TISSUE PLASMINOGEN ACTIVATOR (T-PA) PRODUCTION BY HUMAN EMBRYONIC FIBROBLASTS, IMR-90, STIMULATED BY PROTEOSE PEPTONE

Abstract

In order to study the mechanisms by which t-PA production by IMR-90 cells are induced, lactalbumin hydrolysates, yeast extracts, and peptones were tested for their ability to induce t-PA production by IMR-90 cells. IMR-90 cells were grown to confluency in Dulbecco's modified Eagle's medium(DMEM) supplemented with 10% fetal calf serum at 37°C in 5% CO2 in air. And the cells were maintained in serum free medium containing 1% of each additive. The plasminogen activator activity was determined by fibrin plate method, using urokinase or t-PA from WHO as a standard. It was found that proteose peptone (Difco) and neopeptone (Difco) strongly induced the t-PA production by IMR-90 cells. The t-PA production in DMEM containing 1% proteose peptone reached approx. 200IU/ml after incubation at 37°C for 6 days and was from twenty to fifty times higher than that in DMEM only (control medium). The t-PA production by IMR-90 cells stimulated by proteose peptone was strongly inhibited by RNA synthesis inhibitor(actinomycin D) or prorein synthesis inhibitor (cycloheximide). Hence, t-PA production by IMR-90 cells stimulated by proteose peptone was mediated by de novo synthesis. Chelating reagent (EGTA), Ca2+ entry blocker (verapamil), inhibitor of phospholipase A2 (quinacrine) and inhibitor of lipoxygenase (NDGA) strongly inhibited the t-PA production by IMR-90 cells stimulated by proteose peptone. Inhibitor of cyclooxygenase (indomethacin) was inert. On the contrary, activators of phospholipase A2(Ca2+,melittin) and hydroxy-unsaturated fatty acid (5-HETE) derived from arachidonic acid by lipoxygenase strongly enhanced t-PA production by IMR-90 cells stimulated by proteose peptone. These results suggest that the t-PA production by IMR-90 cells stimulated by proteose peptone is mediated by arachidonate cascade involving the following pathway; (1) proteose peptone stimulates the membrane of IMR-90, (2) this stimulus causes Ca2+ influx, (3) Ca2+ ion activates phopholipase A2, (4) activated phospholipase A2 liberates arachidonic acid from phospholipids in ceil membrane and (5) lipoxygenase converts arachidonic acid into the hydroxy-unsaturated fatty acid.