Intacellular mechanism of pepsinogen secretion

我们研究了肌球蛋白轻链激酶（MLCK）和蛋白激酶C（PKC）在豚鼠胃主细胞分泌胃蛋白酶原中的作用。为此，我们尝试建立了豚鼠主细胞单层培养体系和特异性的胃蛋白酶原酶免疫分析体系。用胶原酶和GEDTA从豚鼠胃底粘膜中分离主细胞，Percoll液离心，悬浮于Dulbecco 'sMEM/Ham' sF-12培养基中，建立单层培养体系（1/1含10%胎牛血清），用DEAE-Sephacel和Sephacryl S-200柱从豚鼠胃底粘膜中纯化胃蛋白酶原。用纯化的胃蛋白酶原免疫家兔，获得了抗胃蛋白酶原的抗体。用β-半乳糖苷酶标记Fab'抗体，建立了双位点酶联免疫分析系统。EIA系统对1.5ng以上的gui测量显示出灵敏度 ...更多信息 nea猪胃蛋白酶原，并且单层培养系统对cardachol（一种Ca^2+信使系统激动剂）、TPA（一种PKC刺激剂）、forskolin（一种cAMP信使系统激动剂）和ionomycin（一种钙离子载体）反应良好。为探讨MLCK和PKC在胃蛋白酶原分泌中的作用，用上述两种系统评价MLCK抑制剂H-7、PKC抑制剂H-7对卡巴胆碱、TPA、毛喉素和离子霉素刺激胃蛋白酶原分泌的影响。此外，用Ca^2+分析仪测定了ML-9和H-7对卡巴胆碱和离子霉素引起的细胞内游离Ca^2+浓度（[Ca^2+]i）升高的影响。ML-9显着减少由卡巴胆碱和离子霉素刺激的胃蛋白原分泌，但不是由TPA或毛喉素。H-7显着降低卡巴胆碱或TPA刺激，但不是由毛喉素和离子霉素。ML-9显著减少基础胃蛋白酶原分泌，然而，H-7没有减少。ML-9和H-7均不能增加单层培养的主细胞上的[Ca^<2+>] i。结论：1）MLCK在基础和刺激的胃蛋白酶原分泌中起重要作用。2)MLCK参与Ca^<2+>依赖的细胞内过程，但不参与c-AMP依赖的细胞内过程。3)PKC不参与MLCK的激活。4）PKC独立地作用于c-AMP和[Ca^<2+>]i的增加。少

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 … More 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