Evidence that the cell wall of Bacillus subtilis is protonated during respiration.

Several independent experiments suggest that cell walls of Bacillus subtilis are protonated during growth. When cells were grown in the presence of fluorescein-labeled dextran to saturate the cell walls, centrifuged, and suspended in PBS, fluorescence-activated cell sorter analyses revealed the bacteria were only poorly fluorescent. In contrast, when the bacteria were purged with N(2) to dissipate protonmotive force (pmf) fluorescence became intense. Upon reconstitution of the pmf with phenazine methosulfate, glucose, and oxygen, fluorescence declined. Another approach used pH-dependent chemical modification of cell walls. The walls of respiring B. subtilis cells were amenable to carboxylate modification by [(14)C]ethanolamine and 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide. The carbodiimide activation of carboxylate groups occurs only in acidic conditions. Upon dissipation of pmf the walls were refractory to chemical modification. Ammonium groups can be condensed with FITC in alkaline medium, but the condensation is very slow in acidic buffers. It was found that the derivatization of the walls with FITC could occur in the absence of pmf. The use of pH-dependent fluorophores and pH-dependent chemical modification reactions suggest that cell walls of respiring B. subtilis cells have a relatively low pH environment. This study shows a bacterium has a protonated compartment. Acidification of cell walls during growth may be one means of regulating autolytic enzymes.

The uptake and metabolism of fructose-1,6-diphosphate in rat cardiomyocytes.

Fructose-1,6-diphosphate (FDP) is a glycolytic intermediate which has been theorized to increase the metabolic activity of ischemic tissues. Here we examine the effects of externally applied FDP on cardiomyocyte uptake and metabolism. Adult rat cardiomyocytes were isolated and exposed to varying concentrations (0, 5, 25 and 50 mM) of FDP for either 1, 16 or 24 h of hypoxia (95% N2/5% CO2), each time period followed by a 1 h reoxygenation (95% air/5% CO2). The uptake of FDP by rat cardiomyocytes was more concentration-dependent than time-dependent. Furthermore, the uptake of FDP by the cardiomyocytes was similar in the hypoxia and normoxia treated cells. Alamar Blue, a redox indicator that is sensitive to metabolic activity, was used to monitor the effects of the FDP on cardiomyocyte metabolism. In the 1 h hypoxia or normoxia group, the 5, 10 and 25 mM FDP showed a significant increase in metabolism compared to the control cells. When the length of hypoxia was extended to 16 h, all doses of FDP were greater than control. And at the 24 h hypoxia or normoxia time period, only the 10, 25 and 50 mM FDP groups were greater than control. The results indicate a non-linear trend between the external concentration of FDP and the changes noted in metabolism. The findings from this study indicate that a narrow concentration range between 5-10 mM augments cardiomyocyte metabolism, but higher or lower doses may have little additional affect.

Membrane permeability of fructose-1,6-diphosphate in lipid vesicles and endothelial cells.

Fructose-1,6-diphosphate (FDP) is a glycolytic intermediate which has been used an intervention in various ischemic conditions for two decades. Yet whether FDP can enter the cell is under constant debate. In this study we examined membrane permeability of FDP in artificial membrane bilayers and in endothelial cells. To examine passive diffusion of FDP through the membrane bilayer, L-alpha-phosphatidylcholine from egg yolk (Egg PC) (10 mM) multi-lamellar vesicles were created containing different external concentrations of FDP (0, 0.5, 5 and 50 mM). The passive diffusion of FDP into the vesicles was followed spectrophotometrically. The results indicate that FDP diffuses through the membrane bilayer in a dose-dependent fashion. The movement of FDP through Egg PC membrane bilayers was confirmed by measuring the conversion of FDP to dihydroxyacetone-phosphate and the formation of hydrozone. FDP (0, 0.5, 5 or 50 mM) was encapsulated in Egg PC multilamellar vesicles and placed in a solution containing aldolase. In the 5 and 50 mM FDP groups there was a significant increase in dihydroxyacetone/hydrazone indicating that FDP crossed the membrane bilayer intact. We theorized that the passive diffusion of FDP might be due to disruption of the membrane bilayer. To examine this hypothesis, small unilamellar vesicles composed of Egg PC were created in the presence of 60 mM carboxyfluorescein, and the leakage of the sequestered dye was followed upon addition of various concentrations of FDP, fructose, fructose-6-phosphate, or fructose-1-phosphate (0, 5 or 50 mM). These results indicate that increasing concentrations of FDP increase the leakage rate of carboxyfluorescein. In contrast, no concentration of fructose, fructose-6-phosphate, or fructose-1-phosphate resulted in any significant increase in membrane permeability to carboxyfluorescein. To examine whether FDP could pass through cellular membranes, we examined the uptake of 14C-FDP by endothelial cells cultured under hypoxia or normoxia for 4 or 16 h. The uptake of FDP was dose-dependent in both the normoxia and hypoxia treated cells, and was accompanied by no significant loss in endothelial cell viability. Our results demonstrate that FDP can diffuse through membrane bilayers in a dose-dependent manner.

Docosahexaenoic acid reverses cyclosporin A-induced changes in membrane structure and function.

The use of a fish oil vehicle for cyclosporin A (CsA) can decrease the toxic effects of CsA but the mechanism is unclear. Here we examine the mechanism by which docosahexaenoic acid (DHA), a fish oil-derived polyunsaturated fatty acid, can alter the toxic effects of CsA on mouse organ function, endothelial macromolecular permeability, and membrane bilayer function. Mice given CsA and fish oil showed increased liver toxicity, kidney toxicity, incorporation of DHA, and evidence of oxidized fatty acids compared to control animals. We hypothesized that the toxic effects of CsA were primarily a result of membrane perturbation, which could be decreased if DHA were not oxidized. The presence of CsA (10 mol%) alone increased dipalmitoylphosphatidylcholine membrane permeability by seven fold over control (no CsA, no DHA). However, if non-oxidized DHA (15 mol%) and CsA were added to the membrane, the permeability returned to control levels. Interestingly, if the DHA was oxidized, the antagonistic effect of DHA on CsA was completely lost. While CsA alone increased endothelial permeability to albumin, the combination of non-oxidized DHA and CsA had no effect on endothelial macromolecular permeability. However the combination of oxidized DHA and CsA was no different than the effects of CsA only. CsA increased the fluorescence anisotropy of DPH in the liquid crystalline state of DPPC, while DHA decreased fluorescence anisotropy. However the combination of CsA and DHA was no different than DHA alone. We conclude that non-oxidized DHA can reverse the membrane perturbing effects of CsA, and the increases in endothelial macromolecular permeability, which may explain how fish oil is capable of decreasing the toxicity of CsA.

Fructose-1,6-diphosphate and a glucose-free solution enhances functional recovery in hypothermic heart preservation.

BACKGROUND: Fructose-1,6-diphosphate (FDP) has been shown to protect tissue during hypoxia under various ischemic conditions, including isolated heart perfusion. We tested the hypothesis that adding FDP to St. Thomas solution can extend hypothermic heart preservation time. METHODS: Sixteen adult Sprague-Dawley rats were used. Under general anesthesia, the hearts were removed and preserved at 4 degrees C in St. Thomas solution (30 ml/kg) for 12 hours. FDP (5 mM) was added to the St. Thomas solution in the study group (n = 8), whereas no FDP was used in the control group (n = 10). The hearts were reperfused after 12 hours of preservation using a working heart model. RESULTS: In the study group, cardiac output ranged from 13.00 +/- 2.34 to 17.66 +/- 1.71 ml/min, maximum aortic flow was 3.40 +/- 1.99 to 9.26 +/- 1.72 ml/min, left ventricular stroke volume ranged from 0.074 +/- 0.014 to 0.092 +/- 0.009 ml, left ventricular stroke work ranged from 6.22 +/- 0.39 to 7.95 +/- 0.44 ml/mmHg, and maximum left ventricular generated power was 14.38 +/- 2.94 to 20.16 +/- 2.49 Joules/min. All of these parameters were higher than those in the control group (p < 0.001). Coronary vascular resistance and myocardial tissue wet/dry weight ratio were lower in the study group than in the control group (p < 0.05). CONCLUSIONS: Heart function was better preserved when FDP was added to St. Thomas solution during hypothermic rat heart preservation. The mechanism is not totally clear, but enhancement of high-energy phosphate production during ischemia is possible. Key words: heart, procurement, hypothermia, fructose-1,6-diphosphate.

Bradykinin and alpha-thrombin increase human umbilical vein endothelial macromolecular permeability by different mechanisms.

Bradykinin and alpha-thrombin both increase endothelial macromolecular permeability, however the mechanism for this effect is unclear. Human umbilical vein endothelial cell (HUVEC) permeability to human serum albumin was increased by 1 microM alpha-thrombin (AT) or bradykinin (BK), but the kinetics of the permeability response were different. Intracellular calcium mobilization of HUVEC by AT was increased, yet BK had no effect on intracellular calcium. Distribution of F-actin and content was increased by AT as early as 10 minutes after administration, yet BK had no affect on F-actin when compared to control. We hypothesized that BK may increase HUVEC permeability by producing matrix metalloproteinase-2 (MMP-2). The AT-treated HUVEC produced an intermediate 64 kDa MMP-2, whereas BK-treated HUVEC increased the intermediate 64 kDa MMP-2 and also an active 62 kDa MMP-2. Pre-treatment of the HUVEC with tissue inhibitor of matrix metalloproteinase-2 slightly decreased the AT-induced increase in macromolecular permeability and completely inhibited the BK-induced increase in macromolecular permeability.

A new continuous monitoring system for simultaneous measurements of intracellular calcium and either endothelial permeability or leukocyte extravasation.

OBJECTIVE AND DESIGN: We have developed a continuous monitoring system that will quantify lymphocyte extravasation or intercellular permeability with intracellular calcium measurements in a single preparation. MATERIALS: Human microvascular endothelial cells, human lymphocytes and histamine. TREATMENT: Endothelial barrier function and intracellular calcium were examined upon application of either human lymphocytes (1 x 10(5) cells/ml) or histamine (1 microM). METHODS: Endothelial cells labeled with FURA-2 were examined for both changes in endothelial barrier function and mobilization of intracellular calcium using fluorescence spectroscopy and ratio imaging, respectively. RESULTS: Increases in endothelial intracellular calcium occur in the first minute and are followed by increases in endothelial intercellular permeability to albumin (5-7 min) or lymphocytes (15-20 min). Approximately 60 min following calcium mobilization, there was a second and larger extravasation. CONCLUSION: The continuous monitoring system expands our understanding of the time course of the permeability and extravasation events to mobilization of endothelial intracellular calcium.

Enhancement of hypothermic heart preservation with fructose 1, 6-diphosphate.

BACKGROUND: We hypothesized that the addition of fructose 1, 6-diphosphate (FDP) to a hypothermic heart preservation solution could improve metabolic recovery because it has several beneficial effects. MATERIALS AND METHODS: Twenty adult Sprague-Dawley rats were used to study hypothermic heart preservation. The hearts were removed under general anesthesia and preserved at 4 degrees C in Euro-Collins solution (30 ml/kg) for 8 h. In the study group (N = 10), FDP (5 mM) was added to the Euro-Collins solution. In the control group (N = 10), no FDP was added. Heart function was studied after preservation using a working heart model. The ability of various concentrations of fructose 1,6-phosphate to passively diffuse through an egg phosphatidylcholine multilamellar vesicle (MLV) membrane bilayer was examined. RESULTS: Cardiac output ranged from 17.0 +/- 1.9 to 24.9 +/- 1.6 ml/min in the study group vs 2.0 +/- 1.0-12.3 +/- 1.7 ml/min for controls, average aortic flow was 10. 8 +/- 1.4 ml/min in the study group vs -1.3 +/- 1.6 ml/min for controls, and maximum LV generated power was 22.8 +/- 1.7 J/min vs 10.1 +/- 1.6 J/min for controls. Coronary flow, left ventricular stroke volume and stroke work, and myocardial oxygen consumption were much higher in the study group than in the control group. Coronary vascular resistance was lower in the study group than in the control group. Electron microscopic study indicated that many myocytes displayed patches of swollen mitochondria in the control group, but was rarely observed in the study group. The addition of 50 mM FDP caused substantial changes in MLV permeability. No dose of sucrose buffers outside the vesicles resulted in a significant changes of MLV permeability. CONCLUSIONS: Our results indicate that the addition of FDP to Euro-Collins solution significantly improves hypothermic rat heart preservation, and FDP appeared to cross the membrane bilayer.

Quantitative image analysis of F-actin in endothelial cells.

OBJECTIVE: Filamentous actin (F-actin) plays a central role in maintaining endothelial barrier function. Thrombin and histamine, two inflammatory mediators that increase endothelial permeability, can alter F-actin production and distribution. In this study, we use a newly developed image analysis technique to show that these two inflammatory mediators differentially alter F-actin structure. METHODS: Human umbilical vein endothelial cells were grown to confluence and treated with either histamine (1 microM), thrombin (1 microM) or the agonist's vehicle. The endothelium was stained with BODIPY-phallodin, and digitized images were taken of the treated cells. The digitized images of individual human umbilical vein endothelial cells (HUVEC) were imported into a F-actin image analysis program (FAAP) and converted to layers, each one pixel thick. The program then determined the mean gray level (which corresponded to the amount of F-actin) in each layer starting from the outside of the cell (layer 1) and progressing in one pixel layer increments towards the center of the cell (layer 32). RESULTS: Both inflammatory mediators increased endothelial F-actin production, however, the distribution of the actin was different. Thrombin increased the presence of stress fibers, while also decreasing peripheral banding actin. In contrast, histamine had no effect on peripheral actin compared to control, but did increase the presence of F-actin stress fibers. CONCLUSIONS: These results establish that thrombin and histamine alter endothelial F-actin production in different locations within the cell, which can be quantified using an image analysis program.

An increase in endothelial intracellular calcium and F-actin precedes the extravasation of interleukin-2-activated lymphocytes.

OBJECTIVE: Interleukin-2 (IL-2) induces protein leakage from the microcirculation and activates lymphocytes; yet it is unclear how it alters endothelial barrier function. Here, we report of a new continuous monitoring system that allows for the continuous measurement and correlation of endothelial calcium, permeability to albumin, and extravasation of lymphocytes. METHODS: IL-2 activated lymphocytes (IL-2 LYMPH) or unstimulated lymphocytes (LYMPH) were co-incubated with human microvascular endothelial cells (HMVEC). Endothelial albumin permeability, lymphocyte extravasation intracellular calcium mobilization, and f-actin distribution were examined using a new continuous monitoring system. RESULTS: The clearance rate of fluorescein isothiocyanate-labeled-human serum albumin (FITC-HSA) in the presence of IL-2 LYMPH peaked at 20 minutes, whereas the clearance rate of LYMPH peaked at 40 minutes. Approximately 40 minutes after the peak in the clearance rate to albumin, extravasation of carboxyfluorescein-labeled lymphocytes was detected. Peak clearance rates for the extravasation of IL-2 LYMPH occurred at approximately 40 minutes after the addition of the lymphocytes to the HMVEC, whereas the peak clearance rate for the LYMPH occurred at 60 minutes after their addition. Both FITC-HSA and lymphocyte extravasation were measured concurrent to endothelial intracellular calcium mobilization by FURA-2. There was an increase in calcium activation after the addition of IL-2 stimulated lymphocytes (71 +/- 5.1 nmol/L to 185 +/- 18.9 nmol/L) compared with unstimulated lymphocytes (71 +/- 5.1 nmol/L to 110 +/- 12.2 nmol/L). The addition of IL-2 had little or no effect on endothelial actin, whereas the unstimulated lymphocytes and, to a greater extent, IL-2 LYMPH increased the presence of transversing stress fibers and decreased peripheral actin. CONCLUSIONS: The findings reported here suggest that the permeability and extravasation events that occur upon addition of lymphocytes proceeds by a calcium- and actin-dependent mechanism and that incubation of lymphocytes with IL-2 enhances normal lymphocyte mechanisms of extravasation.

Bradykinin antagonizes the effects of alpha-thrombin.

alpha-Thrombin (AT) and bradykinin (BK) are endogenous mediators that are released during an inflammatory response, and could have a synergistic effect on endothelial permeability. Human umbilical vein endothelial cells (HUVEC) were grown on Transwell membranes and then tested for alterations in permeability to fluorescein isothiocyanate-labeled human serum albumin. Addition of 1 microM AT produced a significant increase in the permeability coefficient at 30 minutes from control levels of 1.59 x 10(-6) cm/sec to 4.92 x 10(-6) cm/sec. BK (1 microM) produced a similar increase to 4.46 x 10(-6) cm/sec. For both compounds, permeability remained elevated for 90 minutes. Pre-treatment of the HUVEC with the bradykinin receptor antagonist, Na-adamantaneacetyl-bradykinin (NA-BK) (1 microM), prior to addition of AT, reduced the AT permeability coefficient to 2.69 x 10(-6) cm/sec. Addition of NA-BK (1 microM) for 5 minutes, then BK (1 microM) for 5 minutes, inhibited the effect of BK and of AT (1 microM on permeability, decreasing the permeability coefficient of the endothelial monolayer to control levels (1.62 x 10(-6) cm/sec). AT (1 microM) increased HUVEC intracellular calcium mobilization, as monitored by FURA-2, to 245 nM from control (70 nM), however, pre-treatment with either BK or the bradykinin receptor antagonist decreased the AT induced intracellular calcium mobilization compared to AT alone. Pre-treatment of the HUVEC with bradykinin (1 microM) for 2 minutes also inhibited the effects of alpha-thrombin (1 microM) on f-actin distribution examined by BODIPY-phallodin staining and increased the clotting times for an alpha-thrombin dependent fibrinogen to fibrin clotting assay. However, incubation of bradykinin (1 microM) with alpha-thrombin (1 microM) for either 10 minutes or 100 minutes produced no detectable hydrolysis products. These data strongly suggest that the inflammatory mediators alpha-thrombin and bradykinin when released together, rather than being synergistic, are antagonistic.

Effect of docosahexaenoic acid on mouse mitochondrial membrane properties.

Long-chain polyunsaturated (n-3) fatty acids have been proposed to be involved in a wide variety of biological activities. In this study, mitochondrial docosahexaenoic acid (DHA) levels were increased by either dietary manipulation or by fusing the mitochondria with phospholipid vesicles made from 1-stearoyl-2-docosahexaenoyl-sn-glycero-3-phosphocholine (18:0/22:6 PC). The fused mitochondria exhibited a DHA-induced decrease in respiratory control index (RCI) and membrane potential and an increase in proton movement. The modified mitochondria also demonstrated an increase in fluidity (as detected by 1,6-diphenyl-1,3,5-hexatriene anisotropy) and changes in membrane structure detected by the fluorescence probes MC540 and pyrene decanoate. Proton movement in lipid vesicles made from mitochondrial lipid extracts was shown to be enhanced by incorporated 18:0/22:6 PC. Mitochondria were isolated from young (5-mon) and old (24-mon) mice which were maintained on either a diet rich in saturated fats (hydrogenated coconut oil) or rich in n-3 polyunsaturated fats (menhaden oil). Mitochondrial bioenergetic function was followed by RCI, state 3 respiration, ATP level, and phosphate uptake. In addition, lipid composition, phospholipid area/molecule and extent of lipid peroxidation were also determined. Decreases in RCI for the menhaden oil diet-modified mitochondria paralleled those in which DHA levels were enhanced by fusion with phospholipid vesicles. RCI reductions are attributed to DHA-induced increases in H+ movement, producing diminished mitochondrial membrane potentials. One purpose of this project was to determine if the deleterious effects of aging on mitochondrial bioenergetic function could be reversed by addition of n-3 fatty acids. The experiments reported here indicate that incorporation of long-chain polyunsaturated n-3 fatty acids into mitochondrial membranes does not appear likely to reverse the effects of age on mitochondrial function.

Oxidized low-density lipoprotein increases endothelial intracellular calcium and alters cytoskeletal f-actin distribution.

Central to the pathogenesis of atherosclerosis is an abnormally functioning endothelium and a consequent loss of vascular integrity. These abnormalities may be induced by haemodynamic factors, biochemical substances, and also by oxidatively modified low-density lipoprotein (LDL). To understand the mechanism by which oxidized LDL causes endothelial dysfunction, human umbilical vein endothelial cells (HUVECs) were loaded with FURA-2, and intracellular calcium mobilization was studied in acute (seconds after LDL was injected) or chronic (24 h after LDL was injected) preparations. Our results demonstrate that 100 microg mL(-1) oxidized LDL increases HUVEC intracellular calcium. In contrast, native LDL at this same concentration had no effect. In addition, chronic exposure (24 h) of HUVECs to oxidized LDL significantly increases HUVEC intracellular calcium. Fluorescent photomicrographs of HUVECs stained with BODIPY-phalloidin f-actin indicates that oxidized LDL causes a reorganization of microfilaments. The results of this study demonstrate that the mechanism by which oxidized LDL causes a loss of vascular integrity could be through activation of endothelial cells to increase cytosolic calcium, which alters the endothelial barrier by reorganizing the cytoskeleton.

Mechanisms of alpha-thrombin, histamine, and bradykinin induced endothelial permeability.

alpha-Thrombin, bradykinin, and histamine are endogenous mediators that increase endothelial permeability. We examined the mechanism by which these three vasoactive mediators could alter permeability to albumin of human umbilical vein endothelial cells (HUVEC). HUVEC were grown to confluence on Transwell membranes and we monitored the flux of fluorescein isothiocyanate-labeled human serum albumin across the membrane from the upper to lower chamber of the Transwell. Addition of alpha-thrombin, bradykinin, or histamine increased the permeability coefficient of the HUVEC monolayer. At 30 min the permeability coefficient for alpha-thrombin was 4.92 x 10(-6) cm/sec while histamine was 4.47 x 10(-6) cm/sec. Maximum changes in the permeability coefficient were about three-fold control baseline values (1.59 x 10(-6) cm/sec). There was also a temporal difference in the magnitude of the permeability coefficient. alpha-Thrombin and bradykinin induced HUVEC permeability was increased for the first 90 min after which it returned to control levels. In contrast, histamine increased the permeability of the HUVEC monolayer throughout the 2 h experiment. To determine a possible intracellular mechanism of the altered permeability coefficients, HUVEC were labeled with FURA-2 and intracellular calcium was monitored by digital fluorescence ratio imaging. Maximum intracellular calcium in HUVEC was increased by alpha-thrombin (245 +/- 20 nM) and histamine (210 +/- 22 nM), but not by bradykinin (70 +/- 7 nM) as compared to control (69 +/- 10). Fluorescent photomicrographs of HUVEC stimulated with the three agonists indicated that alpha-thrombin and histamine substantially altered HUVEC f-actin arrangement, while bradykinin had no effect on HUVEC f-actin distribution. These data support previous in vitro and in vivo studies demonstrating increased permeability by all three agonists. These data also show, for the first time, that histamine and alpha-thrombin increased permeability by calcium-dependent intracellular pathways, but bradykinin operates through a calcium-independent mechanism.

Docosahexaenoic acid-induced alteration of Thy-1 and CD8 expression on murine splenocytes.

Here we test whether the incorporation of docosahexaenoic acid (DHA, 22:6), an (n-3) fatty acid, into lymphocyte membranes affects the expression of the surface proteins Thy-1.2 and CD8. DHA was incorporated into splenocytes by three methods: feeding mice diets containing menhaden (fish) oil, fusing splenocytes with DHA-containing phosphatidylcholine vesicles, and culturing splenocytes with DHA. Thy-1.2 and CD8 expression were measured by flow cytometry and complement-mediated lysis using a panel of monoclonal antibodies. As (n-3) fatty acid incorporation into the lymphocytes increased, the expression of one Thy-1.2 epitope and one CD8 epitope decreased; the expression of two CD8 epitopes increased. Although diet-induced changes in surface protein expression may result from selective migration of cell populations or the diet's effect on protein biosynthesis, fusion with lipid vesicles demonstrated that DHA-containing phospholipids can mediate a direct and immediate effect. The decrease in Thy-1.2 expression was sustained for more than a week after removal of (n-3) fatty acids from the diet, most likely due to retention of membrane-bound (n-3) fatty acids. Because Thy-1.2 and CD8 participate in T cell activation, modulation of their expression by DHA suggests that DHA, when serving as a membrane structural element, may alter immune function.

Cholesterol condensation of alpha-linolenic and gamma-linolenic acid-containing phosphatidylcholine monolayers and bilayers.

Cholesterol is demonstrated to condense phosphatidylcholine (PC) monolayers and bilayers containing stearic acid in the sn-1 position and alpha-linolenic acid in the sn-2 position (18:0, alpha-18:3 PC) but has no effect when gamma-linolenic acid occupies the sn-2 position (18:0,gamma-18:3 PC). Cholesterol-induced condensation is measured by area/molecule determinations made on monolayers using a Langmuir trough, while condensation in bilayers is followed by the fluorescent dyes merocyanine (MC540) and dansyllysine. Permeability to erythritol is also demonstrated to be diminished by cholesterol for the condensable 18:0,alpha-18:3 PC bilayer membranes but not the 18:0,gamma-18:3 PC membranes. alpha- and gamma-linolenic acid are isomers containing 18 carbons and three unsaturations. Both fatty acids have unsaturations at positions 9 and 12 and differ only in the location of the third unsaturation, at either position 6 for gamma-linolenic acid (an omega-6 fatty acid) and at position 15 for alpha-linolenic acid (an omega-3 fatty acid). Here lipid-cholesterol interaction is used to distinguish the effect of position of unsaturation on membrane structure.

Aged lymphocyte proliferation following incorporation and retention of dietary omega-3 fatty acids.

T cell activation involves events at the plasma membrane; therefore, molecules such as long chain omega-3 fatty acids that alter the structure of the plasma membrane may affect the activation of aged T cells. In this project we investigated whether the incorporation of omega-3 fatty acids (from fish oil), in the presence of vitamin E, improves age-diminished T cell proliferation. Young and old mice were fed diets rich in either fish (menhaden) oil or saturated fat for various lengths of time. Splenocytes were harvested from these mice and stimulated in culture with either mitogen or the antigen keyhole limpet hemocyanin (for a secondary response); proliferation was estimated by [3H]thymidine incorporation. We found no discernible effect of dietary omega-3 fatty acids (with vitamin E supplementation) on lymphocyte proliferation stimulated by the mitogens concanavalin A or phytohemagglutinin. We did, however, find that the saturated fat diet and the menhaden oil diet in young mice lowered protein kinase C activities in the particulate fractions of spleen cells when compared to chow-fed mice. Middle-aged and old mice were less affected by the experimental diets than young mice, but they demonstrated decreased protein kinase C activity as well. These alterations did not affect the ability of splenocytes to respond to mitogenic stimulation. Fatty acid analysis revealed that lymphocytes from mice fed saturated fat for 8.5 months retained significant amounts of the omega-3 fatty acid docosahexaenoic acid, despite the lack of dietary omega-3 fatty acids. However, when aged (but not young) lymphocytes were clonally expanded by antigen in vivo in the presence of dietary omega-3 fatty acids, they produced a greater secondary proliferative response than old lymphocytes expanded during a saturated fat diet. Although our results suggest that omega-3 fatty acids may enhance aged lymphocyte proliferation, the tenacious retention of these fatty acids makes comparison with omega-3-depleted lymphocytes difficult.

2H NMR studies of isomeric omega 3 and omega 6 polyunsaturated phospholipid membranes.

The properties of aqueous multilamellar dispersions of [2H31]16:0-alpha 18:3 PC (1-[2H31]palmitoyl-2-cis,cis,cis-octa-9,12,15- trienoylphosphatidylcholine) and of [2H31]16:0-gamma 18:3 PC (1-[2H31]palmitoyl-2-cis,cis,cis-octa-6,9,12-trienoylphosphatid ylcholine) were compared by broadline 2H NMR spectroscopy. These isomeric phospholipids differ only in the location of the unsaturations in the sn-2 chain. The alpha 18:3 chain has double bonds at delta 9, 12, and 15 positions whereas in the gamma 18:3 chain they are at positions delta 6, 9, and 12. Moment analysis of spectra recorded as a function of temperature reveals dramatically distinct phase behavior for the two isomers. The gel to liquid crystalline transition for [2H31]16:0-alpha 18:3 PC membranes exhibits broad hysteresis which is characterized by a mid point temperature of -9 degrees C and -20 degrees C on heating and cooling, respectively. In contrast, the phase transition of [2H31]16:0-gamma 18:3 PC membranes does not exhibit hysteresis and occurs over a lower temperature range centred on -27 degrees C. Appreciably different molecular ordering also exists within the membranes in the liquid crystalline state. Average order parameters SCD are smaller in [2H31] 16:0-alpha 18:3 PC than in [2H31]16:0-gamma 18:3 PC by 10% at the same temperature and by 20% at equal reduced temperature. Smoothed order parameter profiles generated from depaked spectra clarify the nature of the difference.(ABSTRACT TRUNCATED AT 250 WORDS)

Use of merocyanine (MC540) in quantifying lipid domains and packing in phospholipid vesicles and tumor cells.

The fluorescent probe merocyanine (MC540) reports qualitatively on several membrane events. Here we demonstrate that MC540 fluorescence can quantify the degree of coexisting liquid-crystalline and gel states in mixed monotectic phosphatidylcholine (PC) bilayers. The probe exhibits disparate fluorescence wavelength maximas and and intensities when incorporated into liquid-crystalline and gel state membranes. The fluorescence measurements partitioning of the EPR spin probe TEMPO between the aqueous environment and the membrane fluid phase. While both techniques can accurately assess the phase transition of synthetic PCs, only MC540 can distinguish between liquid-crystalline phases of different composition. MC540 fluorescence for single-component PC bilayers correlates quantitatively with estimates of the area/molecule determined from surface area/pressure isotherms of lipid monolayers, whereas partitioning of TEMPO fails to assess the relative degree of lipid packing in various fluid state membranes. Additionally, MC540 fluorescence characterizes the interaction of cholesterol with membranes made from condensable (18:0, 18:1-PC) and non-condensable (18:0, 22:6-PC) lipids. Finally MC540 distinguishes tumor cell membranes differing only in the amount of docosahexaenoic acid (DHA). Thus we conclude that MC540 can be used quantitatively to study phospholipid packing and membrane phases with lipid vesicles and to sense subtle differences in the arrangement of phospholipids in biological membranes.

Docosahexaenoic acid increases permeability of lipid vesicles and tumor cells.

Docosahexaenoic acid (DHA), a long-chain polyunsaturated omega 3 fatty acid, is tested to determine its mode of action as an anti-cancer agent. We demonstrate that DHA can increase the permeability of phospholipid vesicles, as monitored by vesicle swelling in isosmolar erythritol and leakage of sequestered carboxyfluorescein, and T27A tumor cells, as monitored by swelling in isosmolar erythritol and release of sequestered 51Cr. DHA was incorporated into lipid vesicles as either the free fatty acid or as 1-stearoyl-2-docosahexaenoyl-sn-glycero-3-phosphocholine. DHA was incorporated into the tumor cells by fusion with vesicles made from the mixed-chain phosphatidylcholines. DHA is demonstrated here to be much more effective in increasing permeability than is oleic acid, the major unsaturated fatty acid normally found in tumor plasma membranes. It is proposed that incorporation of DHA makes tumor plasma membranes substantially more permeable, which may explain, in part, its anti-tumor properties.