| Budget Amount *help |
¥2,100,000 (Direct Cost: ¥2,100,000)
Fiscal Year 1992: ¥800,000 (Direct Cost: ¥800,000)
Fiscal Year 1991: ¥1,300,000 (Direct Cost: ¥1,300,000)
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| Research Abstract |
We evaluated the role of myosin light-chain kinase (MLCK) and protein kinase C(PKC) in pepsinogen secretion from guinea pig gastric chief cells. For this purpose we tried to establish a monolayer culture system of guinea pig chief cells and an enzyme immunoassay (EIA) system specific for guinea pig pepsinogen. A monolayer culture system was established by the methods in which dispersed chief cells were obtained from gastric fundic mucosa of a guinea pig by using collagenase and GEDTA, and by centrifugating in Percoll solution, suspended into Dulbecco's MEM/Ham s F-12 (1/1 containing 10% fetal calf serum), and cultured for 70 hr. Pepsinogen was purified from gastric fundic mucosa of a guinea pig by using DEAE-Sephacel and Sephacryl S-200 columns. Antibody to pepsinogen was raised by immunizing rabbit with the purified pepsinogen. A two-site EIA system was then establish-ed by using beta-galactosidase labeled Fab' antibody. The EIA system showed sensitivity to measure above 1.5 ng of gui
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nea pig pepsinogen, and the monolayer culture system responded well to cardachol (a Ca^<2+> messenger system agonist), TPA (a PKC stimulator), forskolin (a cAMP messenger system agonist) and ionomycin (a calcium ionophore). To study the role of MLCK and PKC in pepsinogen secretion, the effect of ML-., a MLCK inhibitor, H-7, a PKC inhibitor on pepsinogen secretion stimulated by carbachol, TPA, forskolin and ionomycin was evaluated by using above-mentioned two systems. Furthermore, the effect of ML-9 and H-7 on intracellular free Ca^<2+> concentration ([Ca^<2+>]i) elevated by carbachol and ionomycin was evaluated by using a Ca^<2+> analyzer. ML-9 significantly reduced pepsionogen secretion stimulated by carbachol and ionomycin but not by TPA or forskolin. H-7 significantly reduced that stimulated by carbachol or TPA, but not by forskolin and ionomycin. ML-9 significantly reduced basal pepsinogen secretion, however, H-7 did not reduced that. Both ML-9 and H-7 failed to increase in [Ca^<2+>] i on a monolayer cultured chief cell. We concluded 1) MLCK plays an important role in both basal and stimulated pepsinogen secretion. 2) MLCK is involved in Ca^<2+> dependent intracellular process, but not in c-AMP dependent ones. 3) PKC is not involved in activation of MLCK.4) PKC acts independently on increases in c-AMP and [Ca^<2+>]i. Less
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