Search

P388D(1) macrophages prelabeled with [(3)H]arachidonic acid (AA) respond to bacterial lipopolysaccharide (LPS) by mobilizing AA in a process that takes several hours and is mediated by the concerted actions of the group IV cytosolic phospholipase A(2) and the group V secretory phospholipase A(2) (sPLA(2)). Here we show that when the LPS-activated cells are prelabeled with [(3)H]oleic acid (OA), they also mobilize and release OA to the extracellular medium. The time and concentration dependence of the LPS effect on OA release fully resemble those of the AA release. Experiments in which both AA and OA release are measured simultaneously indicate that AA is released 3 times more efficiently than OA. Importantly, LPS-stimulated OA release is strongly inhibited by the selective sPLA(2) inhibitors 3-(3-acetamide-1-benzyl-2-ethylindolyl-5-oxy)propane sulfonic acid and carboxymethylcellulose-linked phosphatidylethanolamine. The addition of exogenous recombinant sPLA(2) to the cells also triggers OA release. These data implicate a functionally active sPLA(2) as being essential for the cells to release OA upon stimulation with LPS. OA release is also inhibited by methyl arachidonyl fluorophosphonate but not by bromoenol lactone, indicating that the group IV cytosolic phospholipase A(2) is also involved in the process. Together, these data reveal that OA release occurs during stimulation of the P388D(1) macrophages by LPS and that the regulatory features of the OA release are strikingly similar to those previously found for the AA release.

The effect of hydrostatic pressure at levels applied in diving or hyperbaric treatment (thus considered "physiological") on the order of lipid domains in human red blood cell (RBC) membrane was studied. Membrane order was determined by measuring 1) the fluorescence anisotropy (FAn) of lipid probes, 2) the resonance energy transfer from tryptophan to lipid probes, and 3) spectral shifts in Laurdan fluorescence emission. It was found that the application of mild pressure (< 15 atm) 1) increased, selectively, the FAn of lipid probes that monitor the membrane lipid core, 2) increased the tryptophan FAn, 3) increased the resonance energy transfer from tryptophan to lipid probes residing in the lipid core, and 4) induced changes in the Laurdan fluorescence spectrum, which corresponded to reduced membrane hydration. It is proposed that the application of pressure of several atmospheres increases the phase order of membrane lipid domains, particularly in the proximity of proteins. Because the membrane lipid order ("fluidity") of RBCs plays an important role in their cellular and rheological functions, the pressure-induced alterations of the RBC membrane might be pertinent to microcirculatory disorders observed in humans subjected to elevated pressure.

To explore the contribution of red blood cell (RBC) deformability and interaction with endothelial cells (ECs) to circulatory disorders, these RBC properties were modified by treatment with hydrogen peroxide ([H.sub.2][O.sub.2]), and their effects on vascular resistance were monitored following their infusion into rat mesocecum vasculature. Treatment with 0.5 mM H202 increased RBC/EC adherence without significant alteration of RBC deformability. At 5.0 mM [H.sub.2][O.sub.2], RBC deformability was considerably reduced, inducing a threefold increase in the number of undeformable cells, whereas RBC/EC adherence was not further affected by the increased [H.sub.2][O.sub.2] concentration. This enabled the selective manipulation of RBC adherence and deformability and the testing of their differential effect on vascular resistance. Perfusion of RBCs with enhanced adherence and unchanged deformability (treatment with 0.5 mM [H.sub.2][O.sub.2]) increased vascular resistance by about 35% compared with untreated control RBCs. Perfusion of 5.0 mM [H.sub.2][O.sub.2]-treated RBCs, with reduced deformability (without additional increase of adherence), further increased vascular resistance by about 60% compared with untreated control RBCs. These results demonstrate the specific effects of elevated adherence and reduced deformability of oxidized RBCs on vascular resistance. These effects can be additive, depending on the oxidation conditions. The oxidation-induced changes applied in this study are moderate compared with those observed in RBCs in pathological states. Yet, they caused a considerable increase in vascular resistance, thus demonstrating the potency of RBC/EC adherence and RBC deformability in determining resistance to blood flow in vivo. erythrocyte rheology; aggregability; adhesion; circulatory disorders

The pathophysiology of infiammatory bowel disease (IBD) involves the production of diverse lipid mediators, namely eicosanoids, lysophospholipids, and platelet-activating factor, in which phospholipase [A.sub.2] (PL[A.sub.2]) is the key enzyme. Accordingly, it has been postulated that control of lipid mediator production by inhibition of PL[A.sub.2] would be useful for the treatment of IBD. This hypothesis was tested in the present study by examining the therapeutic effect of a novel extracellular PL[A.sub.2] inhibitor (ExPLI), composed of carboxymethylcellulose-linked phosphatidylethanolamine (CMPE), on trinitrobenzenesulfonic acid-induced colitis. Intraperitoneal administration of CMPE suppressed the colitis as measured by mortality rate, intestinal permeability, plasma PL[A.sub.2] activity, intestinal myeloperoxidase activity, and histological morphometry. Current therapeutic approaches for inflammatory conditions focus on the selective control of a lipid mediator(s) (e.g., prostaglandins or leukotrienes). The present study supports the concept that inclusive control of lipid mediator production by PL[A.sub.2] inhibition is a plausible approach to the treatment of colitis and introduces the ExPLIs as a prototype of a novel NSAID for the treatment of intestinal inflammation. inflammatory bowel disease; colitis; nonsteroidal anti-inflammatory drugs

Glycerol reduces the apparent specific volume (V) and adiabatic compressibility (K) of protein interiors but enhances it for amino acids. Protein interiors in water exhibit V and K values of 0.784 plus or minus 0.026 mL/g and 12.8 plus or minus 2.5 x 10 to the power -6 mL/g atmosphere. Glycerol dehydrates the protein, decreases the thermal motion of the polypeptide chain, and increases protein stability. The decrease in V and K is probably due to a disintegration of voids in the protein core after the glycerol-stimulated depletion of lubricant water.

Parameters of red blood cell aggregation as correlates of the inflammatory state. Am J Physiol Heart Circ Physiol 280: H1982-H1988, 2001.--To identify clinically relevant parameters of red blood cell (RBC) aggregation, we examined correlations of aggregation parameters with C-reactive protein and fibrinogen in unstable angina (UA), acute myocardial infarction (AMI), and bacterial infection (BI). Aggregation parameters were derived from the distribution of RBC population into aggregate sizes (cells per aggregate) and changing of the distribution by flow-derived shear stress. Increased aggregation was observed in the following order: UA, AMI, and BI. The best correlation was obtained by integration of large aggregate fraction as a function of shear stress. To differentiate plasmatic from cellular factors in RBC aggregation, we determined the aggregation in the presence and absence of plasma and formulated a 'plasma factor' (PF) ranging from 0 to 1. In AMI the enhanced aggregation was entirely due to PF (PF = 1), whereas in UA and BI it was due to both plasmatic and cellular factors (0 [is less than or equal to] PF [is less than or equal to] 1). It is proposed that clinically relevant parameters of RBC aggregation should express both RBC aggregate size distribution and aggregate resistance to disaggregation and distinguish between plasmatic and cellular factors. acute-phase response; aggregate size distribution; shear stress

Background: The pathophysiology of asthma involves the action of inflammatory/allergic lipid mediators formed following membrane phospholipid hydrolysis by phospholipase A2 (PLA2). Cysteinyl leukotrienes are considered potent inducers of bronchoconstriction and airway remodelling. Ovalbumin (OVA) induced bronchoconstriction in rats is associated with increased secretory PLA2 (sPLA2) activation and cysteinyl leukotriene production, together with suppression of cytosolic PLA2 and prostaglandin E2. These processes are reversed when the animals are pretreated systemically with an extracellular cell impermeable sPLA2 inhibitor which also suppresses the early allergic reaction to OVA challenge. In this study we examine the capacity of the sPLA2 inhibitor to ameliorate inflammatory and allergic manifestations (early and late bronchoconstriction) of OVA induced allergic bronchitis in rats when the inhibitor was administered by inhalation to confine it to the airways. Methods: Rats sensitised with OVA were treated with the sPLA2 inhibitor hyaluronic acid-linked phosphatidyl ethanolamine (HyPE). The rats were divided into four groups (n = 10 per group): (1) naïve controls (no sensitisation/no treatment); (2) positive controls (sensitisation + challenge with OVA inhalation and subcutaneous injection of 1 ml saline before each challenge; (3) sensitisation + challenge with OVA and HyPE inhalation before every challenge; and (4) sensitisation + challenge with OVA and treatment with subcutaneous dexamethasone (300 μg) before each challenge as a conventional reference. Another group received no treatment with HyPE during the sensitisation process but only before or after challenge of already sensitised rats. Pulmonary function was assessed and changes in the histology of the airways, levels of cysteinyl leukotrienes in BAL fluid, and the production of nitric oxide (No) and tumour necrosis factor α (TNFα) by BAL macrophages were determined. Results: Inhalation of HyPE markedly suppressed OVA induced early and late asthmatic reactions as expressed by bronchoconstriction, airway remodelling (histology), cysteinyl leukotriene level in BAL fluid, and production of TNFα and NO by BAL macrophages. OVA induced bronchoconstriction in sensitised non-pretreated rats was also inhibited by inhalation of HyPE either before or after the challenge. Conclusions: These findings confirm the pivotal role of sPLA2 in the pathophysiology of both the immediate allergic response and the inflammatory asthmatic process. Control of airway sPLA2 may be a new therapeutic approach to the treatment of asthma. [ABSTRACT FROM AUTHOR]

The pathophysiology of inflammatory bowel disease (IBD) involves the production of diverse lipid mediators, namely eicosanoids, lysophospholipids, and platelet-activating factor, in which phospholipase A[sub 2] (PLA[sub 2]) is the key enzyme. Accordingly, it has been postulated that control of lipid mediator production by inhibition of PLA[sub 2] would be useful for the treatment of IBD. This hypothesis was tested in the present study by examining the therapeutic effect of a novel extracellular PLA[sub 2] inhibitor (ExPLI), composed of carboxymethylcellulose-linked phosphatidylethanolamine (CMPE), on trinitrobenzenesulfonic acid-induced colitis. Intraperitoneal administration of CMPE suppressed the colitis as measured by mortality rate, intestinal permeability, plasma PLA[sub 2] activity, intestinal myeloperoxidase activity, and histological morphometry. Current therapeutic approaches for inflammatory conditions focus on the selective control of a lipid mediator(s) (e.g., prostaglandins or leukotrienes). The present study supports the concept that inclusive control of lipid mediator production by PLA[sub 2] inhibition is a plausible approach to the treatment of colitis and introduces the ExPLIs as a prototype of a novel NSAID for the treatment of intestinal inflammation. [ABSTRACT FROM AUTHOR]