Tadahisa Miyamoto

Experimental and clinical studies on epidermal growth factor for gastric mucosal protection and healing of gastric ulcers

In experimental studies, 0.6 N HCl-induced gastric mucosal injury was significantly severe in submandibularec-tomized rats (SMR rats) than that in either SMR rats receiving exogenous mouse EGF (SMR + EGF rats) or controls. This was also true in gastric injury induced by 0.4 N HCl under pretreatment with indomethacin to reduce gastric mucosal prostaglandins (PGs). Somatostatin (SLI), PGE2, and PAS-stained mucus in the corpus were significantly reduced in SMR rats in comparison to SMR + EGF and control rats. In clinical studies, salivary EGF secretion was much higher in peptic ulcer patients than healthy controls. β-Urogastrone was effective in the treatment of gastric ulcers. On the basis of experimental studies, we conclude that the protective effect of EGF on the gastric mucosa is, in part, mediated indirectly by increases in SLI, PGE2, and mucus production. However, endogenous, as well as exogenous, EGF has an important direct, cytoprotective effect on the gastric mucosa. From the clinical studies, we also conclude that salivary EGF secretion in ulcer patients increases in a homeostatic response to the presence of an ulcer, facilitating ulcer healing. Furthermore, we believe that β-urogastrone, human EGF, might prove to be an effective drug in the clinical treatment of gastric ulcers.

Role of PAF in Ischemia-Reperfusion Injury in the Rat Stomach

Ischemia-reperfusion induced extensive gastric mucosal injury and an increase in chemiluminescence activity of neutrophils obtained from the portal vein in hemorrhagic shock rats. CV-3988, a selective antagonist of platelet activating factor (PAF), significantly reduced the gross and histologic gastric damage, and the increase in chemiluminescence activity of neutrophils. These results suggest that PAF generated on hypoxia might stimulate oxygen radical production by neutrophils, resulting in the occurrence of gastric injury in hemorrhagic shock rats.

Nicotine stimulates pepsinogen secretion from guinea pig gastric chief cells in monolayer culture

We evaluated the effects of nicotine on pepsinogen secretion in vitro, using a monolayer culture system of guinea pig gastric chief cells. Pepsinogen secretion was increased by above 5 mM nicotine in a dose-dependent manner, as was the elevation of intracellular free calcium concentration ([Ca2+]i). The pepsinogen secretion stimulated by 10 mM nicotine was inhibited by above 1 mM d-tubocurarine, a nicotinic receptor antagonist, but not by same concentrations of scopolamine hydrobromide monohydrate or pirenzepine, a muscarinic receptor antagonist. The elevation of [Ca2+]i induced by 5 mM nicotine was also reduced by 10 mM d-tubocurarine, but not by 10 mM pirenzepine. A calmodulin inhibitor, N-(6-aminohexyl)-5-chloro-1-naphthalene-sulfonamide (W-7), at the concentration of 10(-6) M and a myosin light-chain kinase inhibitor, 1-(5-chloronaphthalene-1-sulfonyl)-1H-hexahydro-1,4-diazepine (ML-9), at concentrations above 10(-7) M also significantly blocked 10 mM nicotine-induced pepsinogen secretion. These finding indicate that nicotine directly stimulates pepsinogen secretion probably via nicotinic receptors on the gastric chief cells, and that the Ca(2+)-mediated messenger system, including calmodulin and myosin light-chain kinase, is involved in this event.

Mediation of pepsinogen secretion from guinea pig chief cells by Ca2+/calmodulin-dependent protein kinase II.

In the presence of Ca 2 ÷ bound to calmodulin, Ca 2 +/calmodulin-dependent protein kinase II (CaMK II) exhibits an intramolecular autophosphorylation and modulates many cell functions. In this study, the role of CaMK II in pepsinogen secretion was investigated in cultured guinea pig chief cells by using a specific CaMK II inhibitor, 1-[N,O-Bis(5-isoquinolinesulfonyl)-N-methyl-L-tyrosyl]-4-phenylpiperazine (KN-62), and an antibody for the Thr-286-autophospholylated a subunit of CaMK II which specifically recognized the autophosphorylated form of CaMK II. KN-62 inhibited the pepsinogen secretion stimulated by carbamylcholine chloride, cholecystokinin octapeptide, and ionomycin in a dose-dependent manner without affecting intracellular Ca 2+ concentrations, but had no effect on the secretion by 12-O-tetradecanoyl phorbol-13-acetate (TPA) and forskolin. Heavy staining with the antibody for autophosphorylated CaMK II was observed in the cytoplasm of chief cells treated with carbamylcholine chloride or ionomycin, but only light staining was seen in cells treated with TPA or forskolin. Thus, CaMK II and its autophosphorylation may be a critical step in the intracellular pathway by which Ca 2+ causes pepsinogen secretion from guinea pig chief cells.

Carcinoid of the esophagus located in lamina propria

Abstract: We report a carcinoid tumor in the mucosal layer of the esophagus of a 63-year-old man. Barium X-ray and endoscopy indicated the tumor to be a polypoid lesion in the lower esophagus.Endoscopic ultrasonography (EUS) demonstrated the lesion to be a sharply demarcated hyperechoic tumor in the mucosal layer. Biopsy yielded a diagnosis carcinoid of the esophagus. In the resected specimen of the esophagus, the tumor was 11 mm in longest dimension with a shallow depression on it smooth surface. Histologically, the tumor was located in the mucosal layer, as shown by EUS, and was composed of small round cells which were positive for argyrophil, but not argentaffine. Carcinoid tumor of the esophagus found at an early stage, and localized in the lamina propria layer, is very rare. The present case is the second report in Japan.

Role of myosin light-chain kinase and protein kinase C in pepsinogen secretion from guinea pig gastric chief cells in monolayer culture.

We evaluated the role of myosin light-chain kinase (MLCK) and protein kinase C (PKC) in pepsinogen secretion from guinea pig gastric chief cells using a monolayer culture system of chief cells and an enzyme immunoassay system for guinea pig pepsinogen. An MLCK inhibitor, 1-(5-chloronaphthalene-1-sulfonyl)-1H-hexahydro-1,4-diazepine (ML-9), significantly inhibited both the basal pepsinogen secretion and the secretion by carbamylcholine chloride (carbachol) or ionomycin without affecting intracellular free Ca2+ concentration ([Ca2+]i), but not by 12-O-tetradecanoylphorbol-13-acetate (TPA) or forskolin. A PKC inhibitor, 1-(5-isoquinolinesulfonyl)-2-methylpiperazine (H-7), significantly reduced the pepsinogen secretion by carbachol or TPA, but not by forskolin or ionomycin, and did not affect the basal secretion and the [Ca2+]i elevated by carbachol or ionomycin. We concluded that: (1) MLCK plays an important role in basal and drug-stimulated pepsinogen secretion, (2) MLCK is involved in the Ca2+-dependent intracellular pathway but not in the cyclic adenosine monophosphate (cAMP) dependent pathway, (3) PKC is irrelevant to activation of MLCK, and (4) increases in cAMP and [Ca2+]i are independent of activation of PKC.

An enzyme immunoassay for pepsinogen II(PGII): Chronological changes in serum PGII concentrations

A new two-site enzyme immunoassay for pepsinogen II(PGII) has been developed. The detection limit of PGII was 0.15 ng/ml (15 pg/tube) and the optimal assay range was 1.5 to 150 ng/ml (150 pg to 15 ng/tube). Mean serum PGII concentrations in normal males and females were 17.2 and 14.6 ng/ml, respectively. The chronological analysis among 5 age groups from 20 to 69 yr, males and females together, revealed that the serum level was low (11.9 ng/ml) between 20-39 yr, gradually increased from 40 yr of age and attained the maximum (22.5 ng/ml) in the 50-yr group. Total gastrectomy markedly lowered the serum level to 1.9 ng/ml, suggesting that the majority of PGII in serum was derived from the gastric mucosa. This assay makes it possible to measure trace amounts of PGII derived from extragastric as well as gastroduodenal tissues.

Regulatory Mechanism of Pepsinogen Gene Expression in Monolayer Cultured Guinea Pig Gastric Chief Cells

We have investigated the effects of dibutyrylcAMP, forskolin, carbamylcholine chloride (carbachol),ionomycin, and 12-O-tetradecanoylphorbol-13-acetate(TPA) on the expression of guinea pig pepsinogen mRNA in monolayer cultured gastric chief cells.After exposure of the cells to each of these compoundsfor 4 to 24 hr, and at 48 hr after primary culture,total cellular RNA was isolated using acidguanidium-phenol-chloroform and then was reverse transcribed to cDNA.Obtained cDNA was amplified by polymerase chain reaction(PCR) using primers detecting guinea pig pepsinogen mRNAand human β-actin mRNA as an internal standard. The PCR products were separated and quantifiedusing capillary electrophoresis. Dibutyryl cAMP andforskolin significantly increased pepsinogen mRNA, butcarbachol, ionomycin, and TPA failed to increase that. These findings suggested that pepsinogengene expression was up-regulated by intracellular cAMP,but not by intracellular calcium or protein kinase C inguinea pig chief cells.

A Case of Choledochocele Associated with Carcinoma of the Pancreas

A‐59‐year old woman was admitted with lumbago and back pain. An abdominal ultrasonography and abdominal computed tomography demonstrated a tumor of the body of the pancreas and multiple space occupying lesions in the liver. Endoscopic retrograde chorangiopancreatography revealed a 11 mm cystic dilatation of the terminal portion of the common bile duct protruding into the duodenal lumen and an obstruction of the pancreatic duct in the body portion. Celiac artery angiography showed an irregularity in the wall of the splenic artery. In line with these findings, the patient was diagnosed as suffering from choledochocele associated with carcinoma of the pancreas. As she already had multiple metastatic liver tumors, her treatment was limited to amelioration of her symptoms. Increasing stenosis of the common bile duct, however, required endoscopic sphinctomy and endoscopic retrograde biliary drainage. Type 3 Choledochocele of Alonso‐Lejs classification is very rare, and only a few cases have been reported in association with malignant bile duct tumors. We believe this case to be the first one in our country linked with carcinoma of the pancreas.

An enzyme immunoassay of slow moving protease (SMP) in human gastric mucosa

A two-site enzyme immunoassay for slow moving protease (SMP) of human gastric mucosa has been developed. The detection limit of SMP was 50 pg/tube and the optimal assay range was 0.5 to 50 ng/tube. This assay system made it possible to measure a trace amount of gastric mucosal SMP in endoscopic biopsy-specimens. Mean gastric mucosal SMP contents of the antrum, the angles, and the distal and proximal corpuses were 4.5, 3.7, 2.6, and 2.5 micrograms/mg protein, respectively. The antral mucosa contained a larger amount of SMP than the gastric mucosa of other area. The present enzyme immunoassay system is sufficiently sensitive for the clinical study of SMP in gastrointestinal tract.