Vu Mai Phuong and Dr. John Bell, Physiology and Developmental Biology
Secretory phospholipase A2 (sPLA2) is a secretory enzyme thought to mainly function in hydrolyzing membrane phospholipids in certain conditions of signaling induced by the immune system or hormone activity. sPLA2 is known to be implicated in inflammatory diseases, sepsis, adult respiratory distress syndrome, ischemia, cancer, atherosclerosis, pre-eclampsia, and gall stones; therefore, understanding its mechanism of action is an important step in helping other researchers determine therapeutic means for these diseases.
However not all cells are susceptible, healthy cells are resistant to the hydrolytic action of sPLA2. From previous researches cellular apoptosis (programmed cell death) is suggested to be an indicative condition for sPLA2 susceptibility. Identifying membrane changes associated with sPLA2 susceptibility, as cells undergo apoptotic stress can open new doors to developing better techniques for interpretation of membrane dynamics in relationship with sPLA2 activity. Our goal in this project was to gather information about the biophysical properties of the lymphocyte membrane that allows the cell to become susceptible to sPLA2. We used various probes to monitor and analyze any changes in the biological membranes of lymphocytes during apoptosis.
Fluorescent probes like merocyanine 540 (MC540), laurdan, or bis-pyrene are widely used in many researches as satellites (or probes) to scan and measure different changes in the bilayer phospholipids. Depending on their chemical structures and specific chemical interactions with the phospholipids (surface or in the bilayer depth), these three molecules seem to report very specific pieces of information. MC540 is a probe that intercalates between the phospholipid heads in the outer leaflet of cell membranes, therefore it is sensitive to changes in the phospholipid packing. Because it sits at the level of the glycerol backbones laurdan’s emission spectrum is highly sensitive to the solvent relaxation effect; as consequence, it was used to monitor the degree to which lipid molecules are ordered within the membrane. In contrast, bis-pyrene detects changes deep in the bilayer, where membrane order is reflected by alterations in its tendency to form intramolecular excimers. When membrane lipids become more disordered, the probability of excimer fluorescence rises. Our analysis was based on the fluorescence information changes of these probes during the apoptotic process before and after sPLA2 binding.
S49 lymphocytes were incubated with dexamethasone for periods of time ranging from 0 to 48 hours; dexamethasone is a drug which induces apoptosis in lymphocytes. At the end of this time period probes were added. Data with MC540 (300 nM final) were acquired as emission spectra (excitation, 540 nm; emission, 550-700 nm). Spectra were acquired before the addition of the probe, after equilibration (5 min), and 10 min following addition of dexamethasone or DMSO (negative control). Laurdan and bis-pyrene fluorescence emission was acquired at dual wavelengths in real time. After initiating data acquisition, laurdan (50 nM final; excitation, 350 nm; emission 435 and 500 nm) or bis-pyrene (625 nM final; excitation, 344 nm; emission 376 and 480 nm) was added at 100 s. Dexamethasone or DMSO was included after initial equilibration of cells with the probe (laurdan, 10 min; bis-pyrene, 30 min).
Cell preparation for spectral analysis was done by transfering an aliquot of cells (1.0-3.0 x 106 cells/ml in MBSS) to a quartz fluorometer cuvette and allowed 5 min to equilibrate in the spectrofluorometer. Temperature and sample homogeneity were maintained as described in Harris et al., 2001. For enzyme binding experiments, cells were resuspended in MBSS with 20 mM BaCl2 in order to block sPLA2 hydrolytic activity without preventing binding. We equilibrated and incubated S49 lymphocytes solution after probe addition at 37 °C; then we measured the background fluorescence spectra of the cells so that it can be subtracted from probe fluorescence measurements. The fluorescence difference of the probe was assayed as a function of treatment time with dexamethasone in a photon counting spectrofluorometer (Fluoromax, Jobin Yvon, Edison, NJ). Each time course experiment was consisted of measuring the initial background, adding the probe, and later adding sPLA2. Data was compiled and analyzed using Excel and a software program called Prism.
Treatment of cells with dexamethasone resulted in a significant increase in MC540 total fluorescence. The spectral changes in MC540 fluorescence suggested that treatment with the drug increased the spacing among lipids. It was clear that Merocyanine 540 bound more easily to the membrane, while laurdan emission was shifted, consistent with increased penetration of water molecules between the glycerol backbones, and that the rate of phospholipid extraction from the outer leaflet was enhanced in conditions of apoptotic stress. These three observations suggest that the degree to which neighboring phospholipids interact with one another decrease leading to disruptions and their movement to the binging site of sPLA2. The experiments conducted with bis-pyrene allowed us to examine whether changes in solvent access to laurdan (reflected by laurdan GP) correlate with the level of phospholipid chain order. Interestingly, the probe did not detect any change associated with dexamethasone treatment. Therefore, the average level of phospholipid chain order in the membrane remains stable, and the apparent penetration of water molecules into the membrane surface identified by laurdan presumably occured by different means. It was agreeable that our observations suggested the alterations in physical properties to be confined to the level of the phospholipid head region of the membrane rather than acyl chain order.
In my research I found several trouble-shooting difficulties such as planning times throughout the day for dexamethasone treatment which required commitment to wake up as early as 5 am, and staying as late as 11 am to run experiments and conform to the schedule. However, this did not force on me the impossible; on the contrary the results, the knowledge, and yet the continuing learning experience have given me confidence as researcher, and the persistence to repeat experiments for consistency and even for experimental errors. Moreover, the far more rewarding experience for me is to be able to participate in this coming March 2007 annual Biophysical Society in Baltimore, where our lab will present posters including the findings from this project. I am also optimistic that these results will also contribute in writing our up-coming paper which will be published in the Biophysics Journal.
Reference
polarization to distinguish between changes in membrane fluidity and phospholipid order. Biochim. Biophys. Acta. 1565:123–128.