Expression of rat kallikrein and epithelial polarity in transfected Madin-Darby canine kidney cells.
Expression of rat kallikrein and epithelial polarity in transfected Madin-Darby canine kidney cells.
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转染的 Madin-Darby 犬肾细胞中大鼠激肽释放酶和上皮极性的表达。
DOI：
10.1161/01.hyp.26.6.891
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发表时间：
1995
期刊：
影响因子：
--
通讯作者：
Erdös,EG
中科院分区：
文献类型：
--
作者：
Abe,M;Nakamura,F;Tan,F;Deddish,PA;Colley,KJ;Becker,RP;Skidgel,RA;Erdös,EG
尿激肽释放酶的许多性质已被很好地描述，但细胞内处理原激肽释放酶并由肾细胞释放的机制尚不清楚。本文报道了转导大鼠颌下腺激肽释放酶基因的Madin-Darby犬肾(MDCK)细胞中激肽释放酶原的合成及其在MDCK细胞和富含肝高尔基膜制剂中的激活作用。转导激肽释放酶基因的MDCK细胞仅在顶端和基底外侧分泌激肽释放酶原，比例约为4：1，而反向转导激肽释放酶基因的细胞或未经处理的细胞仅释放微量的激肽释放酶。在培养液或匀浆的MDCK细胞中，原激肽释放酶可被胰酶完全激活，但仅被热裂解酶激活44%。原激肽释放酶以较高的速率被合成并释放到培养液中：5×106细胞在24小时内分泌的酶切割46 nmol/mind-Val-Leu-Arg-7-amino-4-methylcoumarin，激活后释放63 ng/min的缓激肽。免疫细胞学显示，在转基因细胞中，原激肽释放酶与高尔基体有关。大鼠尿激肽释放酶抗血清在条件培养液的Western印迹中检测到一条单一条带，并对该酶进行免疫沉淀。抑肽酶抑制激活的激肽释放酶。虽然MDCK细胞释放激肽释放酶原，但它们的匀浆在pH 5.5和7.5时都激活了激肽释放酶原。原激肽释放酶也可被高度浓缩的肝脏高尔基膜组分和内质网制剂激活，但高尔基体制剂的活性是前者的38倍。丝氨酸蛋白酶的抑制剂可显著抑制该激活，而半胱氨酸蛋白酶抑制剂的阻断作用较小。因此，转基因的MDCK细胞合成和释放前激肽释放酶，而不从顶端(80%)和基底侧(20%)激活它。
Many properties of urinary kallikrein are well characterized, but the intracellular processing of prokallikrein and release by kidney cells have yet to be clarified. We report here on the synthesis of prokallikrein in Madin-Darby canine kidney (MDCK) cells transfected with rat submaxillary gland kallikrein cDNA and on its activation by MDCK cells and by an enriched liver Golgi membrane preparation. Transfected MDCK cells secreted only prokallikrein at both the apical and basolateral sides in about a 4:1 ratio, but cells transfected with kallikrein cDNA in reverse orientation or untreated cells released only traces of the enzyme. Prokallikrein, in culture medium or in homogenized MDCK cells, was fully activated by trypsin but activated only to 44% by thermolysin. Prokallikrein was synthesized and released into the medium at a high rate: the enzyme secreted by 5×106cells in 24 hours cleaved 46 nmol/mind-Val-Leu-Arg-7-amino-4-methylcoumarin and liberated 63 ng/min bradykinin after activation. Immunocytology indicated the association of prokallikrein with the Golgi apparatus in the transfected cells. Antiserum to rat urinary kallikrein detected a single band in a Western blot of conditioned medium and also immunoprecipitated the enzyme. Aprotinin inhibited activated prokallikrein. Although MDCK cells released prokallikrein, their homogenates activated prokallikrein at both pH 5.5 and 7.5. Prokallikrein was also activated by a highly enriched liver Golgi membrane fraction and by an endoplasmic reticulum preparation, but the Golgi preparation was 38-fold more active. The activation was blocked significantly by inhibitors of serine proteases and less by cysteine protease inhibitors. Thus, transfected MDCK cells synthesize and release prokallikrein without activating it from the apical (80%) and the basolateral (20%) sides.