Poor glycaemic control in type 2 diabetes patients reduces endothelial progenitor cell number by influencing SIRT1 signalling via platelet-activating factor receptor activation.

AIMS/HYPOTHESIS: Downregulation of levels of endothelial progenitor cells (EPCs) during in-vitro short-term exposure to high glucose concentrations relates to reduced activity of silent information regulator 1 (SIRT1) and increased synthesis of platelet-activating factor (PAF). We investigated the possible relationship between PAF and SIRT1 pathways in EPCs during altered glucose homeostasis. METHODS: SIRT1 and PAF receptor (PAF-R) levels were determined by western blot, RT-PCR and confocal laser-scanning microscopy. In-vivo experiments were performed on 48 type 2 diabetic patients (25 with poor glycaemic control and 23 with good glycaemic control) and 20 control individuals. In-vitro experiments with the PAF-R antagonist CV3988 were performed on EPCs isolated from leucocyte-rich buffy coat of healthy human donors. RESULTS: Decreased SIRT1 protein levels were observed in EPCs from type 2 diabetic patients compared with control individuals (p < 0.01). Notably, the SIRT1 level was consistently lower in patients with poor glycaemic control than in those with good glycaemic control (p < 0.01). Diabetic patients also showed an upregulation of PAF-Rs; this response occurred to a greater extent in individuals with poor glycaemic control than in those with good glycaemic control. In-vitro experiments confirmed that EPCs respond to PAF stimulation with decreased SIRT1 protein and SIRT1 mRNA levels. Moreover, reduction of SIRT1 levels and activity were abolished by CV3988. CONCLUSIONS/INTERPRETATION: These findings unveil a link between PAF and SIRT1 pathways in EPCs that contributes to the deleterious effect of hyperglycaemia on the functional properties of EPCs, crucial in diabetes and peripheral vascular complications.

Analysis of pulegone and its enanthiomeric distribution in mint-flavoured food products.

A procedure for the extraction and determination of pulegone enanthiomers in mint essential oils and mint products (syrups, dried leaves, toothpaste, lozenges, candy and chewing-gum) was developed. The compounds were recovered from the food matrices by employing a simultaneous distillation-extraction (SDE) technique with a Likens-Nickerson apparatus using dichloromethane as an extraction solvent. The analyses were performed by capillary gas chromatography mass spectrometry (GC/MS). Experiments on food products spiked at different pulegone concentrations showed recoveries ranging from 95 to 106%. The detection limit was about 5?mg?l(-1) for both pulegone enanthiomers and good linearity was found in the concentration range 0.5-25?mg?l(-1). In a number of repeated analyses, the pulegone peak height repeatability (RSD) was 0.2%. The pulegone enanthiomers were separated and quantified by enanthioselective multidimensional gas chromatography. The results of analyses conducted on essential mint oils and mint-flavoured food products are reported.

Lycopene in association with alpha-tocopherol or tomato lipophilic extracts enhances acyl-platelet-activating factor biosynthesis in endothelial cells during oxidative stress.

Lipophilic compounds contained in tomato can prevent cardiovascular diseases by modulating the atherogenic processes in vascular endothelium mediated by oxidized low-density lipoproteins (LDLs). We investigated the effects of lycopene on the metabolism of platelet-activating factor (PAF) and its much less biologically active acyl analog, acyl-PAF, known to prevent LDL oxidation. Lycopene, or lycopene in association with alpha-tocopherol, or whole tomato lipophilic extracts (containing more than 80% lycopene) were used in experiments in which endothelial cells (ECs) are known to synthesize PAF following H(2)O(2)-induced oxidative stress. The results indicated that in each case H(2)O(2)-stimulated PAF biosynthesis in ECs, which is catalyzed by acetyl-CoA acetyltransferase (AT), appeared strongly inhibited. However, acyl-PAF biosynthesis, which also occurs through the PAF-dependent transacetylase (TA), was significantly increased by lycopene only when it was in association with alpha-tocopherol or with the minor compounds present in the whole lipophilic tomato extract. These findings suggest that alpha-tocopherol or lipophilic compounds present in tomato juice potentiate the effects of lycopene on the modulation of PAF and acyl-PAF biosynthesis in ECs during oxidative stress.

Pectin methylesterase inhibitor.

Pectin methylesterase (PME) is the first enzyme acting on pectin, a major component of plant cell wall. PME action produces pectin with different structural and functional properties, having an important role in plant physiology. Regulation of plant PME activity is obtained by the differential expression of several isoforms in different tissues and developmental stages and by subtle modifications of cell wall local pH. Inhibitory activities from various plant sources have also been reported. A proteinaceous inhibitor of PME (PMEI) has been purified from kiwi fruit. The kiwi PMEI is active against plant PMEs, forming a 1:1 non-covalent complex. The polypeptide chain comprises 152 amino acid residues and contains five Cys residues, four of which are connected by disulfide bridges, first to second and third to fourth. The sequence shows significant similarity with the N-terminal pro-peptides of plant PME, and with plant invertase inhibitors. In particular, the four Cys residues involved in disulfide bridges are conserved. On the basis of amino acid sequence similarity and Cys residues conservation, a large protein family including PMEI, invertase inhibitors and related proteins of unknown function has been identified. The presence of at least two sequences in the Arabidopsis genome having high similarity with kiwi PMEI suggests the ubiquitous presence of this inhibitor. PMEI has an interest in food industry as inhibitor of endogenous PME, responsible for phase separation and cloud loss in fruit juice manufacturing. Affinity chromatography on resin-bound PMEI can also be used to concentrate and detect residual PME activity in fruit and vegetable products.

Conformational changes and stabilization induced by phosphate binding to 5'-methylthioadenosine phosphorylase from the thermophilic archaeon Sulfolobus solfataricus.

The effect of phosphate, its analogues, and other substrates on structural features of recombinant 5'-methylthioadenosine phosphorylase from Sulfolobus solfataricus (SsMTAP) was investigated. Phosphate was found to exert a significant stabilizing effect on the protein against the inactivation caused by temperature, sodium dodecyl sulfate (SDS), urea, and proteolytic enzymes. In the presence of 100 mM phosphate: (i) the apparent transition temperature (Tm) of recombinant SsMTAP increased from 111 degrees to 118 degrees C; and (ii) the enzyme still retained 40% and 30% activity, respectively, after 30 min of incubation at 90 degrees C with 2% SDS or 8 M urea. The structure modification of SsMTAP by phosphate binding was probed by limited proteolysis with subtilisin and proteinase K and analysis of polypeptide fragments by SDS-PAGE. The binding of the phosphate substrate protected SsMTAP against protease inactivation, as proven by the disappearance of a previously accessible proteolytic cleavage site that was localized in the N-terminal region of the enzyme. The conformational changes of SsMTAP induced by phosphate and ribose-1-phosphate were analyzed by fluorescence spectroscopy, and modifications of the protein intrinsic fluorophore exposure, as a consequence of substrate binding, were evidenced.

Kiwi protein inhibitor of pectin methylesterase amino-acid sequence and structural importance of two disulfide bridges.

A protein acting as a powerful inhibitor of plant pectin methylesterase was isolated from kiwi (Actinidia chinensis) fruit. The complete amino-acid sequence of the pectin methylesterase inhibitor (PMEI) was determined by direct protein analysis. The sequence comprises 152 amino-acid residues, accounting for a molecular mass of 16 277 Da. The far-UV CD spectrum indicated a predominant alpha-helix conformation in the secondary structure. The protein has five cysteine residues but neither tryptophan nor methionine. Analysis of fragments obtained after digestion of the protein alkylated without previous reduction identified two disulfide bridges connecting Cys9 with Cys18, and Cys74 with Cys114; Cys140 bears a free thiol group. A database search pointed out a similarity between PMEI and plant invertase inhibitors. In particular, the four Cys residues, which in PMEI are involved in the disulfide bridges, are conserved. This allows us to infer that also in the homologous proteins, whose primary structure was deduced only by cDNA sequencing, those cysteine residues are engaged in two disulfide bridges, and constitute a common structural motif. The comparison of the sequence of these inhibitors confirms the existence of a novel class of proteins with moderate but significant sequence conservation, comprising plant proteins acting as inhibitors of sugar metabolism enzymes, and probably involved in various steps of plant development.

Increased blood levels of platelet-activating factor in insulin-dependent diabetic patients with microalbuminuria.

BACKGROUND: Platelet-activating factor (PAF), a phospholipid mediator of inflammation, may induce an enhanced size- and charge-dependent glomerular permeability in experimental animals. Studies on the role of PAF in enhanced glomerular permeability in the early phase of diabetic nephropathy are still lacking. METHODS: We evaluated the intravascular levels of PAF and its main catabolic enzyme, the PAF-specific plasma acetyl-hydrolase (PAF-AH), in basal conditions and after exercise, in normo- or micro-albuminuric insulin-dependent diabetic (IDD) patients and in normal subjects. RESULTS: The results obtained indicate that the concentration of PAF in whole blood was significantly enhanced in basal conditions, during and after exercise in all microalbuminuric IDD patients, but not in normoalbuminuric IDD or in control subjects. The increased concentration of PAF did not correlate with changes in the activity of PAF-AH, suggesting an enhanced production rather than a decreased catabolism of PAF. CONCLUSIONS: These results indicate an association between increased production of PAF and enhanced glomerular permeability in microalbuminuric IDD patients.

Analysis of free and esterified ergosterol in tomato products.

A new extraction and chromatographic procedure to quantify free and esterified ergosterol in tomato products was devised. The extraction solution was composed of a dichloromethane/methanol mixture in a 2:1 (v/v) ratio. This extraction solvent allowed for higher ergosterol recovery from tomato products (an average of 25% more) compared to hexane, which is frequently employed for ergosterol extraction. Both free and esterified ergosterol were determined by HPLC reverse-phase chromatography employing a Nova-Pak C-18 column (300 x 3.9 mm), filled with 4 mm average particle size and a guard column of the same material. The elution was performed at a flow rate of 1 mL. min(-1) with a linear gradient of solvent A (methanol/water, 80:20, v/v) and solvent B (dichloromethane). The gradient, starting at sample injection, was from 0 to 50% B for 20 min for the free ergosterol analysis and additional 15 min at 50% B to analyze the ergosterol esters. This technique has proven to be more sensitive for ergosterol determination than other reported chromatographic procedures. Moreover, ergosterol esters, extracted from various fungal sources, separated well and were easily quantified.

2-aminoanthracene as an analytical tool with the acetylation reaction catalyzed by arylamine N-acetyltransferase.

The polynuclear aromatic amine, 2-aminoanthracene, was found to be acetylated with high efficiency in the presence of acetyl-CoA by pigeon liver arylamine N-acetyltransferase (EC 2.3.1.5). As a consequence of acetylation the fluorescence properties of the compound dramatically change and the reaction time course can be easily followed fluorometrically at the emission wavelength of 425 nm upon excitation at 360 nm. When 2-aminoanthracene is employed with pigeon arylamine N-acetyltransferase, as the ultimate acceptor of the acetyl group in coupled fluorometric assays, it is possible to measure enzymatic activities, such as pyruvate dehydrogenase or carnitine acetyltransferase, in continuous assays rapidly and with high sensitivity or to determine with as much sensitivity important metabolites such as acetylcarnitine or acetyl-CoA.

The role of platelet-activating factor-dependent transacetylase in the biosynthesis of 1-acyl-2-acetyl-sn-glycero-3-phosphocholine by stimulated endothelial cells.

Acyl analogs of platelet-activating factor (PAF) (1-acyl-2-acetyl-sn-glycero-3-phosphocholine, acylacetyl -GPC) are the predominant products synthesized during thrombin or ionophore A23187-mediated activation of endothelial cells. However, the biosynthetic pathway responsible for the production of acylacetyl-GPC is not well understood. In the present investigation, we have demonstrated that the acyl analogs of PAF are also the major products from calf pulmonary artery endothelial cells in response to a time-dependent stimulation of ATP (10(-3) M), bradykinin (10(-8) M), or ionophore A23187 (2 microM). In addition, we have found that the CoA-independent PAF:acyllyso-GPC transacetylase recently identified by us is concurrently and transiently induced with maximal 4-fold enhancement at 5 min and returned to near basal level by 10 min treatment of endothelial cells with ATP. Acid phosphatase reduces the increased PAF:acyllyso-GPC transacetylase activity from the homogenates of ATP-activated endothelial cells. Reduced PAF:acyllyso-GPC transacetylase activity can be restored by incubating the acid phosphatase-treated homogenates with ATP (5 mM) and Mg2+ (10 mM). Furthermore, okadaic acid, a protein phosphatase 1 and 2A inhibitor, incubated with endothelial cells in a dose-dependent manner (1-100 nM) for 10-min potentiates and sustained the stimulation of PAF:acyllyso-GPC transacetylase activity by ATP. On the other hand, genistein, tyrphostin-25 (inhibitors of tyrosine-specific protein kinase), and calphostin C (an inhibitor of protein kinase C) block the activation of PAF:acyllyso-GPC transacetylase by ATP. These results are consistent with the notion that ATP regulates the transacetylase activity by reversible activation and inactivation via the phosphorylation and dephosphorylation cycle. ATP also augments the activities of alkyllyso-GPC/acyllyso-GPC:acetyl-CoA acetyltransferase. However, the activation of the acetyltransferases precedes that of the transacetylase with peak activation occurring at 1-2 min of the ATP treatment. In addition, sodium vanadate, also an inhibitor of protein phosphatase, stimulates the increase in the incorporation of [3H]acetate into acyl[3H]acetyl-GPC of the ATP-treated endothelial cells. Collectively, our data show that both acetyltransferases and transacetylase participate in and contribute to the biosynthesis of acyl analogs of PAF in a coordinate fashion in endothelial cells.

Simultaneous determination of lysophospholipids by high-performance liquid chromatography with fluorescence detection.

A high-performance liquid chromatography (HPLC) procedure for the separation of choline lysophospholipids including 1-acyl-lysophosphatidylcholines and 1-O-alkyl-lysophosphatidyl- cholines, like the lysoform of the platelet activating factor (2-lysoPAF), is described. The lysophospholipids are derivatized at the sn-2 position of the hydroxyl group by 7-diethylaminocoumarin-3-carbonylazide, which converts them into the corresponding carbamoyl derivatives. The derivatized compounds were well separated by reversed-phase HPLC and quantified by fluorimetric detection. This method shows a high sensitivity and allows the separation and quantification of mixtures of lysophospholipids at picomolar level. The method was applied to assay enzyme activities, like phospholipase A2 and PAF-acetylhydrolase, on single phospholipids or their mixtures.

Determination of residual pectin methylesterase activity in food products.

A method for the determination of a low level of pectin methylesterase activity from vegetable products is described. The method is based on an affinity chromatography technique that employs a resin-bound pectin methylesterase inhibitor, purified from kiwi fruit, which selectively binds the pectin methylesterase. The resin has the capacity to concentrate the enzyme, allowing measurement of enzyme activities too low to be determined by commonly employed techniques and commensurate with those found in pasteurized food products. The enzyme is eluted from the resin at alkaline pH (9.5) and assayed by a pH-stat method. Depulped orange juices containing different amounts of pectin methylesterase were prepared and used to determine enzyme recovery. The results show a recovery of 90% with a standard deviation of 6.8%.

Measurement of platelet-activating factor acetylhydrolase activity by quantitative high-performance liquid chromatography determination of coumarin-derivatized 1-O-alkyl-2-sn-lysoglyceryl-3-phosphorylcholine.

A sensitive method for determining platelet-activating factor acetylhydrolase (PAF-AH) activity in human serum, using high-performance liquid chromatography (HPLC) with a fluorimetric detection, is described. The method is based on the derivatization with 7-diethylaminocoumarin-3-carbonylazide of the 2-lyso-PAF, by-product of PAF-AH activity, extracted from the reaction mixture by phase partition into organic solvents. After 3 h of derivatization, the fluorescent derivatives were analyzed by HPLC on a reversed-phase column. The mobile phase was made up with a gradient between head solvent, composed of methanol:water (80:20, v/v) containing 0.25 g/liter choline chloride, and chloroform. Fluorescence detection was at excitation wavelength of 400 nm and at emission wavelength of 480 nm. The described chromatographic procedure is able to resolve and simultaneously quantitate the fluorescent derivatives of the C:18 and C:16 2-lysoPAF. The comparison with the classical radiometric determination of PAF-AH activity demonstrates that the herein described procedure is suitable for study of enzyme kinetics and changes occurring in physiological conditions such as pregnancy.

A glycoprotein inhibitor of pectin methylesterase in kiwi fruit. Purification by affinity chromatography and evidence of a ripening-related precursor.

The pectin methylesterase inhibitor from kiwi fruit (Actinidia chinensis) was purified by a single-step procedure based on affinity chromatography. Partially purified tomato pectin methylesterase was covalently bound to Sepharose. The affinity resin strongly and selectively binds the inhibitor, which could be eluted in high yield as a single, homogeneous and sharp peak by high salt concentration at pH 9.5 without loss of inhibitory activity. The purified protein possesses a molecular mass of 18 kDa, as estimated by SDS/PAGE, whereas by gel filtration under native conditions, its molecular mass appears to be 25 kDa. The inhibitor interacts with pectin methylesterase, forming a 1:1 complex, as demonstrated by gel-filtration experiments. The inhibitor was glycosylated. Its glycidic portion can be removed by digestion with N-glycosidase F after protein denaturation and, to a minor extent, by digestion with N-glycosidase H. No glycidic residue could be removed by digesting the native protein with those N-glycosidases. Antibodies against pectin methylesterase inhibitor were raised in rabbits and used to evidence protein expression during fruit ripening. The results showed that the inhibitor is present in the unripe fruit as an inactive precursor with a higher molecular mass (30 kDa) and is transformed into the active protein, most likely by proteinase action, during the course of the ripening process.

Interaction studies between elongation factor Tu and anthraniloyl-fluorescent analogues of guanyl nucleotides.

A fluorescent analogue of GDP, the 3'-O-anthraniloyl-GDP (anl-GDP) was demonstrated to bind to the elongation factor Tu (EF-Tu) with an affinity even higher than that of the parent nucleotide. As a consequence of the binding, an increase in fluorescence anisotropy and an emission band arising from non-radiative energy transfer among the protein intrinsic fluorophores and the labelled nucleotide were observed. Therefore, it was possible to study the exchange kinetics and the equilibrium between the protein-bound labelled GDP and the natural nucleotide through modifications, occurring during the course of the reaction, of fluorescence anisotropy and non-radiative energy transfer. In this way, it was also easily proven that, in the presence of aurodox (N-methylkirromycin), an antibiotic impairing EF-Tu biological function, the exchange kinetics between the protein-bound labeled GDP and the natural nucleotide was faster. Moreover, it was also found that the labelled nucleotide is recognized as a substrate by pyruvate kinase, being converted by this enzyme, in the presence of phosphoenolpyruvate, into anl-GTP. Pyruvate kinase is also able to convert, in the presence of phosphoenolpyruvate, the complex EF-Tu.anl-GDP into the complex EF-Tu.anl-GTP. The fluorescence properties of the 3'-O-anthraniloyl-labeled guanyl nucleotides and their feature as excellent acceptors of fluorescence arising from protein intrinsic fluorophores, may make these compounds useful for structural and binding studies on guanosine-nucleotide-binding proteins.

tRNA fluorescent labeling at 3' end inducing an aminoacyl-tRNA-like behavior.

A fluorescent tRNA derivative labeled at 3'-O position of the ultimate adenosine residue by reaction, under mild conditions, of tRNA with isatoic anhydride [3,1-benzoxazine-2,4(1H)-dione] was obtained. The labeling selectivity was determined by several criteria: digestion with RNase, followed by HPLC of the digest, produces only one labeled nucleoside, identified as 3'-O-anthraniloyladenosine; the ratio of the absorbance at 260 nm to 332 nm also suggests a 1:1 molar ratio between the nucleic acid and the fluorophore; finally, the incapacity of the labeled tRNA to be charged by the specific aminoacyltransferase further demonstrates the engagement of the 3'-O position. Although the 3'-O-anthraniloyl-labeled tRNA does not seem to be functionally active, as far as the aminoacyl charging activity is concerned, surprisingly we found that it is able to form the ternary complex with elongation factor Tu (EF-Tu) and GTP with an affinity consistently higher than uncharged tRNA. From fluorescence anisotropy measurements the ternary complex dissociation constant was estimated as 73 nM for Escherichia coli and 140 nM for yeast anthraniloyl-tRNA(Phe). These results may be interpreted in terms of the particular structure of the anthraniloyl group that makes the labeled tRNA similar to an aminoacyl-tRNA.

Role of calcium in chloride secretion mediated by cAMP pathway activation in rabbit distal colon mucosa.

Effects of Ca2+ on adenosine 3',5'-cyclic monophosphate (cAMP)-mediated Cl- secretion were investigated in intact mucosa and isolated crypt cells of rabbit descending colon. Addition of 10 microM prostaglandin (PG)E2 or forskolin to tissues incubated in Ca(2+)-free medium increased the size of short-circuit current (Isc) and Cl- secretion as estimated by unidirectional 36Cl flux measurements (net flux = -2.31 +/- 0.24 vs. -1.22 +/- 0.10 mueq.h-1.cm-2, n = 4, P < 0.001). Addition of 10 microM PGE2 to tissues incubated in 1.2 mM Ca2+ Ringer induced a 7-fold increase in mean cAMP level, whereas it produced an 11-fold increase in tissues exposed to Ca(2+)-free medium. Membrane preparations from whole mucosa incubated in Ca(2+)-free medium displayed a cyclic nucleotide phosphodiesterase activity significantly lower than controls (18.76 +/- 0.54 vs. 31.20 +/- 0.39 pmol cAMP. mg protein-1.min-1, means +/- SE, n = 4, P < 0.001). Ca2+ removal also affected adenylate cyclase (AC) responsiveness to agonists; AC activity increased in controls by 54 and 226% after stimulation with 10 microM PGE2 and forskolin, respectively, but it increased more (77 and 325%, respectively) after incubation in Ca(2+)-free solutions.(ABSTRACT TRUNCATED AT 250 WORDS)

Porins and lipopolysaccharide stimulate platelet activating factor synthesis by human mesangial cells.

Porins, a family of hydrophobic proteins located in the outer membrane of the cell wall of gram-negative bacteria and lipopolysaccharide (LPS), were shown to stimulate the synthesis of platelet activating factor (PAF), a phospholipid mediator of inflammation and endotoxic shock, by cultured human glomerular mesangial cells (MC). The synthesis of PAF induced by porins was rapid (peak at 20 min) and independent either from contamination by LPS or from generation of an endotoxin-induced cytokine such as tumor necrosis factor (TNF) since it was not prevented by cycloheximide, an inhibitor of protein synthesis or anti-TNF blocking antibodies. LPS also stimulated PAF synthesis by MC. However, the kinetic of PAF synthesis induced by LPS was biphasic with an early and transient peak at 10 minutes and a second and sustained peak at three to six hours. This second peak required an intact protein synthesis and was prevented by anti-TNF antibodies, suggesting the dependency on LPS-induced synthesis of TNF. Experiments with labeled precursors demonstrated that in MC, either after stimulation with porins or LPS, PAF was synthesized via the remodeling pathway that involves acetylation of 1-0-alkyl-sn-glyceryl-3-phosphorylcholine (2-lyso-PAF) generated from 1-0-alkyl-2-acyl-sn-glyceryl-3-phosphorylcholine by phospholipase A2 (PLA2) activity. Porins and LPS, indeed, induced PLA2-dependent mobilization of [14C]-arachidonic acid that was inhibited by p-bromodiphenacylbromide (PBDB). PBDB, an inhibitor of PLA2, also blocked PAF synthesis by preventing the mobilization of 2-lyso-PAF, the substrate for PAF-specific acetyltransferase.(ABSTRACT TRUNCATED AT 250 WORDS)

Platelet-activating factor biosynthesis by cultured mesangial cells is modulated by proteinase inhibitors.

Rat mesangial cells stimulated with calcium ionophore A23187 and phagocytosis were shown to produce platelet-activating factor (PAF), a mediator of inflammation and endotoxic shock. In the study presented here, the cultured human mesangial but not epithelial cells synthetized PAF not only in response to calcium ionophore A23187 and phagocytosis of immunoglobulin G-coated latex beads, but also after stimulation with cytokines such as tumor necrosis factor-alpha and interleukin-1 beta. PAF synthetized after stimulation with A23187 and to a lesser extent with phagocytosis was partially released. In contrast, PAF synthesized by stimulation with tumor necrosis factor-alpha and interleukin-1 beta remained cell associated. Experiments with labeled precursors demonstrated that PAF was synthetized via the remodeling pathway that involves the activation of phospholipase A2 and of an acetyl-coenzymeA:2-lyso-PAF acetyltransferase. Synthetic inhibitors of serine proteases as well as plasma alpha 1-proteinase inhibitor inhibited the activation of phospholipase A2 detected as release of (14C) arachidonic acid and the activation of acetyl-CoA:2-lyso-PAF acetyltransferase at concentrations 100-fold lower than those present in plasma. This raises the question about the ability of mesangial cells to synthetize PAF in vivo. However, the inhibitory effect of plasma alpha 1-proteinase inhibitor may be abrogated by oxidative inactivation due to a concomitant stimulation of mesangial cell respiratory burst or in zones of close contact among cells or matrix, which have been shown to exclude antiproteinases.

Mechanism of arachidonic acid transport across rabbit distal colonic mucosa.

The initial rate of [1-14C]arachidonic acid (AA) entry in the serosal side of rabbit distal colonic mucosa mounted in Ussing-type chambers is linear and independent of intracellular metabolism. When the maximal AA uptake was plotted as a function of medium AA concentration in ranges between 50 and 500 nM, saturation of the AA uptake with increasing concentrations was observed. The time course of the uptake of oleic acid and palmitic acid was similar to that observed with AA, and their separate addition to incubation medium strongly reduced the AA uptake. The influx of arachidonate was largely inhibited by ouabain and by incubation with mucosal sodium-free solution and amiloride, while it was increased when colonic mucosa was exposed to luminal amphotericin B. However, voltage-clamp studies showed that the AA entry rate appeared to be linearly related (r = 0.99) to transepithelial potential difference (PD) and suggested that the sodium dependence of AA translocation is an indirect effect of the changes in transepithelial PD induced by sodium transport shifts. These features provide evidence that there is a common entry pathway for AA and other long-chain free fatty acids mediated by a mechanism of facilitated diffusion driven by transmembrane PD.