Determination of cytochrome P450 metabolites of arachidonic acid in coronary venous plasma during ischemia and reperfusion in dogs

Arachidonic acid (AA) can be metabolized by cytochrome P450 enzymes to many biologically active compounds including 5,6-, 8,9-, 11,12-, and 14,15-epoxyeicosatrienoic acids (EETs), their corresponding dihydroxyeicosatrienoic acids (DHETs), as well as 19- and 20-hydroxyeicosatetraenoic acids (HETEs). These eicosanoids are potent regulators of vascular tone. However, their role in the ischemic myocardium has not been well investigated. In this study, we used a gas chromatographic-mass spectrometric technique to analyze total EETs, DHETs, and 20-HETE released into coronary venous plasma during coronary artery occlusion and reperfusion in anesthetized dogs. Pentafluorobenzyl esters (PFB-esters) of EETs and PFB-esters/trimethylsilyl ethers (TMS-ethers) of DHETs and 20-HETE were detected in the negative ion chemical ionization (NICI) using methane as a reagent gas. Under the conditions used, all four regioisomers of EET eluted from the capillary gas chromatographic column at similar retention times while four regioisomers of DHETs and 20-HETE eluted separately. The detection limits in plasma samples are 5 pg for total EETs, 40 pg for DHET, and 15 pg for 20-HETE. 14,15-DHET is the major regioisomer detected in the plasma samples while other regioisomers of DHETs are probably present at too low a concentration for detection. During the first 5 to 15 min of coronary occlusion, a slight decrease in the concentration of EETs, 14,15-DHET, and 20-HETE from the control values was observed in coronary venous plasma. At 60 min of occlusion, their concentrations significantly increased and remained elevated during 5 to 60 min of reperfusion. The concentrations decreased at 120 min of reperfusion. The NICI GC-MS was successfully used as a sensitive technique to determine cP450 metabolites of AA in plasma during prolonged occlusion-reperfusion periods. Furthermore, the results indicate that these metabolites may play a role in mediating ischemic-reperfusion injury.     

Cyclic AMP-dependent modulation of cardiac L-type Ca2+ and transient outward K+ channel activities by epoxyeicosatrienoic acids

The three major enzyme systems, cyclo-oxygenase, lipoxygenase, and cytochrome P450 (P450/CYP), metabolize arachidonic acid (AA) to biologically active compounds. P450 and its associated monooxygenase activities have been identified in mammalian cardiac tissue, including humans. The four regioisomeric eicosanoids, 5,6-, 8,9-, 11,12-, and 14,15-epoxyeicosatrienoic acids (EETs) of AA metabolites derived by P450 epoxygenases have shown to possess potent biological effects in numerous tissues. In the coronary circulation the EETs are leading candidates for endothelial-derived hyperpolarizing factors that hyperpolarize vascular smooth muscle cells by opening Ca2+-activated K+ channels. Recently, the effects of the CYP pathways and their metabolites on cardiac ischemia-reperfusion injury have been evaluated in animal models. Some of these AA metabolites are cardioprotective and some are detrimental. However, EETs appear to be cardioprotective in CYP2J2 transgenic mice and in a canine ischemic model. Multiple effects of EETs on cardiac ion channels have been observed, such as activation of ATP-sensitive K+ channels and L-type Ca2+ channels in cardiomyocytes and inhibition of cardiac Na+ channels and L-type Ca2+ channels reconstructed in planar lipid bilayers. This brief review summarizes EET-induced modulation of cardiac ion channels.     

Regulation of prostaglandin synthesis by protein kinase C in mouse peritoneal macrophages.

Resident mouse peritoneal macrophages synthesized and released prostaglandins (PGs) when challenged with 12-O-tetradecanoylphorbol 13-acetate (TPA) or 1,2-dioctanoyl-sn-glycerol (DiC8). Both stimuli were found to activate Ca2+/phospholipid-dependent protein kinase C (PKC). 1-(5-Isoquinolinesulphonyl)-2-methylpiperazine ('H-7') and D-sphingosine, known to inhibit PKC by different mechanisms, were able to decrease the PKC activity of macrophages in a dose-dependent manner. Addition of either PKC inhibitor decreased PG synthesis and also the release of arachidonic acid (AA) from phospholipids induced by TPA or DiC8. Simultaneously TPA or DiC8 also decreased incorporation of free AA into membrane phospholipids of macrophages. AA incorporation could be restored, however, by pretreatment with the PKC inhibitors. Our results demonstrate an involvement of PKC in the regulation of PG synthesis in mouse peritoneal macrophages and provide further evidence that reacylation of released fatty acids may be an important regulatory step.     

Glycerylprostaglandin synthesis by resident peritoneal macrophages in response to a zymosan stimulus

Cyclooxygenase (COX)-2 oxygenates arachidonic acid (AA) and 2-arachidonylglycerol (2-AG) to endoperoxides, which are subsequently transformed to prostaglandins (PGs) and glycerylprostaglandins (PG-Gs). PG-G formation has not been demonstrated in intact cells treated with a physiological agonist. Resident peritoneal macrophages, which express COX-1, were pretreated with lipopolysaccharide to induce COX-2. Addition of zymosan caused release of 2-AG and production of the glyceryl esters of PGE2 and PGI2 over 60 min. The total quantity of PG-Gs (16 +/- 6 pmol/10(7) cells) was much lower than that of the corresponding PGs produced from AA (21,000 +/- 7,000 pmol/10(7) cells). The differences in PG-G and PG production were partially explained by differences in the amounts of 2-AG and AA released in response to zymosan. The selective COX-2 inhibitor, SC236, reduced PG-G and PG production by 49 and 17%, respectively, indicating a significant role for COX-1 in PG-G and especially PG synthesis. Time course studies indicated that COX-2-dependent oxygenation rapidly declined 20 min after zymosan addition. When exogenous 2-AG was added to macrophages, a substantial portion was hydrolyzed to AA and converted to PGs; 1 microm 2-AG yielded 820 +/- 200 pmol of PGs/10(7) cells and 78 +/- 41 pmol of PG-Gs/10(7) cells. SC236 reduced PG-G and PG production from exogenous 2-AG by 88 and 76%, respectively, indicating a more significant role for COX-2 in the utilization of exogenous substrate. In conclusion, lipopolysaccharide-pretreated macrophages produce PG-Gs from endogenous 2-AG during zymosan phagocytosis, but PG-G formation is limited by substrate hydrolysis and inactivation of COX-2.     

Cytochrome P450 induction and mutagenicity of 2-aminoanthracene (2AA) in rat liver and gut.

The aim of our study was to establish a relationship between the ability of rat liver and gut to activate 2-aminoanthracene (2AA) into mutagens and their P450 enzyme composition. Rats were orally pretreated with beta-naphthoflavone (beta NF), phenobarbital (PB), dexamethasone (DEX) or acetone (AT). Mutagenic activation of 2AA was detected in the Ames test. P450IA1, IA2, IIB1/B2 and IIE1 were immunochemically quantified by Western blots. All the results were compared to those obtained in untreated rats. In all tissues, beta NF treatment considerably increased the mutagenicity of 2AA. PB treatment significantly reduced the mutagenicity of 2AA in the liver but not in the intestine. By contrast, AT treatment significantly decreased the number of revertants in the duodenum but not in the liver whereas DEX treatment significantly decreased the number of revertants in both tissues. 2AA appears to be metabolized by various P450s in both organs. In the liver, reactive metabolites may be produced after metabolism by the P450IA subfamily. The other P450 enzyme seems to play a part in the metabolism of 2AA leading to formation of either mutagenic or non-mutagenic metabolites.     

Determination of bioactive eicosanoids in brain tissue by a sensitive reversed-phase liquid chromatographic method with fluorescence detection

Arachidonic acid (AA) is metabolized to prostaglandins (PGs) via cyclooxygenases (COX) catalysis, and to epoxyeicosatrienoic acids (EETs), dihydroxyeicosatrienoic acids (DiHETrEs), and hydroxyeicosatetraenoic acids (HETEs) via cytochrome P450 (CYP450) enzymes. A reliable and robust fluorescence based HPLC method for these eicosanoids was developed. A new selective reverse-phase solid phase extraction (SPE) procedure was developed for PG, DiHETrEs, HETE, and EETs of interest from rat cortical brain tissue. The eicosanoids were derivatized with 2-(2,3-naphthalimino)ethyl-trifluoromethanesulphonate (NE-OTf), followed by separation and quantification at high sensitivity using reverse-phase HPLC with fluorescent detection, and further identified via LC/MS. The derivatization was studied and optimized to obtain reproducible reactions. Various PGs, DiHETrEs, HETEs, EETs, and AA were sensitively detected and baseline resolved simultaneously. LC/MS under positive electrospray ionization selected ion monitoring (SIM) mode was developed to further identify the peaks of these eicosanoids in cortical brain tissue. The method was applied in the traumatic brain injured rat brain.     

Zymosan-induced glycerylprostaglandin and prostaglandin synthesis in resident peritoneal macrophages: roles of cyclo-oxygenase-1 and -2

COX [cyclo-oxygenase; PG (prostaglandin) G/H synthase] oxygenates AA (arachidonic acid) and 2-AG (2-arachidonylglycerol) to endoperoxides that are converted into PGs and PG-Gs (glycerylprostaglandins) respectively. In vitro, 2-AG is a selective substrate for COX-2, but in zymosan-stimulated peritoneal macrophages, PG-G synthesis is not sensitive to selective COX-2 inhibition. This suggests that COX-1 oxygenates 2-AG, so studies were carried out to identify enzymes involved in zymosan-dependent PG-G and PG synthesis. When macrophages from COX-1-/- or COX-2-/- mice were treated with zymosan, 20-25% and 10-15% of the PG and PG-G synthesis observed in wild-type cells respectively was COX-2 dependent. When exogenous AA and 2-AG were supplied to COX-2-/- macrophages, PG and PG-G synthesis was reduced as compared with wild-type cells. In contrast, when exogenous substrates were provided to COX-1-/- macrophages, PG-G but not PG synthesis was reduced. Product synthesis also was evaluated in macrophages from cPLA(2alpha) (cytosolic phospholipase A2alpha)-/- mice, in which zymosan-induced PG synthesis was markedly reduced, and PG-G synthesis was increased approx. 2-fold. These studies confirm that peritoneal macrophages synthesize PG-Gs in response to zymosan, but that this process is primarily COX-1-dependent, as is the synthesis of PGs. They also indicate that the 2-AG and AA used for PG-G and PG synthesis respectively are derived from independent pathways.     

Copper modulation of macrophage cyclooxygenase metabolite synthesis

The effect of copper on the release of cyclooxygenase metabolites from starch elicited, rat, peritoneal macrophages was investigated. Copper sulphate, in the range 10(-6)-10(-5) M, inhibited the formation of prostaglandin (PG) E2 and thromboxane (Tx) B2, the stable metabolite of TxA2, in a dose dependent manner but had no effect on the production of 6-keto-PGF1 alpha, the stable product of prostacyclin. At higher concentrations (5 x 10(-5) and 10(-4) M) the synthesis of all three metabolites of arachidonic acid (AA) was stimulated as was the release of radioactivity from macrophages prelabelled with 14C AA. Copper had no effect on the metabolism of exogenous AA however. At 10(-4) M copper also stimulated secretion of the lysosomal enzyme, beta-glucuronidase (GUR). Copper nitrate (10(-4) M), but not zinc sulphate, also stimulated eicosanoid formation and lysosomal enzyme release. Our results are consistent with the idea that copper stimulates eicosanoid formation via an effect on PL activity.     

Formation of epoxyeicosatrienoic acids from arachidonic acid by cultured rat aortic smooth muscle cell microsomes.

The vasodilatory effect of epoxyeicosatrienoic acids (EpETrE), especially 5(6)-EpETrE, has been reported recently and a role of P-450-dependent arachidonic acid monooxygenase metabolites was suggested in vasoregulation. Accordingly, the presence of P-450-dependent arachidonic acid monooxygenase was investigated in rat aortic smooth muscle cells. Incubation of the microsomes of rat cultured aortic smooth muscle cells with 14C-arachidonic acid in the presence of 1 mM NADPH resulted in the formation of oxygenated metabolites. The metabolites were separated and purified by reverse phase and straight phase high performance liquid chromatography and identified by gas chromatography-mass spectrometry. Identified metabolites were 5(6)-EpETrE, 5,6-dihydroxyeicosatrienoic acid (DiHETrE), and 14,15-DiHETrE. The formation of these metabolites was totally dependent on the presence of NADPH, and inhibitors of cytochrome P-450-dependent enzymes, SKF-525A and metyrapone, reduced the formation of these metabolites. This is the first report that cytochrome P-450-dependent arachidonic acid metabolites, especially 5(6)-EpETrE and 14(15)-EpETrE, can be produced in the microsomes of vascular smooth muscle cells of rats.     

Role of cytochrome P450 in phospholipase A2- and arachidonic acid-mediated cytotoxicity

Phospholipases A2 (PLA2) comprise a set of extracellular and intracellular enzymes that catalyze the hydrolysis of the sn-2 fatty acyl bond of phospholipids to yield fatty acids and lysophospholipids. The PLA2 reaction is the primary pathway through which arachidonic acid (AA) is released from phospholipids. PLA2s have an important role in cellular death that occurs via necrosis or apoptosis. Several reports support the hypothesis that unesterified arachidonic acid in cells is a signal for the induction of apoptosis. However, most of the biological effects of arachidonic acid are attributable to its metabolism by mainly three different groups of enzymes: cytochromes P450, cyclooxygenases, and lipoxygenases. In this review we will focus on the role of cytochrome P450 in AA metabolism and toxicity. The major pathways of arachidonic acid metabolism catalyzed by cytochrome P450 generate metabolites that are subdivided into two groups: the epoxyeicosatrienoic acids, formed by CYP epoxygenases, and the arachidonic acid derivatives that are hydroxylated at or near the omega-terminus by CYP omega-oxidases. In addition, autoxidation of AA by cytochrome P450-derived reactive oxygen species produces lipid hydroperoxides as primary oxidation products. In some cellular models of toxicity, cytochrome P450 activity exacerbates PLA2- and AA-dependent injury, mainly through the production of oxygen radicals that promote lipid peroxidation or production of metabolites that alter Ca2+ homeostasis. In contrast, in other situations, cytochrome P450 metabolism of AA is protective, mainly by lowering levels of unesterified AA and by production of metabolites that activate antiapoptotic pathways. Several lines of evidence point to the combined action of phospholipase A2 and cytochrome P450 as central in the mechanism of cellular injury in several human diseases, such as alcoholic liver disease and myocardial reperfusion injury. Inhibition of specific PLA2 and cytochrome P450 isoforms may represent novel therapeutic strategies against these diseases.     

Induction of tumor cell resistance to macrophage-mediated lysis by preexposure to non-activated macrophages

Thioglycollate-elicited peritoneal exudate (non-activated) macrophages do not lyse tumor cells and in contrast to activated macrophages bind less target cells. However, a non-lethal encounter of tumor cells with non-activated macrophages resulted in a pronounced effect on the subsequent tumor cell binding to and lysis by activated macrophages. Our results have shown that binding of tumor cells by non-activated macrophages was Ca2+ and temperature dependent; had a requirement for a Pronase-sensitive structure on macrophage surface membranes; was saturable; and was 2-3X less than that observed for activated macrophages. Experiments were conducted in which syngeneic tumor cells were incubated with a monolayer of non-activated macrophages and then assayed for selective binding and sensitivity to lysis. The important observations were that as a result of a 3-hr incubation with non-activated macrophages at an EC: TC ratio of 5:1 there was an increase in the number of tumor cells that bound to both activated and non-activated macrophages; a loss of selective binding in which the ratio of tumor cells bound to activated/non-activated macrophages (normally greater than 2) was lowered to 1.0; and a concomitant decrease in the susceptibility of tumor cells to macrophage-mediated cytolysis. The induction of tumor cell resistance to macrophage kill required an exposure to an excess number of non-activated macrophages, was reversible upon culturing with or without macrophages for 24 hr and required cell-cell contact. Our results reinforce the importance of selective binding between tumor cells and activated macrophages as an initial phase in tumor cell killing and also illustrates an active role for non-activated macrophages in vivo in allowing tumor cells to escape the immune surveillance by activated macrophages.     

Inhibition of ATP binding to the carboxyl terminus of Kir6.2 by epoxyeicosatrienoic acids

Epoxyeicosatrienoic acids (EETs), the cytochrome P450 metabolites of arachidonic acid (AA), are potent and stereospecific activators of cardiac ATP-sensitive K(+)(K(ATP)) channels. EETs activate K(ATP) channels by reducing channel sensitivity to ATP. In this study, we determined the direct effects of EETs on the binding of ATP to K(ATP) channel protein. A fluorescent ATP analog, 2,4,6-trinitrophenyl (TNP)-ATP, which increases its fluorescence emission significantly upon binding with proteins, was used for binding studies with glutathione-S-transferase (GST) Kir6.2 fusion proteins. TNP-ATP bound to GST fusion protein containing the C-terminus of Kir6.2 (GST-Kir6.2C), but not to the N-terminus of Kir6.2, or to GST alone. 11,12-EET (5 muM) did not change TNP-ATP binding K(D) to GST-Kir6.2C, but B(max) was reduced by half. The effect of 11,12-EET was dose-dependent, and 8,9- and 14,15-EETs were as effective as 11,12-EET in inhibiting TNP-ATP binding to GST-Kir6.2C. AA and 11,12-dihydroxyeicosatrienoic acid (11,12-DHET), the parent compound and metabolite of 11,12-EET, respectively, were not effective inhibitors of TNP-ATP binding to GST-Kir6.2C, whereas the methyl ester of 11,12-EET was. These findings suggest that the epoxide group in EETs is important for modulation of ATP binding to Kir6.2. We conclude that EETs bind to the C-terminus of K(ATP) channels, inhibiting binding of ATP to the channel.     

Epoxyeicosatrienoic acids induce growth inhibition and calpain/caspase-12 dependent apoptosis in PDGF cultured 3T6 fibroblast

Previous studies have demonstrated that arachidonic acid (AA) metabolites released by the cyclooxygenase pathway is involved in serum-induced 3T6 fibroblast cycle progression and proliferation. However, these results also suggest that other AA cascade pathways might be involved. Recently, we also described the role of hydroxyeicosatetraenoic acids, which are produced by cytochrome P450 monooxygenases (CYP), in 3T6 fibroblast growth. AA can be also metabolized by the epoxygenase activity of CYP-producing epoxyeicosatrienoic acids (EETs). Finally, the cytosolic epoxide hydrolases catalyze the hydration of the EETs, transforming them into dihydroxyeicosatetraenoic acids (DHETEs). In this work, we have studied the role of the EETs/DHETEs on 3T6 fibroblasts growth. Our results show that PDGF stimulates 3T6 fibroblast proliferation and [³H]thymidine incorporation, while the addition of 5,6-EET, 8,9-EET, 11,12-EET or 14,15-EET (0.1–1 μM) inhibit these processes. Furthermore, 5,6-DHETE and 11,12-DHETE (0.1–1 μM) also inhibit cell proliferation and DNA synthesis. Interestingly, this growth inhibition was correlated with an induction of apoptosis. Thus, we observed that in the presence of PDGF, EETs or DHETEs (0.1–1 μM) induce phosphatidylserine externalization (as measured by annexin V-binding) and DNA fragmentation (as quantified using a TUNEL assay). Our results show that calpain, as well as caspase-12 and caspase-3, are involved in these events. Therefore, EETs and DHETEs have anti-proliferative and pro-apoptotic effects on PDGF-stimulated 3T6 fibroblasts.     

Characterization of Anopheles minimus CYP6AA3 expressed in a recombinant baculovirus system

Metabolism by cytochrome P450 monooxygenases is a major mechanism implicated in resistance of insects to insecticides, including pyrethroids. We previously isolated the cytochrome P450 CYP6AA3 from deltamethrin-selected resistant strain of Anopheles minimus mosquito, a major malaria vector in Thailand. In the present study, we further investigated the role of CYP6AA3 enzyme in deltamethrin metabolism in vitro. The CYP6AA3 was expressed in Spodoptera frugiperda (Sf9) insect cells via baculovirus-mediated expression system. The enzymatic activity of CYP6AA3 in deltamethrin metabolism was characterized after being reconstituted with An. minimus NADPH-cytochrome P450 reductase and a NADPH-regenerating system. The contribution of CYP6AA3 responsible for deltamethrin metabolism was determined by measurement of deltamethrin disappearance following the incubation period and deltamethrin-derived compounds were detected using combined gas chromatography mass spectrometry analysis. 3-Phenoxybenzaldehyde was a major product of CYP6AA3-mediated deltamethrin metabolism. Deltamethrin degradation and formation of metabolites were NADPH-dependent and inhibited by piperonyl butoxide. Deltamethrin was catalyzed by CYP6AA3 with an apparent K(m) of 80.0 +/- 2.0 and V(max) of 60.2 +/- 3.6 pmol/min/pmol P450. Furthermore, deltamethrin cytotoxicity assays by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and trypan blue dye exclusion were examined in Sf9 insect cells, with and without expression of CYP6AA3. Results revealed that CYP6AA3 could play a role in detoxifying deltamethrin in the cells. Thus, the results of this study support the role of CYP6AA3 in deltamethrin metabolism.     

Metabolism of eicosapentaenoic and docosahexaenoic acids by recombinant human cytochromes P450

Epoxidation and hydroxylation of arachidonic acid (AA) are both catalyzed by cytochromes P450s (CYPs). The oxidized metabolites are known to be involved in the regulation of vascular tone and renal function. By using a panel of 15 human recombinant CYPs, this study demonstrates that other polyunsaturated long-chain fatty acids (PUFA-LC), especially the ω3 fatty acids eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), are also epoxidised. The regioselectivity of epoxidation of four PUFA-LC by CYPs was investigated. Among the several CYPs tested, CYP2C9/2C19 and 1A2 were the most efficient in EPA and DHA epoxidations. It ensued that 10 μM of these two ω3 fatty acids decreased by more than 80% and 60%, respectively, the formation by CYP2C9 of AA-epoxidised derivatives. These findings suggest that some physiological effects of ω3 fatty acids may be due to a shift in the generation of active epoxidised metabolites of AA through CYP-mediated catalysis.     

Arachidonic acid inhibits inflammatory responses by binding to myeloid differentiation factor-2 (MD2) and preventing MD2/toll-like receptor 4 signaling activation

Arachidonic acid (AA) plays a fundamental role in the function of all cells. Metabolites of AA contribute to inflammation as well as for resolving inflammation. Although AA-derived metabolites exhibit well-substantiated bioactivity, it is not known whether AA regulates inflammatory responses independent of its metabolites. With the recent discovery that saturated fatty acids activate toll-like receptor-4 (TLR4), we tested the hypothesis that AA directly regulates inflammatory responses through modulating the activity of TLR4. In cultured cardiomyocytes and macrophages, we found that AA prevents saturated fatty acid-induced TLR4 complex formation with accessory proteins and the induction of proinflammatory cytokines. We discovered that AA directly binds to TLR4 co-receptor, myeloid differentiation factor 2 (MD2) and prevents saturated fatty acids from activating TLR4 pro-inflammatory signaling pathway. Similarly, AA reduced lipopolysaccharide (LPS)-induced inflammation in macrophages and septic death in mice through binding to MD2. In high-fat diet mouse model of obesity and LPS-induced model of acute lung injury, both mediating inflammatory responses through TLR4, treatment with AA prevented MD2/TLR4 dimerization, induction of inflammatory factors, and tissue injuries. In summary, we have discovered that AA interacts with MD2 and disrupts TLR4 activation by LPS and saturated fatty acids. These findings provide experimental evidence for a direct mechanism of AA-induced regulation of inflammation.     

Multiple eicosanoid-activated nonselective cation channels regulate B-lymphocyte adhesion to integrin ligands

Arachidonic acid (AA) is a substrate for a variety of proinflammatory mediators, which are generated by cyclooxygenases (COXs), lipoxygenases (LOXs), and cytochrome P-450 (CYP450) enzymes. COX (e.g., PGs and prostacyclins) and LOX (e.g., leukotrienes) products have well-established proinflammatory roles; however, little is known about the functions of CYP450 products in leukocytes. We previously found that mechanical strain generated by subjecting lymphocytes to hypotonic challenge triggered AA production and that two CYP450 products of AA, 5,6-epoxyeicosatrienoic acid (5,6-EET) and 20-hydroxyeicosatetraenoic acid (20-HETE), as well as a product of LOX, 5-(S)-hydroperoxyeicosatetrenoic acid (5-HPETE), induced Ca²⁺ entry into primary B cells. The main goal of the present studies, therefore, was to define the biophysically properties of eicosanoid-activated channels responsible for Ca²⁺ entry and the physiological consequences of activating these channels, including their role in mechanical signaling. We found that 5,6-EET, 20-HETE, and 5-HPETE each activated distinct Ca²⁺-permeant nonselective cation channels (NSCCs) in primary B cells. These NSCCs each regulate plasma membrane potential and B-cell adhesion to integrin ligands ICAM-1 and VCAM-1. Thus our data demonstrate that proinflammatory mediators produced in response to osmotic and/or physical stress play a direct role in regulating the B-cell membrane potential and their adhesion to specific ECM proteins. These results not only have important implications for understanding normal mechanisms of B-cell activation, differentiation, and trafficking but also point to novel targets for modulating the pathogenesis of B-cell-mediated inflammatory diseases. calcium; arachidonic acid; membrane potential; hypotonicity; cytochrome P-450     

Regional biosynthesis of prostaglandins and hydroxyeicosatetraenoic acids from arachidonic acid in the rat stomach tissue

The present study was conducted to determine regional differences in the biosynthesis of prostaglandins (PGs) and hydroxyeicosatetraenoic acids (HETEs) in the rat stomach tissue (fundus, corpus and pyloric antrum) from radioactive arachidonic acid (AA). The radioactive metabolites were validated by RP-HPLC using non-radioactive AA as substrate. PGE(2) was the major prostanoid in the tissue(.) The relative ratio of PGE(2):PGF(2)alpha:PGD(2) in the whole stomach was 1:0.5:0.1. Regionally, the fundus biosynthesized the largest amount of all three cyclo-oxygenase products. Among the lipoxygenase metabolites, 15S-HETE was the predominant product, while 12S-HETE was found to be the lowest. The relative ratio of 15S-HETE:5S-HETE:12S-HETE in the whole stomach was 1:0.6:0.4. Interestingly, the generation of lipoxygenase products was the highest in the pyloric antrum when compared to fundus or corpus. Thus, the regional differences in the biosyntheses of gastric PGs and monohydroxy fatty acids may be relevant to our understanding of corresponding differences in mucosal resistance or susceptibility to gastric disease.     

Characterization of arachidonic acid metabolism in Watanabe heritable hyperlipidemic (WHHL) and New Zealand white (NZW) rabbit aortas

WHHL rabbits develop progressive atherosclerosis. There are no visible signs of the disease at 1 month, however, by 12 months, the formation of aortic plaques is extensive. This study characterized arachidonic acid (AA) metabolism in 1 and 12 month old WHHL and NZW rabbit aortas. Vessels incubated with 14C-AA and A23187 metabolized AA to a number of oxygenated products as identified by high pressure liquid chromatography. The major AA metabolites produced by WHHL and NZW aortas were 6-keto PGF1 alpha, PGE2, 12- and 15-hydroxyeicosatetraenoic acids (HETEs). The structures of the HETEs were confirmed by gas chromatography-mass spectrometry. Indomethacin blocked the synthesis of prostaglandins (PGs) but not HETEs whereas ETYA, NDGA or removal of the endothelium attenuated the production of both PGs and HETEs. Measurement of 6-keto PGF1 alpha, 12- and 15-HETE by specific radioimmunoassays indicated that as the rabbits aged and as atherosclerosis progressed, aortas lost the ability to synthesize 6-keto PGF1 alpha and 15-HETE. Prior to the development of atherosclerosis, 1 month old WHHL aortas produced 70% less 15-HETE than did NZW aortas. Atherosclerotic aortas from 12 month old WHHLs synthesized 60% less 6-keto PGF1 alpha during stimulation with AA or A23187 than did 12 month old NZW aortas. We conclude that the development and expression of atherosclerosis in WHHL rabbits impairs the ability of aortas to metabolize AA to both PGs and HETEs.