Involvement of adenosine monophosphate activated kinase in interleukin-6 regulation of steroidogenic acute regulatory protein and cholesterol side chain cleavage enzyme in the bovine zona fasciculata and zona reticularis.

In bovine adrenal zona fasciculata (ZF) and NCI-H295R cells, interleukin-6 (IL-6) increases cortisol release, increases expression of steroidogenic acute regulatory protein (StAR), cholesterol side chain cleavage enzyme (P450scc), and steroidogenic factor 1 (SF-1) (increases steroidogenic proteins), and decreases the expression of adrenal hypoplasia congenita-like protein (DAX-1) (inhibits steroidogenic proteins). In contrast, IL-6 decreases bovine adrenal zona reticularis (ZR) androgen release, StAR, P450scc, and SF-1 expression, and increases DAX-1 expression. Adenosine monophosphate (AMP) activated kinase (AMPK) regulates steroidogenesis, but its role in IL-6 regulation of adrenal steroidogenesis is unknown. In the present study, an AMPK activator (AICAR) increased (P < 0.01) NCI-H295R StAR promoter activity, StAR and P450scc expression, and the phosphorylation of AMPK (PAMPK) and acetyl-CoA carboxylase (PACC) (indexes of AMPK activity). In ZR (decreased StAR, P450scc, SF-1, increased DAX-1) (P < 0.01) and ZF tissues (increased StAR, P450scc, SF-1, decreased DAX-1) (P < 0.01), AICAR modified StAR, P450scc, SF-1 and DAX-1 mRNAs/proteins similar to the effects of IL-6. The activity (increased PAMPK and PACC) (P < 0.01) of AMPK in the ZF and ZR was increased by AICAR and IL-6. In support of an AMPK role in IL-6 ZF and ZR effects, the AMPK inhibitor compound C blocked (P < 0.01) the effects of IL-6 on the expression of StAR, P450scc, SF-1, and DAX-1. Therefore, IL-6 modification of the expression of StAR and P450scc in the ZF and ZR may involve activation of AMPK and these changes may be related to changes in the expression of SF-1 and DAX-1.

Interleukin-6 increases the expression of key proteins associated with steroidogenesis in human NCI-H295R adrenocortical cells.

Mechanisms of interleukin-6 (IL-6)-induced cortisol release (CR) were investigated by exposing H295R cells to IL-6 and determining mRNA/protein expression (PCR/western blots) for steroidogenic enzymes (SE), steroidogenic acute regulatory protein (StAR), steroidogenic factor-1 (SF-1) (enhances SE/StAR expression), activator protein 1 (AP-1) (regulates SE/StAR expression) and adrenal hypoplasia congenita-like protein (DAX-1) (inhibits SE/StAR expression). Promoter activity of StAR (SPA) was measured by a luciferase-coupled promoter. Cortisol release was increased by 10ng/mL IL-6 (24h P<0.01). Proteins/mRNAs (StAR, cholesterol side chain cleavage enzyme, SF-1, AP-1) and SPA were increased by IL-6 (60min 1-50ng/mL IL-6; 5ng/mL IL-6 30-120min P<0.05). Four other SE proteins/mRNAs were also increased by 10ng/mL IL-6 (60min P<0.01). Protein/mRNA for DAX-1 was decreased by IL-6 (60min 1-50ng/mL IL-6; 5ng/mL IL-6 30-120min P<0.01). Phosphorylation of Janus kinase (JAK) and signal transducer and activator of transcription (STAT) was increased by IL-6 (JAK2 60min 1-50ng/mL IL-6; 10ng/mL IL-6 5-60min P<0.05; STAT1 and STAT3 60min 10ng/mL IL-6 P<0.01). Inhibition of JAK/STAT with AG490 (10µM) or piceatannol (50µM) blocked (P<0.01 10ng/mL IL-6vs. IL-6 plus AG490 or piceatannol) IL-6-induced increases in SPA and StAR mRNA. In summary, IL-6-induced CR may be facilitated by increased StAR and SE mediated by increased SF-1 and AP-1, decreased DAX-1, and increased phosphorylation of JAK/STAT.

Cigarette smoke increases cardiomyocyte ceramide accumulation and inhibits mitochondrial respiration.

BACKGROUND: Cigarette smoking is a common and lethal worldwide habit, with considerable mortality stemming from its deleterious effects on heart function. While current theories posit altered blood lipids and fibrinogen metabolism as likely mediators, none have explored the role of the sphingolipid ceramide in exacerbating heart function with smoke exposure. Ceramide production is a consequence of cigarette smoke in the lung, and considering ceramide's harmful effects on mitochondrial function, we sought to elucidate the role of ceramide in mediating smoke-induced altered heart mitochondrial respiration. METHODS: Lung cells (A549) were exposed to cigarette smoke extract (CSE) and heart cells (H9C2) were exposed to the lung-cell conditioned medium. Adult male mice were exposed sidestream cigarette smoke for 8 wk with dietary intervention and ceramide inhibition. Ceramides and heart cell or myocardial mitochondrial respiration were determined. RESULTS: Lung cell cultures revealed a robust response to cigarette smoke extract in both production and secretion of ceramides. Heart cells incubated with lung-cell conditioned medium revealed a pronounced inhibition of myocardial mitochondrial respiration, though this effect was mitigated with ceramide inhibition via myriocin. In vivo, heart ceramides increased roughly 600% in adult mice with long-term sidestream cigarette smoke exposure. This resulted in a significant ceramide-dependent reduction in left myocardial mitochondrial respiration, as heart mitochondria from the mice exposed to both smoke and myriocin injections respired normally. CONCLUSIONS: These results suggest ceramide to be an important mediator of altered myocardial mitochondrial function with cigarette smoke exposure. Thus, anti-ceramide therapies might be considered in the future to protect heart mitochondrial function with smoke exposure.

Ionomycin causes susceptibility to phospholipase A2 while temperature-induced increases in membrane fluidity fail: possible involvement of actin fragmentation.

A diminution in the order of membrane lipids, which occurs during apoptosis, has been shown to correlate with increased membrane susceptibility to hydrolysis by secretory phospholipase A2. Studies with artificial membranes, however, have demonstrated that the relationship between membrane order and hydrolysis is more complex than suggested thus far by cell studies. To better resolve this relationship, this study focused on comparisons between increasing temperature and calcium ionophore as means of decreasing membrane order in S49 cells. Although these two treatments caused comparable changes in apparent membrane order as detected by steady-state fluorescence measurements, only ionophore treatment enhanced phospholipase activity. Experiments with exogenously-added phosphatidylserine indicated that the difference was not due to the presence of that anionic phospholipid in the outer membrane leaflet. Instead, analysis of the equilibration kinetics of various cationic membrane probes revealed that the difference could relate to the spacing of membrane lipids. Specifically, ionophore treatment increased that spacing while temperature only affected overall membrane order and fluidity. To consider the possibility that the distinction with ionophore might relate to the actin cytoskeleton, cells were stained with phalloidin and imaged via confocal microscopy. Ionophore caused disruption of actin fibers while increased temperature did not. This apparent connection between membrane hydrolysis and the cytoskeleton was further corroborated by examining the relationship among these events during apoptosis stimulated by thapsigargin.

Membrane properties involved in calcium-stimulated microparticle release from the plasma membranes of S49 lymphoma cells.

This study answered the question of whether biophysical mechanisms for microparticle shedding discovered in platelets and erythrocytes also apply to nucleated cells: cytoskeletal disruption, potassium efflux, transbilayer phospholipid migration, and membrane disordering. The calcium ionophore, ionomycin, disrupted the actin cytoskeleton of S49 lymphoma cells and produced rapid release of microparticles. This release was significantly inhibited by interventions that impaired calcium-activated potassium current. Microparticle release was also greatly reduced in a lymphocyte cell line deficient in the expression of scramblase, the enzyme responsible for calcium-stimulated dismantling of the normal phospholipid transbilayer asymmetry. Rescue of the scrambling function at high ionophore concentration also resulted in enhanced particle shedding. The effect of membrane physical properties was addressed by varying the experimental temperature (32-42°C). A significant positive trend in the rate of microparticle release as a function of temperature was observed. Fluorescence experiments with trimethylammonium diphenylhexatriene and Patman revealed significant decrease in the level of apparent membrane order along that temperature range. These results demonstrated that biophysical mechanisms involved in microparticle release from platelets and erythrocytes apply also to lymphocytes.

Synergistic effects of secretory phospholipase A2 from the venom of Agkistrodon piscivorus piscivorus with cancer chemotherapeutic agents.

Healthy cells typically resist hydrolysis catalyzed by snake venom secretory phospholipase A2. However, during various forms of programmed cell death, they become vulnerable to attack by the enzyme. This observation raises the question of whether the specificity of the enzyme for dying cells could be used as a strategy to eliminate tumor cells that have been intoxicated but not directly killed by chemotherapeutic agents. This idea was tested with S49 lymphoma cells and a broad range of antineoplastic drugs: methotrexate, daunorubicin, actinomycin D, and paclitaxel. In each case, a substantial population of treated cells was still alive yet vulnerable to attack by the enzyme. Induction of cell death by these agents also perturbed the biophysical properties of the membrane as detected by merocyanine 540 and trimethylammonium-diphenylhexatriene. These results suggest that exposure of lymphoma cells to these drugs universally causes changes to the cell membrane that render it susceptible to enzymatic attack. The data also argue that the snake venom enzyme is not only capable of clearing cell corpses but can aid in the demise of tumor cells that have initiated but not yet completed the death process.

Molecular details of membrane fluidity changes during apoptosis and relationship to phospholipase A(2) activity.

Secretory phospholipase A(2) exhibits much greater activity toward apoptotic versus healthy cells. Various plasma membrane changes responsible for this phenomenon have been proposed, including biophysical alterations described as "membrane fluidity" and "order." Understanding of these membrane perturbations was refined by applying studies with model membranes to fluorescence measurements during thapsigargin-induced apoptosis of S49 cells using probes specific for the plasma membrane: Patman and trimethylammonium-diphenylhexatriene. Alterations in emission properties of these probes corresponded with enhanced susceptibility of the cells to hydrolysis by secretory phospholipase A(2). By applying a quantitative model, additional information was extracted from the kinetics of Patman equilibration with the membrane. Taken together, these data suggested that the phospholipids of apoptotic membranes display greater spacing between adjacent headgroups, reduced interactions between neighboring lipid tails, and increased penetration of water among the heads. The phase transition of artificial bilayers was used to calibrate quantitatively the relationship between probe fluorescence and the energy of interlipid interactions. This analysis was applied to results from apoptotic cells to estimate the frequency with which phospholipids protrude sufficiently at the membrane surface to enter the enzyme's active site. The data suggested that this frequency increases 50-100-fold as membranes become susceptible to hydrolysis during apoptosis.

Role of membrane oxidation in controlling the activity of human group IIa secretory phospholipase A(2) toward apoptotic lymphoma cells.

The membranes of healthy lymphocytes normally resist hydrolysis by secretory phospholipase A(2). However, they become susceptible during the process of apoptosis. Previous experiments have demonstrated the importance of certain physical changes to the membrane during cell death such as a reduction in membrane lipid order and exposure of phosphatidylserine on the membrane surface. Nevertheless, those investigations also showed that at least one additional factor was required for rapid hydrolysis by the human group IIa phospholipase isozyme. This study was designed to test the possibility that oxidation of membrane lipids is the additional factor. Flow cytometry and confocal microscopy with a fluorescent probe of oxidative potential suggested that oxidation of the plasma membrane occurs during apoptosis stimulated by thapsigargin. When oxidative potential was high, the activity of human group IIa secretory phospholipase A(2) was enhanced 30- to 100-fold compared to that observed with conditions sufficient for maximal hydrolysis by other secretory phospholipase A(2) isoforms. Direct oxidation of cell membranes with either of two oxidizing agents also stimulated hydrolysis by secretory phospholipase A(2). Both oxidizers caused externalization of phosphatidylserine, but a change in lipid order did not always occur. These results demonstrated that membrane oxidation strongly stimulates human group IIa secretory phospholipase A(2) activity toward apoptotic cells. Interestingly, the change in membrane order, previously thought to be imperative for high rates of hydrolysis, was not required when membrane lipids were oxidized. Whether phosphatidylserine exposure is still necessary with oxidation remains unresolved since the two events could not be deconvoluted.

Investigation into the role of phosphatidylserine in modifying the susceptibility of human lymphocytes to secretory phospholipase A(2) using cells deficient in the expression of scramblase.

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 100s. 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.

Relationship between membrane permeability and specificity of human secretory phospholipase A(2) isoforms during cell death.

During apoptosis, a number of physical changes occur in the cell membrane including a gradual increase in permeability to vital stains such as propidium iodide. This study explored the possibility that one consequence of membrane changes concurrent with early modest permeability is vulnerability to degradation by secretory phospholipase A(2). The activity of this hydrolytic enzyme toward mammalian cells depends on the health of the cell; healthy cells are resistant, but they become susceptible early during programmed death. Populations of S49 lymphoma cells during programmed death were classified by flow cytometry based on permeability to propidium iodide and susceptibility to secretory phospholipase A(2). The apoptotic inducers thapsigargin and dexamethasone caused modest permeability to propidium iodide and increased staining by merocyanine 540, a dye sensitive to membrane perturbations. Various secretory phospholipase A(2) isozymes (human groups IIa, V, X, and snake venom) preferentially hydrolyzed the membranes of cells that displayed enhanced permeability. In contrast, cells exposed briefly to a calcium ionophore showed the increase in cell staining intensity by merocyanine 540 without accompanying uptake of propidium iodide. Under that condition, only the snake venom and human group X enzymes hydrolyzed cells that were dying. These results suggested that cells showing modest permeability to propidium iodide during the early phase of apoptosis are substrates for secretory phospholipase A(2) and that specificity among isoforms of the enzyme depends on the degree to which the membrane has been perturbed during the death process. This susceptibility to hydrolysis may be important as part of the signal to attract macrophages toward apoptotic cells.

Kinetic evaluation of cell membrane hydrolysis during apoptosis by human isoforms of secretory phospholipase A2.

Some isoforms of secretory phospholipase A(2) (sPLA(2)) distinguish between healthy and damaged or apoptotic cells. This distinction reflects differences in membrane physical properties. Because various sPLA(2) isoforms respond differently to properties of artificial membranes such as surface charge, they should also behave differently as these properties evolve during a dynamic physiological process such as apoptosis. To test this idea, S49 lymphoma cell death was induced by glucocorticoid (6-48 h) or calcium ionophore. Rates of membrane hydrolysis catalyzed by various concentrations of snake venom and human groups IIa, V, and X sPLA(2) were compared after each treatment condition. The data were analyzed using a model that evaluates the adsorption of enzyme to the membrane surface and subsequent binding of substrate to the active site. Results were compared temporally to changes in membrane biophysics and composition. Under control conditions, membrane hydrolysis was confined to the few unhealthy cells present in each sample. Increased hydrolysis during apoptosis and necrosis appeared to reflect substrate access to adsorbed enzyme for the snake venom and group X isoforms corresponding to weakened lipid-lipid interactions in the membrane. In contrast, apoptosis promoted initial adsorption of human groups V and IIa concurrent with phosphatidylserine exposure on the membrane surface. However, this observation was inadequate to explain the behavior of the groups V and IIa enzymes toward necrotic cells where hydrolysis was reduced or absent. Thus, a combination of changes in cell membrane properties during apoptosis and necrosis capacitates the cell for hydrolysis differently by each isoform.

The influence of membrane physical properties on microvesicle release in human erythrocytes.

Exposure of human erythrocytes to elevated intracellular calcium causes fragments of the cell membrane to be shed as microvesicles. This study tested the hypothesis that microvesicle release depends on microscopic membrane physical properties such as lipid order, fluidity, and composition. Membrane properties were manipulated by varying the experimental temperature, membrane cholesterol content, and the activity of the trans-membrane phospholipid transporter, scramblase. Microvesicle release was enhanced by increasing the experimental temperature. Reduction in membrane cholesterol content by treatment with methyl-beta-cyclodextrin also facilitated vesicle shedding. Inhibition of scramblase with R5421 impaired vesicle release. These data were interpreted in the context of membrane characteristics assessed previously by fluorescence spectroscopy with environment-sensitive probes such as laurdan, diphenylhexatriene, and merocyanine 540. The observations supported the following conclusions: 1) calcium-induced microvesicle shedding in erythrocytes relates more to membrane properties detected by diphenylhexatriene than by the other probes; 2) loss of trans-membrane phospholipid asymmetry is required for microvesicle release.PACS Codes: 87.16.dj, 87.16.dt.

Sequence of physical changes to the cell membrane during glucocorticoid-induced apoptosis in S49 lymphoma cells.

During apoptosis, physical changes in the plasma membrane prepare the cell for clearance by phagocytes and hydrolysis by secretory phospholipase A(2) (sPLA(2)). The relationships among these changes have not been adequately established, especially for hormone-stimulated apoptosis. This study addresses these issues for glucocorticoid-induced apoptosis in S49 lymphoma cells. Flow cytometry, microscopy, and fluorescence spectroscopy were used to assess merocyanine 540 emission, laurdan generalized polarization, phosphatidylserine exposure, caspase activation, and membrane permeability to propidium iodide in the absence and presence of sPLA(2). The earliest event observed was activation of cellular caspases. Results with membrane probes suggest that interlipid spacing also increases early during apoptosis and precedes transbilayer migration of phosphatidylserine, DNA fragmentation, and a general increase in lipid order associated with blebbing and dissolution of the cells. The activity of sPLA(2) appeared to be linked more to lipid spacing than to loss of membrane asymmetry. The early nature of some of these events and their ability to promote activity of a proinflammatory enzyme suggests the possibility of an inflammatory response during T-lymphocyte apoptosis.

Leukemia inhibitory factor protein and receptors are expressed in the bovine adrenal cortex and increase cortisol and decrease adrenal androgen release.

The release of adrenal steroids during acute stress is primarily regulated by adrenocorticotropic hormone (ACTH). In contrast, during chronic inflammatory stress additional factors are involved in regulating adrenal function. Leukemia inhibitory factor (LIF) is a pleiotropic cytokine that increases ACTH release from the pituitary. In addition, LIF and LIF receptors (LIFR) are expressed in the human adrenal cortex and the human adrenocortical tumor cell line H295R. Furthermore, LIF increases basal and ACTH-stimulated cortisol release from H295R cells. However, the expression of LIF and LIFR in non-human adrenal glands and the effects of LIF on the release of cortisol from adrenal cells of non-human species have not been determined. Furthermore, the effects of LIF on adrenal androgen release from all species are unknown. In this study, immunohistochemistry, Western blots, RT-PCR, and nucleotide sequencing was utilized to demonstrate that LIF and its receptor are expressed throughout the bovine adrenal cortex. Although LIF did not modify basal cortisol release from dispersed cells isolated from the bovine adrenal zona fasciculate, this cytokine increased ACTH-stimulated release of cortisol from these cells in a manner dependent on the LIF concentration and exposure interval. In contrast, LIF in a concentration-dependent and time-dependent manner decreased basal and ACTH-stimulated adrenal androgen release from dispersed cells isolated from the bovine adrenal zona reticularis. Because LIF release increases during inflammatory stress and this cytokine stimulates adrenal cortisol release and inhibits adrenal androgen release, this cytokine may play an important role in regulating the release of adrenal steroids during inflammatory stress.

Effects of cholesterol on physical properties of human erythrocyte membranes: impact on susceptibility to hydrolysis by secretory phospholipase A2.

The ability of secretory phospholipase A(2) (sPLA(2)) to hydrolyze cell membranes is highly dependent on the physical properties of the membrane. The effects of cholesterol on these properties have been characterized in artificial bilayers and found to alter sPLA(2) activity significantly. It is hypothesized that the natural difference in cholesterol content between erythrocytes and leukocytes is in part responsible for their differing susceptibility to hydrolysis by sPLA(2). To test this hypothesis, defined amounts of cholesterol were removed from erythrocyte membranes using methyl-beta-cyclodextrin. Treatment of cells with methyl-beta-cyclodextrin increased the hydrolysis rate and total substrate hydrolyzed by sPLA(2). In general, this effect of cholesterol removal was more pronounced at higher temperatures. Comparison of the level of membrane order (assessed with the fluorescent probe laurdan) with hydrolysis rate revealed that sPLA(2) activity was greatly enhanced upon significant reductions in lipid order. Additional treatment of the cells with calcium ionophore further enhanced the hydrolysis rate and altered the relationship with membrane order. These data demonstrated that interactions with sPLA(2) observed in artificial bilayers apply to biological membranes. It is also proposed that the high level of cholesterol in erythrocyte membranes is a protective mechanism to guard against hydrolytic enzymes.

Interleukin-4 increases cortisol release and decreases adrenal androgen release from bovine adrenal cells.

ACTH is the primary regulator of adrenal function during acute stress. However, during chronic inflammatory stress additional factors play a major role in the regulation of adrenal secretion. Many cytokines circulate in the blood and are synthesized and released from adrenal tissue. Furthermore, these peptides modify adrenal function. Recently, interleukin-4 (IL-4) was demonstrated to be released from a human adrenal tumor cell line. Therefore, we hypothesized that normal bovine adrenocortical cells could express IL-4 and that this cytokine may modify adrenal function. We determined that IL-4 and IL-4 receptors (IL-4R) are expressed in the bovine adrenal cortex whereas the expression of IL-4 and IL-4R in the adrenal medulla was not apparent. Exposure of dispersed bovine adrenocortical cells isolated from the zona fasciculate to IL-4 did not modify basal release of cortisol. However, the ACTH-stimulated release of cortisol from the bovine adrenal cells was augmented by IL-4. IL-4 exposure had no affect on adrenal androgen release from bovine zona reticularis cells, but IL-4 inhibited the ACTH-stimulated release of adrenal androgens from these cells. The effects of IL-4 on ACTH-stimulated cortisol and adrenal androgen release were dependent upon the IL-4 incubation interval and the IL-4 concentration. Because communication between the immune and endocrine systems is important in inflammatory conditions, IL-4 may play a role in coordinating the adrenal response to inflammatory stress.

Relationship between membrane physical properties and secretory phospholipase A2 hydrolysis kinetics in S49 cells during ionophore-induced apoptosis.

During apoptosis, changes occur in lymphocyte membranes that render them susceptible to hydrolysis by secretory phospholipase A(2) (sPLA(2)). To study the relevant mechanisms, a simplified model of apoptosis using a calcium ionophore was applied. Kinetic and flow cytometry experiments provided key observations regarding ionophore treatment: the initial rate of hydrolysis was elevated at all enzyme concentrations, the total amount of reaction product was increased fourfold, and adsorption of the enzyme to the membrane surface was unaltered. Analysis of these results suggested that susceptibility during calcium-induced apoptosis is limited by availability of substrate rather than adsorption of enzyme. Fluorescence experiments identified three membrane alterations during apoptosis that might affect substrate access to the sPLA(2) active site. First, intercalation of merocyanine 540 into the membrane was improved, suggesting an increase in lipid spacing. Second, laurdan detected increased solvation of the lower headgroup region of the membrane. Third, the rate at which fluorescent lipids could be removed from the membrane by albumin was enhanced, implying greater vertical mobility of phospholipids. Thus, it is proposed that the membranes of apoptotic cells become susceptible to sPLA(2) through a reduction in lipid-neighbor interactions that facilitates migration of phospholipids into the enzyme active site.

Use of steady-state laurdan fluorescence to detect changes in liquid ordered phases in human erythrocyte membranes.

In artificial phospholipid bilayers, dual measurements of laurdan steady-state anisotropy and emission spectra can be used to identify the presence of liquid ordered phases. Human erythrocytes were used as a model to test whether similar measurements could be applied to biological samples. Specifically, laurdan anisotropy and emission spectra were obtained from native erythrocytes before and after treatment with calcium ionophore and from the microvesicles (known to be enriched in liquid ordered domains) shed from the cells during calcium entry. Spectral and anisotropy data were consistent with an increased order and reduced fluidity of erythrocyte membrane lipids upon ionophore treatment. Microvesicle membranes appeared more ordered than native erythrocytes and similar to ionophore-treated cells based on laurdan emission. In contrast, the anisotropy value was lower in microvesicles compared to ionophore-treated cells, suggesting greater probe mobility. Parallel measurements of diphenylhexatriene anisotropy corroborated the laurdan data. These results were consistent with the liquid ordered property of microvesicle membranes based on comparisons to behavior in artificial membranes. Two-photon microscopy was used to examine the distribution of laurdan fluorescence along the surface of erythrocyte membranes before and after ionophore treatment. A dual spatial analysis of laurdan anisotropy, as revealed by the distribution of laurdan emission spectra, and intensity excited by polarized light suggested that the plasma membranes of ionophore-treated erythrocytes may also exhibit elevated numbers of liquid ordered domains.

Mechanisms governing the level of susceptibility of erythrocyte membranes to secretory phospholipase A2.

Although cell membranes normally resist the hydrolytic action of secretory phospholipase A(2) (sPLA(2)), they become susceptible during apoptosis or after cellular trauma. Experimentally, susceptibility to the enzyme can be induced by loading cells with calcium. In human erythrocytes, the ability of the calcium ionophore to cause susceptibility depends on temperature, occurring best above approximately 35 degrees C. Considerable evidence from experiments with artificial bilayers suggests that hydrolysis of membrane lipids requires two steps. First, the enzyme adsorbs to the membrane surface, and second, a phospholipid diffuses from the membrane into the active site of the adsorbed enzyme. Analysis of kinetic experiments suggested that this mechanism can explain the action of sPLA(2) on erythrocyte membranes and that temperature and calcium loading promote the second step. This conclusion was further supported by binding experiments and assessment of membrane lipid packing. The adsorption of fluorescent-labeled sPLA(2) was insensitive to either temperature or ionophore treatment. In contrast, the fluorescence of merocyanine 540, a probe sensitive to lipid packing, was affected by both. Lipid packing decreased modestly as temperature was raised from 20 to 60 degrees C. Calcium loading enhanced packing at temperatures in the low end of this range, but greatly reduced packing at higher temperatures. This result was corroborated by measurements of the rate of extraction of a fluorescent phosphatidylcholine analog from erythrocyte membranes. Furthermore, drugs known to inhibit susceptibility in erythrocytes also prevented the increase in phospholipid extraction rate. These results argue that the two-step model applies to biological as well as artificial membranes and that a limiting step in the hydrolysis of erythrocyte membranes is the ability of phospholipids to migrate into the active site of adsorbed enzyme.

Divalent cations increase lipid order in erythrocytes and susceptibility to secretory phospholipase A2.

Elevated concentrations of intracellular calcium in erythrocytes increase membrane order and susceptibility to secretory phospholipase A2. We hypothesize that calcium aids the formation of domains of ordered lipids within erythrocyte membranes by interacting directly with the inner leaflet of the cell membrane. The interface of these domains with regions of more fluid lipids may create an environment with weakened neighbor-neighbor interactions that would facilitate phospholipid migration into the active site of bound secretory phospholipase A2. This hypothesis was investigated by determining the effects of seven other divalent ions on erythrocyte membrane properties. Changes in membrane order were assessed with steady-state fluorescence spectroscopy and two-photon microscopy with an environment-sensitive probe, laurdan. Each ion increased apparent membrane order in model membranes and in erythrocytes when introduced with an ionophore, suggesting that direct binding to the inner face of the membrane accounts for the effects of calcium on membrane fluidity. Furthermore, the degree to which ions affected membrane properties correlated with the ionic radius and electronegativity of the ions. Lastly, erythrocytes became more susceptible to enzyme hydrolysis in the presence of elevated intracellular levels of nickel and manganese, but not magnesium. These differences appeared related to the ability of the ions to induce a transition in erythrocyte shape.