Susceptibility of S49 lymphoma cell membranes to hydrolysis by secretory phospholipase A(2) during early phase of apoptosis.

During cell death, plasma membranes of cells become vulnerable to attack by extracellular secretory phospholipase A(2). The purpose of this study was to identify the timing of this phenomenon relative to other events that occur during the process of cell death. Death was induced in S49 murine lymphoma cells by treatment with dexamethasone, dibutyryl cAMP, ionomycin, thapsigargin, or heat shock (1 h at 43 degrees C). The appearance of membrane susceptibility to secretory phospholipase A(2) was compared to the following apoptotic events: loss of mitochondrial membrane potential, phosphatidylserine exposure in the outer leaflet of the cell membrane, early DNA damage assessed by the comet assay, and changes in cell size and internal complexity assessed by flow cytometry. Each inducer of death was distinct in the time course of events produced. Although dead cells were susceptible to the action of phospholipase A(2), live cells (impermeable to propidium iodide) also became vulnerable to the enzyme during characteristic time courses after exposure to each inducer. In fact, susceptibility to sPLA(2) was observed in each case prior to or concurrent with the earliest of the markers of apoptosis. These results demonstrate that the onset of susceptibility to sPLA(2) is an early event in apoptosis suggesting that changes in membrane structure may be relevant to initial aspects of the apoptotic process.

Mechanisms by which elevated intracellular calcium induces S49 cell membranes to become susceptible to the action of secretory phospholipase A2.

Exposure of S49 lymphoma cells to exogenous group IIA or V secretory phospholipase A2 (sPLA2) caused an initial release of fatty acid followed by resistance to further hydrolysis by the enzyme. This refractoriness was overcome by exposing cells to palmitoyl lysolecithin. This effect was specific in terms of lysophospholipid structure. Induction of membrane susceptibility by lysolecithin involved an increase in cytosolic calcium and was duplicated by incubating the cells with calcium ionophores such as ionomycin. Lysolecithin also activated cytosolic phospholipase A2 (cPLA2). Inhibition of this enzyme attenuated the ability of lysolecithin (but not ionomycin) to induce susceptibility to sPLA2. Lysolecithin or ionomycin caused concurrent hydrolysis of both phosphatidylethanolamine and phosphatidylcholine implying that transbilayer movement of phosphatidylethanolamine occurred upon exposure to these agents but that susceptibility is not simply due to exposure of a preferred substrate (i.e. phosphatidylethanolamine) to the enzyme. Microvesicles were apparently released from the cells upon addition of lysolecithin or ionomycin. Both these vesicles and the remnant cell membranes were susceptible to sPLA2. Together these data suggest that lysolecithin induces susceptibility through both cPLA2-dependent and -independent pathways. Whereas elevated cytosolic calcium was required for both pathways, it was sufficient only for the cPLA2-independent pathway. This cPLA2-independent pathway involved changes in cell membrane structure associated with transbilayer phospholipid migration and microvesicle release.

Definition of the specific roles of lysolecithin and palmitic acid in altering the susceptibility of dipalmitoylphosphatidylcholine bilayers to phospholipase A2.

Bilayers composed of phosphatidylcholine initially resist catalysis by phospholipase A2. However, after a latency period, they become susceptible when sufficient reaction products (lysolecithin and fatty acid) accumulate in the membrane. Temperature near the main bilayer phase transition and calcium concentration modulate the effectiveness of the reaction products. The purpose of this study was to examine the individual contributions of lysolecithin and palmitic acid to the susceptibility of dipalmitoylphosphatidylcholine vesicles and to rationalize the effects of temperature and calcium. Various fluorescent probes (Prodan, Laurdan, pyrene-labeled fatty acid, and dansyl-labeled phospholipid) were used to assess changes in the ability of the reaction products to perturb the bilayer and to affect the interactions with the enzyme. Un-ionized palmitic acid decreased bilayer polarity and perturbed the membrane surface exposing some of the Prodan to bulk water. Lysolecithin increased bilayer polarity and the rate of dipolar relaxation in response to the excited states of Laurdan and Prodan. A combination of the individual contributions of each product was observed when palmitic acid and lysolecithin were present together at low calcium, and the effects of lysolecithin dominated at high calcium. Palmitic acid, but not lysolecithin, promoted the binding of phospholipase A2 to the bilayer surface in the absence of calcium. Lysolecithin reduced the ability of fatty acid to enhance binding apparently by altering the structure of fatty acid domains in the membrane. Furthermore, increased temperature and ionization of the fatty acid tended to cause segregation of bound phospholipase A2 into domains poor in phospholipid content which presumably impeded bilayer hydrolysis. In contrast, un-ionized palmitic acid and lysolecithin promoted hydrolysis by augmenting a step distal to the adsorption of enzyme to the bilayer. This kinetic response to lysolecithin was calcium-dependent. A model accounting for these varied influences of the reaction products is presented.