Multiple Transport Mechanisms Involved in the Intestinal Absorption of Metformin: Impact on the Nonlinear Absorption Kinetics.

The aim of this study was to investigate the contributions of multiple transport mechanisms to the intestinal absorption of metformin, focusing on OCT3, PMAT, THTR2, SERT and OCTN2. We also assessed the impact of these transporters on the nonlinear absorption of metformin. Uptake studies with MDCKII cells expressing OCT3, PMAT, THTR2 or SERT confirmed that metformin is a substrate of these transporters. Decynium22 strongly inhibited metformin uptake mediated by all the transporters. 7-Cyclopentyl inhibited OCT3- and THTR2-mediated uptake of metformin. AG835, thiamine and paroxetine specifically inhibited PMAT-, THTR2- and SERT-mediated uptake of metformin, respectively. Using these inhibitors, the relative contributions of OCT3, PMAT, THTR2, SERT, OCTN2 and others to the intestinal permeation of metformin across Caco-2 cells were estimated to be 9.77%, 9.68%, 22.2%, 1.52%, 0% and 0.66%, respectively. Concentration-dependent analysis of metformin uptake by Caco-2 cells revealed nonlinear kinetics with the similar Km(app) value to the value for THTR2. Further in situ absorption study demonstrated that rat intestinal permeability of metformin was significantly decreased in the presence of decynium22, 7-cyclopentyl and thiamine. The present study indicated that THTR2 is the major determinant of the nonlinear absorption of metformin, although multiple transport mechanisms contribute to its intestinal absorption.

NNC 55-0396, a T-type calcium channel blocker, protects against the brain injury induced by middle cerebral artery occlusion and reperfusion in mice.

We tested whether NNC 55-0396 (NNC), a T-type calcium channel (T-channel) blocker, reduces the brain injury caused by middle cerebral artery occlusion and reperfusion (MCAO/R) in mice. NNC, administered i.c.v. before the occlusion, greatly reduced the MCAO/R-induced brain infarct and neurological dysfunctions, although it, given toward the end of occlusion, was less effective. Systemic administration of NNC before the occlusion also attenuated the infarct and neurological dysfunctions. Our data imply that blood-brain-barrier-permeable T-channel blockers such as NNC are capable of reducing MCAO/R-induced brain damage, and that T-channels are involved in neuronal damage induced by ischemia rather than reperfusion.

Involvement of ERK in NMDA receptor-independent cortical neurotoxicity of hydrogen sulfide.

Hydrogen sulfide (H(2)S), a gasotransmitter, exerts both neurotoxicity and neuroprotection, and targets multiple molecules including NMDA receptors, T-type calcium channels and NO synthase (NOS) that might affect neuronal viability. Here, we determined and characterized effects of NaHS, an H(2)S donor, on cell viability in the primary cultures of mouse fetal cortical neurons. NaHS caused neuronal death, as assessed by LDH release and trypan blue staining, but did not significantly reduce the glutamate toxicity. The neurotoxicity of NaHS was resistant to inhibitors of NMDA receptors, T-type calcium channels and NOS, and was blocked by inhibitors of MEK, but not JNK, p38 MAP kinase, PKC and Src. NaHS caused prompt phosphorylation of ERK and upregulation of Bad, followed by translocation of Bax to mitochondria and release of mitochondrial cytochrome c, leading to the nuclear condensation/fragmentation. These effects of NaHS were suppressed by the MEK inhibitor. Our data suggest that the NMDA receptor-independent neurotoxicity of H(2)S involves activation of the MEK/ERK pathway and some apoptotic mechanisms.

[Comparative study of oral tracheal tube extubation force in a baby manikin model: taping methods and tube diameters].

BACKGROUND: Adhesive tape is the standard method for securing tracheal tubes. There have been several studies about extubation forces in adult models. However, there have been few in pediatric models. Therefore, we examined the force required to extubate tracheal tube from a manikin using three methods. METHODS: A baby simulation manikin was orally intubated. The tracheal tube (3.0 to 4.5 mm internal diameter) was fixed at the right oral corner. The tracheal tube was secured with adhesive tape with three different methods as follows: 1) 0.63 cm-wide tape coiled around the tube twice, 2) 1.25 cm-wide tape coiled around the tube twice, 3) 1.25 cm-wide tape coiled up three times around the tube ("puttee style"). Adhesive tape was attached 10 cm on both sides of the tube along the lips. The tracheal tube was connected to a force-measuring device and pulled vertically until the tube was displaced 1 cm distally from the initial position. The required force was defined as the "extubation force". Each method was repeated 5 times. RESULTS: The extubation force was larger when the tape used was wider, and the contact area between the tracheal tube and tape was larger. The force was also larger as the tube diameter became larger. CONCLUSIONS: To fix a tracheal tube securely, adhesive tape should be wider and the contact area between the tube and the tape should be larger. In this regard, "puttee style" fixation seems to be effective.

Hydrogen sulfide inhibits activity of three isoforms of recombinant nitric oxide synthase.

To clarify the presence of cross-talk between H(2)S and NO, we investigated effect of NaHS, an H(2)S donor, on activity of recombinant NO synthase (NOS) isoforms. Activity of all nNOS, iNOS and eNOS was inhibited by NaHS (IC(50): 0.13-0.21 mM). In contrast, Na(2)SO(3), L-cysteine and threo-1,4-dimercapto-2,3-butanediol, a reductant, exerted poor inhibition of NOS activity. Increasing concentrations of tetrahydrobiopterin (BH(4)) reversed the NaHS inhibition of nNOS and eNOS, but not iNOS. Our data thus demonstrate inhibition of three NOS isoforms by NaHS/H(2)S, and suggest involvement of interaction of NaHS/H(2)S with BH(4) in inhibition of nNOS and eNOS, but not iNOS.

Hydrogen sulfide causes relaxation in mouse bronchial smooth muscle.

We investigated the effects of NaHS, a hydrogen sulfide (H(2)S) donor, on the tension of isolated mouse and guinea-pig bronchial rings. NaHS at 0.01 - 10 mM had no effect on the tone of those preparations without precontraction. When the preparation was precontracted with carbachol, NaHS at 0.1 - 3 mM strongly relaxed the mouse rings, but produced only slight relaxation in the guinea-pig rings. The NaHS-induced relaxation in the mouse bronchus was resistant to inhibitors of ATP-sensitive K(+) channels, soluble guanylyl cyclase, cyclooxygenase (COX)-1 or COX-2, and antagonists of tachykinin receptors. Thus, NaHS evokes bronchodilation in mice, suggesting a possible role for H(2)S in the respiratory system.

Effects of tributyltin on the immune system of Japanese flounder (Paralichthys olivaceus).

Effects of tributyltin (TBT) which has been used for antifouling paint of ship's hulls and fishing nets on the immune system in Japanese flounder (Paralichthys olivaceus) were investigated. After short-term exposure to a high level of TBT, leucocytes in the head kidney from 1-year-old flounder were examined for the proportion of neutrophils in total leucocytes. Also examined were their respiratory burst activities using flow cytometry, the reduction of nitroblue tetrazolium (NBT) and lysozyme activities. Furthermore, long-term exposures to a relatively low level of TBT using young flounder were also carried out. The proportion of neutrophils in total leucocytes prepared from head kidney in each fish exposed to TBT at 20 microg/L for 5 days and the reduction of NBT by leucocytes prepared from the same experimental conditions increase compared to the control group. The contents were 42.0+/-6.8 and 52.5+/-6.3%, respectively. Significant differences of the NBT reduction were observed between 0 and 20 microg/L TBT exposure groups. On the other hand, the respiratory burst activity of cells in the exposure group clearly showed a tendency to decrease compared to the control group. Furthermore, high level of TBT also inhibited lysozyme activity which plays an important role for the bacteriocidal procedures. However, similar results were not obtained in the exposure group with a relatively low level of TBT. To determine the immunotoxic effects of TBT, infection experiments using pathogens which are naturally occurring should be further investigated.

Direct inhibition of endothelial nitric oxide synthase by hydrogen sulfide: contribution to dual modulation of vascular tension.

We characterized actions of hydrogen sulfide (H(2)S) on tension of isolated rat and mouse aortae, and then examined if H(2)S could directly modulate activity of endothelial nitric oxide (NO) synthase (eNOS). Isometric tension was recorded in rat and mouse aortic rings. Activity of recombinant bovine eNOS was determined as conversion of [(3)H]-arginine into [(3)H]-citrulline. NaHS, a H(2)S donor, caused contraction at low concentrations and relaxation at high concentrations in both rat and mouse aortae precontracted with phenylephrine. The contractile and relaxant effects of NaHS were enhanced and partially blocked, respectively, by the K(+)(ATP) channel inhibitor glibenclamide in the rat, but not mouse, aortae. In the KCl-precontracted rat aorta, NaHS produced glibenclamide-resistant contraction and relaxation. NaHS produced only relaxation, but not contraction, in the endothelium-denuded aortae, and also in the endothelium-intact aortae in the presence of inhibitors of NOS or soluble guanylate cyclase. NaHS pretreatment greatly attenuated the relaxation induced by acetylcholine, but not by an NO donor, in the tissues. Finally, we found that NaHS inhibited the conversion of [(3)H]-arginine into [(3)H]-citrulline by recombinant eNOS. NaHS thus causes contraction and relaxation in rat and mouse aortae. K(+)(ATP) channels are considered to contribute only partially to the NaHS-evoked relaxation. Most interestingly, our data demonstrate direct inhibition of eNOS by NaHS, probably responsible for its contractile activity, being evidence for a novel function of H(2)S.

Concentrations of antifouling biocides in sediment and mussel samples collected from Otsuchi bay, Japan.

Organotin compounds (OTs) and representative booster biocides were measured in sediment and mussels from Otsuchi Bay, Japan. The mean amounts of tributyltin (TBT) and triphenyltin (TPT) compounds in sediment were 13 microg kg(-1) dry and 3 microg kg(-1) dry, respectively. Representative booster biocides (Sea-Nine 211, Diuron, Dichlofluanid, Irgarol 1501, M1, which is a degradation compound of Irgarol 1051, and Copper pyrithione) were also detected in sediment from Otsuchi Bay. OT concentrations were higher than those of the measured booster biocides. Otsuchi Bay was divided into four parts by cluster analysis based on OT concentrations in sediment sampled from the bay. These areas included the vicinity of a shipyard, a small fishing port, the closed inner area of the bay, and the mouth of the bay. Higher concentrations of TBT and TPT and a higher ratio of TBT to total BTs were observed in the vicinity of the shipyard. A higher concentration of TPT in comparison with TBT was detected in a small fishing port. Furthermore, OT concentrations in the mouth of the bay were higher than those in the closed-off section. OT concentrations in mussels decreased with distance from the shipyard. Otsuchi Bay was then divided into three parts by cluster analysis based on the concentrations of representative booster biocides found in the bay's sediment. These areas included the vicinity of a shipyard, a small fishing port, and other sites. Concentrations of Diuron and Irgarol 1051 in the vicinity of a shipyard and a small fishing port were dramatically high in comparison with the other sites. Copper pyrithione and Dichlofluanid in addition to Diuron and Irgarol 1051 were also detected in the area of a small fishing port. The concentrations of antifouling biocides were highest in the water in front of the shipyard and showed a marked decrease with distance from the shipyard.

Evaluation of the Ishikawa cell line bioassay for the detection of estrogenic substances from sediment extracts.

This study examines the application of Ishikawa human endometrial adenocarcinoma cells to measure the estrogenic activity of fractionated extracts of sediments from Tokyo Bay, Japan. Estrogen stimulates alkaline phosphatase activity in this cell line. The results of these assays were compared with those of a yeast estrogen screen (YES) assay. The Ishikawa cell line bioassay showed higher sensitivity to 17beta-estradiol (median effective concentration [EC50], 10.7 pM) than did the YES assay (EC50, 480 pM). Fractionation of sediment extracts (all samples collected from 5 sites) showed that the nonpolar fraction was poisonous to yeast cells; the estrogenic activity of this fraction, therefore, could not be measured by YES. However, the nonpolar fraction did not kill the Ishikawa cells. The 17beta-estradiol-equivalent values of 15 extracts (3 fractions from each of 5 sediment samples) ranged from 5.7 to 697 pg/g dry weight according to the Ishikawa cell line bioassay. Chemical analysis using gas chromatography-mass spectrometry revealed that the highest concentrations of endocrine-disrupting chemicals were observed at the sampling station near the sewage treatment plant. The results support that the Ishikawa cell line bioassay is suitable for measuring the estrogenic activity of sediment samples.

Suppressive effect of a hot water extract of adzuki beans (Vigna angularis) on hyperglycemia after sucrose loading in mice and diabetic rats.

A hot water extract obtained by boiling adzuki beans (Vigna angularis) to produce bean paste for Japanese cake showed inhibitory activity against alpha-glucosidase, alpha-amylase, maltase, sucrase, and isomaltase after HP-20 column chromatography. The IC(50) values for each hydrolylase were 0.78 mg/ml (alpha-amylase), 2.45 mg/ml (maltase), 5.37 mg/ml (sucrase), and 1.75 mg/ml (isomaltase). The active fraction showed potential hypoglycemic activity in both normal mice and streptozotocin (STZ)-induced diabetic rats after an oral administration of sucrose, but did not show any effect on the blood glucose concentration after glucose administration, suggesting that the active fraction suppressed the postprandial blood glucose level by inhibiting alpha-glucosidase and alpha-amylase, irrespective of the endogenous blood insulin level.