Investigation into the Role of Phosphatidylserine in Modifying the Susceptibility of Human Lymphocytes to Secretory Phospholipase A2 using Cells Deficient in the Expression of Scramblase.

Abstract

Normal human lymphocytes resisted the hydrolytic action of secretory phospholipase A 2 but became susceptible to the enzyme following treatment with a calcium ionophore, ionomycin. To test the hypothesis that this susceptibility requires exposure of the anionic lipid phosphatidylserine on the external face of the cell membrane, experiments were repeated with a human Burkitt's lymphoma cell line (Raji cells). In contrast to normal lymphocytes or S49 mouse lymphoma cells, most of the Raji cells (83%) did not translocate phosphatidylserine to the cell surface upon treatment with ionomycin. Those few that did display exposed phosphatidylserine were hydrolyzed immediately upon addition of phospholipase A 2. Interestingly, the remaining cells were also completely susceptible to the enzyme but were hydrolyzed at a slower rate and after a latency of about 100 s. In contradistinction to the defect in phosphatidylserine translocation, Raji cells did display other physical membrane changes upon ionomycin treatment that may be relevant to hydrolysis by phospholipase A 2. These changes were detected by merocyanine 540 and trimethylammonium diphenylhexatriene fluorescence and were common among normal lymphocytes, S49 cells, and Raji cells. The levels of these latter effects corresponded well with the relative rates of hydrolysis among the three cell lines. These results suggested that while phosphatidylserine enhances the rate of cell membrane hydrolysis by secretory phospholipase A 2 , it is not an absolute requirement. Other physical properties such as membrane order contribute to the level of membrane susceptibility to the enzyme independent of phosphatidylserine.

Membrane Properties Involved in Calcium-Stimulated Microparticle Release from the Plasma Membranes of S49 Lymphoma Cells

“…data and experiments with a pharmacological inhibitor of scramblase (R5421, [35]) demonstrated in erythrocytes and platelets that the activity of that enzyme is important for microparticle shedding [19,29,36]. Since a scramblase inhibitor is no longer available, the requirement for migration of PS from the inner to the outer leaflet of the cell membrane was tested here for lymphoma cells by using the Raji lymphoma line, which is deficient in scramblase activity [37][38][39][40].…”

“…The ionomycin-stimulated change in light scatter intensity in Raji cells was only 16% of that observed in S49 cells. Furthermore, incubation of Raji cells with a higher dose of ionomycin sufficient to restore some of the ability of the cells to translocate PS [40] generated a twofold enhancement of apparent rate of microparticle release (2.1 ± 0.4, = 0.01, = 10). These data argued that PS exposure is also important for microparticle release in nucleated cells.…”