Hisayuki Fukutomi

RGM1, A CELL LINE DERIVED FROM NORMAL GASTRIC MUCOSA OF RAT

The epithelium of gastric mucosa undergoes constant renewal. However, the mechanisms of regulation of epithelial cell proliferation and differentiation in the gastric mucosa are poorly understood. Many investigators have used primary cultures of gastric mucosal cells to investigate cell physiology of gastric mucosal epithelium (5,8,14). Because this technique resulted in mixed cell populations of the stomach (5,15), the results may be influenced by complex cell-tocell interactions and are difficult to interpret. For this reason, a cell line made of a single type of cells is more ideal for precise investigation of proliferation or differentiation of gastric mucosal cells. However, cell lines from normal gastric mucosa have not been established. Recently, Matsui and Ohno (in preparation) established a new cell line, RGM1 (rat gastric mucosal cell first), from a rat glandular stomach by a method modified from Matsuoka et ah (7). In brief, the stomach was harvested from an anesthetized Wistar rat aged 4 wk and inverted. After washing the mucosa with phosphate-buffered saline (PBS) at 4 ~ C, the inverted pouch was immersed in a 0.2% pronase E solution at 37 ~ C. The solution was changed every 15 rain and centrifuged to collect the exfoliated gastric cells. Thereafter, the c.ells were washed twice with PBS, and cultivated in a 1:1 mixture of Dulbeccos modified Eagles medium (DMEM) and Hams F-12 medium supplemented with 20% fetal calf serum (FCS). When the cells were passaged to the 10th generation, this cell line was named RGM1. We examined character and usefulness of RGM1 cells using 3 0 4 0 times passaged cells. RGM1 cells are homogeneous epitheliallike cells with large oval nuclei and a polygonal shape (Fig. 1 A). They grow as monolayers of closely opposed cells, and many granules are visible in the cytoplasm. These cells were stained with periodic acid-Schiff (PAS) reagent and were negative for Bowie staining for chief cells (3) and succinic dehydrogenase activity for parietal cells (9). The cells were positive for cytokeratin in cytoplasm by immunohistochemistry. Transmission electron microscopy demonstrated that RGM1 cells had some microvilli on the surface. In the cytoplasm, a few homogeneous, dense cytoplasmic granules, presumably secretory granules, were observed. Junctional complexes were seen at the cell boundaries (Fig. 1 B and C). These data indicate that RGM 1 cells are epithelial in origin and like gastric mucous epithelial cells or mucous neck cells. RGM1 cells grew in an exponential manner with a population doubling time of 15.7 h, with saturation density of 1.97 + 0.38 X

Transforming growth factor-alpha (TGF alpha)-producing gastric carcinoma with acanthosis nigricans: an endocrine effect of TGF alpha in the pathogenesis of cutaneous paraneoplastic syndrome and epithelial hyperplasia of the esophagus.

A case of well-differentiated adenocarcinoma (Borrmann type 3) of the stomach in a 76-year-old man associated with the typical skin manifestations of acanthosis nigricans and with multiple protruding lesions showing epithelial hyperplasia of the esophagus is reported. The advanced tumor was located in the cardiac region of the stomach, and measured approximately 8cm in diameter, with partial invasion to the esophagus. The associated cutaneous lesions were characterized by hyperpigmentation and by protruding verrucous papules on the torso, head, face, neck, upper extremities, perineum, and inguinal region. Histologically, the protruding skin lesions showed keratinocytes proliferation throughout the epidermis, resulting in diffhyperkeratosis, papillomatosis, and acanthosis of the skin. Immunohistological analysis showed coexpression of transforming growth factor alpha (TGF-a) and epidermal growth factor (EGF) receptors in the tumor from the stomach. It is reasonable to conclude from this evidence that gastric carcinoma cells secrete TGF α in an autocrine for auto-stimulation. EGF receptor expression was also noted on the papillomatous hyperplasia of the cutaneous lesion. Serum level of TGF α, determined by an enzyme-linked immunosorbent assay, was high (144pg/ml; normal, 22.0 ±16pg/ml (Mean±SD)). Serum TGF α abruptly decreased to 49pg/ml on day 7 after the total gastrectomy, and then gradually increased to 77pg/ml within 28 days. Amelioration of the cutaneous lesions and the protruding lesions in the esophagus was observed after surgical resection of the gastric carcinoma. This suggests that the TGF α stimulates the proliferation of keratinocytes involved with EGF receptor. Large amounts of circulating TGF α in the blood over a long period released by the primary tumor seem to act as an endocrine-like mechanism causing epidermal and esophageal epithelial cells to proliferate. There is a possible link in the pathogenesis of the acanthosis nigricans as a cutaneous paraneoplastic syndrome, and epithelial hyperplasia of the esophagus.

Endoscopic classification of chronic gastritis based on a pilot study by the research society for gastritis

Background: Various types of classification of gastritis have been proposed, but no plausible classification has been available until now. The Research Society for Gastritis performed a pilot study to establish an endoscopic classification, taking into consideration the following: (i) ease of use; (ii) permitting everyone the common image; and (iii) presence of histopathological evidence.

NATURAL HISTORY OF A SPONTANEOUS DISSECTING ANEURYSM OF THE PROXIMAL SUPERIOR MESENTERIC ARTERY : REPORT OF A CASE

We report herein the case of a 44-year-old man in whom an asymptomatic dissecting aneurysm was found in the proximal part of the superior mesenteric artery (SMA) during a preoperative evaluation for colon cancer. The patient was managed conservatively with blood pressure control during the perioperative period of the colon resection as the false lumen of the dissecting aneurysm was revealed to be completely occluded by thrombus. The thrombus in the false lumen continued to be absorbed until 1 month after surgery. The patient is currently well 4 years after his operation without any evidence of recurrence of the aneurysm.

Bosentan, a novel synthetic mixed-type endothelin receptor antagonist, attenuates acute gastric mucosal lesions induced by indomethacin and HCl in the rat: Role of endogenous endothelin-1

Endothelin-1 has been reported to be responsible for gastric mucosal damage in various experimental models. We evaluated the role of endogenous endothelin-1 in the pathogenesis of gastric mucosal damage induced by indomethacin and HCl in the rat. Rats were given indomethacin (25 mg/kg) subcutaneously, and 15 min later, 0.2N HCl intragastrically. Gastric mucosal damage, gastric endogenous endothelin-1, and gastric mucosal hemodynamics were measured. The effects of bosentan, a mixed endothelin receptor antagonist, on gastric mucosal integrity and hemodynamics were assessed. Gastric endogenous endothelin-1 was significantly elevated at 20min, gastric mucosal blood flow began to decrease significantly at 25 min, and gastric damage occupied 52.2% of the total glandular mucosa at 135 min after injection of indomethacin. Intragastric pretreatment with bosenthan (5, 10, 30, and 60 mg/kg) significantly attenuated gastric damage, to 26.1%, 7.7%, 3.6%, and 1.6%, respectively, of the total glandular mucosa. Bosentan (60 mg/kg) prevented the initial decrease of blood flow and, even at 135 min, improved blood flow and hemoglobin oxygen saturation significantly. We suggest that indomethacin-induced endogenous endothelin-1 diminishes gastric mucosal blood flow and tissue oxygenation and ultimately causes gastric damage. Endogenous endothelin-1 may play an important role in the pathogenesis of the acute gastric mucosal lesions induced by indomethacin and HCl.

Gastric mucosal injury induced by local ischemia-reperfusion in rats : Role of endogenous endothelin-1 and free radical

We investigated the role of an endogenousvasoconstrictor peptide endothelin-1 (ET-1) and freeradicals in local gastric ischemia-reperfusion injury inrats. Local gastric ischemia was induced by clamping the left gastric artery for 15 min andreperfusion was done for 10-30 min in the presence of150 mM exogenous HCl intragastrically. Local gastricischemia and reperfusion resulted in significantmacroscopic and microscopic gastric mucosal damage togetherwith elevation of gastric tissue ET-1 concentration.Gastric tissue ET-1 was found to increase after 15 minof ischemia alone and also with 30 min of reperfusion. A novel nonpeptide endothelin receptorantagonist, bosentan, or a combination of radicalscavengers (superoxide dismutase, catalase, anddeferoxamine) both attenuated gastric mucosal injury.However, the greater protection observed with bosentan thanwith radical scavengers might reflect a preferentialrole of endothelin-1 in this type of injury.

Immunocytochemical localization of cathepsins B, H, and L in the rat gastro-duodenal mucosa

SummaryCathepsins B, H, and L are representative cysteine proteinases in lysosomes of a large variety of cells. Previous immunochemical studies indicated the presence of these enzymes also in the gastrointestinal wall. Using specific antisera, the cellular and subcellular distribution of cathepsins B, H, and L in rat gastric (oxyntic and pyloric part) and duodenal mucosa was investigated by light and electron microscopical immunocytochemistry. The subtypes of cathepsins were distributed differently in the cellular constituents of the epithelia: Cathepsin B was localized to lysosomes of all cells except goblet cells. Cathepsin H was found predominantly in gastric parietal cells (lysosomes) and in secretion granules of pyloric gastrin and duodenal cholecystokinin cells. Cathepsin L immunoreactivities were weak and restricted to a minority of cells (gastric mucous cells, enterocytes). Interstitial cells of the lamina propria immunoreactive for cathepsins H and L were identified as macrophages. The present findings suggest a dual function of cathepsins in the gastro-duodenal mucosa. They (1) cleave enzymatically proteins and peptides ingested in lysosomes, and (2) they may be involved in the processing of biologically active peptides (enteric hormones) from their precursor proteins.

Phosphoramidon, an endothelin converting enzyme inhibitor attenuates local gastric ischemia-reperfusion injury in rats

Recently increased production of endothelin-1 has been implicated in the pathogenesis of gastric ischemia-reperfusion injury. We have investigated the effects of endothelin converting enzyme inhibition on local gastric ischemia-reperfusion injury in rats by using two metalloprotease inhibitors, phosphoramidon and thiorphan. In presence of exogenous 0.15M HCI intragastrically, local ischemia was induced by the clamping of left gastric artery for 15 min and reperfusion was done for 30 min. In separate group of rats, phosphoramidon (10-60 mg/kg) or thiorphan (60 mg/kg) were given as i.v. bolus injection immediately before the induction of ischemia. Phosphoramidon dose dependently attenuated the macroscopic and microscopic mucosal injuries while thiorphan did not. These results indicate that phosphoramidon-sensitive endothelin converting enzyme activity is highly present in stomach and phosphoramidon, by inhibiting the conversion of big endothelin-1 to endothelin-1 attenuated the gastric mucosal damage in this model.

Bosentan antagonizes the effects of endothelin-1 on rat gastric blood flow and mucosal integrity

Bosentan, a new type of orally effective, mixed (ETA+ETB) endothelin receptor antagonist has been recently introduced and tested in a variety of experimental models. We studied the effect of bosentan on the changes in gastric mucosal hemodynamics and mucosal integrity, induced by the exogenous application of endothelin-1, in rats. Bosentan (10 mg/kg iv) pretreated rats were injected with endothelin-1 (500-1000-2000 pmol/kg, iv) and gastric mucosal hemodynamics were monitored. After combined oral (30 mg/kg) and systemic pretreatment with bosentan we studied the effects of submucosal injection of endothelin-1 (50 pmol) on blood flow and gastric mucosa. Bosentan antagonized the vasodilator, vasoconstrictor and ulcerogenic effects of endothelin-1 in the rat gastric mucosa. These results show that bosentan can be a useful probe in the study of endogenous endothelin in the gastrointestinal tract.

Venous tumor thrombosis and cavernous transformation of the portal vein in a patient with gastric carcinoma

A case of extensive extra-and intrahepatic portal tumor thrombosis, with no metastatic foci in liver parenchyma, secondary to advanced gastric carcinoma in a 69-year-old man is reported. The portal tumor thrombosis was characterized by enlargement of the thrombosed segment of the vein, decreased density mass without intraluminal enhancement of the involved vein, nonvisualization of the portal venous branch in the involved lobe, and the so-called cavernous transformation of the portal vein. The surgically resected gastric specimen showed Borrmann type 3 advanced papillary adenocarcinoma. The portal tumor thrombus is presumed to have arisen from vascular invasion in the primary foci of gastric carcinoma, and then to have permeated the portal vein without invasion of liver parenchyma.