title: Internalization of recombinant tissue-type plasminogen activator by isolated rat hepatocytes is via coated pits.
abstract: 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.
authors.affiliation: Department of Experimental Pathology, Smith Kline and French Laboratories, King of Prussia, PA 19406.
authors.forename: G J
authors.initials: GJ
authors.lastname: Marks
authors.forename: T K
authors.initials: TK
authors.lastname: Hart
authors.forename: G F
authors.initials: GF
authors.lastname: Rush
authors.forename: S T
authors.initials: ST
authors.lastname: Hoffstein
authors.forename: K L
authors.initials: KL
authors.lastname: Fong
authors.forename: P J
authors.initials: PJ
authors.lastname: Bugelski
mesh_terms: D000165:Acridine Orange; D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D003034:Coated Pits, Cell-Membrane / Q000378:metabolism*; D011992:Endosomes / Q000378:metabolism*; D007150:Immunohistochemistry; D066298:In Vitro Techniques; D007457:Iodine Radioisotopes; D007700:Kinetics; D008099:Liver / Q000378:metabolism*; D008854:Microscopy, Electron; D011188:Potassium / Q000378:metabolism; D051381:Rats; D011994:Recombinant Proteins / Q000378:metabolism; D013696:Temperature; D010959:Tissue Plasminogen Activator / Q000378:metabolism*
chemical_list: D007457:Iodine Radioisotopes; D011994:Recombinant Proteins; D000255:Adenosine Triphosphate; D010959:Tissue Plasminogen Activator; D000165:Acridine Orange; D011188:Potassium