Yoshikazu Yonei

Role in nitric oxide in Kupffer cell–mediated hepatoma cell cytotoxicity in vitro and ex vivo

The metabolic change in tumor cells (AH70, a rat hepatoma cell line) cocultured with isolated rat Kupffer cells were visualized and analyzed by a laser scanning confocal imaging system. When AH70 cells were cocultured with Kupffer cells, fluorescence intensity of rhodamine 123 (Rh123) decreased, indicating the reduction of mitochondrial function. The reduction in Rh123 was eliminated by NG-monomethyl-L-arginine (L-NMMA), an analogue of L-arginine, suggesting the involvement of nitric oxide (NO). Two hour after the cells were cocultured, membrane compromised AH70 cells which were observed as propidium from 2.8% to 25%. This increase was also attenuated by L-NMMA, suggesting that Kupffer cell-mediated injury of tumor cells largely depends on NO. The concentrations of NO-2 + NO-3 in the culture medium markedly increased after coculture of AH70 cells with Kupffer cells. Moreover, NO synthase (NOS) activity in Kupffer cells significantly increased after coculture. These in vitro results suggest that NO mediates Kupffer cell-induced tumor cell damage characterized by reduced mitochondrial function and diminished barrier function. In the ex vivo study of the perfused liver to which AH70 cells were injected via the catheter inserted into the portal vein, some AH70 cells were arrested in the upper stream of sinusoid and the fluorescence intensity of Rh123 in adherent AH70 cells decreased in a time-dependent manner within 2 hours. The number of PI-positive AH70 cells also increased 2 hours after the injection of AH70 cells. These changes were inhibited by either administration of N omega-L-nitroarginine-methylester (L-NAME) to perfusate or pretreatment of the rat liver with GdCl3, which is known to deplete Kupffer cell function. Thus, the present study suggests that NO from Kupffer cells induces mitochondrial dysfunction in tumor cells followed by membrane barrier dysfunction in the liver sinusoid.

Muscarinic acetylcholine receptors in rat gastric mucosa

SummaryThe muscarinic cholinergic innervation of the rat gastric mucosa was investigated by localizing the muscarinic receptors using a tritiated muscarinic antagonist, pirenzepine. Radioautography was performed by freeze drying stomach tissue, which was then embedded in Epon and wet sectioned with ethylene glycol, and dry mounting on emulsion film by the wire-loop method to prevent loss of the labelled substance during fixation and the radioautographic procedure. Light and electron microscopy showed that the specific pirenzepine-binding sites were localized predominantly on parietal cells, chief cells and perivascular plexuses. Analysis of the grain distribution on parietal cells revealed that the silver grains corresponding to the pirenzepine-binding sites were mainly on the basolateral plasma membrane. On the other hand, the surface mucous or mucous neck cells had few pirenzepine-binding sites.

Rat Kupffer cell-derived nitric oxide suppresses proliferation and induces apoptosis of syngeneic hepatoma cells

BACKGROUND & AIMSnEvidence increasingly indicates that nitric oxide plays an important role in antitumor mechanisms. The aim of this study was to investigate the role of NO in the mechanisms regulating the proliferation and death of hepatoma cells cocultured with Kupffer cells.nnnMETHODSnKupffer cells were isolated from male Wistar rats and cocultured with rat hepatoma AH70 cells. Proliferation was determined by calculating the number of total and 5-bromodeoxyuridine-positive AH70 cells. Apoptosis was assessed by electron-microscopic and fluorescence-microscopic observations and in situ nick end labeling method. Immunofluorescence and in situ hybridization studies were performed to investigate the induction of inducible NO synthase (iNOS).nnnRESULTSnKupffer cells reduced proliferation and induced apoptosis of AH70 cells, which were attenuated by the NO synthesis inhibitors NG-monomethyl-L-arginine and aminoguanidine. Increased inductions of iNOS messenger RNA and iNOS were observed in Kupffer cells cocultured with AH70 cells. Addition of monoclonal antibody directed against either rat CD18 or intercellular adhesion molecule 1 also attenuated the increased NO production of Kupffer cells and the alterations of AH70 cells.nnnCONCLUSIONSnKupffer cell-derived NO suppresses proliferation and induces apoptosis of hepatoma cells. The CD18 intercellular adhesion molecule 1-dependent adhesive interaction with hepatoma cells triggers NO production by Kupffer cells.

Autoradiographic demonstration of gastrin binding sites in rat gastric mucosa.

The location of 125I-iodotyrosyl gastrin I binding sites in rat gastric mucosa was studied. Peptide specificity was demonstrated by competitive binding studies through the addition of a large dose of cold human gastrin I or cholecystokinin-octapeptide. Autoradiography of the stomach tissue was carried out by freeze-drying, embedding in Epon, wet-sectioning with ethylene glycol, and dry-mounting the emulsion film by means of the wire-loop method to prevent loss of the labeled substance. Specific binding sites for gastrin were found on parietal and chief cells, whereas few binding sites were seen on the surface mucous or mucous neck cells. Binding sites on the parietal cells were dispersed in the cytoplasm, while those on the chief cells were found near the basal plasma membrane.

Nitric oxide mediates mitochondrial dysfunction in hepatoma cells induced by non-activated Kupffer cells: Evidence implicating ICAM-1-dependent process

The metabolic changes in a rat hepatoma cell line, AH70 cells, after co‐culture with rat Kupffer cells (KC) were visualized and analysed using a fluorescence microscope equipped with a silicon intensified target camera and a laser scanning confocal microscopic system. Kupffer cells were isolated from male Wistar rats, and cultured without any stimuli. The non‐activated KC reduced the mitochondrial energization in the cocultured AH70 cells within 2 h, which was indicated by decreased rhodamine 123 (Rh123) fluorescence. Either Ng‐monomethyl‐l‐arginine or dexamethasone significantly attenuated the KC‐induced mitochondrial dysfunction in AH70 cells, suggesting the involvement of nitric oxide (NO) derived from inducible‐type nitric oxide synthase (iNOS). Administration of monoclonal antibody (mAb) directed against rat ICAM‐1 also prevented the decrease in Rh123 fluorescence. Electron microscopy revealed that the membrane‐to‐membrane attachment between KC and AH70 cells occurred within 2 h. A laser scanning confocal microscopic observation using mAb against ICAM‐1 presented that the ICAM‐1 expression on AH70 cells and KC increased after the co‐culture. It is therefore concluded that the KC‐mediated mitochondrial dysfunction of hepatoma cells largely depends on NO production by iNOS. Furthermore, the present study supports a scenario that the NO production and release from KC is triggered by the close contact with hepatoma cells through adhesion molecules such as ICAM‐1.

Pathological findings of lymphangiectasia of the large intestine in a patient with protein-losing enteropathy

Lymphangiectasia of the large intestine associated with protein-losing enteropathy is reported. A 33-yr-old man suffered from diarrhea, sometimes mixed with blood. Colonoscopic study revealed reddish and edematous mucosa with multiple flat elevated lesions and giant folds in the localized segment of the rectosigmoid. An intestinal clearance test of alpha 1-antitrypsin revealed the association of protein-losing enteropathy. Operation was performed successfully, resulting in a marked improvement of symptoms and laboratory data, especially serum total protein, albumin, IgG, and Leu-2a-positive cells (suppressor/cytotoxic T cells). Giant folds consisted of the submucosal edema and hyperplasia of the epithelial glands with cystic dilatation of glands, and flat elevated lesions consisted of mucosal and submucosal edema associated with intestinal lymphangiectasia, adipose tissue, and blood capillaries. The population of the Leu-2a-positive cells in the lamina propria and intraepithelial layer was decreased and that of the Leu-3a-positive cells (helper/inducer T cells) in the lamina propria was increased.

CD18/ICAM-1-dependent nitric oxide production of Kupffer cells as a cause of mitochondrial dysfunction in hepatoma cells: Influence of chronic alcohol feeding

The present study was designed to monitor the process for hepatoma cell injury induced by Kupffer cells. The non-activated Kupffer cells isolated from male Wistar rats reduced the mitochondrial membrane potential in the cocultured AH70 cells, which was indicated by the decreased rhodamine 123 (Rh123) fluorescence. Increased level of nitrite and nitrate in the medium and induction of iNOS in Kupffer cells were observed after coculture with AH70 cells. Incubation with either NG-monomethyl-L-arginine or aminoguanidine attenuated the increased nitric oxide (NO) production of Kupffer cells and the decreased Rh123 fluorescence of AH70 cells. Fluo-3, a calcium-sensitive probe, fluorescence in Kupffer cells increased after coculture with AH70 cells. Addition of TMB-8, a calcium inhibitor, or monoclonal antibody directed against ICAM-1 or CD18 prevented the increases in fluo-3 fluorescence and NO production of Kupffer cells and Kupffer cell-induced mitochondrial dysfunction in AH70 cells, suggesting the involvement of calcium mobilization and CD18/ICAM-1. It is therefore suggested that the Kupffer cell-mediated mitochondrial dysfunction of hepatoma cells largely depends on NO production by iNOS, and that the NO production by Kupffer cells is triggered by CD18/ICAM-1-dependent interaction with hepatoma cells and subsequent calcium mobilization. In other series of experiments, male Wistar rats fed ethanol for 4 weeks were used. The NO production and calcium mobilization of Kupffer cells and reduction of the mitochondrial membrane potential in cocultured hepatoma cells were diminished in the case of Kupffer cells isolated from chronically ethanol-fed rats, while CD18 and ICAM-1 expression was still observed. Thus, the present study further suggests that NO-dependent anti-hepatoma cell activity of Kupffer cells is suppressed in chronically ethanol-fed animals.

Alterations in Gastric Mucosal Microvascular Endothelium in a Stressed Condition-Relevance to Gastric Ulcerogenesis

The present paper describes the morphological and functional alterations of the gastric mucosal microvascular endothelium under restraint-stressed condition. On the basis of the direct cholinergic innervation of capillaries and non-muscular venules in the gastric mucosa, these endothelial changes would be caused by the stress-induced overstimulation of the cholinergic nerves and modified by the degranulation of mast cells, contributing to the stress-induced ulcer formation as schematically illustrated in Fig. 10.

A case of appendiceal mucocele showing massive mucous production with concomitant colonic cancer

SummaryA 58-year-old male patient presented with abnormal discharge of 200 ml transparent fecal mucus. Irregular protuberance of the ascending colon into the adjacent ileocecal region was observed by colonofiberscopy, and a cystic lesion in the ileocecal region was suggested by computerized axial tomography. Pale yellowish semitransparent jellied substances were observed exudating from the site of ileocecal resection. The diagnosis established was moderately differentiated colonic adenocarcinoma and a mucocele of the appendix. The mucus discharge disappeared after resection. Biochemical analysis of the mucus suggested the mucocele as a source of the discharged mucus. We report an extremely rare case of mucocele of the appendix that may be related to the cause of mucous stool.