The effect of various vitamin E derivatives on the urokinase-plasminogen activator system of ovine macrophages and neutrophils

Abstract

The effect of vitamin E derivatives on the urokinase-plasminogen activator (u-PA) system of resting and phorbol myristate acetate (PMA)-activated ovine macrophages and neutrophils were investigated. Blood monocyte -macrophages and neutrophils were isolated from twentyfour animals. Macrophages or neutrophils were cultured in vitro for 3 or 24 h with or without various vitamin E derivatives: free a-tocopherol (a-T), a-tocopheryl acetate (a-TA), or a-tocopheryl succinate (a-TS). Following incubation, cells were stimulated with 80 mM-PMA. Total cell-associated u-PA, membrane-bound u-PA and free u-PA binding sites were determined before and after stimulation with PMA. Results showed that none of the vitamin E derivatives had any effect (P.0·05) on the u-PA system of resting monocyte -macrophages or neutrophils. In contrast, a-TS, but not a-TA or a-T, increased (P,0·01) total cell-associated u-PA and membrane-bound u-PA of PMA-stimulated macrophages and neutrophils. a-TS had no effect (P. 0·05) on total u-PA and membrane-bound u-PA activities of macrophages and neutrophils cultured in the presence of 4-phorbol 12,13 didecanoate, a phorbol ester that does not activate protein kinase (PK) C. Addition of H7 (1-(5-isoquinolinesulfonyl)-2-methylpiperazine dihydrochloride), which is a potent inhibitor of both PK A and C, completely abolished the effect of a-TS on total cell-associated u-PA and membrane-bound u-PA of PMA-activated macrophages and neutrophils. Addition of HA1004 (N-(2-quanidinoethyl)-5-isoquinoline sulfonamide hydrochloride), which is a potent PK A but a weak PK C inhibitor, had no effect (P.0·05) on total cell-associated u-PA and membrane-bound u-PA of PMA-activated macrophages and neutrophils cultured in the presence of a-TS. Thus, PK C modulates the effect of a-TS on the u-PA system of ovine macrophages and neutrophils.

Physiological role of indigenous milk enzymes: An overview of an evolving picture

“…The conversion of plasminogen to plasmin is modulated by PA (Politis, 1996), two types of which exist in mammals: urokinasetype PA (u-PA) and tissue-type PA (t-PA) (Heegaard, Rasmussen, & Andreasen, 1994;Politis, 1996). Plasmin, plasminogen, and t-PA are closely associated with the casein micelles (Politis, 1996), whereas u-PA is associated with neutrophils (Politis, Voudouri, Bizelis, & Zervas, 2003;Politis, Zavizion, Cheli, & Baldi, 2002) and inhibitors of PA and plasmin are in the milk serum (Precetti, Oria, & Nielsen, 1997;Politis, 1996). The close proximity of plasmin to its substrate ensures that hydrolysis is an efficient process (Korycka-Dahl, Dumas, Chene, & Martal, 1983) and it is the primary agent of proteolysis in goodquality milk (Kelly & McSweeney, 2003).…”

Proteolytic Systems in Milk: Perspectives on the Evolutionary Function within the Mammary Gland and the Infant

“…Ostensibly, the presence of the plasminogen activator inhibitors on the micelle prevents more extensive casein micelle degradation in the mammary gland. The other main type of plasminogen activator in milk, urokinase-type plasminogen activator is associated only with the human milk somatic cell fraction (50), specifically the neutrophils (51, 52). …”

Effect of Vitamin E Supplementation on Various Functional Properties of Macrophages and Neutrophils Obtained from Weaned Piglets

“…Cells were then lysed by addition of 500 μ l of 1 m m sodium bicarbonate, centrifuged at 12 000 × g for 3 min, aliquoted, and stored at −80°C. Activity of u‐PA in aliquots of lysed cells was determined (Politis et al., 2003).…”

“…Cells (2 × 10 6 /ml) were resuspended in 500 μ l of HBSS containing 20 m m HEPES and 81 m m PMA. After incubation for 30 min at 37°C, neutrophils were washed three times with HBSS and resuspended in 500 μ l of HBSS plus 20 m m HEPES containing purified u‐PA (10 U/ml; Sigma) to fully saturate all u‐PA binding sites on the cell membrane of neutrophils (Politis et al., 2003). After incubation for another 30 min at 37°C, neutrophils were washed three times with HBSS, and then resuspended at various cell concentrations in 250 μ l of 100 m m Tris buffer (pH 8.0) for the determination of the membrane‐bound u‐PA as described above.…”