Epoxyeicosatrienoic acids increase intracellular calcium concentration in vascular smooth muscle cells.

Epoxyeicosatrienoic acids (EETs) are cytochrome P450-derived metabolites of arachidonic acid. They are potent endogenous vasodilator compounds produced by vascular cells, and EET-induced vasodilation has been attributed to activation of vascular smooth muscle cell (SMC) K(+) channels. However, in some cells, EETs activate Ca(2+) channels, resulting in Ca(2+) influx and increased intracellular Ca(2+) concentration ([Ca(2+)](i)). We investigated whether EETs also can activate Ca(2+) channels in vascular SMC and whether the resultant Ca(2+) influx can influence vascular tone. The 4 EET regioisomers (1 micromol/L) increased porcine aortic SMC [Ca(2+)](i) by 52% to 81%, whereas arachidonic acid, dihydroxyeicosatrienoic acids, and 15-hydroxyeicosatetraenoic acid (1 micromol/L) produced little effect. The increases in [Ca(2+)](i) produced by 14,15-EET were abolished by removal of extracellular Ca(2+) and by pretreatment with verapamil (10 micromol/L), an inhibitor of voltage-dependent (L-type) Ca(2+) channels. 14,15-EET did not alter Ca(2+) signaling induced by norepinephrine and thapsigargin. When administered to porcine coronary artery rings precontracted with a thromboxane mimetic, 14,15-EET produced relaxation. However, when administered to rings precontracted with acetylcholine or KCl, 14,15-EET produced additional contractions. In rings exposed to 10 mmol/L KCl, a concentration that did not affect resting ring tension, 14,15-EET produced small contractions that were abolished by EGTA (3 mmol/L) or verapamil (10 micromol/L). These observations indicate that 14,15-EET enhances [Ca(2+)](i) influx in vascular SMC through voltage-dependent Ca(2+) channels. This 14,15-EET-induced increase in [Ca(i)(2+)] can produce vasoconstriction and therefore may act to modulate EET-induced vasorelaxation.

14,15-Epoxyeicosatrienoic acid inhibits prostaglandin E2 production in vascular smooth muscle cells.

14,15-Epoxyeicosatrienoic acid (EET), a cytochrome P-450 epoxygenase product of arachidonic acid (AA), reduced PGE2 formation by 40-75% in porcine aortic and murine brain microvascular smooth muscle cells. The inhibition was reversed 6-10 h after removal of 14,15-EET from the medium and was regioisomeric specific; 8,9-EET produced a smaller effect, whereas 11,12- and 5,6-EET were ineffective. Although the cells converted 14,15-EET to 14, 15-dihydroxyeicosatrienoic acid (14,15-DHET), 14,15-DHET did not inhibit PGE2 formation, and the 14,15-EET-induced inhibition was potentiated by 4-phenylchalcone oxide, an epoxide hydrolase inhibitor. The inhibition occurred when substrate amounts of AA were used and was not accompanied by enhanced production of other PGs, suggesting an effect on PGH synthase; however, in murine cells, 14, 15-EET did not reduce PGH synthase mRNA or protein. Moreover, the 14, 15-EET-induced decrease in PGE2 production was overcome by increasing the concentration of AA, but not oleic acid (which is not a substrate for PGH synthase). These findings suggest that 14,15-EET competitively inhibits PGH synthase activity in vascular smooth muscle cells. The 14,15-EET-induced inhibition of PGE2 production resulted in potentiation of platelet-derived growth factor-induced smooth muscle cell proliferation, suggesting that the competitive inhibition of PGH synthase by 14,15-EET can affect growth responses in smooth muscle cells.

Pseudomonas pyocyanin increases interleukin-8 expression by human airway epithelial cells.

Pseudomonas aeruginosa, an opportunistic human pathogen, causes acute pneumonia in patients with hospital-acquired infections and is commonly associated with chronic lung disease in individuals with cystic fibrosis (CF). Evidence suggests that the pathophysiological effects of P. aeruginosa are mediated in part by virulence factors secreted by the bacterium. Among these factors is pyocyanin, a redox active compound that increases intracellular oxidant stress. We find that pyocyanin increases release of interleukin-8 (IL-8) by both normal and CF airway epithelial cell lines and by primary airway epithelial cells. Moreover, pyocyanin synergizes with the inflammatory cytokines tumor necrosis factor alpha and IL-1alpha. RNase protection assays indicate that increased IL-8 release is accompanied by increased levels of IL-8 mRNA. The antioxidant n-acetyl cysteine, general inhibitors of protein tyrosine kinases, and specific inhibitors of mitogen-activated protein kinases diminish pyocyanin-dependent increases in IL-8 release. Conversely, inhibitors of protein kinases C (PKC) and PKA have no effect. In contrast to its effects on IL-8 expression, pyocyanin inhibits cytokine-dependent expression of the monocyte/macrophage/T-cell chemokine RANTES. Increased release of IL-8, a potent neutrophil chemoattractant, in response to pyocyanin could contribute to the marked infiltration of neutrophils and subsequent neutrophil-mediated tissue damage that are observed in Pseudomonas-associated lung disease.

L- and D-S-nitroso-beta,beta-dimethylcysteine differentially increase cGMP in cultured vascular smooth muscle cells.

We examined the effects of the L- and D-isomers of S-nitroso-beta,beta-dimethylcysteine (L- and D-S-nitrosopenicillamine, 10(-7)-10(-5) M) on the guanosine 3',5'-cyclic monophosphate (cGMP) content of cultured porcine aortic smooth muscle cells and the decomposition of these stereoisomers to nitric oxide (NO). L-S-nitrosopenicillamine was a more potent generator of cGMP than D-S-nitrosopenicillamine although both stereoisomers equally decomposed to NO. The 10(-7) M concentration of L- or D-S-nitrosopenicillamine did not generate detectable amounts of NO although 10(-7) M L-S-nitrosopenicillamine but not D-S-nitrosopenicillamine generated significant amounts of cGMP. This study shows that the stereoisomeric configuration of S-nitrosopenicillamine is an important factor in its biological potency. The data suggest that the extracellular or intracellular generation of NO is not the only mechanism by which this S-nitrosothiol generates cGMP in vascular smooth muscle.

Functional and ultrastructural effects of essential fatty acid deficiency in kidney epithelial cells.

Madin-Darby canine kidney (MDCK) epithelial cells were grown in culture medium supplemented with 1% fetal bovine serum (FBS) to provide a cell culture model of essential fatty acid deficiency (EFAD). 5,8,11-Eicosatrienoic acid (20:3n-9) accumulated in cellular phospholipids, and arachidonic acid (20:4) decreased. A large increase in cellular cholesterol/phospholipid ratio was observed. Hemicyst formation was greatly reduced from normal levels in the EFAD-MDCK cells. Scanning and transmission electron microscopy revealed that EFAD-MDCK were much flatter than their normal counterparts. They had much less dense surface microvilli, mitochondria and other organelles were very sparse, except in the perinuclear area, and much of the peripheral cytoplasm was amorphous. The EFAD was rapidly reversed by the addition of as little as 10 microM linoleic or arachidonic acid to the medium. Cells supplemented with 10% FBS, the usual culture condition, displayed borderline EFAD, with intermediate levels of 20:3n-9 and 20:4 and hemicyst formation. These studies suggest that EFAD reduces water and electrolyte transport in renal tubular epithelium.

13-HODE increases intracellular calcium in vascular smooth muscle cells.

13-Hydroxyoctadecadienoic acid (HODE) (2 microM) consistently increased porcine aortic and pulmonary artery smooth muscle cell calcium concentrations ([Ca2+]i), whereas 9-HODE and linoleic acid had no significant effect in the aortic cells and a much lesser effect in the pulmonary artery cells. A transient increase in [Ca2+]i occurred with as little as 50 nM 13-HODE. Structural specificity for elevation of [Ca2+]i also was seen with the monohydroxyeicosatetraenoic acids (HETEs), with 12-HETE but not 5- or 15-HETE increasing [Ca2+]i. 13-HODE, but not 9-HODE, increased smooth muscle cell guanosine 3',5'-cyclic monophosphate (cGMP) levels. The [Ca2+]i increase produced by 13-HODE was dependent on extracellular calcium and was inhibited by the calcium channel blockers verapamil and nifedipine and by KT-5823, a cGMP-dependent kinase inhibitor. A similar increase in [Ca2+]i was produced by 8-bromo-cGMP. These results suggest that 13-HODE, a 15-lipoxygenase product formed from linoleic acid, can act as a lipid mediator in vascular smooth muscle. It raises smooth muscle cGMP, causing a secondary increase in [Ca2+]i due to Ca2+ influx through a cGMP kinase-dependent L-type channel.

Platelet-activating factor may stimulate both receptor-dependent and receptor-independent increases in [Ca2+] in human airway epithelial cells.

Platelet-activating factor (PAF) is a potent mediator which produces a wide range of biological responses by binding to specific, high affinity receptors on the target cell surface. In addition, we and others have observed cellular responses to PAF which are not receptor-mediated. We report here that in HBE-16 human bronchial epithelial cells, PAF produces a biphasic increase in [Ca2+]i consisting of a rapid initial increase due to release from intracellular stores followed by a gradual, sustained phase caused by influx of extracellular Ca2+. Under certain conditions, the PAF receptor antagonist L-659,989 completely blocks the release of Ca2+ from intracellular stores, suggesting a complete block of the receptor-mediated response. Under these same conditions, a residual influx of extracellular Ca2+ is observed, suggesting a possible receptor-independent response. HBE-16 cells partially metabolize PAF to 1-O-alkyl-2-acetyl-sn-glycerol (AAG), a bioactive diacylglycerol analog. Moreover, AAG stimulates Ca2+ influx in these cells; the response to AAG is at least 100-fold more potent than that to PAF. Taken together, these results suggest that PAF may stimulate Ca2+ influx in HBE-16 cells through a receptor-independent pathway mediated by AAG. Thus these studies suggest a previously unrecognized dual-pathway regulatory mechanism for PAF in the airway.

Lysophosphatidylcholine causes cGMP-dependent verapamil-sensitive Ca2+ influx in vascular smooth muscle cells.

Lysophosphatidylcholine (lyso-PC) is a vasoactive phospholipid present in oxidized low-density lipoprotein. We used a coculture model of the vascular wall to study its interaction with endothelial cells (EC) and vascular smooth muscle cells (SMC). Lyso-PC was taken up readily by SMC and gradually acylated to phosphatidylcholine. Low concentrations (< or = 1 microM) of lyso-PC present in the interstitial medium of an EC-SMC coculture system were taken up primarily by the SMC. Lyso-PC produced a rapid two- to three-fold increase in SMC guanosine 3',5'-cyclic monophosphate (cGMP) levels, reaching a maximum in 1 min. This increase was associated with decreased SMC proliferation and increased calcium influx. The increase in intracellular calcium was inhibited by verapamil and KT5823, a specific cGMP-dependent kinase inhibitor, while a similar increase was produced by the membrane-permeant cGMP analogue 8-bromoguanosine 3',5'-cyclic monophosphate. These studies suggest that SMC are the primary target for the biological effects of lyso-PC present in the vessel wall and that the responses are mediated by calcium influx, possibly due to opening of a verapamil-sensitive cGMP kinase-dependent channel.

Interaction of lysophosphatidylcholine with aortic endothelial cells.

To better understand the vascular actions of lysophosphatidylcholine (lysoPC), we studied the interaction of [1-14C]palmitate-labeled lysoPC with bovine aortic endothelial cells. These cells took up lysoPC from media containing albumin, low-density lipoproteins (LDL), or acetyl-LDL. Uptake occurred faster than conversion to phosphatidylcholine (PC), leading to some lysoPC accumulation in endothelial lipids. Endothelial cell monolayers grown on micropore filters took up lysoPC from both apical and basolateral surfaces, preventing substantial amounts from passage across the endothelial monolayer. However, lysoPC present in the interstitial medium of an endothelial-smooth muscle coculture was incorporated primarily by the smooth muscle cells. Endothelial cells grown on filters released lysoPC into both the apical and basolateral medium in the presence of albumin or lipoproteins. Exposure to 50 microM lysoPC produced no evidence of endothelial cytotoxicity, but prostaglandin (PG)I2 production was reduced. These studies suggest that the endothelium can participate in the processing of circulating lysoPC and, through basolateral uptake, can facilitate the removal of lysoPC formed within the arterial wall. By decreasing PGI2 output, however, exposure to high concentrations of lysoPC may reduce the antithrombotic and vasodilator capacity of the endothelium.

Effect of antineoplastic agents on smooth muscle cell proliferation in vitro: implications for prevention of restenosis after transluminal angioplasty.

The effect of several antineoplastic agents on vascular smooth muscle cell proliferation was studied in vitro. Both fluorouracil and cytarabine produced significant concentration-dependent inhibition of smooth muscle cell proliferation in cultured porcine pulmonary artery in vitro, while cyclophosphamide stimulated growth. For fluorouracil, inhibition was near maximal at a concentration of 13.0 microgram/mL and was seen with both coincubation and 2-hour preincubation of fluorouracil with quiescent cells. Fluorouracil is a promising agent for inhibition of intimal proliferation. Further work is warranted to determine its effect in vivo.

Effect of differentiation on platelet-activating factor metabolism in HL-60 cells.

The formation and metabolism of 1-O-alkyl-2-acetyl-sn-glycerol (AAG), a protein kinase C (PKC) activator formed from platelet-activating factor (1-O-alkyl-2-acetyl-sn-glycero-3- phosphocholine; PAF), was studied in HL-60 cells to determine whether differentiation may influence this process. HL-60 cells differentiated to macrophages (HL-60/M phi) with a phorbol ester convert added [3H]PAF to AAG; 22% of the incorporated radioactivity is converted to AAG within 15s. By contrast, neither undifferentiated HL-60 cells (HL-60/U) nor HL-60 cells differentiated to granulocytes (HL-60/GN) with retinoic acid produce AAG from PAF. The HL-60/M phi rapidly convert radiolabeled AAG to 1-O-alkyl-sn-glycerol and, subsequently, to two other unidentified metabolites. However, some apparently unmodified AAG persists in the cell lipids for at least 6 h. The HL-60 subtypes which do not convert PAF to AAG can nevertheless catabolize AAG; HL-60/U and HL-60/GN produce alkylglycerol and the other AAG metabolites. These findings demonstrate that differentiation can alter the processing of PAF in a human leukocyte cell line. Furthermore, they suggest that PAF may produce at least some of its biological effects in macrophages by conversion to AAG.

1-O-alkyl-2-acetyl-sn-glycerol: a platelet-activating factor metabolite with biological activity in vascular smooth muscle cells.

Platelet-activating factor (1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine; PAF) is a potent vasoactive ether lipid produced by activated blood cells and endothelial cells. Vascular smooth muscle cells partially convert exogenous PAF to 1-O-alkyl-2-acetyl-sn-glycerol (AAG), a biologically active diacylglycerol analogue. AAG is formed rapidly (less than 15 s) after exposure of the smooth muscle cells and does not appear to be a substrate for diacylglycerol kinase in these cells. Although most of the compound is metabolized to 1-O-alkyl-sn-glycerol, a small quantity remains as AAG for greater than or equal to 6 h. AAG inhibits phorbol ester binding, and it is as effective an activator of protein kinase C as diolein in an in vitro assay. Furthermore, AAG and PAF produce the same pattern of effects on smooth muscle cell proliferation. These observations suggest that at least some of the actions of PAF in vascular smooth muscle may be mediated through the formation of AAG, a stable, bioactive metabolite that appears to function as a diacylglycerol analogue.

Interaction of platelet-activating factor with endothelial and vascular smooth muscle cells in coculture.

Platelet-activating factor (1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine [PAF]) is a vasoactive ether lipid produced by activated blood cells. To examine the molecular traffic and sites of metabolism of PAF released in the vascular wall, we used a coculture system in which endothelial cells are grown on micropore filters suspended over confluent cultures of vascular smooth muscle cells. The endothelial cells took up PAF 5-7 times more readily from the apical than from the basolateral surface, converting it to 1-O-alkyl-2-acyl-sn-glycero-3-phosphocholine (2-acyl-PAF) and other minor metabolites. Intact endothelial monolayers effectively shielded the underlying smooth muscle cells from PAF present in the apical fluid; after a 30-min incubation with [3H]-PAF, only 1% of the radioactivity was transferred to the interstitial fluid. By contrast, PAF readily entered the interstitial fluid when the endothelial monolayers were injured by exposure to xanthine and xanthine oxidase. PAF did not significantly increase the permeability of endothelial monolayers to albumin. Smooth muscle cells took up and metabolized interstitial PAF more quickly and more completely than did endothelial cells; 65% was converted to 2-acyl-PAF in 15 min by the smooth muscle cells. PAF enhanced the proliferative effect of PDGF on smooth muscle cells, as assessed by [3H]-thymidine incorporation. These findings suggest that endothelial cells form a barrier to PAF released at the luminal surface, but PAF released in the vascular intima interacts primarily with smooth muscle cells, possibly stimulating proliferation in these cells.

Polarity of arachidonic acid metabolism by bovine aortic endothelial cell monolayers.

Monolayers of bovine aortic endothelial cells cultured on micropore filters were used to determine the polarity of endothelial uptake, release, and transfer of arachidonic acid and some of its metabolites. Uptake and spontaneous release of arachidonic acid were more rapid at the luminal than at the interstitial surface. Transfer of arachidonic acid was more rapid from the luminal to the interstitial compartment than from the interstitial to the luminal compartment. After stimulation with the ionophore A23187, monolayers released arachidonate metabolites, including prostacyclin, to both the luminal and the interstitial compartments. The ability of the endothelium to rapidly take up and release arachidonic acid from the luminal surface and the ability to release biologically active eicosanoids to both the lumen and interstitium could be important for endothelial modulation of vascular events.

Lipid transfer between endothelial and smooth muscle cells in coculture.

A coculture system was employed to study the interactions between endothelium and vascular smooth muscle cells in arachidonic acid metabolism. Bovine aortic endothelial cells grown on micropore filters impregnated with gelatin and coated with fibronectin are mounted on polystyrene chambers and suspended over confluent smooth muscle cultures. The endothelial basal laminae are oriented toward the underlying smooth muscle, and the two layers are separated by only 1 mm. Each cell layer was assayed individually: apical and basolateral fluid also was collected separately for assay. Fatty acids, including arachidonic acid, are readily transferred between the endothelial and smooth muscle cells in this system. Distribution of the incorporated fatty acids among the lipids of each cell is the same as when the fatty acid is added directly to the culture medium. Arachidonic acid released from endothelial cells is available as a substrate for prostaglandin production by smooth muscle. In addition, fatty acids released from the smooth muscle cells can pass through the endothelium and accumulate in the fluid bathing the endothelial apical surface. These fatty acid interchanges may be involved in cell-cell signaling within the vascular wall, the clearance of lipids from the vascular wall, or the redistribution of arachidonic acid and other polyunsaturated fatty acids between adjacent cell types. Furthermore, the findings suggest that prostaglandin production by smooth muscle cells can occur in response to stimuli that cause arachidonic acid release from endothelial cells.

Free fatty acid release from endothelial cells.

Cultured bovine aortic endothelial cells that have been previously enriched with fatty acid are able to release free fatty acid (FFA) into the extracellular fluid. No stimulus other than the presence of albumin in the medium is needed to elicit the FFA release. Intracellular triglycerides appear to be the source of most of the FFA that is released. The released FFA is composed of a mixture of fatty acids, with the fatty acid used to enrich the cells contributing about half of the total. Under certain conditions sufficient fatty acid can be released to increase the FFA concentration of the extracellular fluid. Cells enriched initially with arachidonic acid released 1.7- to 2.9-times more FFA as compared to cells enriched with corresponding amounts of oleic acid. Neither prostaglandins nor lipoxygenase products contributed appreciably to the amount of FFA released from cells enriched with arachidonic acid. Porcine pulmonary artery endothelial cells also can release net amounts of FFA. These findings indicate that endothelial cells have the capacity to release fatty acid in the form of FFA. This process could possibly play a role in the transfer of fatty acids, particularly arachidonic acid, across the endothelium.

Changes in serum influence the fatty acid composition of established cell lines.

The fatty acid composition of different kinds of commercially available serum used to supplement cell culture media differs widely. As compared with fetal bovine serum, horse and bovine calf serum have a very high content of linoleic acid (18:2) and are low in arachidonic acid (20:4). (Fatty acids are abbreviated as number of carbon atoms:number of double bonds). Swine serum contains substantial amounts of both 18:2 and 20:4. Only fetal bovine serum contains more than 1% docosahexaenoic acid (22:6). Considerable differences in fatty acid composition occur when cells are grown in media containing any of these different serum supplements. The 18:2 and 20:4 content of 3T3 mouse fibroblast phospholipids is highest when the medium contains horse serum, intermediate with bovine calf serum, and lowest with swine or fetal bovine serum. Likewise, the highest phospholipid 18:2 content in Madin-Darby canine kidney cells (MDCK) occurs when the medium contains horse serum. With MDCK cells, however, growth in swine serum produces the highest 20:4 content. The 3T3 cell phospholipids accumulate more than 1% 22:6 only when the medium contains fetal bovine serum, whereas in no case do the MDCK cell phospholipids accumulate appreciable amounts of 22:6. The fact that the cellular fatty acid composition is likely to change should be taken into account when changes are contemplated in the serum used to grow established cell lines.

Prostaglandin production by 3T3-L1 cells in culture.

Rapidly growing cultures of 3T3-L1 preadipocytes produce large quantities of prostaglandins when they are either stimulated with the calcium ionophore A23187 or incubated with arachidonic acid. The main prostaglandin produced under all conditions was prostaglandin E2. Prostaglandin production in response to ionophore stimulation or incubation with arachidonic acid decreased markedly, however, as the cultures approached confluence, were maintained in the confluent state, or were stimulated to differentiate. Enrichment of confluent, differentiated cultures with arachidonic acid did not enhance prostaglandin production. Recovery of prostaglandin production occurred when logarithmic growth was reinstituted by reseeding confluent cultures at low cell densities, but sparse cultures maintained in a low-growth phase did not recover the ability to produce large amounts of prostaglandin E2. Therefore, the decline in prostaglandin synthetic capacity appears to be associated with the decrease in growth rate as the cells approach confluence. Media conditioned by confluent cells reduced prostaglandin E2 production when added to rapidly growing cells, suggesting that an inhibitor of prostaglandin synthesis may be formed by the confluent cultures. Nondifferentiating 3T3 fibroblasts, which similarly release mainly prostaglandin E2, also exhibited a decrease in prostaglandin production as the cultures became confluent. The amounts of prostaglandins produced by 3T3 cells in the confluent state were much greater, however, than those produced by confluent or differentiated 3T3-L1 cultures. These findings suggest that the low capacity to produce prostaglandins may be involved in either the induction or maintenance of differentiation in 3T3-L1 cells.

Accumulation of (n-9)-eicosatrienoic acid in confluent 3T3-L1 and 3T3 cells.

The 3T3-L1 preadipocyte cultured cell line accumulates the n-9 class of eicosatrienoic acid (20:3 n-9) after becoming confluent when the medium contains 10% fetal bovine serum. Accumulation of 20:3 n-9 also occurred in confluent, nondifferentiating 3T3 cells but not in eight other cultured cell lines that were tested. Radioisotope experiments indicate that confluent 3T3-L1 cells synthesized 20:3 n-9 from [1-14C]oleic acid. 20:3 n-9 comprised 9.8% of the cell phospholipid fatty acids, and its accumulation was associated with a 40% reduction in the phospholipid arachidonic acid content. The highest percentage of 20:3 n-9 was present in the ethanolamine and inositol glycerophospholipid fractions. Eicosatrienoic acid also accumulated in 3T3-L1 cells that were treated with dexamethasone, methylisobutylxanthine, and insulin to achieve adipocyte morphology. Supplementation of the culture medium with 0.03 mumol/ml of either linoleic or arachidonic acid prevented 20:3 n-9 accumulation. Partial reversal of the 20:3 n-9 accumulation occurred when confluent cells were either replated at a low density to initiate logarithmic growth or supplemented with 0.03 mumol/ml of arachidonic acid. The accumulation of 20:3 n-9, a polyunsaturate that ordinarily is found only in essential fatty acid deficiency, may be an important variable in studies involving 3T3-L1 and 3T3 cells.