of interleukin-1 beta, tumor necrosis factor-alpha, and lipopolysaccharide in a concentration-dependent manner. The inhibitory effect of ET-3 was less potent than that of ET-1. A selective ETA antagonist, BQ-485, and an ETA and ETB antagonist, TAK-044, abolished the inhibitory effects of ET-1, whereas the selective ETB antagonist BQ-788 had no effect on the inhibition produced by ET-1. These observations indicate that ET-1 inhibits cytokine-stimulated nitrite accumulation through the ETA receptor. Western blot analysis showed that the suppression of nitrite accumulation was...
The nature of the pharmacodynamic interactions of drugs is influenced by the drugs׳ mechanisms of... more The nature of the pharmacodynamic interactions of drugs is influenced by the drugs׳ mechanisms of action. It has been hypothesized that drugs with different mechanisms are likely to interact synergistically, whereas those with similar mechanisms seem to produce additive interactions. In this review, we describe an extensive investigation of the published literature on drug combinations of anticonvulsants, the nature of the interaction of which has been evaluated by type I and II isobolographic analyses and the subthreshold method. The molecular targets of antiepileptic drugs (AEDs) include Na(+) and Ca(2+) channels, GABA type-A receptor, and glutamate receptors such as NMDA and AMPA/kainate receptors. The results of this review indicate that the nature of interactions evaluated by type I isobolographic analyses but not by the two other methods seems to be consistent with the above hypothesis. Type I isobolographic analyses may be used not only for evaluating drug combinations but al...
Glutathione (GSH) was discovered in yeast cells in 1888. Studies of GSH in mammalian cells before... more Glutathione (GSH) was discovered in yeast cells in 1888. Studies of GSH in mammalian cells before the 1980s focused exclusively on its function for the detoxication of xenobiotics or for drug metabolism in the liver, in which GSH is present at its highest concentration in the body. Increasing evidence has demonstrated other important roles of GSH in the brain, not only for the detoxication of xenobiotics but also for antioxidant defense and the regulation of intracellular redox homeostasis. GSH also regulates cell signaling, protein function, gene expression, and cell differentiation/proliferation in the brain. Clinically, inborn errors in GSH-related enzymes are very rare, but disorders of GSH metabolism are common in major neurodegenerative diseases showing GSH depletion and increased levels of oxidative stress in the brain. GSH depletion would precipitate oxidative damage in the brain, leading to neurodegenerative diseases. This review focuses on the significance of GSH function,...
Systemic administration of lipopolysaccharide (LPS), which causes endotoxemia and systemic inflam... more Systemic administration of lipopolysaccharide (LPS), which causes endotoxemia and systemic inflammation, has been reported to induce expression of the gene for type II inducible nitric oxide synthase (iNOS) in peripheral organs. This study was carried out to examine whether intraperitoneally injected LPS elicits the expression of iNOS messenger ribonucleic acid (mRNA) in the rat brain. We also investigated whether intraperitoneal treatment with dexamethasone (DEX) prevents this induction. To determine levels of iNOS mRNA, a quantitative reverse transcription-polymerase chain reaction (RT-PCR) method was employed. Treatment with LPS induced the expression of iNOS mRNA in various brain regions, accounting for approximately 1 x 10(5) to 4 x 10(5) molecules per micrograms of poly A+ RNA, and these inductions were markedly suppressed by DEX. The results suggest that, during systemic inflammation, iNOS mRNA induction occurs in brain through a DEX-sensitive mechanism.
Glutathione (GSH) is a tripeptide consisting of glutamate, cysteine, and glycine; it has a variet... more Glutathione (GSH) is a tripeptide consisting of glutamate, cysteine, and glycine; it has a variety of functions in the central nervous system. Brain GSH depletion is considered a preclinical sign in age-related neurodegenerative diseases, and it promotes the subsequent processes toward neurotoxicity. A neuroprotective mechanism accomplished by increasing GSH synthesis could be a promising approach in the treatment of neurodegenerative diseases. In neurons, cysteine is the rate-limiting substrate for GSH synthesis. Excitatory amino acid carrier 1 (EAAC1) is a neuronal cysteine/glutamate transporter in the brain. EAAC1 translocation to the plasma membrane promotes cysteine uptake, leading to GSH synthesis, while being negatively regulated by glutamate transport associated protein 3-18 (GTRAP3-18). Our recent studies have suggested GTRAP3-18 as an inhibitory factor for neuronal GSH synthesis. Inhibiting GTRAP3-18 function is an endogenous mechanism to increase neuron-specific GSH synthesis in the brain. This review gives an overview of EAAC1-mediated GSH synthesis, and its regulatory mechanisms by GTRAP3-18 in the brain, and a potential approach against neurodegeneration. compared to other organs . Under normal physiological conditions, antioxidant mechanisms function efficiently in the brain to overwhelm the lethal insults to neurons by reactive oxygen species (ROS). However, disequilibrium due to increased ROS production or decreased antioxidant defense systems would cause oxidative stress, which is considered a causal factor for neurodegeneration . Glutathione (GSH) is the most abundant thiol-containing molecule and one of the most important substances in the brain for neuroprotection. GSH is a tripeptide synthesized from glutamate, cysteine and glycine via two enzymatic steps. Previous studies focused on the enzymatic regulation of GSH synthesis, and the regulatory mechanisms for the neuronal transport system of the rate-limiting substrate, cysteine, have not been clarified. Our recent studies have helped reveal the regulatory mechanisms for cysteine uptake leading to neuronal GSH synthesis. In the present review, we provide an overview of the key molecular mechanisms underlying cysteine uptake leading to increase neuronal GSH content in the brain.
Stem cell factor (SCF) known as the c-kit ligand is a two disulfide bridge-containing cytokine in... more Stem cell factor (SCF) known as the c-kit ligand is a two disulfide bridge-containing cytokine in the regulation of the development and function of hematopoietic cell lineages and other cells such as mast cells, germ cells, and melanocytes. The secreted soluble form of SCF exists as noncovalently associated homodimer and exerts its activity by signaling through the c-Kit receptor. In this report, we present the high level expression of a soluble recombinant human SCF (rhSCF) in Escherichia coli. A codon-optimized Profinity eXact™-tagged hSCF cDNA was cloned into pET3b vector, and transformed into E. coli BL21(DE3) harboring a bacterial thioredoxin coexpression vector. The recombinant protein was purified via an affinity chromatography processed by cleavage with sodium fluoride, resulting in the complete proteolytic removal the N-terminal tag. Although almost none of the soluble fusion protein bound to the resin in standard protocol using 0.1M sodium phosphate buffer (pH 7.2), the use of binding buffer containing 0.5M l-arginine for protein stabilization dramatically enhanced binding to resin and recovery of the protein beyond expectation. Also pretreatment by Triton X-114 for removing endotoxin was effective for affinity chromatography. In chromatography performance, l-arginine was more effective than Triton X-114 treatment. Following Mono Q anion exchange chromatography, the target protein was isolated in high purity. The rhSCF protein specifically enhanced the viability of human myeloid leukemia cell line TF-1 and the proliferation and maturation of human mast cell line LAD2 cell. This novel protocol for the production of rhSCF is a simple, suitable, and efficient method.
Effects of intraluminal pressure on cultured intestinal epithelial cells were assessed by measuri... more Effects of intraluminal pressure on cultured intestinal epithelial cells were assessed by measuring cell proliferation and DNA synthesis after exposure to various pressures. Pressures of 40 to 120 mm Hg promoted cell proliferation and DNA synthesis. Pressure-induced DNA synthesis was further enhanced by the addition of interleukin-2, suggesting the regulation of intestinal epithelial growth by pressure in coordination with cytokines. Pretreatment with either a phospholipase C inhibitor or protein kinase C inhibitor significantly inhibited DNA synthesis promoted by pressure and interleukin-2. This study demonstrates a novel mechanism whereby pressure regulates cell growth in intestinal epithelial cells, possibly via activation of phospholipase C and protein kinase C.
Ursodeoxycholic acid (UDCA) has been used to treat patients with cholestatic and autoimmune liver... more Ursodeoxycholic acid (UDCA) has been used to treat patients with cholestatic and autoimmune liver diseases. Several studies have addressed whether UDCA can inhibit graft rejection in experimental and clinical transplantation, but the results have varied. We investigated the effect of UDCA and the mechanism of its effect on alloimmune responses in a murine model of cardiac transplantation. CBA mice underwent transplantation of a C57BL/10 heart and received a single dose of UDCA. Survival times of the allografts were recorded. An adoptive transfer study was conducted to determine whether regulatory cells were generated. The effects on graft survival of adding FK506 or cyclosporine A (CyA) to UDCA treatment were assessed. Histologic, cell proliferation, and cytokine assessments were performed. CBA recipients given UDCA (25 mg/kg) had indefinite allograft survival (median survival time [MST], >100 days). UDCA also suppressed proliferation of splenocytes and production of interleukin ...
7-Nitroindazole (NI) is a widely used inhibitor of neuronal nitric-oxide synthase (nNOS) used to ... more 7-Nitroindazole (NI) is a widely used inhibitor of neuronal nitric-oxide synthase (nNOS) used to study the role of the neuronal NO pathway in the nervous system. 7-NI prevents convulsions, including 2-amino-4-methylphosphinobutyric acid (glufosinate)-induced convulsions, in experimental models. Herein, we examined nNOS involvement in glufosinate-induced convulsions and the specificity of 7-NI for nNOS. Another nNOS inhibitor, 1-[2-(trifluoromethyl)phenyl]imidazole (TRIM), inhibited NOS activity in vivo, and it prevented glufosinate-induced convulsions. In contrast, an endothelial NOS inhibitor, N 5-(1-iminoethyl)-L-ornithine, inhibited NOS activity in vivo, but it did not prevent the convulsions. These results suggest the involvement of nNOS in glufosinate-induced convulsions. However, a nonspe-cific NOS inhibitor, N-nitro-L-arginine methyl ester, inhibited NOS activity in vivo, but it failed to prevent glufosinate-induced convulsions. 6-NI and indazole, which did not inhibit NOS activity in vivo, suppressed glufosinate-induced convulsions. Moreover, glufosinate elicited convulsions in nNOS-deficient mice. These results suggest the anticonvulsant effects of 7-NI and TRIM on glufosinate-induced convulsions do not involve nNOS inhibition, instead possibly being related to an undefined property of nitrogen-containing chemical structures.
The authors previously demonstrated that 5HT recognition sites in rat aorta are coupled to a phos... more The authors previously demonstrated that 5HT recognition sites in rat aorta are coupled to a phosphoinositide (PI)-specific phospholipase C. Further, they showed that the mechanism of contraction elicited by 5HT is a complicated scenario involving receptor-mediated activation of calcium channels and a phospholipase C. They now report that in rat aorta, the 5HT-induced contraction and PI turnover are modulated by biologically active phorbol esters. In rat aorta the 5HT-induced contraction and PI hydrolysis (ECââ = 10 +/- 3 ..mu..M) were highly correlated (r = 0.95; p < 0.01). Also, the inhibitory potency of a variety of 5HTâ antagonists was correlated with binding to the brain 5HTâ receptor (r = 0.90; p < 0.05). Further, the tumor-promoting phorbol ester, phorbol dibutyrate (PDB) inhibited 5HT-induced PI turnover at low nM concentrations (ICââ approx. = to 30 nM), while the biologically inactive substance 4-..cap alpha..-phorbol was ineffective. Pretreatment of rat aortic rings with PDB at concentrations which desensitized 5HT-induced PI turnover also attenuated the aortic contraction induced by 5HT in the presence of a calcium channel blocker nitrendipine. Tge results suggest that phorbol esters desensitize 5HT receptor-mediated PI turnover and contraction, probably by activation of protein kinase C.
A peptide was partially purified from bovine forebrain which specifically inhibits the binding of... more A peptide was partially purified from bovine forebrain which specifically inhibits the binding of [3H]-ketanserin to the 5HT2 recognition site. The peptide has a MW of approximately 6,000 daltons and is partially destroyed by limited proteolysis. We suggest that the peptide is a candidate for an endacoid for the 5HT2 recognition site.
Journal of Pharmacology and Experimental Therapeutics
The mechanisms of 5-hydroxytryptamine (5-HT)-induced contraction of rat aorta were investigated i... more The mechanisms of 5-hydroxytryptamine (5-HT)-induced contraction of rat aorta were investigated in vitro. The 5-HT-induced contraction could be analyzed into two distinct components (phasic and tonic) by the use of appropriate inhibitors; nifedipine, an inhibitor of voltage-dependent Ca++ channels, inhibited only the phasic component of 5-HT-induced contraction while totally blocking the KCl-induced contraction. 2-Nitro-4-carboxyphenyl-N,N-diphenylcarbamate, an inhibitor of phospholipase C, inhibited the tonic components of 5-HT-induced contraction as well as the 5-HT-induced stimulation of phosphoinositide hydrolysis in rat aorta. This component of contraction was mimicked by a protein kinase C activator 12-O-tetradecanoylphorbol-13-acetate. These results suggest that 5-HT2 receptors differentially regulate a voltage-dependent Ca++ channel and phospholipase C activity; the voltage-dependent Ca++ channel is involved in the phasic component of contraction whereas the phosphoinositide hydrolysis that results in the activation of protein kinase C and calcium mobilization by inositol triphosphate plays a physiologically important role in the tonic component of the aortic contraction.
The effects of interleukin-1 alpha, tumor necrosis factor-alpha and dexamethasone on the inductio... more The effects of interleukin-1 alpha, tumor necrosis factor-alpha and dexamethasone on the induction of nitric oxide synthase mRNA in rat aortic smooth muscle cells were studied. Neither interleukin-1 alpha (up to 100 U/ml) nor tumor necrosis factor-alpha (up to 5000 U/ml) was capable of inducing nitrite/nitrate production and nitric oxide synthase mRNA in smooth muscle cells. In contrast, treatment for 12 hr or longer with a combination of the two synergistically induced nitrite/nitrate and cyclic GMP production in cell culture media and nitric oxide synthase mRNA, both of which were prevented by dexamethasone. Contamination with bacterial lipopolysaccharide, which may affect the induction of nitric oxide synthase, was below 30 pg/ml in all experiments. Our findings show that dexamethasone and these cytokines regulate the induction of nitric oxide synthase at the mRNA level in vascular smooth muscle cells.
Journal of Pharmacology and Experimental Therapeutics
Effects of various alpha adrenergic agents on insulin release and cyclic 3&#39;:5&#39;-ad... more Effects of various alpha adrenergic agents on insulin release and cyclic 3&#39;:5&#39;-adenosine monophosphate (cAMP) accumulation in pancreatic islets were investigated. Clonidine, epinephrine, alpha-methylnorepinephrine and norepinephrine were most potent and methoxamine and phenylephrine least potent in inhibiting the glucose-stimulated insulin release. Yohimbine and phentolamine were the most effective and prazosin was the least effective in antagonizing the epinephrine-inhibited insulin release. Clonidine markedly inhibited the glucagon-stimulated cAMP accumulation, whereas methoxamine showed weak inhibition. Yohimbine markedly increased cAMP accumulation in the presence of epinephrine, whereas prazosin showed little effect. The effects of alpha adrenergic agents on rabbit aorta contraction were also examined for comparison with alpha adrenergic receptors in pancreatic islets. In the aorta, the order of agonist potency was norepinephrine greater than phenylephrine greater than epinephrine greater than methoxamine greater than alpha-methylnorepinephrine and that of antagonist potency was prazosin greater than WB-4101 greater than phentolamine greater than dihydroergotamine greater than phenoxybenzamine greater than yohimbine. These orders of potencies were markedly different from those in pancreatic islets. These results clearly demonstrate that the alpha adrenergic receptors in rat pancreatic islets are different from those on rabbit aorta (alpha-1) and are typical postsynaptic alpha-2 adrenergic receptors.
Using a new method which combines a brain microdialysis technique and measurement of nitrite/nitr... more Using a new method which combines a brain microdialysis technique and measurement of nitrite/nitrate levels by the Griess reaction, it has been proven that activation of N-methyl-D-aspartate (NMDA) receptors in the cerebelli of rats which had been under non-anesthetic and freely moving conditions induces the release of nitric oxide (NO). Since L-NG-monomethylarginine (L-NMMA), which competitively blocks NO synthesis from L-arginine, significantly inhibited the release of nitrite/nitrate from the rat cerebellum, these results indicate that the new method is capable of measuring NO formation from L-arginine following the stimulation of NMDA receptors. This method should prove useful for investigating the relation between brain functions such as behavior, learning and memory and NO in the central nervous system.
Interleukin-1 augments release of norepinephrine, dopamine, and serotonin in the rat anterior hy... more Interleukin-1 augments release of norepinephrine, dopamine, and serotonin in the rat anterior hypothalamus. J Neurosci 13: 3574 -3581 36. Linthorst AC, Flachskamm C, Muller-Preuss P, Holsboer F, Reul JM 1995 Effect of bacterial endotoxin and interleukin-1 on hippocampal serotonergic neurotransmission, behavioral activity, and free corticosterone levels: an in vivo microdialysis study. J Neurosci 15:2920 -2934 37. Weidenfeld J, Abramsky O, Ovadia H 1989 Evidence for the involvement of the central nervous adrenergic system in interleukin-1-induced adrenocortical response.
The Journal of Neuroscience : The Official Journal of the Society for Neuroscience
We investigated the effects of interleukin-1 beta (IL-1 beta), administered directly into the rat... more We investigated the effects of interleukin-1 beta (IL-1 beta), administered directly into the rat anterior hypothalamus (AHY), on monoamine release in the same region by using a brain microdialysis technique and an HPLC-electrochemical detection system. First, to study the local effects of IL-1 beta, we used a microdialysis probe equipped with a microinjection tube for administering IL-1 beta in the same region into which the probe had been inserted. IL-1 beta (1 ng) injected directly into the AHY elicited release of norepinephrine (NE), dopamine (DA), and 5-HT, as well as increases in their metabolites, 4-hydroxy-3-methoxyphenylglycol, 3,4-dihydroxyphenylacetic acid, 4-hydroxy-3-methoxyphenylacetic acid, and 5-hydroxyindole-3-acetic acid, in the AHY. Vehicle alone exerted no effect on monoamine release. Although the elevated levels of NE and DA persisted for more than 6 hr after injection of IL-1 beta, the elevated levels of 5-HT were transient. Second, in order to investigate whet...
To investigate the interaction between endothelin (ET) and the nitric oxide system, we examined t... more To investigate the interaction between endothelin (ET) and the nitric oxide system, we examined the effects of ET-1 and ET-3 on the induction of inducible nitric oxide synthase (iNOS) and guanosine triphosphate cyclohydrolase I (GTP:CHI), the rate-limiting enzyme of de novo synthesis of the cofactor tetrahydrobiopterin (BH4), in rat mesangial cells. ET-1 inhibited the nitrite accumulation induced by a combination of interleukin-1 beta, tumor necrosis factor-alpha, and lipopolysaccharide in a concentration-dependent manner. The inhibitory effect of ET-3 was less potent than that of ET-1. A selective ETA antagonist, BQ-485, and an ETA and ETB antagonist, TAK-044, abolished the inhibitory effects of ET-1, whereas the selective ETB antagonist BQ-788 had no effect on the inhibition produced by ET-1. These observations indicate that ET-1 inhibits cytokine-stimulated nitrite accumulation through the ETA receptor. Western blot analysis showed that the suppression of nitrite accumulation was...
The nature of the pharmacodynamic interactions of drugs is influenced by the drugs׳ mechanisms of... more The nature of the pharmacodynamic interactions of drugs is influenced by the drugs׳ mechanisms of action. It has been hypothesized that drugs with different mechanisms are likely to interact synergistically, whereas those with similar mechanisms seem to produce additive interactions. In this review, we describe an extensive investigation of the published literature on drug combinations of anticonvulsants, the nature of the interaction of which has been evaluated by type I and II isobolographic analyses and the subthreshold method. The molecular targets of antiepileptic drugs (AEDs) include Na(+) and Ca(2+) channels, GABA type-A receptor, and glutamate receptors such as NMDA and AMPA/kainate receptors. The results of this review indicate that the nature of interactions evaluated by type I isobolographic analyses but not by the two other methods seems to be consistent with the above hypothesis. Type I isobolographic analyses may be used not only for evaluating drug combinations but al...
Glutathione (GSH) was discovered in yeast cells in 1888. Studies of GSH in mammalian cells before... more Glutathione (GSH) was discovered in yeast cells in 1888. Studies of GSH in mammalian cells before the 1980s focused exclusively on its function for the detoxication of xenobiotics or for drug metabolism in the liver, in which GSH is present at its highest concentration in the body. Increasing evidence has demonstrated other important roles of GSH in the brain, not only for the detoxication of xenobiotics but also for antioxidant defense and the regulation of intracellular redox homeostasis. GSH also regulates cell signaling, protein function, gene expression, and cell differentiation/proliferation in the brain. Clinically, inborn errors in GSH-related enzymes are very rare, but disorders of GSH metabolism are common in major neurodegenerative diseases showing GSH depletion and increased levels of oxidative stress in the brain. GSH depletion would precipitate oxidative damage in the brain, leading to neurodegenerative diseases. This review focuses on the significance of GSH function,...
Systemic administration of lipopolysaccharide (LPS), which causes endotoxemia and systemic inflam... more Systemic administration of lipopolysaccharide (LPS), which causes endotoxemia and systemic inflammation, has been reported to induce expression of the gene for type II inducible nitric oxide synthase (iNOS) in peripheral organs. This study was carried out to examine whether intraperitoneally injected LPS elicits the expression of iNOS messenger ribonucleic acid (mRNA) in the rat brain. We also investigated whether intraperitoneal treatment with dexamethasone (DEX) prevents this induction. To determine levels of iNOS mRNA, a quantitative reverse transcription-polymerase chain reaction (RT-PCR) method was employed. Treatment with LPS induced the expression of iNOS mRNA in various brain regions, accounting for approximately 1 x 10(5) to 4 x 10(5) molecules per micrograms of poly A+ RNA, and these inductions were markedly suppressed by DEX. The results suggest that, during systemic inflammation, iNOS mRNA induction occurs in brain through a DEX-sensitive mechanism.
Glutathione (GSH) is a tripeptide consisting of glutamate, cysteine, and glycine; it has a variet... more Glutathione (GSH) is a tripeptide consisting of glutamate, cysteine, and glycine; it has a variety of functions in the central nervous system. Brain GSH depletion is considered a preclinical sign in age-related neurodegenerative diseases, and it promotes the subsequent processes toward neurotoxicity. A neuroprotective mechanism accomplished by increasing GSH synthesis could be a promising approach in the treatment of neurodegenerative diseases. In neurons, cysteine is the rate-limiting substrate for GSH synthesis. Excitatory amino acid carrier 1 (EAAC1) is a neuronal cysteine/glutamate transporter in the brain. EAAC1 translocation to the plasma membrane promotes cysteine uptake, leading to GSH synthesis, while being negatively regulated by glutamate transport associated protein 3-18 (GTRAP3-18). Our recent studies have suggested GTRAP3-18 as an inhibitory factor for neuronal GSH synthesis. Inhibiting GTRAP3-18 function is an endogenous mechanism to increase neuron-specific GSH synthesis in the brain. This review gives an overview of EAAC1-mediated GSH synthesis, and its regulatory mechanisms by GTRAP3-18 in the brain, and a potential approach against neurodegeneration. compared to other organs . Under normal physiological conditions, antioxidant mechanisms function efficiently in the brain to overwhelm the lethal insults to neurons by reactive oxygen species (ROS). However, disequilibrium due to increased ROS production or decreased antioxidant defense systems would cause oxidative stress, which is considered a causal factor for neurodegeneration . Glutathione (GSH) is the most abundant thiol-containing molecule and one of the most important substances in the brain for neuroprotection. GSH is a tripeptide synthesized from glutamate, cysteine and glycine via two enzymatic steps. Previous studies focused on the enzymatic regulation of GSH synthesis, and the regulatory mechanisms for the neuronal transport system of the rate-limiting substrate, cysteine, have not been clarified. Our recent studies have helped reveal the regulatory mechanisms for cysteine uptake leading to neuronal GSH synthesis. In the present review, we provide an overview of the key molecular mechanisms underlying cysteine uptake leading to increase neuronal GSH content in the brain.
Stem cell factor (SCF) known as the c-kit ligand is a two disulfide bridge-containing cytokine in... more Stem cell factor (SCF) known as the c-kit ligand is a two disulfide bridge-containing cytokine in the regulation of the development and function of hematopoietic cell lineages and other cells such as mast cells, germ cells, and melanocytes. The secreted soluble form of SCF exists as noncovalently associated homodimer and exerts its activity by signaling through the c-Kit receptor. In this report, we present the high level expression of a soluble recombinant human SCF (rhSCF) in Escherichia coli. A codon-optimized Profinity eXact™-tagged hSCF cDNA was cloned into pET3b vector, and transformed into E. coli BL21(DE3) harboring a bacterial thioredoxin coexpression vector. The recombinant protein was purified via an affinity chromatography processed by cleavage with sodium fluoride, resulting in the complete proteolytic removal the N-terminal tag. Although almost none of the soluble fusion protein bound to the resin in standard protocol using 0.1M sodium phosphate buffer (pH 7.2), the use of binding buffer containing 0.5M l-arginine for protein stabilization dramatically enhanced binding to resin and recovery of the protein beyond expectation. Also pretreatment by Triton X-114 for removing endotoxin was effective for affinity chromatography. In chromatography performance, l-arginine was more effective than Triton X-114 treatment. Following Mono Q anion exchange chromatography, the target protein was isolated in high purity. The rhSCF protein specifically enhanced the viability of human myeloid leukemia cell line TF-1 and the proliferation and maturation of human mast cell line LAD2 cell. This novel protocol for the production of rhSCF is a simple, suitable, and efficient method.
Effects of intraluminal pressure on cultured intestinal epithelial cells were assessed by measuri... more Effects of intraluminal pressure on cultured intestinal epithelial cells were assessed by measuring cell proliferation and DNA synthesis after exposure to various pressures. Pressures of 40 to 120 mm Hg promoted cell proliferation and DNA synthesis. Pressure-induced DNA synthesis was further enhanced by the addition of interleukin-2, suggesting the regulation of intestinal epithelial growth by pressure in coordination with cytokines. Pretreatment with either a phospholipase C inhibitor or protein kinase C inhibitor significantly inhibited DNA synthesis promoted by pressure and interleukin-2. This study demonstrates a novel mechanism whereby pressure regulates cell growth in intestinal epithelial cells, possibly via activation of phospholipase C and protein kinase C.
Ursodeoxycholic acid (UDCA) has been used to treat patients with cholestatic and autoimmune liver... more Ursodeoxycholic acid (UDCA) has been used to treat patients with cholestatic and autoimmune liver diseases. Several studies have addressed whether UDCA can inhibit graft rejection in experimental and clinical transplantation, but the results have varied. We investigated the effect of UDCA and the mechanism of its effect on alloimmune responses in a murine model of cardiac transplantation. CBA mice underwent transplantation of a C57BL/10 heart and received a single dose of UDCA. Survival times of the allografts were recorded. An adoptive transfer study was conducted to determine whether regulatory cells were generated. The effects on graft survival of adding FK506 or cyclosporine A (CyA) to UDCA treatment were assessed. Histologic, cell proliferation, and cytokine assessments were performed. CBA recipients given UDCA (25 mg/kg) had indefinite allograft survival (median survival time [MST], >100 days). UDCA also suppressed proliferation of splenocytes and production of interleukin ...
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Papers by Toshio Nakaki