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)在豚鼠胃主细胞胃蛋白酶原分泌中的作用。为此，我们试图建立豚鼠主细胞的单层培养体系和豚鼠胃蛋白酶原的酶免疫分析(EIA)体系。用胶原酶和乙二胺四乙酸二钠从豚鼠胃底粘膜中分离出分散的主细胞，Percoll液离心，悬浮于Dulbecco‘s MEM/Ham S F-12(1/1含10%胎牛血清)中，培养70h，建立单层培养体系。用DEAESephacel和Sephacryl S-200柱从豚鼠胃底粘膜中分离纯化胃蛋白酶原。用纯化的胃蛋白酶原免疫兔，获得抗胃蛋白酶原抗体。用β-半乳糖苷酶标记的Fab‘抗体建立了双位点EIA体系。环境影响评估系统对超过1.5nggui…的测量显示出灵敏度。单层培养体系对Cardacol(钙离子信使系统激动剂)、TPA(PKC激动剂)、Forsklin(cAMP信使系统激动剂)和离子霉素(钙离子载体)的反应良好。为了研究MLCK和PKC在胃蛋白酶原分泌中的作用，用上述两种系统观察了MLCK抑制剂ML-1、PKC抑制剂H-7对卡巴胆碱、TPA、Forsklin和离子霉素刺激的胃蛋白酶原分泌的影响。用钙离子分析仪检测ML-9和H-7对卡巴胆碱和离子霉素引起的细胞内游离钙离子浓度([Ca^&lt；2+&gt；]i)的影响。ML-9可显著抑制卡巴胆碱和离子霉素刺激的胃泌素分泌，但不能抑制TPA和Forsklin刺激的胃泌素分泌。H-7能显著降低卡巴胆碱或TPA的刺激强度，但不能抑制Forskolin和离子霉素的刺激作用。ML-9显著减少基础胃蛋白酶原的分泌，而H-7不能减少基础胃蛋白酶原的分泌。在单层培养的主细胞上，ML-9和H-7均不能使[Ca~(2+)]i升高。我们的结论是：1)MLCK在基础和刺激胃蛋白酶原分泌中都起着重要作用。2)MLCK参与钙依赖的细胞内过程，而不参与c-AMP依赖的细胞内过程。3)PKC不参与MLCK的激活。4)PKC独立作用于c-AMP和[Ca^&lt；2+&gt；]i.

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