Regulatory volume increase after secretory volume decrease in colonic epithelial cells under muscarinic stimulation

To address the question of whether colonic secretory cells change their volume in response to carbachol (CCh) stimulation and, if so, the mechanisms involved therein, we used two-photon laser scanning microscopy to measure the volume of individual epithelial cells in the fundus region of crypts isolated from the guinea-pig distal colon. We also measured the volume of human colonic epithelial T84 cells using an electronic sizing technique. Both types of colonocytes responded to stimulation by CCh with shrinkage and then underwent a regulatory volume increase (RVI), even during continued stimulation by CCh. The secretory volume decrease (SVD) induced by CCh was antagonized by atropine, BAPTA loading and niflumic acid, a blocker of Ca²⁺-activated Cl^– channels. An increase in the intracellular free [Ca²⁺] was observed with fura-2 during these volume responses to CCh. Removal of all Na⁺ or K⁺ or of most of the Cl^– from the extracellular solution abolished the RVI, but not the preceding SVD. The RVI, but not the preceding SVD, was abolished by bumetanide, a blocker of the Na⁺-K⁺-2Cl^– cotransporter. We conclude that guinea-pig crypt colonocytes and human T84 cells exhibit a cytosolic Ca²⁺-dependent SVD and undergo a subsequent RVI that is dependent on the operation of Na⁺-K⁺-2Cl^– cotransporters.     

Dioxin health risk to infants using simulated tissue concentrations

Dioxin concentrations in infant and child were simulated using physiologically based pharmacokinetic (PBPK) models developed for these groups. The infant model was validated by comparing the simulated concentration with the measured concentration from the literature, and they showed good agreement. Simulations with our PBPK model showed temporal patterns in concentrations in various tissues. For risk assessment, estimated concentrations of 29 dioxins in the liver were summed up in a toxic equivalency (TEQ) basis to be compared with actual 2,3,7,8-TCDD concentrations in rat liver associated with toxicity. Maximum liver concentrations in breast-fed and formula-fed infants were 16.8pg TEQ/g and 3.5pg TEQ/g, respectively. The level in breast-fed infant liver was approximately 1/300 of the level associated with hepatocellular carcinoma and 1/5 of the level found in maternal rat liver associated with alterations in reproductive organs in the next generation. Based on our analysis, the present contamination level is not safe enough, but further dose-response data is required for a quantitative risk assessment.     

Activity of docetaxel in paclitaxel-resistant ovarian cancer cells

Purpose The aim of this study was to determine the behavior of docetaxel (DTX) in ovarian cancer cells resistant to paclitaxel (PTX).Methods We used human ovarian adenocarcinoma cell lines KF, KFTx (PTX-resistant KF), SK-OV-3, and HAC-2. The sensitivity of the cells to PTX or DTX was determined by the MTT assay. Cellular accumulation of PTX and DTX was measured by high-performance liquid chromatography. mRNA of MDR-1 was detected by RT-PCR. Cell cycle distribution was determined by flow cytometry after exposure to the IC₅₀ of each drug. Bcl-2 phosphorylation was determined by Western blot analysis. Activity for tubulin polymerization of each drug was examined by a -tubulin polymerization assay.Results KFTx cells had a 5.5-fold greater resistance to PTX and a 7.3-fold greater resistance to DTX than KF cells, indicating that KFTx cells had acquired cross-resistance to DTX. SK-OV-3 cells were sensitive and HAC-2 cells were resistant to both PTX and DTX. The gene expression of MDR-1 increased after exposure to DTX in KF and KFTx cells. Residual cellular accumulation of PTX and DTX was significantly lower in KFTx cells than in KF cells. In contrast, MDR-1 expression was not detected in SK-OV-3 and HAC-2 cells. Flow cytometric analysis indicated no differences in alterations of cell cycle distribution following exposure to the two drugs. Bcl-2 phosphorylation occurred after exposure to DTX at a concentration equivalent to the clinical dose, but did not occur after exposure to PTX in KFTx cells. In HAC-2 cells, Bcl-2 phosphorylation was not detected after exposure to DTX or PTX at concentrations equivalent to the clinical doses. DTX showed greater tubulin polymerization activity than PTX in KFTx cells. -tubulin polymerization did not correlate with the concentration of PTX or DTX, suggesting that alteration in the tubulin reaction might contribute to the resistance in HAC-2 cells.Conclusions The present study suggests that the mechanisms involved in cytotoxicity of and resistance to PTX and DTX do not differ, but DTX has a greater cytotoxic potential in PTX-resistant cells with an efflux system.     

A case of postoperative pelvic metastasis and multiple liver metastases of the rectal cancer successfully treated by arterial infusion therapy with 5-FU/leucovorin

A 55-year-old man underwent a rectal amputation for rectal cancer in 1994. As the tumor marker was elevated in 2002, we performed an abdominal CT scan and detected local and multiple liver recurrences. We treated the patient with intra-arterial infusion of 5-FU/LV via the internal iliac artery and the hepatic artery. The chemotherapy was performed on a weekly basis; it consisted of 5-FU (500 mg/body), administered for 5 hours to bilateral reservoirs through an infusion pump and l-leucovorin (400 mg/body), administered intravenously for 2 hours. After 18 administrations of this regimen during a hospital stay and after a discharge from the hospital as an outpatient, the multiple liver metastases that were observed have disappeared. Further, the local recurrences showed a partial reduction in tumor size with a decrease in perineal pain. Subsequently, the patient did not require further doses of morphine. He exhibited no severe side effects except for grade 1 nausea, and his QOL was also good. Therefore, local intra-arterial infusion chemotherapy with 5-FU/LV appears to have been effective for rectal cancer recurrences.     

Ischemia-induced enhancement of CFTR expression on the plasma membrane in neonatal rat ventricular myocytes

Pathophysiological functions of cardiac cystic fibrosis transmembrane conductance regulator (cCFTR) in ischemia are not well known. Using neonatal rat ventricular cardiomyocytes in primary culture in this study, we thus examined whether the CFTR protein is expressed and is functioning as a cAMP-activated anion channel on the plasma membrane under ischemic conditions. After the cells were subjected to simulated ischemia (O(2) and glucose deprivation), an up-regulation of the CFTR expression was transiently observed in the membrane fraction by Western blot. A peak expression of mature CFTR protein was found at 3 h of ischemia, and thereafter the signal diminished gradually. In contrast, the results of Northern blot indicated that the expression level of CFTR mRNA changed little until 3 h of ischemia, whereas the level slightly decreased after 8 h of ischemia. An immunohistochemical examination showed, in agreement with the results of Western blot analysis, that the expression of CFTR protein on the plasma membrane became most prominent at 3 h of ischemia, whereas the plasmalemmal CFTR signal was markedly reduced after 8 h of ischemia. Whole-cell recordings showed that the cardiomyocytes responded to cAMP with an activation of time- and voltage-independent currents that contained an anion-selective component sensitive to CFTR Cl(-) channel blockers (NPPB and glibenclamide) but not to a stilbene-derivative conventional Cl(-) channel blocker (SITS). This cAMP-activated Cl(-) channel current was found to be enhanced after an application of ischemic stress for 3 to 4 h. These findings indicate that a plasmalemmal expression of CFTR is transiently enhanced under glucose-free hypoxic conditions presumably because of a posttranslational control.     

Dysgerminoma in a patient with a tumor of the neck. Empiric treatment of stage IV dysgerminoma

The evolution of therapy for malignant ovarian germ cell tumors is one of the true success stories in oncology. Treatment outcome has improved greatly thanks to cisplatin-based combination chemotherapy. According to the well-established treatment guidelines for advanced cases, we treated a case of stage IV undifferentiated germ cell tumor in which we were able to preserve the patient's fertility. We concluded that the PEP regimen is an effective treatment for the patient with metastatic germ cell tumor.     

A common Ile 823 Met variant of ATP-binding cassette transporter A1 gene (ABCA1) alters high density lipoprotein cholesterol level in Japanese population

Recently, variants in ATP-binding cassette transporter A1 (ABCA1) were demonstrated to be associated with increased level of high density lipoprotein cholesterol (HDL-C) and decreased risk of coronary artery disease (CAD) in Caucasians. However, this is not universally applicable due to the ethnic or environmental differences. In this context, to clarify the effect of ABCA1 in Japanese, we evaluated the phenotypic effects of I/M 823 and R/K 219 variants on the plasma level of HDL-C in 410 patients recruited in our hospital. Subjects with M 823 allele had significantly higher level of HDL-C than those without M823 allele (49.0+/-15.1 vs. 44.9+/-11.5 mg/dl, respectively, P<0.05). This statistical significance did not change even after multiple regression analysis. In contrast, there was no difference in HDL-C level among the genotypes in R/K 219 polymorphism. Further, in our study population an inverse relationship was shown to exist between HDL-C level and incidence of CAD. However, no positive association was observed between those variants and susceptibility to CAD. In this study, we provide evidence that I/M 823 variant, not R/K 219 variant, in ABCA1 is one of the determinants of HDL-C level, suggesting the importance of this gene on lipid metabolism in Japanese.