Enhancement of endogenous production of 12-L-hydroxy-5,8,10,14-eicosatetraenoic acid in aortic smooth muscle cells by platelet-derived growth factor

Platelet-derived growth factor (PDGF) has a chemotactic effect on smooth muscle cells, which is inhibited by lipoxygenase inhibitor caffeic acid. In order to study the role of endogenous lipoxygenase products of arachidonic acid on the chemotactic action of PDGF, effects of PDGF on the lipoxygenase pathway in smooth muscle cells were examined. Lipoxygenase products were analyzed by high-performance liquid chromatography. 15-, 5- and 12-lipoxygenase activities, in order of magnitude, were found in smooth muscle cell homogenate. However, when the lipoxygenase products were analyzed using intact cells prelabelled with [14C]arachidonic acid, only 12-L-hydroxy-5,8,10,14-eicosatetraenoic acid (HETE) was found to be produced endogenously. In addition, 12-HETE was not released into the medium. Treatment of the cells with PDGF increased the endogenous production of 12-HETE. The amounts of intracellular 12-HETE in PDGF-treated cells were 126, 132 and 146% at 1, 3, and 10 hr's after the initiation of PDGF treatment, respectively, when control value at each time point was considered as 100%. Caffeic acid (10(-4) M) completely inhibited the PDGF effect on 12-HETE production. However, PDGF treatment did not significantly alter the 12-lipoxygenase activity. These results suggest that the stimulatory effect of PDGF on 12-HETE production was not mediated by the activation of 12-lipoxygenase activity. Since 12-HETE itself is a potent chemoattractant for smooth muscle cells, the present dat strongly suggest that 12-HETE could be an important intracellular mediator of the chemotactic action of PDGF on aortic smooth muscle cells.     

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.     

Effect of elastase on prostacyclin synthesis in aortic smooth muscle cells

The effects of elastase on prostacyclin biosynthesis in cultured rat aortic smooth muscle cells were investigated. Prostacyclin is the major product formed from arachidonic acid by aortic smooth muscle cells. When intact cells were incubated with elastase, a significant stimulatory effect on prostacyclin biosynthetic activity in cells was evident. However, the addition of elastase directly to the cell-free homogenates did not show any effects on prostacyclin biosynthesis. The maximal effect of elastase on the stimulation of prostacyclin biosynthesis without any cellular damage was observed at a concentration of 50 unit/ml elastase. Elastase also caused a marked release of arachidonic acid. At higher concentrations of elastase (75-100 units/ml), the release of arachidonic acid and prostacyclin synthesis was observed, but, at these concentrations of elastase, cells were slightly damaged. On the other hand, the releases of prostacyclin and arachidonic acid were markedly enhanced, when cells were preincubated with elastase (1 unit/ml) for 3 days. These results indicate that elastase, even at low concentrations, causes the releases of arachidonic acid and prostacyclin, especially when aortic smooth muscle cells are pre-treated with elastase.     

15-Hydroxyeicosatetraenoic acid (15-HETE) protects pulmonary artery smooth muscle cells from apoptosis via inducible nitric oxide synthase (iNOS) pathway

15-Hydroxyeicosatetraenoic acid (15-HETE), one of many important metabolic products of arachidonic acid (AA) catalyzed by 15-lipoxygenase, plays an important role in pulmonary vascular smooth muscle remodeling. We have previously shown its unsubstituted effects on the apoptotic responses of pulmonary artery smooth muscle cells (PASMCs), but the underlying mechanisms are still poorly manifested. Previous studies have shown that inducible nitric oxide synthase (iNOS) plays an important protective role against sepsis-induced pulmonary apoptosis. Therefore, the purpose of this study is to determine whether 15-HETE anti-apoptotic process is mediated through the iNOS pathway in rat PASMCs. To test this hypothesis, we studied the contribution of iNOS to the 15-HETE induced anti-apoptotic responses using cell viability measurement, Western blot, mitochondrial potential analysis, nuclear morphology determination and TUNEL assay. Our results showed that both exogenous and endogenous 15-HETE up-regulated iNOS protein and mRNA expression and 15-HETE enhanced the cell survival, attenuated mitochondrial depolarization, up-regulated the expression of Bcl-2 and procaspase-3 in PASMCs under serum-deprived condition. These effects were reversed by iNOS inhibitor SMT or l-canavanine. Taken together, our data indicates that iNOS is a novel signaling transduction pathway, which is necessary for the effects of 15-HETE in protection PASMCs from apoptosis and may be an important mechanism underlying the treatment of pulmonary artery hypertension and also provides a novel therapeutic insight in future.     

Decreased prostaglandin production in cultured smooth muscle cells from atherosclerotic rabbit aorta

Prostaglandin synthesis in aortic smooth muscle cells originating from healthy an atherosclerotic rabbits was studied by incubating [14C]arachidonic acid with intact confluent cells and cell homogenates. In spite of a reduced 6-keto prostaglandin F1 alpha formation, no potentiating effect on the prostaglandin E2 generation occurred. Indeed, both cyclooxygenase and prostaglandin I2 synthetase activities appear to be reduced. These results suggest that an impaired arachidonic acid utilisation in aortic smooth muscle cells may be involved in the course of the atherosclerotic process.     

11,12,15-Trihydroxyeicosatrienoic acid mediates ACh-induced relaxations in rabbit aorta

Rabbit aortic endothelium metabolizes arachidonic acid (AA) by the 15-lipoxygenase pathway to vasodilatory eicosanoids, hydroxyepoxyeicosatrienoic acids (HEETAs), and trihydroxyeicosatrienoic acids (THETAs). The present study determined the chemical identity of the vasoactive THETA and investigated its role in ACh-induced relaxation in the rabbit aorta. AA caused endothelium-dependent, concentration-related relaxations of the rabbit aorta. Increasing the extracellular KCl concentration from 4.8 to 20 mM inhibited the relaxations to AA by approximately 60%, thereby implicating K+-channel activation in the relaxations. In addition, AA caused an endothelium-dependent hyperpolarization of aortic smooth muscle from -39.6 +/- 2.7 to -56.1 +/- 3.4 mV. In rabbit aortic rings, [14C]AA was metabolized to prostaglandins, HEETAs, THETAs, and 15-hydroxyeicosatetraenoic acid. Additional purification of the THETAs by HPLC resolved the mixture into its 14C-labeled products. Gas chromatography/mass spectrometry identified the metabolites as isomers of 11,12,15-THETA and 11,14,15-THETA. The 11,12,15-THETA relaxed and hyperpolarized the rabbit aorta, whereas 11,14,15-THETA had no vasoactive effect. The relaxations to 11,12,15-THETA were blocked by 20 mM KCl. In aortic rings pretreated with inhibitors of nitric oxide and prostaglandin synthesis, ACh caused a concentration-related relaxation that was completely blocked by 20 mM KCl. Pretreatment with the phospholipase A2 inhibitors mepacrine and 7,7-dimethyl-5,8-eicosadienoic acid, the lipoxygenase inhibitors cinnamyl-3,4-dihydroxy-alpha-cyanocinnamate, nordihydroguaiaretic acid, and ebselen, or the hydroperoxide isomerase inhibitors miconazole and clotrimazole also blocked ACh-induced relaxations. ACh caused a threefold increase in THETA release. These studies indicate that AA is metabolized by endothelial cells to 11,12,15-THETA, which activates K+ channels to hyperpolarize the aortic smooth muscle membrane and induce relaxation. Additionally, this lipoxygenase pathway mediates the nonnitric oxide, nonprostaglandin relaxations to ACh in the rabbit aorta by acting as a source of an endothelium-derived hyperpolarizing factor.     

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.     

15-HETE protects rat pulmonary arterial smooth muscle cells from apoptosis via the PI3K/Akt pathway

15-Hydroxyeicosatetraenoic acid (15-HETE), a metabolic product of arachidonic acid (AA), plays an important role in pulmonary vascular smooth muscle remodeling. Although its effects on the apoptotic responses are known, the underlying mechanisms are still poorly understood. Since Akt is a critical regulator of cell survival and vascular remodeling, there may be a crosstalk between 15-HETE anti-apoptotic process and PI3K/Akt survival effect in rat pulmonary arterial smooth muscle cells (PASMCs). To test this hypothesis, we studied the effect of 15-HETE on cell survival and apoptosis using Western blot, cell viability measurement, nuclear morphology determination, TUNEL assay and mitochondrial potential analysis. We found that activation of the PI3K/Akt signaling system was necessary for the 15-HETE to suppress PASMC apoptosis and improve cell survival. Our results indicated that 15-HETE inhibited the apoptotic responses of PASMCs, including morphological alterations, mitochondrial depolarization and the expression apoptosis-specific proteins. These effects were likely to be mediated through the activation of PI3K/Akt. Two downstream signal molecules of PI3K/Akt were identified. Both FasL and Bad were down-regulated by 15-HETE and 15-HETE phosphorylated Bad. These changes depended on the PI3K/Akt signaling pathway in PASMCs. Thus a signal transduction pathway was demonstrated which is necessary for the effects of 15-HETE in protection PASMCs from apoptosis.     

Characterization of 11-HETE and 15-HETE, together with prostacyclin, as major products of the cyclooxygenase pathway in cultured rat aorta smooth muscle cells

Arachidonic acid is the precursor of several potent derivatives that regulate physiological functions in the cardiovascular system. Thromboxane (TXA2) and prostacyclin (PGI2) are synthesized by the cyclooxygenase enzyme. The proaggregatory and vasoconstrictive TXA2 produced by platelets is opposed in vivo by the antiaggregatory and vasodilating activity of PGI2 synthesized by blood vessels. Arachidonic acid is also converted via a 5-lipoxygenase to leukotrienes, the vasoconstrictive components of SRSA. We have shown that this latter pathway is regulated by 15-HETE, a product of the 15-lipoxygenase present in lymphocytes. Confluent cultures of rat aorta smooth muscle cells (RSM) were superfused briefly with [14C]arachidonic acid. The products were isolated and analyzed by thin-layer chromatography-radioautography, high performance liquid chromatography, and gas-liquid chromatography-mass spectrometry. Prostacyclin (PGI2) was identified as the major product both by its biological properties in a platelet aggregation assay and by the mass spectrum of its tetra-trimethylsilylether-methyl ester derivative. Minor quantities of PGE2, PGD2, and PGF2 alpha were also synthesized. Three other compounds with chromatographic properties of mono-hydroxy eicosanoic acids were also formed in major amounts. These were shown to be cyclooxygenase products since their synthesis, together with that of prostacyclin, was blocked by the cyclooxygenase inhibitors aspirin (0.2 mM) and indomethacin (10 microM). Quantities of the hydroxy-eicosanoids were isolated from large scale incubations by silicic acid chromatography. Following methylation and reduction with platinum oxide/H2, the compounds were converted to their trimethylsilylether derivatives and analyzed by gas-liquid chromatography-mass spectrometry. The compounds were identified as 11-hydroxy-5,8,12,14-eicosatetraenoic acid (11-HETE), 15-hydroxy-5,8,11,13-eicosatetraenoic acid (15-HETE), and hydroxy-5,8,10-heptadeca-trienoic acid (HHT) by simultaneous ion monitoring of characteristic ions at M/e ratios of 287, 258, 229 for 11-HETE and 343, 314, 173 for 15-HETE, and by comparison with the mass spectra of authentic samples. Rat smooth muscle cells, prelabeled by 24-hour incubation with [14C]arachidonic acid, released large amounts of prostacyclin together with enhanced amounts of 11- and 15-HETE in response to physiological levels of thrombin (0.5-5 units/ml). These experiments demonstrate that, in addition to the thromboxane antagonist prostacyclin, vascular smooth muscle cells produce significant quantities of the leukotriene inhibitor 15-HETE via the cyclooxygenase pathway in response to physiological stimuli such as thrombin. The release of both prostacyclin and 15-HETE by vascular smooth muscle cells may thus play an important role in vascular homeostasis.     

Aortic smooth muscle cell migration caused by platelet-derived growth factor is mediated by lipoxygenase product(s) of arachidonic acid

The relation between platelet-derived growth factor (PDGF)-induced smooth muscle cell migration, measured in Boyden chambers, and cellular arachidonic acid cascade was studied by using rat aortic smooth muscle cells. Partially purified PDGF stimulated cell migration significantly at a concentration of 1.33-133.0 micrograms/ml. Treatment of the cells with 10(-4)M of 5,8,11,14-eicosatetraynoic acid, an inhibitor of lipoxygenase and cyclooxygenase, and 10(-4)M of caffeic acid, a specific inhibitor of lipoxygenase, caused a significant suppression of PDGF-induced cell migration. Treatment with indomethacin, an inhibitor of cyclooxygenase, did not affect cell migration. These data indicate the involvement of a lipoxygenase product(s) of arachidonic acid in PDGF-associated smooth muscle cell migration.     

15-HETE suppresses K+ channel activity and inhibits apoptosis in pulmonary artery smooth muscle cells

15-Hydroxyeicosatetraenoic acid (15-HETE) is an important hypoxic product from arachidonic acid (AA) in the wall of pulmonary vessels. Although its effects on pulmonary artery constriction are well known, it remains unclear whether 15-HETE acts on the apoptotic responses in pulmonary artery smooth muscle cells (PASMCs) and whether K⁺ channels participate in this process. These hypothesises were validated by cell viability assay, terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling, mitochondrial potentials assay, caspase activity assay and western blot. We found that 15-HETE enhanced cell survival, suppressed the expression and activity of caspase-3, upregulated bcl-2 and attenuated mitochondrial depolarization, prevented chromatin condensation and partly reversed K⁺ channel opener-induced apoptosis in PASMCs under serum-deprived conditions. Our data indicated that 15-HETE inhibits the apoptosis in PASMCs through, at least in part, inactivating K⁺ channels. Yumei Li and Qian Li contributed equally to this work.     

Increase in 12-lipoxygenase activity in platelets of spontaneously hypertensive rats

12-Lipoxygenase activity in platelets of spontaneously hypertensive rats was investigated. Enzyme activity was measured in the absence and the presence of reduced glutathione. In both assay conditions, 12-lipoxygenase activity in platelets of spontaneously hypertensive rats was significantly higher than that in platelets of normotensive rats. Since 12-L-hydroxy-5,8,10,14-eicosatetraenoic acid (12-HETE), a 12-lipoxygenase product of arachidonic acid in platelets, has been reported to be a potent chemoattractant for aortic smooth muscle cells, increase in biosynthesis of 12-HETE in platelets of spontaneously hypertensive rats might contribute to the explanation of pathogenesis of vascular disorder commonly found in hypertension patients.     

Hydrogen peroxide-induced c-fos expression is mediated by arachidonic acid release: role of protein kinase C.

We found previously that stimulation of c-fos and c-myc mRNA expression are early events in hydrogen peroxide-induced growth in rat aortic smooth muscle (RASM) cells. In the present study, we investigated the role of phospholipase A2 (PLA2) and protein kinase C (PKC) in mediating hydrogen peroxide-induced c-fos mRNA expression in RASM cells. Mepacrine and p-bromophenacylbromide, potent inhibitors of PLA2 activity, blocked hydrogen peroxide-induced c-fos mRNA expression. Arachidonic acid, a product of PLA2 activity, stimulated the expression of c-fos mRNA with a time course similar to that of hydrogen peroxide. PKC down-regulation attenuated both hydrogen peroxide and arachidonic acid-induced c-fos mRNA expression by 50%. Nordihydroguaiaretic acid (a lipoxygenase-cytochrome P450 monooxygenase inhibitor) significantly inhibited both hydrogen peroxide and arachidonic acid-induced c-fos mRNA expression, whereas indomethacin (a cyclooxygenase inhibitor) had no effect. Together, these findings indicate that 1) hydrogen peroxide-induced c-fos mRNA expression is mediated by PLA2-dependent arachidonic acid release, 2) both PKC-dependent and independent mechanisms are involved in hydrogen peroxide-induced expression of c-fos mRNA and 3) arachidonic acid metabolism via the lipoxygenase-cytochrome P450 monooxygenase pathway appears to be required for hydrogen peroxide-induced expression of c-fos mRNA.     

Stimulation of prostaglandin cyclooxygenase and prostacyclin synthetase activities by estradiol in rat aortic smooth muscle cells

The effects of estradiol on the arachidonic acid pool and prostacyclin biosynthetic activity in rat aortic smooth muscle cells were studied. Estradiol has no significant effect on the distribution of [14C]arachidonic acid in cells with respect to prostacyclin production assay, the endogenous fatty acid (specifically, arachidonic acid) composition of cellular phospholipid fractions and cellular phospholipase (or/and lipase) activities. However, estradiol significantly stimulates both prostaglandin cyclooxygenase and prostacyclin synthetase activities of cells, and induction of new protein biosynthesis is involved in the effect of estradiol on the stimulation of prostacyclin biosynthetic activity.     

15-Lipoxygenase products affect protein phosphorylation in Friend erythroleukemia cells

Endogenous arachidonic acid metabolism and protein phosphorylation have been examined in Friend erythroleukemia cells in response to the induction of differentiation by dimethyl sulfoxide and hexamethylene bisacetamide. 15-Hydroxyeicosatetraenoic acid levels were elevated in cells differentiated with hexamethylene bisacetamide or dimethyl sulfoxide compared with undifferentiated cells. Protein phosphorylation decreased markedly in differentiated cells compared with undifferentiated cells and the addition of 15-hydroperoxyeicosatetraenoic acid specifically decreased the phosphorylation of a 28-kilodalton protein. These findings indicate that products of 15-lipoxygenase may act as intracellular messengers in Friend erythroleukemia cells by affecting protein phosphorylation.     

Brain Micro vessel 12-Hydroxyeicosatetraenoic Acid Is the (S) Enantiomer and Is Lipoxygenase Derived

12-Hydroxyeicosatetraenoic acid (12-HETE) production from arachidonic acid by cerebral microvessels isolated from perfused adult murine brain was reduced by the lipoxygenase inhibitors baicalein, esculetin, gossypol, nordihydroguaiaretic acid, and quercetin. Except for quercetin and gossypol, the IC50 did not exceed 10 microM. Each inhibitor, except baicalein, also decreased microvessel prostaglandin production when present in concentrations above their IC50 value for 12-HETE. In contrast, inhibitors of the cytochrome P450 monooxygenase system, clotrimazole, metyrapone, and proadifen (SKF-525A), had little effect on microvessel 12-HETE production. Chiral phase HPLC analysis revealed that only the (S) enantiomer of 12-HETE was formed. The major microvessel metabolite of eicosapentaenoic acid co-eluted with 12-hydroxyeicosapentaenoic acid (12-HEPE) on reverse-phase HPLC and the (S) enantiomer of 12-HEPE on chiral phase HPLC. Furthermore, like 12-HETE, 12-HEPE production was blocked by lipoxygenase inhibitors. These studies demonstrate that brain microvessels produce only the (S) enantiomeric 12-hydroxy derivatives of both arachidonic acid and eicosapentaenoic acid by the action of a lipoxygenase that can be selectively inhibited by baicalein. Since arachidonic acid and eicosapentaenoic acid are available to cerebral blood vessels in certain pathological settings, these 12-hydroxy acid lipoxygenase products may mediate some of the cerebrovascular dysfunction that occurs following stroke, brain trauma, or seizures.     

Effects of isolation and culture on prostaglandin synthesis by porcine aortic endothelial and smooth muscle cells

Freshly isolated neonatal porcine aortic tissue (smooth muscle with or without endothelium present) produced approximately 30 ng/mg wet tissue of 6-oxo-prostaglandin F1 alpha (the stable hydrolysis product from prostacyclin) and approximately 15 ng/mg of prostaglandin E2, as measured by radioimmunoassay after 24 h incubation in culture medium. Primary cultures of porcine endothelial and smooth muscle cells (isolated by enzymic digestion of aortic tissue) exhibited the same pattern of prostaglandin production, but absolute values were greater than for fresh tissue, particularly in the case of endothelium. Subcultures of endothelium produced smaller amounts of prostaglandins, although the pattern remained similar. In contrast, subcultures of smooth muscle cells produced a greater total amount of prostaglandins than did primary cultures, and the main product was prostaglandin E2. Experiments with [14C] prostaglandin H2 or [14C]arachidonic acid confirmed that aortic tissue, cultured endothelium, and primary cultures or aortic smooth muscle cells synthesized prostacyclin, and demonstrated that subcultured smooth muscle cells enzymically isomerised prostaglandin H2 to prostaglandin E2. Kinetic studies showed that prostaglandin production by cultured vascular cells was transiently increased by subculture or changing the growth medium, and that production per cell declined with increasing cell density. The change in pattern of prostaglandin production during culture was shown to be due to a rapid decline in the rate of prostacyclin production (which apparently began immediately after tissue isolation), together with a more gradual rise in prostaglandin E2 production. These results indicate that the amounts and ratios of prostaglandins produced by vascular endothelial and smooth muscle cells are greatly affected by the conditions used to isolate and culture the cells; vascular cells in vivo may similarly alter their pattern of prostaglandin production in response to local changes in their environment.     

Incorporation and redistribution of arachidonic acid in diacyl and ether phospholipids of bovine aortic endothelial cells

The present experiments characterized the incorporation and redistribution of arachidonic acid in diacyl and ether phospholipids of bovine aortic endothelial cells. Confluent cultures were either continuously labeled or pulse labeled with [14C]arachidonic acid. Major lipid classes and ether-linked subclasses of phosphatidyl-ethanolamine (PE) and phosphatidylcholine (PC) were separated by high-performance liquid chromatography and thin-layer chromatography. During continuous labeling, total incorporation of arachidonic acid reached a peak at 8 h and was essentially constant up to 24 h. After 8 h, net label in total PC declined, whereas that in total PE continued to rise. In pulse labeling experiments radioactivity in diacyl PC continuously declined with concomitant increases in both diacyl- and alkenylacyl PE. The data demonstrate that transfer of arachidonic acid from diacyl PC to both diacyl- and alkenylacyl PE occurs in endothelial cells. In contrast to previous observations in platelets, transfer of arachidonic acid to alkenylacyl PE did not require agonist stimulation. This pathway may contribute to the enrichment of endothelial cell PE with arachidonic acid with the potential for subsequent metabolism to prostacyclin.     

Stimulation of arachidonic acid metabolism: differences in potencies of recombinant human interleukin-1 alpha and interleukin-1 beta on two cell types

Recombinant human interleukin-l (rIL-1) alpha and beta, which have 26% homology in their amino acid sequence, stimulated arachidonic acid metabolism by squirrel monkey smooth muscle cells and rat liver cells; their relative effectiveness, however, varied with the two cells. Recombinant IL-1 alpha was 3 times more effective than rIL-1 beta at stimulating arachidonic acid metabolism by the primate smooth muscle cells. Recombinant IL-1 alpha was 3 times less effective than rIL-1 beta when measured by their capacity to synergistically stimulate arachidonic acid metabolism of rat liver cells in the presence of palytoxin and anti-diuretic hormone (ADH). The rIL-1 alpha and rIL-1 beta also stimulated the release of radiolabelled arachidonic acid from the smooth muscle cells prelabelled with [3H]arachidonic acid. The two recombinant IL-1s have different heat stabilities, again when measured by their capacity to stimulate arachidonic acid metabolism; IL-1 alpha was more heat stable than IL-1 beta.     

Small GTP binding protein Ras contributes to norepinephrine-induced mitogenesis of vascular smooth muscle cells

Norepinephrine stimulates release of arachidonic acid from tissue lipids. Arachidonic acid metabolites generated through the lipoxygenase and cytochrome P-450 pathways but not cyclooxygenase stimulate mitogen activated protein (MAP) kinase activity and proliferation of vascular smooth muscle cells (VSMC). Moreover, norepinephrine has been shown to activate the Ras/MAP kinase pathway through generation of cytochrome P450 metabolite of arachidonic acid, 20-hydroxyeicosatetraenoic acid (20-HETE). The purpose of this study was to investigate the contribution of Ras in norepinephrine-induced mitogenesis in aortic VSMC. Farnesylation of Ras by farnesyl transferase is required for its full activation. Norepinephrine-induced DNA synthesis, as measured by [3H]-thymidine incorporation, was attenuated by inhibitors of Ras farnesyl transferase FPT III and BMS-191563. These agents also inhibited 20-HETE-stimulated [3H]-thymidine incorporation. In cells transiently transfected with dominant negative Ras (RasN17), norepinephrine, and 20-HETE-induced proliferation of VSMC was attenuated. Both norepinephrine and 20-HETE increased localization of Ras to plasma membrane and MAP kinase activity; FPT III attenuated these effects. These data suggest that VSMC proliferation induced by norepinephrine and 20-HETE is mediated by Ras/MAP kinase pathway.