The urokinase-plasminogen activator system in ovine macrophages and neutrophils

Abstract

The urokinase-plasminogen activator (u-PA) system in resting and activated ovine macrophages and neutrophils was examined. Macrophages and neutrophils were isolated from a total of 28 lactating sheep of the Chios breed. Low amounts of u-PA were found intracellularly or membrane-bound in resting macrophages and neutrophils. However, incubation of resting macrophages or neutrophils with purified u-PA (10 IU/ml) revealed extensive binding of u-PA to cell membranes. Excess amino terminal fragment of the u-PA molecule, a proteolytically inactive fragment of u-PA (amino acids 1–135) blocked binding of u-PA to macrophage or neutrophil cell membrane. These results indicate that the binding of u-PA is specific and that resting neutrophils and macrophages have unoccupied u-PA receptors on their cell membrane. Addition of phorbol myristate acetate (PMA) led to an increase ( P<0.01) in total cell-associated and membrane-bound u-PA activity and a decrease ( P<0.01) in free, unoccupied u-PA binding sites of macrophages or neutrophils. No significant effects on total cell-associated or membrane-bound u-PA were found when macrophages or neutrophils were treated with 4-phorbol-12,13-didecanoate, a phorbol ester that does not activate protein kinase C (PKC). Furthermore, the PMA-induced increase in total cell-associated u-PA activity of sheep macrophages or neutrophils was completely ablated by the PKC inhibitor 1-(5-isoquinolinesulfonyl)-2-methylpiperazine dihydrochloride (H-7, 100 μM) but was unaffected by the cyclic nucleotide-dependent protein kinase inhibitor N-(2-quanidinoethyl)-5-isoquinoline sulfonamide hydrochloride (HA-1004, 100 μM). Thus, PKC plays a role in the modulation of u-PA system by PMA in ovine macrophages and neutrophils.