APPEARANCE OF PHOSPHATIDYLSERINE ON APOPTOTIC CELLS REQUIRES CALCIUM-MEDIATED NONSPECIFIC FLIP-FLOP AND IS ENHANCED BY LOSS OF THE AMINOPHOSPHOLIPID TRANSLOCASE

Phosphatidylserine (PS), ordinarily sequestered in the plasma membrane inner leaflet, appears in the outer leaflet during apoptosis, where i t triggers non-inflammatory phagocytic recognition of the apoptotic ce ll, The mechanism of PS appearance during apoptosis is not well unders tood but has been associated with loss of aminophospholipid translocas e activity and nonspecific flip flop of phospholipids of various class es, The human leukemic cell line HL-60, the T cell line Jurkat, and pe ripheral blood neutrophils, undergoing apoptosis induced either with U V irradiation or anti-Fas antibody, were probed in the cytofluorograph for (i) surface PS using fluorescein isothiocyanate-labeled annexin V , (ii) PS uptake by the aminophospholipid translocase using [6-[(7-nit robenz-2-oxa-1,3-diazol-4-yl)amino] caproyl] (NBD)-labeled PS, (iii) n onspecific uptake of phospholipids (as a measure of transbilayer flip flop) using NBD-labeled phosphatidylcholine, and (iv) the appearance o f hypodiploid DNA, In all three types of cells undergoing apoptosis, t he appearance of PS followed loss of aminophospholipid translocase and was accompanied by nonspecific phospholipid flip-flop, Importantly, h owever, in the absence of extracellular calcium, the appearance of PS was completely inhibited despite DNA fragmentation and loss of aminoph ospholipid translocase activity, the latter demonstrating that loss of the translocase is insufficient for PS appearance during apoptosis, F urthermore, while both the appearance of PS and nonspecific phospholip id uptake demonstrated identical extracellular calcium requirements wi th an ED50 of nearly 100 mu m, the magnitude of PS appearance depended on the level of aminophospholipid translocase activity, Taken togethe r, the data strongly suggest that while nonspecific flip-flop is the d riving event for PS appearance in the plasma membrane outer leaflet, a minophospholipid translocase activity ultimately modulates its appeara nce.