Marks G J, Hart T K, Rush G F, Hoffstein S T, Fong K L, Bugelski P J
Department of Experimental Pathology, Smith Kline and French Laboratories, King of Prussia, PA 19406.
Thromb Haemost. 1990 Apr 12;63(2):251-8.
The uptake and internalization of tissue-type plasminogen activator (t-PA) by freshly isolated rat hepatocytes was investigated. Electron microscopic examination of the uptake of t-PA-colloidal gold conjugates (t-PA-gold) by isolated rat hepatocytes showed that t-PA-gold was internalized via coated pits. This was inhibited with excess t-PA. Uptake of 125I-t-PA by isolated rat hepatocytes was a rapid, saturable, and specific process. The initial rate of specific uptake was 0.1 fmol/10(6) cells per min. The specific uptake plateaued at 1.4 fmol/10(6) cells by 30 min and declined to 0.8 fmol/10(6) cells at 2 h. Depletion of cellular ATP by 85-90% did not affect the initial rate of specific uptake. However, specific uptake by ATP-depleted hepatocytes at 30 min was reduced by 37%. By 2 h specific uptake by ATP-depleted hepatocytes was only 5% lower than by untreated hepatocytes, suggesting that processing of t-PA and/or its receptor is ATP-dependent. Uptake of 125I-t-PA was temperature dependent. Specific uptake was reduced by approximately 20% at 22 degrees C and by 70% at temperatures below 16 degrees C. Finally, inhibition of coated pit formation by K(+)-depletion with nigericin decreased the uptake of 125I-t-PA. This inhibition was shown to be K(+)-specific since treatment with nigericin in the presence of K+ did not inhibit coated pit formation or 125I-t-PA uptake. A threshold K(+)-depletion level for inhibition of coated pit formation was also demonstrated since treatment under conditions that reduced cellular K+ by only 54% had no effect on coated pit formation or 125I-t-PA uptake. These data support our hypothesis that internalization of t-PA by isolated rat hepatocytes is via coated pits and suggest that uptake of t-PA is a receptor-mediated process.
研究了新鲜分离的大鼠肝细胞对组织型纤溶酶原激活剂（t-PA）的摄取和内化过程。对分离的大鼠肝细胞摄取t-PA-胶体金复合物（t-PA-金）进行电子显微镜检查发现，t-PA-金通过有被小窝内化。过量的t-PA可抑制此过程。分离的大鼠肝细胞对125I-t-PA的摄取是一个快速、可饱和且具有特异性的过程。特异性摄取的初始速率为每分钟0.1 fmol/10(6)个细胞。到30分钟时，特异性摄取达到平台期，为1.4 fmol/10(6)个细胞，2小时时降至0.8 fmol/10(6)个细胞。细胞ATP耗竭85 - 90%不影响特异性摄取的初始速率。然而，ATP耗竭的肝细胞在30分钟时的特异性摄取降低了37%。到2小时时，ATP耗竭的肝细胞的特异性摄取仅比未处理的肝细胞低5%，这表明t-PA及其受体的加工过程是ATP依赖的。125I-t-PA的摄取是温度依赖的。在22℃时特异性摄取降低约20%，在低于16℃的温度下降低70%。最后，尼日利亚菌素通过K(+)耗竭抑制有被小窝形成，降低了125I-t-PA的摄取。这种抑制作用显示是K(+)特异性的，因为在K(+)存在下用尼日利亚菌素处理不会抑制有被小窝形成或125I-t-PA摄取。还证明了抑制有被小窝形成的K(+)耗竭阈值水平，因为在仅使细胞K(+)降低54%的条件下处理对有被小窝形成或125I-t-PA摄取没有影响。这些数据支持我们的假设，即分离的大鼠肝细胞对t-PA的内化是通过有被小窝进行的，并表明t-PA的摄取是一个受体介导的过程。
