The effects of cyclic fatty acid monomers on cultured porcine endothelial cells.

The popularity of polyunsaturated oils used in food applications and preparation continues to appreciate as a result of positive health claims. With polyunsaturated oils inherently more susceptible to oxidative and thermal degradation, the formation of new fatty acid species increases considerably. The presence of one species known as cyclic fatty acid monomers (CFAM) has been detected as a component of many oils subjected to various thermal processes including deep-fat frying. The effect of CFAM on metabolic processes has not been fully characterized. In this study, confluent porcine aortic endothelial cells incorporated CFAM into their polar and nonpolar lipid fractions following a 48-h exposure to 31 and 62 ppm CFAM in the culture medium. Subsequently, the influence of CFAM incorporation on various membrane-dependent physical properties and biochemical processes was investigated. CFAM decreased the lipid packing order of the membrane bilayer core but did not alter the lipid packing order of lipid chain segments at or near the lipid-water interface of the membrane. CFAM led to significant reductions in Ca2+ ATPase activity and monolayer integrity while eliciting a significant increase of prostacyclin synthesis and secretion.

Rapid determination of double bond configuration and position along the hydrocarbon chain in cyclic fatty acid monomers.

This study reports the structural elucidation of diunsaturated 5- or 6-membered ring cyclic fatty acid monomers (CFAM) isolated from heated flaxseed oil by complementary gas chromatography (GC)-mass spectrometry (MS) and GC-matrix isolation-Fourier transform infrared spectroscopy (MI-FTIR). Infrared measurements of CFAM were carried out on methyl ester derivatives as well-resolved chromatograms were obtained on a polar 100% cyanopropyl polysiloxane capillary GC column. By contrast, electron ionization MS of methyl ester derivatives was of limited value because of double bond migration during the ionization process in the mass spectrometer. This communication reports definitive MS fragmentation patterns that can confirm ring position and double bond position along the fatty acid chain in 1,2-disubstituted CFAM determined as 2-alkenyl-4,4-dimethyl-oxazoline derivatives. Double bond configuration (cis, trans, or conjugated cis,cis) in CFAM was confirmed by GC-MI-FTIR. The presence of CFAM, degradation products found in used frying oils, is a potential source of dietary toxicity.

Localization of the platelet-activating factor receptor to rat pancreatic microvascular endothelial cells.

Platelet-activating factor (PAF) is a potent lipid autocoid involved in numerous inflammatory processes. Although PAF plays a key role as a mediator of inflammation in acute pancreatitis, the site(s) of action of PAF in the pancreas remains unknown. One of the aims of this study was to identify cell types within the pancreas expressing the PAF receptor using immunohistochemical protocols. Additionally, pancreatic microvascular endothelial cells were isolated and examined for the PAF receptor using immunohistochemistry, reverse transcription-polymerase chain reaction, and intracellular calcium responses to PAF exposure. Immunohistochemical analysis of pancreatic slices using an antibody directed toward the N-terminus of the PAF receptor revealed specific localization to the vascular endothelium with no localization to other pancreatic cell types. Reverse transcription-polymerase chain reaction of RNA isolated from cultured pancreatic islet endothelial cells yielded the predicted amplicon for the PAF receptor. Cultured pancreatic islet endothelial cells responded to PAF as measured by a transient increase in intracellular calcium, which was ameliorated in the presence of a PAF receptor antagonist. The results demonstrate the localization of PAF receptors on the pancreatic vascular endothelium. The presence of PAF receptors on the pancreatic vascular endothelium provides a defined, highly localized target for therapeutic intervention.

Suppression of lipopolysaccharide-induced nitric oxide synthase expression by platelet-activating factor receptor antagonists in the rat liver and cultured rat Kupffer cells.

Excessive nitric oxide (NO) generated by hepatic cells in response to lipopolysaccharide (LPS) and inflammatory substances (e.g., platelet-activating factor [PAF]) is a key contributor to the pathophysiological outcomes observed in the liver during sepsis. In rats subjected to liver-focused endotoxemia, inducible nitric oxide synthase (iNOS) levels in the intact liver were elevated by 6 hours; cell-specific expression of iNOS messenger RNA (mRNA) was Kupffer cells (KCs), endothelial cells, and hepatocytes. Elevated serum alanine transaminase (ALT) levels at 6 hours confirmed hepatic damage. Pretreatment of endotoxemic rats with PAF receptor antagonists BN 50739 or WEB 2170 reduced serum ALT and iNOS mRNA levels in the intact liver. Pretreatment of cultured KCs with BN 50739 or WEB 2170 inhibited both LPS and PAF-induced iNOS mRNA formation. In addition, LPS-induced iNOS protein levels in KCs pretreated with BN 50739 or WEB 2170 were decreased. Exposure of KCs to either LPS or PAF caused the translocation of the p65 subunit of nuclear factor kappa B (NF-kappaB) into the nucleus and this process was attenuated by BN 50739 and WEB 2170. There was concomitant inhibition of LPS-dependent degradation of the inhibitory protein IkappaBalpha and increase in intracellular Ca(2+) in KC treated with BN 50739 or WEB 2170. Also, in KCs, LPS was able to induce iNOS mRNA expression independent of CD14. This response was inhibited by pretreatment of KCs with either BN 50739 or WEB 2170. Our findings indicate that PAF receptor antagonists convey protection against hepatocellular injury accompanied by a decrease in nitric oxide (NO) formation in the livers of endotoxemic rats.

Interaction of platelet-activating factor with rat hepatocytes: uptake, translocation, metabolism, and effects on PAF-acetylhydrolase secretion and protein tyrosine phosphorylation.

In the present study, the interaction of the phospholipid mediator platelet-activating factor (PAF) with rat hepatocytes in primary culture was examined. Following exposure to hepatocytes, exogenous [3H]alkyl-PAF was metabolized rapidly to [3H]lyso-PAF, the content of which was raised in the outer leaflet of the plasma membrane within the initial 5 min of incubation. Thereafter [3H]lyso-PAF was translocated into cells with concomitant reacylation to [3H]alkyl-acyl-glycerophosphocholine. A portion of untransformed [3H]PAF accumulated in the outer leaflet, and only a small amount of the [3H]PAF was translocated into the inner leaflet of the plasma membrane. Detectable levels of [3H]lyso-PAF were found in the medium of hepatocyte cultures at all times of incubation. These findings suggest that at least a portion of the cellular PAF-acetylhydrolase (PAF-AH) activity is located in the outer leaflet of the plasma membrane and can be secreted into the medium. Indeed, rat hepatocytes in culture released PAF-AH into the medium in a time-dependent fashion, Incubation of hepatocytes with exogenous PAF increased secretion of PAF-AH, whereas lyso-PAF and the nonhydrolyzable analog methylcarbamyl-PAF significantly reduced secretion. The structurally related PAF receptor antagonist CV 3988 markedly inhibited the activity of PAF-AH and also diminished its release by hepatocytes. In contrast, BN 50739 amd WEB 2170, thienotriazolodiazepine PAF receptor antagonists, did not affect the PAF-AH activity, but increased its secretion by the cells. A full-length 3.8-kb mRNA encoding the cell surface PAF receptor was absent in hepatocytes as indicated by Northern blot analysis using the rat PAF receptor cDNA, whereas PAF receptor mRNA was readily detected in Kupffer cells. Upon incubation with hepatocytes, PAF induced tyrosine phosphorylation of proteins with molecular masses of 120-130 and 160-180 kDa and dephosphorylation of 80-90-kDa proteins; these responses were not inhibited by WEB 2170 and BN 50739. The protein tyrosine kinase inhibitor genistein abolished the release of free arachidonic acid, suggesting a crucial role for tyrosine phosphorylation in PAF-induced phospholipase A2 activation in rat hepatocytes. Taken together, our data indicate that the interaction of PAF with rat hepatocytes is dependent upon its metabolism, involves protein tyrosine phosphorylation/dephosphorylation and arachidonic acid release, and does not involve the heteromeric G-protein-coupled PAF receptor which has been characterized in Kupffer cells. This metabolically regulated mechanism for PAF action on hepatocytes may be of potential biological importance in the liver under normal and pathological conditions.