Reiko Ishii-Nozawa

Participation of cAMP in the facilitatory action of β,γ-Methylene ATP on the noradrenaline release from rabbit ear artery

Abstract β,γ-Methylene ATP (βγ-mATP) significantly facilitated the electrically (4 Hz) evoked release of noradrenaline (NA) from the rabbit ear artery by activation of prejunctional purinoceptors on the sympathetic nerve terminals. In the present study, we investigated whether intracellular cAMP is involved in the purinoceptor mediated facilitatory mechanisms. Forskolin, an adenylate cyclase activator, and 8-bromo cAMP, a cAMP analogue, significantly enhanced the NA-release. The enhancement of NA-release by βγ-mATP was significantly potentiated by Ro20-1724, a phosphodiesterase inhibitor, but abolished by SQ22536, an adenylate cyclase inhibitor. Both drugs alone had no effect on the NA-release. N-ethylmaleimide and pertussis toxin, inhibitors of Gi-proteins, did not affect the NA-release, or the enhancement of NA-release by βγ-mATP. Alone Cholera toxin (CTX), an activator of Gs-proteins, significantly increased the NA-release, but in the presence of CTX, βγ-mATP could not produce further enhancement of the NA-release. These results suggest that cAMP is closely associated with the facilitatory action of βγ-mATP on NA-release in the rabbit ear artery.

BmDJ-1 Is a Key Regulator of Oxidative Modification in the Development of the Silkworm, Bombyx mori

We cloned cDNA for the Bombyx mori DJ-1 protein (BmDJ-1) from the brains of larvae. BmDJ-1 is composed of 190 amino acids and encoded by 672 nucleotides. Northern blot analysis showed that BmDJ-1 is transcribed as a 756-bp mRNA and has one isoform. Reverse transcriptase (RT)-PCR experiments revealed that the BmDJ-1 was present in the brain, fatbody, Malpighian tubule, ovary and testis but present in only low amounts in the silkgland and hemocyte of day 4 fifth instar larvae. Immunological analysis demonstrated the presence of BmDJ-1 in the brain, midgut, fatbody, Malpighian tubule, testis and ovary from the larvae to the adult. We found that BmDJ-1 has a unique expression pattern through the fifth instar larval to adult developmental stage. We assessed the anti-oxidative function of BmDJ-1 using rotenone (ROT) in day 3 fifth instar larvae. Administration of ROT to day 3 fifth instar larvae, together with exogenous (BmNPV-BmDJ-1 infection for 4 days in advance) BmDJ-1, produced significantly lower 24-h mortality in BmDJ-1 groups than in the control. 2D-PAGE revealed an isoelectric point (pI) shift to an acidic form for BmDJ-1 in BmN4 cells upon ROT stimulus. Among the factors examined for their effects on expression level of BmDJ-1 in the hemolymph, nitric oxide (NO) concentration was identified based on dramatic developmental stage-dependent changes. Administration of isosorbide dinitrate (ISDN), which is an NO donor, to BmN4 cells produced increased expression of BmDJ-1 compared to the control. These results suggest that BmDJ-1 might control oxidative stress in the cell due to NO and serves as a development modulation factor in B. mori.

Size of side-chain at channel pore mouth affects Ca2+ block of P2X2 receptor

Effects of amino acid replacement at the channel pore mouth of P2X(2) receptor/channel on multivalent cation channel block were investigated. When Asn(333) was replaced with various amino acid residues with neutral side chains (Gly, Ala, Val, Leu and Ile), the block by Ca(2+) was attenuated according to the sizes of the side chains. The block by La(3+) was also greatest with the Gly-substituted mutant, but this preference was not found for the block by other multivalent cations tested. The side chain at the channel pore mouth may interfere with the access of Ca(2+) block by steric hindrance.

Intracellular disulfide bond that affects ATP responsiveness of P2X2 receptor/channel

The role of intracellular cysteine residues in P2X(2) receptor/channel was investigated. When dithiothreitol was intracellularly applied, both the maximal response and the sensitivity of the wild-type channel to ATP were decreased. On the other hand, Cu(2+) phenanthroline did not affect the responsiveness. When two intracellular cysteine residues (Cys(9) and Cys(430)) were replaced with alanine, both the maximal response and the sensitivity was decreased with the replacement at Cys(9), whereas no such decrease was observed with the replacement at Cys(430). These results suggest that an intracellular disulfide bond involving Cys(9) regulates the responsiveness of P2X(2) receptor/channel to ATP.

Effect of denuded endothelial cells on arginine amidase activity released from rabbit arteries

1. We examined the secretion of arginine amidase activity from rabbit aorta and ear arteries.

Analysis of factors associated with hiccups based on the Japanese Adverse Drug Event Report database

Hiccups are occasionally experienced by most individuals. Although hiccups are not life-threatening, they may lead to a decline in quality of life. Previous studies showed that hiccups may occur as an adverse effect of certain medicines during chemotherapy. Furthermore, a male dominance in hiccups has been reported. However, due to the limited number of studies conducted on this phenomenon, debate still surrounds the few factors influencing hiccups. The present study aimed to investigate the influence of medicines and patient characteristics on hiccups using a large-sized adverse drug event report database and, specifically, the Japanese Adverse Drug Event Report (JADER) database. Cases of adverse effects associated with medications were extracted from JADER, and Fisher’s exact test was performed to assess the presence or absence of hiccups for each medication. In a multivariate analysis, we conducted a multiple logistic regression analysis using medication and patient characteristic variables exhibiting significance. We also examined the role of dexamethasone in inducing hiccups during chemotherapy. Medicines associated with hiccups included dexamethasone, levofolinate, fluorouracil, oxaliplatin, carboplatin, and irinotecan. Patient characteristics associated with hiccups included a male gender and greater height. The combination of anti-cancer agent and dexamethasone use was noted in more than 95% of patients in the dexamethasone-use group. Hiccups also occurred in patients in the anti-cancer agent-use group who did not use dexamethasone. Most of the medications that induce hiccups are used in chemotherapy. The results of the present study suggest that it is possible to predict a high risk of hiccups using patient characteristics. We confirmed that dexamethasone was the drug that has the strongest influence on the induction of hiccups. However, the influence of anti-cancer agents on the induction of hiccups cannot be denied. We consider the results of the present study to be helpful for the prevention and treatment of hiccups.

Inhibition of Ectopic Arginine Vasopressin Production by Phenytoin in the Small Cell Lung Cancer Cell Line Lu-165

Phenytoin, a voltage-gated sodium channel (NaV channel) antagonist, reportedly inhibits arginine vasopressin (AVP) release from an isolated rat neurohypophysis. So far, it is uncertain whether phenytoin has a direct action on ectopic AVP-producing neuroendocrine tumors. We studied the effect of phenytoin on the release of copeptin, the C-terminal fragment of pro-AVP, and expression of AVP gene in the human small cell lung cancer cell line Lu-165. Cells were maintained in RPMI1640 medium with 10% fetal bovine serum and were used within the fifth passage. Copeptin was detected using a new sandwich immunoassay, and AVP mRNA levels were measured using real-time reverse transcription polymerase chain reaction. Treatment with phenytoin at a concentration of 25 µg/mL, but not at 5 or 10 µg/mL, had an inhibitory effect on copeptin levels in the medium at 48 h. At the same concentration, AVP mRNA levels in Lu-165 cells also decreased. Although a sodium challenge with added sodium at 20 mEq/L increased copeptin levels in the medium, a sodium challenge with added sodium at 10 and 20 mEq/L had no effect on AVP mRNA levels. Phenytoin at a concentration of 25 µg/mL suppressed copeptin levels in the medium under the sodium challenge with added sodium at 10 and 20 mEq/L. Phenytoin at a concentration of 25 µg/mL also decreased AVP mRNA levels in Lu-165 cells under the sodium challenge with added sodium at 10 mEq/L, but not at 20 mEq/L. Among five tested NaV channel subunits, NaV1.3 was highly expressed in Lu-165 cells. However, phenytoin significantly decreased NaV1.3 mRNA levels under the sodium challenge with added sodium at 10 and 20 mEq/L. These results suggest that Lu-165 cells are sensitive to phenytoin and sodium to control of AVP release and its gene expression. Phenytoin might have a direct action on ectopic AVP-producing tumors, suggesting the importance of NaV channels in AVP-producing neuroendocrine tumors.

Influence of Obesity Gene on Pharmacotherapy for Lifestyle-related Vascular Disease

The development of risk factors, such as obesity, for lifestyle-related vascular disease is heterogeneous. In this regard, recent studies have shown that mutation of the β3-adrenoceptor gene, involving replacement of tryptophan by arginine at position 64 (Trp 64 Arg) of encoding amino acid residues (obesity gene), was associated with increased susceptibility to obesity and insulin resistance, and affected obesity treatment in humans. In the present study, we tested for the presence of the obesity gene and examined its influence on pharmacotherapy for lifestyle-related vascular disease.Our study investigated 92 persons receiving medication (depressors, lipid-lowering drugs, oral agents for diabetes; medication group) for a lifestyle-related illness among 2453 persons who consented to β3-adrenoceptor gene determination. There was no difference in the prevalence of the obesity gene between those receiving medicine and those not receiving any. In the medication group, there were no significant differences in the average T-C, TG, LDL-C, RLP-C, glucose, HbAic and BMI measurements among subjects who had the obesity gene. Also, when the minimal cholesterol target following therapy was set to 220 mg or less, this was achieved in all subjects without the obesity gene (wild group). In subjects with the obesity gene, there were many cases in which RLP-C was over 8 mg/dL.These results suggest that the obesity gene would enhance resistance to pharmacotherapy intended to improve lipid levels in the treatment of lifestyle-related vascular disease.