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Aims: This study evaluated drug–drug interactions between the CYP3A4 inhibitor carotegrast methyl and the other CYP3A4 substrates, midazolam, atorvastatin and prednisolone. Methods: A total of 88 healthy volunteers orally received carotegrast methyl 960 mg 3 times daily for 14 days. A single oral (5 mg) or intravenous (0.017 mg kg−1) midazolam, oral (5 mg) prednisolone or oral (10 mg) atorvastatin was administered before, with and after carotegrast methyl treatment. When the 90% confidence interval (CI) for the geometric mean ratios of the pharmacokinetic (PK) parameters with coadministration with carotegrast methyl (Day 14) to those before carotegrast methyl administration was between 0.80 and 1.25, no PK interaction were deemed. Results: The Cmax and AUC0‐t of oral midazolam before administration of carotegrast methyl were 30.9 ± 9.8 ng mL−1 and 74.5 ± 21.9 ng h mL−1, respectively. The geometric mean ratio of the Cmax and AUC0‐t of midazolam on Day 14 to those on Day −1 was 1.86 (90% CI, 1.64–2.11) and 3.07 (90% CI, 2.81–3.35), which did not fall within the range of 0.80–1.25, suggesting that carotegrast methyl had a PK interaction with midazolam. Similar PK interactions were found for intravenous midazolam and atorvastatin, but not for prednisolone. The inhibitory effect of carotegrast methyl on CYP3A4‐mediated metabolism of midazolam and atorvastatin had almost disappeared by 14 days after the end of administration. Conclusion: Carotegrast methyl was classified as a moderate CYP3A4 inhibitor in humans. Carotegrast methyl might enhance the action of drugs that are metabolized by CYP3A4. [ABSTRACT FROM AUTHOR]

Superoxide anion impairs contractility in cultured aortic smooth muscle cells. Am J Physiol Heart Circ Physiol 283: H382-H390, 2002. First published March 28, 2002; 10.1152/ajpheart.00574.2001.--We examined the effects of superoxide anion ([O.sup.-.sub.2]) generated by xanthine plus xanthine oxidase (X/XO) on the intracellular [Ca.sup.2+] concentration ([[[Ca.sup.2+]].sub.i]) and muscle contractility in cultured bovine aortic smooth muscle cells (BASMC). Cells were grown on collagen-coated dish for the measurement of [[[Ca.sup.2+]].sub.i]. Pretreatment with X/XO inhibited ATP-induced [Ca.sup.2]+ transient and [Ca.sup.2+] release-activated [Ca.sup.2+] entry (CRAC) after thapsigargin-induced store depletion, both of which were reversed by superoxide dismutase (SOD). In contrast, [Ca.sup.2+] transients induced by high-[K.sup.+] solution and [Ca.sup.2+] ionophore A-23187 were not affected by X/XO. BASMCembedded collagen gel lattice, which was pretreated with xanthine alone, showed contraction in response to ATP, thapsigargin, high-[K.sup+] solution, and A-23187. Pretreatment of the gel with X/XO impaired gel contraction not only by ATP and thapsigargin, but also by high-[K.sup+] solution and A-23187. The X/XO-treated gel showed normal contraction; however, when SOD was present during the pretreatment period. These results indicate that [O.sup.-.sub.2] attenuates smooth muscle contraction by impairing CRAC, ATP-induced [Ca.sup.2+] transient, and [Ca.sup.2+] sensitivity in BASMC. calcium

Mechanical stress induces auto/paracrine ATP release from various cell types, but the mechanisms underlying this release are not well understood. Here we show that the release of ATP induced by hypotonic stress (HTS) in bovine aortic endothelial cells (BAECs) occurs through volume-regulated anion channels (VRAC). Various VRAC inhibitors, such as glibenclamide, verapamil, tamoxifen, and fluoxetine, suppressed the HTS-induced release of ATP, as well as the concomitant [Ca.sup.2+] oscillations and NO production. They did not, however, affect [Ca.sup.2+] oscillations and NO production induced by exogenously applied ATP. Extracellular ATP inhibited VRAC currents in a voltage-dependent manner: block was absent at negative potentials and was manifest at positive potentials, but decreased at highly depolarized potentials. This phenomenon could be described with a 'permeating blocker model,' in which ATP binds with an affinity of 1.0 [+ or -] 0.5 mM at 0 mV to a site at an electrical distance of 0.41 inside the channel. Bound ATP occludes the channel at moderate positive potentials, but permeates into the cytosol at more depolarized potentials. The triphosphate nucleotides UTP, GTP, and CTP, and the adenine nucleotide ADP, exerted a similar voltage-dependent inhibition of VRAC currents at submillimolar concentrations, which could also be described with this model. However, inhibition by ADP was less voltage sensitive, whereas adenosine did not affect VRAC currents, suggesting that the negative charges of the nucleotides are essential for their inhibitory action. The observation that high concentrations of extracellular ADP enhanced the outward component of the VRAC current in low [Cl.sup.-] hypotonic solution and shifted its reversal potential to negative potentials provides more direct evidence for the nucleotide permeability of VRAC. We conclude from these observations that VRAC is a nucleotide-permeable channel, which may serve as a pathway for HTS-induced ATP release in BAEC. KEY WORDS: ATP release * nitric oxide * hypotonic stress * volume-regulated anion channel * calcium

The synthesis and characterization of the tetranuclear Ru(super II) complex (RuCl(mu-Cl)(P(OMe)3)2(mu,eta-1,eta-2-S2)Ru(P(OMe)3)(mu-P(OMe)3P,O))2 containing a trans-Ru(super II)SSRu(super II) core are presented. A reexamination of the reduction conditions of the complex (RuCl(P(OMe)3)2)2(mu-Cl)2(mu-S2) was carried out and sodium amalgam as a reductant permits quantitative reduction of Ru(III) to Ru(II).

The effects of heat, pressure and CO2 treatment on the inactivation of the spores of five bacteria, including G. stearothermophilus are compared. Findings show that CO2 treatment at temperatures below 100 degree Celsius can effectively activate bacterial spores including those of G. stearothermophilus.

Endothelial migration is one of the major events of pathological neovascularization. We compared the characteristics of [Ca.sup.2+] mobilization in nonconfluent, confluent, and migrating endothelial cells. Migration of endothelial cells was induced by wounding the confluent cell monolayer. The basal intracellular [Ca.sup.2+] concentration was lower in migrating cells and higher in confluent cells than in nonconfluent cells. Thapsigargin (TG)-induced [Ca.sup.2+] leak and TG-evoked [Ca.sup.2+] entry were accelerated in migrating cells, whereas the latter was suppressed in confluent cells. The ATP-induced [Ca.sup.2+] transient was also much larger in migrating cells than in confluent ceils. These alterations were also observed in a cell as an intracellular polarization, i.e., the leading edge showed an acceleration of TG-evoked [Ca.sup.2+] entry and an augmentation of the ATP-induced [Ca.sup.2+] transient. Endothelial migration was significantly suppressed by TG or cyclopiazonic acid. These observations suggest that the alterations of [Ca.sup.2+] store site-related [Ca.sup.2+] mobilizations, i.e., [Ca.sup.2+] sequestration, release, and TG-evoked [Ca.sup.2+] entry, may be involved in the cellular mechanisms of endothelial migration. SERCA; thapsigargin; ATP; intracellular polarization; actin

Impairment of endothelial nitric oxide production by acute glucose overload. Am J Physiol Endocrinol Metab 280: E171-E178, 2001.--We examined the effects of acute glucose overload (pretreatment for 3 h with 23 mM D-glucose) on the cellular productivity of nitric oxide (NO) in bovine aortic endothelial cells (BAEC). We had previously reported (Kimura C, Oike M, and Ito Y. Circ Res, 82: 677-685, 1998) that glucose overload impairs [Ca.sup.2+] mobilization due to an accumulation of superoxide anions ([MATHEMATICAL EXPRESSION NOT REPRODUCIBLE IN ASCII]) in BAEC. In control cells, ATP induced an increase in NO production, assessed by diaminofluorescein 2 (DAF-2), an NO-sensitive fluorescent dye, mainly due to [Ca.sup.2+] entry. In contrast, ATP-induced increase in DAF-2 fluorescence was impaired by glucose overload, which was restored by superoxide dismutase, but not by catalase or deferoxamine. Furthermore, pyrogallol, an [MATHEMATICAL EXPRESSION NOT REPRODUCIBLE IN ASCII] donor, also attenuated ATP-induced increase in DAF-2 fluorescence. In contrast, a nonspecific intracellular [Ca.sup.2+] concentration increase induced by the [Ca.sup.2+] ionophore A-23187, which depletes the intracellular store sites, elevated DAF-2 fluorescence in both control and high D-glucose-treated cells in [Ca.sup.2+]-free solution. These results indicate that glucose overload impairs NO production by the [MATHEMATICAL EXPRESSION NOT REPRODUCIBLE IN ASCII] mediated attenuation of [Ca.sup.2+] entry. calcium; superoxide; diaminofluorescein 2

Oike, Masahiro, Chiwaka Kimura, Tetsuya Koyama, Miyuki Yoshikawa, and Yushi Ito. Hypotonic stress-induced dual [Ca.sup.2+] responses in bovine aortic endothelial cells. Am J Physiol Heart Circ Physiol 279: H630-H638, 2000.--We have investigated the effects of hypotonic stress on intracellular calcium concentration ([[[Ca.sup.2+]].sub.i]) in bovine aortic endothelial cells. Reducing extracellular osmolarity by 5% to 40% elicited a steep [Ca.sup.2+] transient both in normal Krebs and [Ca.sup.2+]-free solutions. The hypotonic stress-induced [Ca.sup.2+] transient was inhibited by phospholipase C inhibitors (neomycin and U-73122), a [P.sub.2]-receptor antagonist (suramin), and an ATP-hydrolyzing enzyme (apyrase), suggesting that the hypotonic stress-induced [Ca.sup.2+] transient is mediated by ATP. A luciferin-luciferase assay confirmed that 40% hypotonic stress released 91.0 amol/cell of ATP in 10 min. When the hypotonic stress-induced fast [Ca.sup.2+] transient was inhibited by neomycin, suramin, or apyrase, a gradual [[[Ca.sup.2+]].sub.i] increase was observed instead. This hypotonic stress-induced gradual [[[Ca.sup.2+]].sub.i] increase was inhibited by a phospholipase [A.sub.2] inhibitor, 4-bromophenacyl bromide. Furthermore, exogenously applied arachidonic acid induced a gradual [[[Ca.sup.2+]].sub.i] increase with an [ED.sub.50] of 13.3 [micro]M. These observations indicate that hypotonic stress induces a dual [Ca.sup.2+] response in bovine aortic endothelial cells, i.e., an ATP-mediated fast [Ca.sup.2+] transient and an arachidonic acid-mediated gradual [Ca.sup.2+] increase, the former being the predominant response in normal conditions. mechanical stress; adenosine 5'-triphosphate; arachidonic acid.

Koyama, Tetsuya, Masahiro Oike, Sohtaro Komiyama, and Yushi Ito. Superoxide anion impairs [Ca.sup.2+] mobilization in cultured human nasal epithelial cells. Am. J. Physiol. 277 (Lung Cell. Mol. Physiol. 21): L1089-L1095, 1999.--We examined the effects of superoxide anion [MATHEMATICAL EXPRESSION NOT REPRODUCIBLE IN ASCII] the intracellular [Ca.sup.2+] concentration in cultured human nasal epithelial cells. The cells were exposed to [MATHEMATICAL EXPRESSION NOT REPRODUCIBLE IN ASCII] by pretreatment with xanthine (X) and xanthine oxidase (XO); control cells were treated with X alone. When [Ca.sup.2+]-containing Krebs solution was reperfused in the thapsigargin-treated, store-depleted cells, reapplication-induced intracellular [Ca.sup.2+] concentration elevation was significantly smaller in X/XO-treated cells than in the control cells, suggesting that [MATHEMATICAL EXPRESSION NOT REPRODUCIBLE IN ASCII] impairs [Ca.sup.2+] release-activated [Ca.sup.2+] entry (CRAC). Bath application of ATP induced a steep [Ca.sup.2+] transient in both control and X/XO-treated cells. However, the concentration-response curve of the ATP-induced [Ca.sup.2+] transient was shifted to a higher concentration in X/XO-treated cells. The impairments of CRAC and ATP-induced [Ca.sup.2+] transient induced by X/XO were reversed by superoxide dismutase. Furthermore, all these X/XO-induced effects were also observed in cells pretreated with pyrogallol, also an [MATHEMATICAL EXPRESSION NOT REPRODUCIBLE IN ASCII] donor. These results indicate that [MATHEMATICAL EXPRESSION NOT REPRODUCIBLE IN ASCII] impairs at least two mechanisms involved in [Ca.sup.2+] mobilization in human nasal epithelial cells, i.e., CRAC and ATP-induced [Ca.sup.2+] release. capacitative calcium entry; adenosine 5'-triphosphate; calcium release