The effects of thiol compounds and ebselen on nitric oxide activity in rat aortic vascular responses.

1. Thiols have been implicated to play a role in a variety of aspects of nitric oxide (NO) generation and activity. Thiol dependence of nitric oxide synthase (NOS) has remained controversial and its mechanism is not clear. This study investigates possible mechanisms between thiol (SH group) and NOS activation, through thiol compounds (glutathione, dithiothreitol, N-acetyl-L-cysteine) and Ebselen [2-phenyl-1,2-benzisoselenazole-3(2H)-one] on rat aortic vascular responses. 2. In rat thoracic aorta, acetylcholine (10(-6)-10(-9) M) induced a relaxation of phenylephrine (PE) (10(-7) M)-induced tone, which was inhibited dose dependently by increasing concentration of ebselen (1-10 microM). 3. In rings of rat thoracic aorta, ebselen and NOS inhibitors (NG-monomethyl-L-arginine, NG-nitro-L-arginine methyl ester) produced an augmentation of phenylephrine (10(-7) M)- induced tone and acetylcholine induced a relaxation of PE (10(-7) M)-induced tone in rat thoracic aorta, which was inhibited by ebselen (10 microM) like NOS inhibitor. 4. The thiol compounds (glutathione, dithiothreitol, and N-acetyl-L-cysteine) alone did not change vascular tone in rat thoracic aorta. Pretreatment with thiol compounds before ebselen treatment, however, reversed the inhibitory effect of ebselen which acts like the NOS inhibitor in rat thoracic aorta. Posttreatment with thiol compounds after ebselen treatment did not reverse the inhibitory effect of ebselen by as much as pretreatment. 5. Calcium ionophore A23187 (10(-7) M)-induced vasodilation was inhibited in ebselen pretreated rat thoracic aorta, but sodium nitroprusside (SNP, 10(-7) M)-induced relaxation was not inhibited by ebselen. This suggests that NOS is involved in the inhibitory effect of ebselen on rat thoracic aorta relaxation. 6. These results suggest that ebselen exerts an inhibitory action on the nitric oxide synthesis in rat thoracic aorta by interacting with thiol groups.

Effects of IY-81149, a newly developed proton pump inhibitor, on gastric acid secretion in vitro and in vivo.

The inhibitory effects of IY-81149 (2-[[(4-methoxy-3-methyl)-2- pyridinyl]methyl-sulfinyl]-5-(1H-pyrol-1-yl)-1H-benzimidazole, CAS 172152-36-2), a newly developed proton pump inhibitor (PPI) on gastric acid secretion were investigated in vitro and in vivo. In rabbit parietal cell preparation, IY-81149 irreversibly inhibited H+/K(+)-ATPase in dose-dependent manner with an IC50 of pump inhibitory activity of 6.0 x 10(-6) mol/l and that of omeprazole (CAS 73590-58-6) was 1 x 10(-4) mol/l at pH 7.4. On cumulation of 14C-aminopyrine in histamine stimulated parietal cells, the IC50 of IY-81149 was 9.0 x 10(-9) mol/l and that of omeprazole was 1.9 x 10(-8) mol/l. The inhibition rates of IY-81149 and omeprazole at a concentration of 1 x 10(-9) mol/l in human parietal cells were 137% and 64%, respectively. In pylorus-ligated rats, IY-81149 showed a 2-3 times stronger inhibitory activity than omeprazole against gastric acid secretion. The ED50 of IY-81149 and omeprazole administered intraduodenally was 1.6 mg/kg and 3.8 mg/kg. In the case of oral administration, the ED50 of IY-81149 and omeprazole was 1.94 mg/kg and 5.64 mg/kg, respectively. But after 24 h administration, the anti-secretory activity of IY-81149 was lower than that of omeprazole at all doses tested. In anesthetized rats, IY-81149 dose-dependently increased gastric pH which was lowered by histamine infusion. In the case of i.v. injection, the ED50 of IY-81149 and omeprazole was 1.2 and 1.4 mg/kg and in the case of i.d. administration, the ED50 of IY-81149 and omeprazole was 3.9 and 4.1 mg/kg, respectively. IY-81149 also significantly inhibited pentagastrin-stimulated gastric secretion. Its ED50 was 2.1 mg/kg and that of omeprazole was 3.5 mg/kg with i.d. administration. In the case of i.v. injection, IY-81149 was equipotent to omeprazole. IY-81149 also inhibited gastric acid secretion strongly in fistular rats. The ED50 of IY-81149 administered intraduodenally was 0.43 mg/kg and that of omeprazole was 0.68 mg/kg. In Heidenhain pouch dogs, the acid output was completely blocked at 0.3 mg/kg, 135 min after i.v. administration. Omeprazole showed a similar effect as IY-81149. The histamine induced increase of acid output in the Heidenhain pouch dog was blocked by 71% 150 min after oral administration of enteric-coated IY-81149 at a dose of 3 mg/kg, and omeprazole showed similar effects. In conclusion, IY-81149 revealed the characteristics as a strong proton pump inhibitor, and its potency against gastric acid secretion was superior to that of the reference drug, omeprazole.

NMDA receptor and NO mediate ET-1-induced behavioral and cardiovascular effects in periaqueductal gray matter of rats.

Endothelin-1 (ET-1), a novel and potent vasoconstrictor in blood vessel, is known to have some functions in the rat central nervous system (CNS). In order to investigate the central functions of ET-1, ET-1 was administered to the periaqueductal gray area (PAG) of anesthetized rats to induce barrel rolling and increase the arterial blood pressure (ABP). ET-1 had a modulatory effect on central cardiovascular and behavioral control. The selective N-methyl-D-aspartate (NMDA) receptor antagonist MK-801 (3 micromol/kg, i.p.) blocked the ET-1 induced responses, and both the nitric oxide synthase (NOS) inhibitor L-NAME (N-nitro-L-arginine methylester 1 mmol/rat) and the nitric oxide (NO) scavenger hemoglobin (15 nmol/rat) had similar effects in reducing the ET-1 (10 pmol/rat)-induced behavioral changes and ABP elevation. However, NO donor sodium nitroprusside (SNP 10 microg, 1 microg/rat) decreased the ET-1 induced ABP elevation, and recovered the ET-1-induced barrel rolling effect that was reduced by MK-801. These results suggest that ET-1 might have neuromodulatory functions such as ABP elevation and barrel rolling induction in the PAG of the rats via the NMDA receptor and NO.

The comparison of vitamin C and vitamin E on the protein oxidation of diabetic rats.

1 We measured the plasma glucose and the glycosylated haemoglobin at the time of sacrifice in streptozotocin-induced diabetic mellitus (DM) rats. 2 In diabetic rats, plasma glucose and glycosylated haemoglobin was increased as compared with normal rats, and vitamin E inhibited the increase of glycosylated haemoglobin level but vitamin C had no effect. 3 The peroxidized proteins and lipids from the diabetic organs such as liver or kidney were measured to assess the oxidative damage. The 2,4-dinitrophenyl-hydrazine (DNPH) incorporation method was used to measure the peroxidized protein. In diabetic rats, DNPH incorporation was increased as compared with normal rats and vitamin E also inhibited the increase of DNPH incorporation but vitamin C had no effect. It suggests that the protein oxidation occurred on the liver in diabetic rats and the oxidative stress is general in the diabetic condition. 4 We measured the systolic arterial pressure and mean arterial pressure in normal rats, nephrectomy (NEPH)-rats, diabetic rats (DM), and NEPH-diabetic rats (NEPH-DM). Blood pressure was significantly increased in DM and NEPH-DM as compared with normal rats. 5 In conclusion, plasma glucose, glycosylated haemoglobin, and the oxidation of proteins or lipid were increased in diabetic rats. Vitamin E decreased the plasma glucose, glycosylated haemoglobin and the oxidation of proteins and lipid, but vitamin C had no effects.

The influences of extremely low frequency magnetic fields on clonidine-induced sleep in 2-day-old chicks.

1. It has been shown that magnetic fields (MFs) affect a variety of biological effects in animal brains. There have been few experiments on the effects of MFs on sleep. Therefore, we investigated whether extremely low frequency (ELF) MFs affect the sleep induced by clonidine, a central alpha(2)-adrenoceptor agonist. Clonidine produced dose-related increase of the sleeping time and dose-related decrease of the onset time in 2-day-old chicks. 2. Exposure of chicks to MFs (5, 10, 20 G; for 3, 6, 9, 12 h) significantly increased the clonidine-induced sleep time as a direct function of intensity and duration of MF application. Clonidine reduced noradrenaline or tyrosine in the brain, an effect which was not further changed in animals exposed to MF. 3. To determine whether the gamma amino butyric acid A (GABA(A))/benzodiazepine (BZD) receptor system is involved in the decrease in clonidine-induced sleep caused by activation of central alpha(2)-adrenergic systems, we examined exposure of chicks to the effects of the BZD receptor antagonist flumazenil (0.5 mg kg(-1), i.p.) and GABA(A) antagonist bicuculline (0.1 mg kg-1, i.p.) on clonidine-induced sleep. Bicuculline and flumazenil inhibited the increase of clonidine-induced sleep time by MFs. Clonidine or MFs did not change GABA levels in the brain. 4. These results suggest that MFs can increase clonidine-induced sleep via a change of GABA(A) and BZD receptor system irrespective of the concentration of GABA or noradrenaline in the brain of 2-day-old chicks.

Endothelium-dependent sensory non-adrenergic non-cholinergic vasodilatation in rat thoracic aorta: involvement of ATP and a role for NO.

The involvement of non-adrenergic non-cholinergic (NANC) transmitters, such as adenosine 5'-triphosphate (ATP) and nitric oxide (NO), in the neurogenic relaxation of rat thoracic aorta was investigated in vessel segments suspended for isometric tension recording by polygraph. Responses to electrical field stimulation (EFS) and exogenous vasodilator were investigated in vessels precontracted with 5-hydroxytryptamine. EFS (100 V, 2-16 Hz, for 10 s at 3-min intervals), in the presence of guanethidine (10 microM) and atropine (10 microM) produced frequency-dependent relaxations. Pretreatment with tetrodotoxin (1 microM) markedly reduced the relaxation and desensitization with capsaicin (10 microM) significantly inhibited the relaxation. Exogenously added ATP caused concentration-dependent relaxations. Mechanical removal of the endothelium significantly inhibited EFS- and ATP-induced relaxation by 30+/-3% and 37+/-2%, respectively. Pretreatment with a P1-purinoceptor antagonist, 8-phenyltheophylline (10 microM) or P2X-purinoceptor antagonist, Evans blue (10 microM) did not influence the relaxations to EFS and exogenously added ATP. In contrast, the P2Y-purinoceptor antagonist, basilen blue (100 microM) markedly reduced the relaxations to EFS by 52+/-4% in the endothelium-intact preparations. However, in the endothelium-denuded preparations and capsaicin-pretreated preparations, basilen blue did not change relaxations elicited by EFS. The NO synthase inhibitor, N(G)-nitro-L-arginine methyl ester (L-NAME, 100 microM) also significantly inhibited the relaxations to EFS and ATP by 40+/-6% and 30+/-2%, respectively, in the endothelium-intact preparations but had no effect on the relaxations in the endothelium-denuded preparations or capsaicin-pretreated preparations. In addition, the EFS-induced relaxations were also inhibited 43+/-7% by pretreatment with 1H-[1,2,4]-oxadiazolo[4,3-alpha]quinoxalin-1-one (ODQ; 1 microM), soluble guanylate cyclase inhibitor. This study suggests that the NANC nerve system is present in the thoracic aorta of rat, mediating vasodilatation by sensory nerves. ATP, as a neurotransmitter released from sensory nerves, activates P2Y-purinoceptors located on the endothelium and stimulates the NO/cyclic GMP pathway, resulting in vasodilatation.

The role of nitric oxide and prostaglandin E2 on the hyperalgesia induced by excitatory amino acids in rats.

The present study was designed to investigate the role of nitric oxide (NO), N-methyl-D-aspartate (NMDA) receptor and prostaglandins on hyperalgesia induced in rats by excitatory amino acids and the possibility that prostaglandins may act as the retrograde messenger in the spinal cord like NO. Nomega-nitro-L-arginine methyl ester (L-NAME; 500 microg/paw, intraplantarly (i.pl.)), MK-801 (10 microg/paw, i.pl.) or indomethacin (300 microg/paw, i.pl.) reduced the duration of phase 2 of the biting/licking and scratching (B/L + S) response induced by formalin injection from 255.6+/-16.7 s to 155.6+/-16.9, 172.25+/-33.3 or 205.6+/-16.7 s, respectively. L-NAME (0.3 mg, i.th.), MK-801 (8 microg, i.th.) or indomethacin (20 microg, i.th) reduced the duration of phase 2 of the B/L + S response induced by saline injection from 288.5+/-7.7s to 207.7+/-19.2, 184.6+/-7.7 or 1923+/-38.5 s, respectively. L-NAME or indomethacin injected into the spinal cord of the rat significantly reduced the hyperalgesia induced by NMDA (1 microg, i.th.) from 43.8+/-4.6% to 12.3+/-3.1 and 19.2+/-2.3%, respectively. It is assumed that NO produced by excitatory amino acids may increase prostaglandin production by cyclooxygenase activation. L-NAME, MK-801 or indomethacin injected into the rat spinal cord significantly reduced the hyperalgesia induced by prostaglandin E2 (PGE2, 25 ng, i.th.) in the tail-flick test from 40.6+/-3.5% to 18.2+/-3.2, 18.8+/-1.8 or 17.6+/-4.1%, respectively, but had little effect on hyperalgesia in the paw pressure test (except for indomethacin). In conclusion, NO and PGE2 affect the hyperalgesia induced by excitatory amino acids. It is suggested that PGE2, like NO, may act as a retrograde messenger in the spinal cord.

Increase of [Ca(2+)]i and release of arachidonic acid via activation of M2 receptor coupled to Gi and rho proteins in oesophageal muscle.

We have previously shown that acetylcholine-induced contraction of oesophageal circular muscle depends on activation of phosphatidylcholine selective phospholipase C and D, which result in formation of diacylglycerol, and of phospholipase 2 which produces arachidonic acid. Diacylglycerol and arachidonic acid interact synergistically to activate protein kinase C. We have therefore investigated the relationship between cytosolic Ca(2+) and activation of phospholipase A(2) in response to acetylcholine-induced stimulation, by measuring the intracellular free Ca(2+) ([Ca(2+)]i), muscle tension, and [3H] arachidonic acid release. Acetylcholine-induced contraction was associated with increased [Ca(2+)]i and arachidonic acid release in a dose-dependent manner. In Ca(2+)-free medium, acetylcholine did not produce contraction, [Ca(2+)]i increase, and arachidonic acid release. In contrast, after depletion of Ca(2+) stores by thapsigargin (3 microM), acetylcholine caused a normal contraction, [Ca(2+)]i increase and arachidonic acid release. The increase in [Ca(2+)]i and arachidonic acid release were attenuated by the M2 receptor antagonist methoctramine, but not by the M3 receptor antagonist p-fluoro-hexahydro siladifenidol. Increase in [Ca(2+)]i and arachidonic acid release by acetylcholine were inhibited by pertussis toxin and C3 toxin. These findings indicate that contraction and arachidonic acid release are mediated through muscarinic M2 coupled to Gi or rho protein activation and Ca(2+) influx. Acetylcholine-induced contraction and the associated increase in [Ca(2+)]i and release of arachidonic acid were completely reduced by the combination treatment with a phospholipase A(2) inhibitor dimethyleicosadienoic acid and a phospholipase D inhibitor pCMB. They increased by the action of the inhibitor of diacylglycerol kinase R59949, whereas they decreased by a protein kinase C inhibitor chelerythrine. These data suggest that in oesophageal circular muscle acetylcholine-induced [Ca(2+)]i increase and arachidonic acid release are mediated through activation of M2 receptor coupled to Gi or rho protein, resulting in the activation of phospholipase A(2) and phospholipase D to activate protein kinase C.

The mechanism of contraction by 2-chloroadenosine in cat detrusor muscle cells.

PURPOSE: Four adenosine receptors (ARs), designated A1AR (A1 adenosine receptor), A2aAR (A2a adenosine receptor), A2bAR (A2b adenosine receptor), and A3AR (A3 adenosine receptor), have been cloned from various species, but the contraction mechanism via A1ARs in cat detrusor muscle cell is not well known. MATERIALS AND METHODS: We examined the cellular mechanism using an A1AR agonist 2-chloroadenosine (2-CA) in cat detrusor cell isolated by enzymatic digestion. To examine which phospholipase mediates the contraction, we used phospholipase inhibitors. RESULTS: The adenosine analog potency order is R-N6-phenylisopropyladenosine (R-PIA) > 5'-N-ethylcarbosamine adenosine (NECA) > 2-chloroadenosine (2-CA) > S-N6-phenylisopropyladenosine (S-PIA). The ratio of equi-effective concentrations of R-PIA/S-PIA was 58.2. 8-cyclopentyl-1,3-dipropylxanthine (DPCPX, 300 nM) shifted to the right the concentration-response curves of 2-CA. These results indicate A1ARs mediate 2-CA induced contraction in cat detrusor muscle. G proteins (Gi1, Gi2, Gi3, Go, Gs, and Gq) in cat detrusor muscle were detected by immunoblot analysis. Pertussis toxin (PTX) inhibited 2-CA induced contraction. In permeabilized cells, antibodies against Galphai3 antagonized 2-CA induced contraction, suggesting that the contraction is mediated by Gi3 protein. A phosphatidylinositol-specific phospholipase C (PLC) inhibitor, neomycin, reduced 2-CA induced contraction, but a phospholipase D (PLD) inhibitor, p-chloromercuribenzoic acid, and a phospholipase A2 (PLA2) inhibitor, dimethyl-eicosa-dienoic acid (DEDA), had no effect. We found the presence of the main PLC isozymes, PLC-beta1, PLC-beta3, and PLC-gamma1. 2-CA induced contraction in permeabilized cells was inhibited by PLC-beta3 but not by PLC-beta1 or PLC-gamma1 antibody. These results imply that A1ARs are coupled to PLC-beta3 via PTX-sensitive Gi3 protein. Sr2+ medium and thapsigargin, which replaces intracellular Ca2+ and deplete intracellular calcium stores respectively, inhibited 2-CA induced contraction. CONCLUSIONS: These results suggest that A1ARs mediating 2-CA induced contraction exist in cat detrusor muscle and the contraction depends on a PTX-sensitive Gi3 protein, PLC-beta3 and the release of intracellular Ca2+.

Effects of extremely low frequency magnetic fields on pain thresholds in mice: roles of melatonin and opioids.

1. We studied the effects of extremely low frequency (ELF, 60 Hz) magnetic fields (MFs) on pain thresholds using the hot plate test. The implication of opioid and benzodiazepine system in the MFs-induced alteration of pain thresholds was also studied. 2. There was an increase at night time and a decrease at daytime of pain thresholds in normal mice. Exposure of MFs (24 h, 20 gauss (G)) inhibited the increase of pain thresholds at night time and even produced hyperalgesia at daytime. 3. The increase of pain thresholds induced by melatonin at daytime was inhibited by exposure to MFs (24 h, 20 G) or opioid antagonist naloxone. The MFs and naloxone synergically inhibited hypoalgesia produced by melatonin. The hyperalgesia at daytime after MFs exposure was potentiated by the benzodiazepine agonist, diazepam, and inhibited by the benzodiazepine antagonist, flumazenil. There was no significant difference in all rotarod performance we tested. 4. From these results, it is suggested that exposure to MFs inhibits the increase of pain thresholds at night time and produces hyperalgesia at daytime with the involvement of opioid and benzodiazepine systems.

Comparison of IY81149 with omeprazole in rat reflux oesophagitis.

1. This study was aimed at evaluating the effects of IY81149[2-[[(4methoxy-3-methyl)-2-pyridinyl]methylsulfinyl]-5-(1H-pyrrol-1-yl)-1H-benzimidazole], a new proton pump inhibitor, on the development of the surgically induced reflux oesophagitis, on gastric secretion and on lipid peroxidation which is a marker of oxidative stress. Omeprazole was used as a reference drug. We furthermore investigated the influence of quercetin and desferrioxamine (DFO) on the development of the surgically induced reflux oesophagitis in rats on gastric secretion and on lipid peroxidation. 2. IY81149 and omeprazole significantly prevented the development of reflux oesophagitis and gastric secretion in a dose-dependent manner. The ED50 values of IY81149 for inhibition of oesophagitis and volume of gastric secretion were lower than of omeprazole (5.7 vs. 14.2 micromol, 15.3 vs. 24.0 micromol, respectively). IY81149 was also more potent in the acid output inhibition with an ED50 of 6.8 micromol compared with 20.8 micromol of omeprazole. 3. Malonyldialdehyde (MDA) content, the end product of lipid peroxidation, increased significantly in the oesophageal mucosa after the induction of reflux oesophagitis. IY81149 and omeprazole significantly and dose-dependently prevented lipid peroxidation. Quercetin (200 mg kg-1, p.o.) and DFO (800 mg kg-1, i.d.) significantly prevented the development of reflux oesophagitis and inhibited the lipid peroxidation independent of their actions on gastric secretion. 4. This result suggests that IY81149 is comparable with omeprazole in the treatment of reflux oesophagitis.

Antigenotoxicity of galangin against N-methyl-N-nitrosourea.

Using the Ames bacterial mutagenicity test and an in vivo micronucleus test, we investigated the antigenotoxic effect of galangin against the genotoxicity of N-methyl-N-nitrosourea (MNU). In the Ames assay, galangin showed an antimutagenic effect towards MNU-induced mutagenicity of Salmonella typhimurium TA 100. In mice, galangin showed an anticlastogenic effect against MNU-induced micronuclei in polychromatic erythrocytes in the MNPCE in mouse bone marrow cells. On the other hand, galangin is neither mutagenic nor clastogenic in both assays. Results from our in vitro and in vivo studies indicate that galangin is capable of suppressing the mutagenicity and clastogenicity of MNU. Therefore, galangin may be a useful chemopreventive agent against potential long-term health effects from genotoxic environmental agents.