Masafumi Wasa

Determinants of glutamine dependence and utilization by normal and tumor‐derived breast cell lines

A continual supply of the amino acid glutamine (GLN) may be necessary for cancerous cell growth. GLN plays a central role in multiple metabolic pathways and has long been considered an essential component of tissue culture media. However, the GLN requirements of tumor cell lines and the factors that determine a cells need for GLN have not been comprehensively studied. Also, it remains unclear how various metabolic pathways contribute to GLN consumption. In the present study, possible determinants of GLN metabolism were examined in seven breast cell lines, two derived from immortalized normal tissue and five of tumor origin. These cells exhibited different dependencies on media GLN concentration for growth and a wide range of GLN utilization rates. GLN uptake was facilitated by a single, common transporter functionally defined as System ASC. However, the affinities for GLN exhibited by this transporter differed appreciably between cell lines. Furthermore, the concentration at which media GLN became a limiting factor for cellular proliferation correlated with transporter affinity. The origin of the cell lines was not a determinant of GLN metabolism because immortalized cells of nontumor origin exhibited GLN dependence and utilization rates comparable to those of tumor‐derived cells. The rates of CO2 production from GLN were similar for each cell lines. Rates of GLN disappearance and glutamate appearance in media were strongly correlated, with 32–80% of media GLN converted to glutamate. Both rates were directly affected by media cystine concentration, suggesting that a large portion of glutamate efflux was coupled with cystine import through the amino acid transport system X  −C These results demonstrated that cell growth is a function of GLN influx and suggest that GLN is used to supply glutamate and cystine, perhaps for glutathione synthesis. J. Cell. Physiol. 176:166–178, 1998.

Glutamine as a regulator of DNA and protein biosynthesis in human solid tumor cell lines.

OBJECTIVE The transport of glutamine by six different human solid tumor-derived cell lines (e.g., breast, colon, liver) was characterized and the impact of glutamine deprivation on rates of tumor cell proliferation and DNA and protein synthesis was assayed. SUMMARY BACKGROUND DATA Glutamine is added routinely to cell culture media and its importance for cellular growth has been established. However, carrier-mediated glutamine transport by solid tumors has not been studied extensively, and the mechanisms by which glutamine contributes to cell growth regulation require further investigation. METHODS In a panel of different human solid tumor-derived cells, sodium-dependent glutamine transport was characterized in vitro and rates of cell proliferation, protein and DNA synthesis, as well as thymidine transport, were correlated with glutamine concentrations in the culture media. RESULTS In all cells, regardless of tissue origin, sodium-dependent glutamine transport was mediated almost exclusively by a single carrier. There was a range of Michaelis constants (Km) and maximal transport velocities (Vmax) for the glutamine transporter in each cell type, but the amino acid inhibition profiles were nearly identical, consistent with uptake by the System ASC family of transporters. Rates of cell growth, DNA and protein synthesis, and thymidine transport correlated with the glutamine concentration in the culture media, indicating the central role of this amino acid in regulating cellular proliferation. CONCLUSIONS These data indicate that glutamine transport by all solid tumors is mediated by the System ASC family of transporters. The variation in Km values suggests that some cancers may be better suited to survive in a low glutamine environment than others. The mechanism by which glutamine supports cell proliferation and regulates cell cycle kinetics involves its modulation of DNA and protein biosynthetic rates.

Alanyl-Glutamine-Supplemented Parenteral Nutrition Increases Luminal Mucus Gel and Decreases Permeability in the Rat Small Intestine

BACKGROUND Effect of supplemental alanyl-glutamine in standard TPN (S-TPN) on luminal mucus gel and small intestinal permeability was investigated. METHODS Thirty Sprague-Dawley rats were divided into group I (n = 10), receiving standard rat diet; group II (n = 10), receiving S-TPN; and group III (n = 10), receiving alanyl-glutamine-supplemented TPN for 1 week. After 1 week, fluorescein isothiocyanate (FITC)-dextran was injected into the small intestine of the rats, and they were killed. A small intestinal sample and portal blood were obtained for morphologic and functional analysis of mucus gel and intestinal permeability. RESULTS In group II, thickness and optical density of mucus gel per millimeter serosal length of intestine were significantly lower than group I (p<.001) and were significantly higher in group III than in group II (p<.001). The number of goblet cells in the villi and in the crypt of the small intestine was significantly lower in group II than in group I (p<.001) and was significantly higher in group III than in group II (p<.001), with the exception of the villi of jejunum. Villous and crypt surface area per millimeter serosal length of intestine was significantly lower in group II than in group I (p<.001) and was significantly higher in group III than in group II (p<.001). Small intestinal permeability to FITC-dextran was significantly higher in group II than in group I (p<.001) and was significantly lower in group III than in group II (p<.001). Glucosamine synthetase level was significantly higher in group III than in group I and ileum of group II (p<.001). CONCLUSIONS Alanyl-glutamine-supplemented TPN prevents a decrease in mucus gel and an increase in small intestinal permeability associated with S-TPN.

Copper deficiency with pancytopenia during total parenteral nutrition.

Anemia and neutropenia are commonly observed hematologic changes in patients with copper (Cu) deficiency, but thrombocytopenia is rarely found. A-69-year-old patient with postoperative small-bowel obstruction underwent laparotomy three times. Because of persistent obstruction, nasoduodenal suction was continued and total parenteral nutrition was instituted. Fifteen months after the initiation of total parenteral nutrition, the patient gradually developed pancytopenia (red blood cell count 222 x 10(4)/mm3, neutrophil count 1254/mm3, and platelet count 9.2 x 10(4)/mm3). The serum Cu level was 10 micrograms/dL and the serum ceruloplasmin level was less than 5 mg/dL. After 2 weeks of Cu supplementation in a daily dose of 20 mumol, the serum Cu level increased to 81 micrograms/dL and the serum ceruloplasmin level to 20 mg/dL. Hematologic values showed a dramatic response: red blood cell count increased to 362 x 10(4)/mm3, neutrophil count to 4819/mm3, and platelet count to 22.1 x 10(4)/mm3. The improvement of pancytopenia could be attributed to Cu supplementation. This is the first case report of Cu deficiency with pancytopenia during total parenteral nutrition.

Total parenteral nutrition decreases luminal mucous gel and increases permeability of small intestine.

The distribution of fluorescein isothiocyanate dextran 70,000 (FITC-dextran) and mucous gel across the lumen of small intestine was observed as an investigation into the role of mucous gel on permeability in total parenteral nutrition (TPN). Thirty-two rats were randomly divided into two groups fed with either TPN or oral rat food. On day 4 or 7, FITC-dextran (750 mg/kg body weight) was given through the gastroduodenal tube. After 1 hour, blood samples were taken by aortic puncture to analyze plasma FITC-dextran by fluorescence spectrometry. Samples of small intestine with luminal contents were frozen and sectioned in a cryostat for fluorescence microscopy; the same sections were placed in a 0.2% celloidin solution for 3 minutes to preserve mucous gel and stained by periodic acid-Schiff reaction for light microscopy. The plasma level of FITC-dextran after 1 hour of this marker injection showed a significant increase (p < .01) in the TPN group compared with the rat food group on days 4 and 7. Morphologic findings on days 4 and 7 were similar in both the jejunum and ileum: The mucous gel filled the spaces between villi and FITC-dextran centered in the lumen in the rat food group, whereas the mucous gel decreased and FITC-dextran filled the spaces between villi in the TPN group. FITC-dextran and mucous gel showed complementary distributions in both groups. These data suggest that TPN decreases luminal mucous gel and increases permeability of small intestine in rats.

TNF-stimulated arginine transport by human vascular endothelium requires activation of protein kinase C

OBJECTIVE The authors determined the endothelial arginine transport mechanism and the potential role of a tumor necrosis factor (TNF)-alpha-mediated signal transduction pathway involving protein kinase C (PKC) in regulating this transport in cultured endothelial cells. SUMMARY BACKGROUND DATA The vascular endothelium metabolizes arginine to generate nitric oxide (NO), and an increase in NO production can be stimulated by several cytokines. The mechanism(s) responsible for the accelerated arginine transport are poorly understood. METHODS Arginine transport was assayed in confluent human umbilical vein endothelial cells in the presence of TNF +/- the PKC inhibitor chelerythrine chloride. RESULTS Carrier-mediated arginine transport was accomplished by two Na(+)-independent transporters, System y+ (80% of total transport) and System b0,+ (20% of transport). Tumor necrosis factor (0.1-2 ng/mL) increased System y(+)-mediated arginine transport in a time- and dose-dependent manner by augmenting System y+ transport maximal capacity (control Vmax = 1325 +/- 60 pmol/mg protein/minute vs. TNF Vmax = 3015 +/- 110 pmol/mg protein/minute, p < 0.01) without affecting transporter affinity (control Km = 30 +/- 1.4 microM vs. 34 +/- 1.3 microM arginine, p = NS). Stimulation was maximal at the 8-hour time point and was inhibited by both actinomycin D and cycloheximide. In addition, inhibition of PKC with chelerythrine abrogated the TNF-augmented arginine transport. Similarly, incubation of cells with the direct PKC activator TPA (phorbol ester 12-myristate 13-acetate) stimulated System y(+)-mediated arginine transport nearly fivefold, secondary to an increase in transporter Vmax (TPA Vmax = 5349 +/- 310 pmol/mg protein/minute, p < 0.001 vs. control), with no change in Km. This TPA-induced stimulation of arginine transport also was blocked by chelerythrine CI, actinomycin D, and cycloheximide. Incubation of TNF-stimulated cells with two NO synthase inhibitors did not reduce transport activity, suggesting that the arginine transporter and the NO synthase enzyme may, in part, be independently regulated.

Endoscopic variceal ligation in the management of gastroesophageal varices in postoperative biliary atresia

BACKGROUND/PURPOSE Gastroesophageal variceal bleeding is a serious and difficult problem in the long-term management of biliary atresia (BA). Recently, endoscopic approaches have been attempted to manage this problem. The authors have attempted endoscopic variceal ligation (EVL), a less invasive procedure than endoscopic sclerotherapy. METHODS In the past 5 years, 66 EVL procedures using standard flexible endoscope with a diameter of 9 mm (type p-30, XQ200, or XQ240; Olympus, Tokyo, Japan) were performed in 30 separate sessions on 11 postoperative BA patients. The mean age of the children was 7.8 (range, 3 to 15) years. The EVL device was a small elastic O-ring or a loop ligator. RESULTS EVL was performed for emergency hemostasis in two patients and prophylaxis for impending rupture in nine with large, blue varices, or with red spots on the variceal surface. During the initial procedure, all varices were ligated successfully, and reduction in size was noted. Of eight patients who were examined 7 to 14 days after treatment, seven (87.5%) had improved. Eight of 11 patients (72.7%) were finally cured or at least had improved after one to seven sessions of EVL. However, three patients did not show improvement after four to seven sessions because of the reappearance of the varices, development of distal lesions such as gastric varices, and acute gastric mucosal lesions. A technical complication encountered was a slippage of the O-ring in one patient. A technical difficulty was seen in ligating the giant gastric varix in one patient. There was no deterioration of liver function induced by EVL in this entire series. CONCLUSIONS EVL is an effective and feasible treatment of gastroesophageal varices in postoperative BA patients. However, reappearance or reactivation of the varices or emergence of the more distal lesions is likely to occur even after repeated EVL.

Prevention of Catheter-Related Sepsis During Parenteral Nutrition: Effect of a NeW Connection Device

A prospective study was carried out to determine the clinical effect of a newly devised catheter connection method (I system) and piggyback access system. Previous studies have demonstrated that the I system avoided bacterial contamination in vitro during tubing change that Luer-Lock connectors did not. The purpose of this study was to investigate the ability of this device coupled with a new closed-system piggyback technique for multipurpose access to reduce catheter-related sepsis in clinical practice. Two hundred and thirty patients receiving total parenteral nutrition were divided into two groups. Group I (n = 106) used the I system connector and group L (n = 124) used a Luer-Lock connector. Catheters in both groups were used for multipurpose access for infusion and blood sampling. In group L, a three-way stopcock and/or pig-gyback system was used for multiple access. In group I, a newly designed closed-system piggyback was used. The incidence of catheter-related sepsis was significantly lower in group I (1.89%/catheter) than in group L (12.10%/catheter) (p < .01, chi 2 analysis), and the average duration of use of each catheter was significantly longer in group I than in Group L (p < .01 by generalized Wilcoxon test). The results of this clinical study suggest that the newly designed connection method and piggyback access system are able to reduce catheter-related sepsis.

EXPRESSION OF ENDOTHELIAL CONSTITUTIVE NITRIC OXIDE SYNTHASE mRNA IN GASTROINTESTINAL MUCOSA AND ITS DOWNREGULATION BY ENDOTOXIN

Endothelial nitric oxide exerts local vasodilatory actions in the gastrointestinal (GI) microvasculature and is proposed to play a role in enteric vasomotor regulation. The aims of this study were to characterize the tissue distribution of mRNA for endothelial nitric oxide synthase (NOS-III) and to examine its response to endotoxin challenge in vivo. We demonstrate the expression of NOS-III mRNA and protein in mucosa throughout the gastrointestinal tract and show for the first time that NOS-III mRNA expression in the GI mucosa was down-regulated in the rats treated with endotoxin. The ubiquitous expression of NOS-III mRNA in digestive tissues is consistent with the proposed role of NOS-III in the physiology of the gastrointestinal tract. The decreased NOS-III mRNA, in parallel to induction of inducible NOS (NOS-II) mRNA, may contribute to the impaired endothelium-dependent relaxation and damaged mucosal integrity during sepsis.

Beneficial effects of growth hormone combined with parenteral nutrition in the management of inflammatory bowel disease: an experimental study.

BACKGROUND Growth hormone (GH) improves net protein anabolism and stimulates wound healing. Although GH is also known to exert the trophic effect on the intestinal tract, its role in the healing of intestinal ulceration is not known. The aim of this study was to evaluate the effects of exogenous GH coinfused with parenteral nutrition (PN) in an experimental model of inflammatory bowel disease in rats. METHODS All rats underwent central venous cannulation and were randomized to two groups after induction of small intestinal ulceration with indomethacin. Both groups received the same PN formula. In addition, the GH group (n = 10) received subcutaneous injections of human GH at a dose of 1.0 IU/kg daily for 4 days, whereas the control group (n = 10) received injections of normal saline solution. Nitrogen balance, macroscopic inflammation score, intestinal myeloperoxidase activity, DNA content, and mucosal permeability were determined for each rat. Insulin-like growth factor-I (IGF-I) mRNA was detected by reverse transcription and polymerase chain reaction. RESULTS Administration of GH significantly improved the cumulative nitrogen balance, ameliorated the gross inflammation score, and decreased intestinal myeloperoxidase activity. Similarly, intestinal permeability was significantly decreased in the GH group as compared with the control group. GH treatment resulted in increased plasma concentration of IGF-I and IGF-I mRNA expressions in both the liver and the small intestine compared with those in the control group. CONCLUSIONS Exogenous GH plays an important role in accelerating intestinal healing in an experimental model of small bowel ulceration in rats. The mechanisms may include the stimulated IGF-I production, which thereafter augments intestinal epithelial cell growth.