Thorne D P, Lockwood T D
Department of Pharmacology and Toxicology, School of Medicine, Wright State University, Dayton, Ohio 45435.
Am J Physiol. 1993 Jul;265(1 Pt 1):E10-9. doi: 10.1152/ajpendo.1993.265.1.E10.
Four distinct processes mediating protein degradation were identified in the Langendorff perfused rat heart. Hearts were biosynthetically labeled in vitro with [3H]leucine for 10 min. The subsequent release of [3H]leucine at 1.5-min intervals (2 mM nonradioactive leucine) was determined from 20 min to 8 h after labeling in rhythmically contracting hearts. Rapid turnover proteins were eliminated during the first 3 h; this degradation was not inhibited by insulin (5 nM) or isoproterenol (0.5 microM). However, the nontoxic thiol reactive agent diamide (100 microM) caused a complete inhibition of the [3H]leucine release from rapidly degraded proteins. After the elimination of rapidly degraded proteins at 3 h, the release of [3H]leucine was inhibited 35-40% by insulin (5 nM) or the lysosomal inhibitor chloroquine (30 microM), thereby defining a second vesicular process. The beta-agonist isoproterenol (0.5 microM) or the nonselective alpha-agonist naphazoline (100 microM) caused 30-35% proteolytic inhibitions, defining a third adrenergic-responsive process. The inhibitory effects of simultaneously combined insulin and chloroquine did not exceed the effect of either agent alone. However, the combined effects of insulin and isoproterenol were additive, inhibiting two-thirds of basal degradation. Beginning at 3 h after labeling a 75% proteolytic inhibition resulted from the thiol reactive agents diamide (100 microM) or N-ethylmaleimide (10 microM); the thiol protease active site inhibitor trans-epoxysuccinly-L-leucylamino-(4-quinidino)butane (50 microM) caused 65% inhibition. The 75% inhibition caused by diamide includes both the insulin-responsive and beta-adrenergic-responsive pathways. A novel fourth proteolytic process (25% of proteolysis) was thereby distinguished from the above three by its resistance to inhibition by insulin, adrenergic agonists, thiol reactive agents, or thiol protease inhibitor. Only the adrenergic-responsive process was correlated with changes in contractile rhythm or fibrillation.
在Langendorff灌注大鼠心脏中鉴定出四种介导蛋白质降解的不同过程。心脏在体外用[3H]亮氨酸进行生物合成标记10分钟。在有节律收缩的心脏中标记后20分钟至8小时，每隔1.5分钟（2 mM非放射性亮氨酸）测定[3H]亮氨酸的后续释放。快速周转蛋白在最初3小时内被清除；这种降解不受胰岛素（5 nM）或异丙肾上腺素（0.5 microM）的抑制。然而，无毒的硫醇反应剂二酰胺（100 microM）完全抑制了快速降解蛋白中[3H]亮氨酸的释放。在3小时快速降解蛋白被清除后，胰岛素（5 nM）或溶酶体抑制剂氯喹（30 microM）抑制了[3H]亮氨酸释放的35 - 40%，从而确定了第二个囊泡过程。β - 激动剂异丙肾上腺素（0.5 microM）或非选择性α - 激动剂萘甲唑啉（100 microM）引起30 - 35%的蛋白水解抑制，确定了第三个肾上腺素能反应过程。胰岛素和氯喹同时联合使用的抑制作用不超过单独使用任何一种药物的效果。然而，胰岛素和异丙肾上腺素的联合作用是相加的，抑制了三分之二的基础降解。从标记后3小时开始，硫醇反应剂二酰胺（100 microM）或N - 乙基马来酰亚胺（10 microM）导致75%的蛋白水解抑制；硫醇蛋白酶活性位点抑制剂反式环氧琥珀酰 - L - 亮氨酰氨基 -（4 - 喹尼丁基）丁烷（50 microM）引起65%的抑制。二酰胺引起的75%抑制包括胰岛素反应性和β - 肾上腺素能反应性途径。由此区分出一种新的第四个蛋白水解过程（占蛋白水解的25%），它对胰岛素、肾上腺素能激动剂、硫醇反应剂或硫醇蛋白酶抑制剂的抑制具有抗性。只有肾上腺素能反应过程与收缩节律或颤动的变化相关。
