Lea Blau

Nonsteroidal antiinflammatory drugs exert differential effects on neutrophil function and plasma membrane viscosity. Studies in human neutrophils and liposomes.

Nonsteroidal antiinflammatory drugs (NSAIDs) inhibit neutrophil functions via mechanisms separate from their capacity to inhibit prostaglandin synthesis. We have studied discrete events in the process of signal transduction: NSAIDs but not a related analgesic drug (acetaminophen), inhibited aggregation in response to the chemoattractants f-Met-Leu-Phe (FMLP), leukotriene B4, and C5a. NSAIDs, but not acetaminophen, inhibited binding of radiolabeled FMLP to purified neutrophil membranes. Gpp(NH)p, a GTPase insensitive analog of GTP, also inhibited the binding of FMLP but, paradoxically, enhanced superoxide anion generation and lysozyme release. The inhibition of ligand binding by NSAIDs did not correlate with their capacity to inhibit FMLP-induced increments in diacylglycerol (DG): piroxicam, but not salicylate effectively inhibited appearance of label ([3H]arachidonate, [14C] glycerol) in DG. Finally, NSAIDs exerted differential effects on the viscosity of neutrophil plasma membranes and multilamellar vesicles (liposomes): membrane viscosity was increased by piroxicam and indomethacin, decreased by salicylate, and unaffected by acetaminophen. Thus, the different effects of NSAIDs on discrete pathways are not due to their shared capacity to reduce ligand binding but rather to a capacity to uncouple postreceptor signaling events that depend upon the state of membrane fluidity.

Permeability behavior of liposomes prepared from fatty acids and fatty acid methyl esters

The permeability properties of liposomes prepared at pH 8.7 from a fatty acid and either methyl oleate or methyl elaidate, with or without cholesterol, were investigated. The fatty acids used were oleic acid, elaidic acid, and the selenium-containing fatty acids 9-selenaheptadecanoic acid and 13-selenaheneicosanoic acid. The liposomes trapped sucrose and carboxyfluorescein. Their volume change resulting from osmotic shock was directly proportional to the change in absorbance (light scattering). Liposomes prepared from oleic acid and either methyl oleate or methyl elaidate underwent osmotic swelling much more slowly than liposomes prepared from elaidic acid and either methyl oleate or methyl elaidate. Incorporation of cholesterol decreased the initial rate of erythritol permeation, especially in liposomes containing methyl oleate. The swelling rates of liposomes prepared with the selenium-containing fatty acids indicated that incorporation of methyl elaidate gave more tightly packed bilayers than did incorporation of methyl oleate. The effect of cholesterol on the initial rate of erythritol influx was greater in oleic acid and elaidic acid liposomes than in selenium-containing fatty acid liposomes, indicating that the large bulk of the selenium heteroatom suppresses the ability of cholesterol to interact with the hydrocarbon chain.

Organization of unesterified cholesterol in high density lipoproteins probed by filipin.

The initial rate of filipin association with unesterified cholesterol in high density lipoproteins (HDL) was measured by stopped-flow spectrophotometry to assess the roles played by apolipoproteins and phospholipids in modulating the surface exposure of cholesterol. The initial rate of filipin-unesterified cholesterol association was enhanced upon hydrolysis of the glycerophospholipids of human HDL3 by phospholipase A2. Rate enhancements were also observed following trypsin-catalyzed hydrolysis of apolipoprotein A-I in canine HDL and of apolipoproteins A-I and A-II in human HDL3. However, the initial rate of filipin-unesterified cholesterol association was not altered upon incubation of HDL3 with polymorphonuclear cells, which causes hydrolysis of apolipoprotein A-II but leaves apolipoprotein A-I intact. These results are consistent with the general structural model of HDL in which unesterified cholesterol, apolipoproteins and glycerophospholipids are presumed to be localized at the surface of the HDL particle. From these studies and from results indicating that the initial rate of filipin-unesterified association was enhanced in canine HDL hybrids in which 50% of the apolipoprotein A-I had been replaced by apolipoprotein A-II, we also conclude that apolipoprotein A-I in HDL is in closer proximity to unesterified cholesterol than apolipoprotein A-II. Thus, it appears that rapid kinetic measurements of filipin-cholesterol association may be useful in assessing the organization of unesterified cholesterol in serum lipoproteins.