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A Masamune

Publications and source records attributed to A Masamune.

18 recordsLinked to original sources
PubMed2000

Ascites of severe acute pancreatitis in rats transcriptionally up-regulates expression of interleukin-6 and -8 in vascular endothelium and mononuclear leukocytes.

The molecular mechanisms that link acute pancreatitis and multiple organ failure remain unknown. We examined the effect of ascitic fluids prepared from rats with experimental necrotizing pancreatitis on the expression of interleukin (IL) -6 and IL-8 in human umbilical vein endothelial cells (HUVEC) and human monocytic THP-1 cells. Incubation of HUVEC or THP-1 cells with the ascitic fluids resulted in a concentration-dependent up-regulation of the cytokine expression with comparable mRNA induction. Electrophoretic mobility shift assay revealed that the ascitic fluids increased the nuclear factor-kappaB (NF-kappaB) and NF-IL6 binding activities. Intraperitoneal injection of ascitic fluids into healthy rats induced the activation of NF-kappaB in the infiltrating leukocytes in the lung. Our results suggested that ascitic fluids may play a role in the pathophysiology of severe acute pancreatitis through the activation of transcription factors and consequent cytokine productions in distant organs.

Animals↗
PubMed2000

Nitric oxide decreases endothelial activation by rat experimental severe pancreatitis-associated ascitic fluids.

To clarify the roles of nitric oxide (NO) in acute pancreatitis (AP), we examined the effects of NO on the endothelial activation induced by ascitic fluids from rats with experimental severe AP. Necrotizing hemorrhagic pancreatitis was induced in male Wistar rats with sodium taurocholate. Six hours later, peritoneal exudates were collected, centrifuged, and human umbilical vein endothelial cells were treated with the supernatants. Then (a) the mRNA level of endothelial-type NO synthase (ecNOS) was examined by reverse transcription-polymerase chain reaction; (b) effects of an NO donor, sodium nitroprusside (SNP) and an inhibitor of NOS, N(omega)-nitro-L-arginine (L-NNA) on the ascitic fluids-induced expression of intercellular adhesion molecule-1, vascular cell adhesion molecule-1, and interleukin-8 were assessed by enzyme-linked immunoassay; (c) nuclear translocation of nuclear factor-kappa B (NF-kappaB) was examined by electrophoretic mobility shift assay; and (d) effects of SNP and L-NNA on the adhesion of U937 cells to endothelial monolayer were assessed. The ecNOS mRNA level was decreased by the ascitic fluids; ascitic fluids-induced expression of adhesion molecules and interleukin-8 as well as the nuclear translocation of NF-kappaB were attenuated by SNP, whereas L-NNA augmented them; and the effects on the endothelial activation were paralleled by the altered adhesion of U937 cells to endothelium. The ability of NO to limit endothelial activation and inhibit leukocyte adhesion might contribute to its antiinflammatory properties in AP.

Acute Disease↗
PubMed1999

Helicobacter pylori-dependent ceramide production may mediate increased interleukin 8 expression in human gastric cancer cell lines.

BACKGROUND & AIMS: Helicobacter pylori adheres to gastric epithelial cells, activates nuclear factor kappaB (NF-kappaB), and stimulates interleukin (IL)-8 production, but the responsible molecular mechanisms remain largely unknown. Because several studies have shown that sphingolipids are involved in a number of signaling pathways, including NF-kappaB activation, we investigated the possible role of sphingolipids in the regulation of IL-8 expression in Kato III and AGS cells. METHODS: IL-8 production in the conditioned media was quantified by enzyme immunoassay. Induction of messenger RNA (mRNA) was assessed by Northern blot analysis. Activation and binding activity of transcription factors were examined by luciferase assay and electrophoretic mobility shift assay, respectively. Intracellular levels of ceramide were quantified by diacylglycerol kinase assay. RESULTS: A cell-permeable ceramide analogue (C2-ceramide) increased IL-8 expression with comparable mRNA induction. This effect was mimicked by sphingomyelinase, but not by phospholipase A2 or phospholipase C. C2-ceramide induced IL-8 gene transcription mainly through activation of NF-kappaB because mutation of the NF-kappaB-binding site completely abrogated the induction of luciferase activity. Direct contact of live H. pylori with epithelial cells increased the intracellular concentration of ceramide. CONCLUSIONS: The results suggest a novel role of the sphingomyelin-ceramide pathway, at least in part through NF-kappaB, in IL-8 production induced by H. pylori infection in gastric epithelial cells.

Ceramides↗
PubMed1999

Specific induction of adhesion molecules in human vascular endothelial cells by rat experimental pancreatitis-associated ascitic fluids.

The molecular mechanisms that link acute pancreatitis (AP) and multiple organ failure remain unknown. To clarify the role of endothelial activation, we examined the effects of ascitic fluids from rats with experimental pancreatitis on the expression of adhesion molecules in human umbilical vein endothelial cells (HUVECs). Necrotizing hemorrhagic pancreatitis was induced with sodium taurocholate. Six and 24 h later, peritoneal exudates were collected, centrifuged and HUVECs were treated with the supernatants. The expression of E-selectin, intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1) was quantified by enzyme-linked immunosorbent assay. Induction of mRNA was assessed by reverse-transcriptase polymerase chain reaction. The activation of transcription factors was examined by electrophoretic mobility shift assay. The expression of ICAM-1 in the tissues was examined immunohistochemically. ICAM-1 and VCAM-1, but not E-selectin expression was upregulated with comparable mRNA induction. Nuclear factor kappaB was activated, while activator protein-1 binding activity was not altered. Immunohistochemically, enhanced ICAM-1 expression was observed in the pancreas and lung, but not in the liver. Ascitic fluids may contain soluble factors responsible for the transcriptional activation of endothelial adhesion molecules, and ICAM-1 may play roles in the pathogenesis of complicated AP.

Animals↗
PubMed1999

Ascitic fluid of experimental severe acute pancreatitis modulates the function of peritoneal macrophages.

Although the pathophysiology of acute pancreatitis appears to be greatly influenced by the production of ascites, little is known about the mechanism. To investigate the effects of pancreatitis-associated ascitic fluid (PAAF) on macrophage function, we examined the effects of PAAF obtained from a rat model of severe acute pancreatitis on the ability of peritoneal macrophages to produce tumor necrosis factor-alpha (TNF-alpha). In addition, we compared the responses of PAAF-treated and PAAF-untreated macrophages to lipopolysaccharide (LPS) by evaluating their TNF-alpha production and nuclear factor-kappaB (NFkappaB) activation. Incubation of peritoneal macrophages with the PAAF led to the rapid and prolonged activation of NF-kappaB and to TNF-alpha production. Pyrrolidine dithiocarbamate, a potent inhibitor of NF-kappaB activation, attenuated the macrophage TNF-alpha production by PAAF. Macrophages produced TNF-alpha in response to LPS, but the cytokine production was significantly reduced when macrophages were pretreated with PAAF. The suppression of TNF-alpha production by PAAF pretreatment accompanied the impairment of NF-kappaB activation in response to LPS. These results indicate that the PAAF of severe acute pancreatitis may play important roles in the pathologic course of this disease through its effects on macrophage function.

Amylases↗
PubMed1999

Fas ligand is frequently expressed in human pancreatic duct cell carcinoma.

Fas ligand (Fas-L) is a key molecule in normal immune development, homeostasis, modulation, and function. Recent reports suggested that tumor cells can evade immune attack by killing lymphocytes through expressing Fas-L on the tumor cell surface. The expression of Fas-L has been demonstrated in pancreatic cancer cell lines and in tissues. The messenger RNA (mRNA) and protein expression of Fas-L was investigated in four pancreatic cancer cell lines and 19 human pancreatic duct cell carcinomas (PDCs) by reverse transcription-polymerase chain reaction (RT-PCR) and immunohistochemistry, respectively. In addition, coculture assay of Fas-L and Fas-expressing PANC-1 cells and Fas-sensitive Jurkat cells was performed. Fas-L mRNA expression was observed in all four cell lines as well as in 10 PDC tissues, and the protein expression was frequently detected in the PDC tissues (16 of 19 cases). The coculture experiments showed that PANC-1 cells induced apoptosis of Jurkat cells, whereas the PANC-1 cells themselves and Jurkat cells cultured without the presence of PANC-1 showed few apoptotic changes. These findings suggest that Fas-L may play an important role in the ability of PDCs to escape from immune surveillance through the induction of apoptosis in tumor-attacking lymphocytes. The frequent expression of Fas-L in PDCs may partly explain the notorious biologic behavior of PDCs.

Apoptosis↗
PubMed1999

Inhibition of nuclear factor-kappaB activation improves the survival of rats with taurocholate pancreatitis.

BACKGROUND: Death in the early stages of severe acute pancreatitis is frequently the result of multiple organ dysfunction, but its mechanism is not clear. AIMS: To investigate the state of nuclear factor-kappaB (NF-kappaB) in macrophages of rats with lethal pancreatitis, and to assess the effectiveness of pyrrolidine dithiocarbamate, an inhibitor of NF-kappaB, on the pathology and mortality. METHODS: Taurocholate pancreatitis was produced in rats, and the severity of the disease, the mortality, and activation of NF-kappaB in peritoneal and alveolar macrophages were compared in rats receiving pyrrolidine dithiocarbamate (PDTC) treatment and those that were not. RESULTS: Taurocholate pancreatitis produced massive necrosis, haemorrhage, and severe leucocyte infiltration in the pancreas as well as alveolar septal thickening in the lung. NF-kappaB was activated in peritoneal and alveolar macrophages six hours after pancreatitis induction. Pretreatment with PDTC dose-dependently attenuated the NF-kappaB activation and improved the survival of the rats, although it did not affect the early increase in serum amylase and histological findings. CONCLUSIONS: Early blockage of NF-kappaB activation may be effective in reducing fatal outcome in severe acute pancreatitis.

Acute Disease↗
PubMed1998

Endogenous glucocorticoids decrease the acinar cell sensitivity to apoptosis during cerulein pancreatitis in rats.

BACKGROUND & AIMS: We recently showed that activation of the hypothalamus-pituitary-adrenal axis may mitigate the progress of acute pancreatitis. To clarify the mechanism, the role of endogenous glucocorticoids in pancreatic acinar cell death was examined. METHODS: The occurrence of apoptosis was studied in adrenalectomized or sham-operated rats with or without cerulein-induced pancreatitis. The effects of RU38486, a glucocorticoid-receptor antagonist, on the survival of cultured acinar cells (AR42J) were also examined. RESULTS: Adrenalectomy caused increases in terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate-biotin nick-end labeling (TUNEL) of acinar nuclei depending on the time after adrenalectomy but not of other cell types in the pancreas and in other digestive organs. Electron microscopy showed the characteristic features of apoptosis in the TUNEL-labeled acinar cells. In cerulein pancreatitis of adrenalectomized rats, the TUNEL-labeled acinar nuclei increased remarkably depending on the time after cerulein infusion. Replacement of glucocorticoids blocked the occurrence of apoptosis in these experiments. RU38486 induced dose dependently the apoptosis of AR42J cells. CONCLUSIONS: These results provide evidence that endogenous glucocorticoids are an important factor for acinar cell survival. Endogenous glucocorticoids may protect acinar cells by decreasing their sensitivity to the induction of cell death during acute pancreatitis.

Acute Disease↗
PubMed1998

Low doses of lipopolysaccharide upregulate acinar cell apoptosis in cerulein pancreatitis.

We have reported previously that administration of a sublethal low dose of lipopolysaccharide (LPS; 50 microg/kg) prior to the induction of cerulein (Cn) pancreatitis mitigates the pathological course. To clarify the mechanism, we examined apoptosis in the pancreas using the same model. Apoptosis was evaluated by terminal deoxynucleotidyl transferase mediated dUTP-biotin nick end labeling (TUNEL) and transitional electron microscopy. LPS pretreatment at a dose of 50 microg/kg increased remarkably the incidence of acinar cell apoptosis in Cn pancreatitis rats compared with LPS-untreated Cn pancreatitis rats. Apoptosis was observed selectively in acinar cells but was not shown in endocrine cells or ductal epithelial cells. Infiltration of inflammatory cells was scarcely observed. These acinar cells showed the characteristic morphological features of apoptosis by electron microscopy. Administration of LPS at a dose of 50 microg/kg alone caused acinar cell apoptosis but the incidence was much lower than that in the LPS-pretreated Cn pancreatitis rats. The TUNEL labeling was significantly increased depending on the dose of LPS and on the interval between the administration of LPS and that of Cn. These results suggest that the pathological features of acute pancreatitis might be modified by the presence of nonfatal endotoxemia through the induction of acinar cell apoptosis.

Animals↗
PubMed1998

Nitric oxide is overproduced by peritoneal macrophages in rat taurocholate pancreatitis: the mechanism of inducible nitric oxide synthase expression.

To investigate the pathobiology of severe acute pancreatitis, we studied the expression of inducible nitric oxide synthase (iNOS) in peritoneal macrophages of experimental pancreatitis. Taurocholate (TCA) pancreatitis and cerulein (CE) pancreatitis were used as models of lethal and self-limited pancreatitis, respectively, and the mechanism of iNOS expression in peritoneal macrophages was studied. Serum nitrate and nitrite (NOx) concentrations increased during the course of TCA pancreatitis, and iNOS-immunoreactivity was detected in the peritoneal macrophages 12 h after the induction of TCA pancreatitis, but these phenomena were not observed in CE pancreatitis. Despite the difference in the iNOS expression, the iNOS messenger RNA (mRNA) and the activation of nuclear factor-kappa B (NF-kappa B) were detected in the peritoneal macrophages of both pancreatitis models. The supernatant of TCA pancreatitis ascites could induce iNOS in the peritoneal macrophages of normal rats in vitro, but the peritoneal lavage fluid of CE pancreatitis rats could not. The results indicated that there may be qualitative or quantitative differences in the macrophage activation between the two types of experimental pancreatitis and suggested that the ascites of rats with lethal acute pancreatitis contains some soluble factors that activate the macrophage/monocyte system and cause an overproduction of NO by the iNOS expression.

Acute Disease↗
PubMed1996

Sphingosine and its methylated derivative N,N-dimethylsphingosine (DMS) induce apoptosis in a variety of human cancer cell lines.

In the study of apoptosis initiated by various signals including ligands binding to cell membrane receptors such as Fas and TNFRI, the sphingomyelin pathway and its resulting metabolites, the sphingolipids, have been suggested to be involved in the signaling pathway. In earlier studies we presented data which indicated that sphingosine (Sph) itself was increased during apoptosis induced by phorbol myristate acetate (PMA) in HL60 cells and tumor necrosis factor (TNF) in neutrophils, and when added exogenously was able to induce apoptosis. We report here that Sph and its methylated derivative N,N,-dimethylsphingosine (DMS) are able to induce apoptosis in cancer cells of both hematopoietic and carcinoma origin. In human leukemic cell lines CMK-7, HL60 and U937, treatment with 20 microM Sph for 6 hr caused apoptosis in up to 90% of cells. Human colonic carcinoma cells HT29, HRT18, MKN74 and COLO205 were shown to be more susceptible to apoptosis upon addition of DMS (>50%) than of Sph (<50%), yet were weakly or not sensitive to N,N,N-trimethylsphingosine (TMS). Under the same conditions, in the presence of serum, neither Sph-1-phosphate nor ceramide analogues C2-, C6- or C8-ceramide were able to induce apoptosis in any cell lines. However, in the absence of serum, ceramide analogues induced apoptosis in leukemia cell lines after 18 hr, yet much less so than Sph or DMS. Furthermore, apoptosis induced by Sph or DMS could not be inhibited by the ceramide synthase inhibitor fumonisin B1. Apoptosis was not induced by sphingolipids in primary culture cells, such as HUVEC or rat mesangial cells, but was apparent in transformed rat mesangial cells. Additionally, apoptosis induced by Sph, DMS or C2Cer was inhibited by protease inhibitors. Our data further support the evidence that the catabolic pathway of sphingomyelin involving Sph and other metabolites is an integral part of the apoptosis pathway.

Animals↗
PubMed1996

Regulatory role of ceramide in interleukin (IL)-1 beta-induced E-selectin expression in human umbilical vein endothelial cells. Ceramide enhances IL-1 beta action, but is not sufficient for E-selectin expression.

Recent studies indicate that sphingolipids mediate several cellular processes. We assessed roles of sphingolipids in the regulation of E-selectin expression in human umbilical vein endothelial cells. All exogenously-added sphingolipids (sphingosine, C2-ceramide, sphingosine 1-phosphate, and N,N-dimethylsphingosine) failed to induce E-selectin expression by themselves. C2-ceramide at 5 micron enhanced interleukin-1 beta (IL-1 beta)-induced E-selectin expression 2.7-fold, whereas other sphingolipids tested had no effects on this process. Sphingomyelinase, but not phospholipases A2, C, or D, mimicked the enhancing effect of C2-ceramide. Northern blot analyses revealed that C2-ceramide and sphingomyelinase increased interleukin-1 beta-induced E-selectin gene transcription levels. C2-ceramide and sphingomyelinase induced NF-kappaB activation by themselves and enhanced activation by IL-1 beta, which is essential for E-selectin expression. Immunological analyses with anti-NF-kappaB antibodies showed that subunit composition of NF-kappaB activated by IL-1 beta differs from that activated by C2-ceramide, suggesting that signaling pathways utilized by these stimuli may be different. Treatment with C2-ceramide or sphingomyelinase did not alter NF-ELAM1 specific binding activity. IL-1 beta induced sphingomyelin hydrolysis to ceramide; intracellular ceramide level increased to 182% of control value at 30 min. Taken together, these findings suggest that (i) sphingomyelin hydrolysis to ceramide does not trigger, but rather enhances cytokine-induced E-selectin expression, in part through NF-kappaB; (ii) sphingomyelin hydrolysis to ceramide does not mediate all the effects of IL-1 beta, although it may play important roles in IL-1 beta signal transduction in human umbilical vein endothelial cells.

Base Sequence↗
PubMed1995

N,N,N-trimethylsphingosine inhibits interleukin-1 beta-induced NF-kappa B activation and consequent E-selectin expression in human umbilical vein endothelial cells.

We examined the effect of N,N,N-trimethylsphingosine (TMS) on the interleukin-1 beta (IL-1 beta)-induced E-selectin expression in human umbilical vein endothelial cells (HUVEC). Incubation of HUVEC with TMS (0.1-10 microM) resulted in a concentration-dependent inhibition of IL-1 beta-induced E-selectin expression. Sphingosine or N,N-dimethylsphingosine had no effects on the expression. Electrophoretic mobility shift assay revealed that TMS inhibited IL-1 beta-induced NF-kappa B activation, which is essential for E-selectin expression. This inhibitory effect of TMS on IL-1 beta-dependent endothelial cell activation may partly explain the known anti-inflammatory or anti-metastatic effect of TMS in vivo.

Base Sequence↗
PubMed1995

Induction of apoptosis by sphingosine in human leukemic HL-60 cells: a possible endogenous modulator of apoptotic DNA fragmentation occurring during phorbol ester-induced differentiation.

The present studies were undertaken to characterize the potential role of sphingosine in the regulation of apoptosis in HL-60 promyelocytic leukemia cells. A 6-h exposure of HL-60 cells to sphingosine or its methylated derivative, N,N-dimethylsphingosine, caused internucleosomal DNA fragmentation and stereotypical morphological changes characteristic of apoptosis (i.e., cell shrinkage, nuclear condensation, and the formation of apoptotic bodies), as well as that to pharmacological inhibitors of protein kinase C such as 1-(5-isoquinolinesulfonyl)-2-methylpiperazine and staurosporine. Apoptosis by sphingosine and N,N-dimethylsphingosine was measured using a flow cytometric method. The percentages of apoptotic cells in cultures treated with sphingosine (10 microM) and N,N-dimethylsphingosine (10 microM) for 6 h were 55.6 +/- 7.8% and 84.2 +/- 11.6%, respectively. HL-60 cells were induced to differentiate toward macrophages by treatment with 5 nM 4 beta-phorbol 12-myristate 13-acetate (PMA). Internucleosomal DNA fragmentation, which was a hallmark of apoptosis, was first detected after 10-h exposure to PMA and increased with longer treatment. Sphingosine concentrations in the cells increased concomitantly with the increasing proportion of apoptotic cells during cell differentiation. Sphingosine level in HL-60 cells differentiated by treatment with PMA for 48 h was about 3.3-fold greater than that in untreated cells. Differentiated HL-60 cells exhibited a markedly increased conversion of exogenously added [3H]ceramide to [3H]sphingosine, indicating elevation of ceramidase activity. Moreover, exposure to sphingosine resulted in down-regulation of c-myc mRNA. These observations suggest the possible role of sphingosine in induction of apoptotic DNA fragmentation during PMA-induced differentiation in myeloid leukemia cells. Sphingosine may function as an endogenous modulator mediating the apoptotic signal.

Apoptosis↗