Signaling mechanisms of nuclear factor-kappab-mediated activation of inflammatory genes by 13-hydroperoxyoctadecadienoic acid in cultured vascular smooth muscle cells.

Oxidatively modified low density lipoprotein (LDL) has been implicated in the pathogenesis of atherosclerosis. LDL oxidation may be mediated by several factors, including cellular lipoxygenases. The lipoxygenase product of linoleic acid, 13-hydroperoxyoctadecadienoic acid (13-HPODE), is a significant component of oxidized LDL and has been shown to be present in atherosclerotic lesions. However, the mechanism of action of these oxidized lipids in vascular smooth muscle cells (VSMCs) is not clear. In the present study, we show that 13-HPODE leads to the activation of Ras as well as the mitogen-activated protein kinases, extracellular signal-regulated kinase 1/2, p38, and c-Jun amino-terminal kinase, in porcine VSMCs. 13-HPODE also specifically activated the oxidant stress-responsive transcription factor, nuclear factor-kappaB, but not activator protein-1 or activator protein-2. 13-HPODE-induced nuclear factor-kappaB DNA binding activity was blocked by an antioxidant, N-acetylcysteine, as well as an inhibitor of protein kinase C. 13-HPODE, but not the hydroxy product, 13-(S)-hydroxyoctadecadienoic acid, also dose-dependently increased vascular cell adhesion molecule-1 promoter activation. This was inhibited by an antioxidant as well as by inhibitors of Ras p38 mitogen-activated protein kinase and protein kinase C. Our results suggest that oxidized lipid components of oxidized LDL, such as 13-HPODE, may play a key role in the atherogenic process by inducing the transcriptional regulation of inflammatory genes in VSMCs via the activation of key signaling kinases.

Functional interaction of calcium-/calmodulin-dependent protein kinase II and cytosolic phospholipase A(2).

Calcium-/calmodulin-dependent protein kinase II (CaM kinase II), a decoder of Ca(2+) signals, and cytosolic phospholipase A(2) (cPLA(2)), an enzyme involved in arachidonate release, are involved in many physiological and pathophysiological processes. Activation of CaM kinase II in norepinephrine-stimulated vascular smooth muscle cells leads to activation of cPLA(2) and arachidonic acid release. Surface plasmon resonance, mass spectrometry, and kinetic studies show that CaM kinase II binds to cPLA(2) resulting in cPLA(2) phosphorylation on Ser-515 and an increase in its enzymatic activity. Phosphopeptide mapping studies with cPLA(2) from norepinephrine-stimulated smooth muscle cells indicates that phosphorylation of cPLA(2) on Ser-515, but not on Ser-505 or Ser-727, occurs in vivo. This novel signaling pathway for arachidonate release is shown to be cPLA(2)-dependent by use of a recently described and highly selective inhibitor of this enzyme.

Ca2+/calmodulin-dependent protein kinase II and cytosolic phospholipase A2 contribute to mitogenic signaling in myeloblastic leukemia U-937 cells.

The signaling mechanisms downstream of growth factor-stimulated proliferation in myeloid leukemia cells have not yet been fully elucidated. Recent evidence suggests that alternate pathways to the mitogen-activated protein kinase cascade are required. We have previously shown that Ca2+/calmodulin-dependent protein kinase II (CaM kinase II) activates cytosolic phospholipase A2 (cPLA2), which is involved in the proliferation of vascular smooth muscle cells. In the present study, the contribution of this pathway was investigated in the proliferation of U-937 myeloid leukemia cells. In U-937 cells, fetal bovine serum (FBS)-induced proliferation was attenuated by CaM kinase II inhibitor KN-93 but not by its inactive analog KN-92. Inhibitors of cPLA2 (methyl arachidonyl fluorophosphonate and arachidonyl trifluoromethyl ketone) also reduced proliferation of U-937 cells. FBS-induced proliferation was also attenuated by cotransfection with cPLA2 antisense oligonucleotides. These results suggest a role for CaM kinase II and cPLA2 in the proliferation of U-937 cells. FBS stimulated CaM kinase II and cPLA2 activities in a time-dependent manner. Moreover, FBS-stimulated phosphorylation and activation of cPLA2 activation was inhibited by KN-93. FBS-stimulated phosphorylation of CaM kinase II was blocked by KN-93 but not by cPLA2 inhibitors, suggesting that CaM kinase II activates cPLA2. The products of phospholipid hydrolysis produced by cPLA2, lysophosphatidylcholine but not arachidonic acid, increased [3H]thymidine incorporation in U-937 cells. These data suggest that exposure of U-937 cells to FBS promotes phosphorylation and activation of CaM kinase II, leading to stimulation of cPLA2 and generation of lysophosphatidylcholine and resultant proliferation of these cells.

Cytosolic phospholipase A2 activation by the p38 kinase inhibitor SB203580 in rabbit aortic smooth muscle cells.

SB203580 [4-(4-fluorophenyl)-2-(4-methylsulfinylphenyl)-5-(4-pyridyl)1H-imidazole] is widely used as a specific inhibitor of p38 mitogen-activated protein kinase (MAPK). Here we report that SB203580, which blocked p38 kinase activation elicited by anisomycin, increased the phosphorylation and activity of cytosolic phospholipase A2 (cPLA2) and arachidonic acid (AA) release in quiescent vascular smooth muscle cells from rabbit aortae. SB203580 also increased the activity of calcium (Ca2+)/camodulin-dependent kinase II (CaMKII) and ERK1/2 MAPK. The increase in CaMKII activity and cPLA2 phosphorylation caused by SB203580 was attenuated by CaMKII inhibitor KN-93, indicating involvement of CaMKII in cPLA2 phosphorylation by this compound. Since KN-93 also inhibited SB203580-induced ERK1/2 activation, it appears that ERK1/2 activation is also mediated by CaMKII. SB203580-induced cPLA2 phosphorylation was inhibited by depletion of Ca2+ from the medium, by the voltage-operated Ca2+ channel blocker nifedipine, and by the calmodulin inhibitor W-7. cPLA2 translocation from cytoplasm to the nuclear envelope caused by SB203580 was also inhibited in the absence of extracellular Ca2+. Other p38 kinase inhibitors, SB202190 and PD169316, failed to alter CaMKII, ERK1/2, and cPLA2 activity or cPLA2 translocation to the nuclear envelope. These data suggest that SB203580 not only inhibits p38 kinase activity but also increases Ca2+ influx through voltage-sensitive Ca2+ channels, which promotes cPLA2 translocation to the nuclear envelope, and by interacting with calmodulin, activates CaMKII and cPLA2 and releases AA.

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.

20-Hydroxyeicosatetraenoic acid mediates angiotensin ii-induced phospholipase d activation in vascular smooth muscle cells.

Angiotensin II (Ang II) activates cytosolic phospholipase A(2) (cPLA(2)) and phospholipase D (PLD) in rabbit vascular smooth muscle cells (VSMCs). Ang II also activates ras/mitogen-activated protein (MAP) kinase in VSMCs; this activation is mediated by 20-hydroxyeicosatetraenoic acid (HETE) and 12(S)-HETE, which are metabolites of arachidonic acid generated by cytochrome P450 4A and lipoxygenase, respectively, produced on activation of cPLA(2). The purpose of this study was to determine if Ang II-induced PLD activation in VSMCs is mediated through the ras/extracellular signal-regulating kinase (ERK) pathway by arachidonic acid metabolites that are generated consequent to cPLA(2) stimulation. Inhibitors of PLD (C(2) ceramide), phosphatidate phosphohydrolase (propranolol), and diacylglycerol lipase (RHC 80267) attenuated Ang II-induced arachidonic acid release. Ang II-induced PLD activation, as measured by [(3)H]phosphatidylethanol production, was inhibited by C(2) ceramide but not by propranolol or RHC 80267. Ang II-induced PLD activation was decreased by the inhibitor methyl arachidonylfluorophosphate (MAFP) and the antisense oligonucleotide of cPLA(2). Inhibitors of lipoxygenases (baicalein) and cytochrome P450 4A (ODYA) attenuated Ang II-induced PLD activation. 20-HETE and 12(S)-HETE increased PLD activity. Inhibitors of ras farnesyltransferase (FPT III and BMS-191563) and MAP kinase kinase (UO126) attenuated the increase in PLD activity elicited by 20-HETE and Ang II. PLD2 was the main isoform activated by Ang II in VSMCs. These data suggest that the CYP4A metabolite 20-HETE, which is generated from arachidonic acid after cPLA(2) activation by Ang II, stimulates the ras/MAP kinase pathway, which in turn activates PLD2 and releases further arachidonic acid for prostaglandin synthesis through the phosphatidate phosphohydrolase/diacylglycerol lipase pathway.

Sphingosylphosphocholine is a naturally occurring lipid mediator in blood plasma: a possible role in regulating cardiac function via sphingolipid receptors.

Blood plasma and serum contain factors that activate inwardly rectifying GIRK1/GIRK4 K+ channels in atrial myocytes via one or more non-atropine-sensitive receptors coupled to pertussis-toxin-sensitive G-proteins. This channel is also the target of muscarinic M(2) receptors activated by the physiological release of acetylcholine from parasympathetic nerve endings. By using a combination of HPLC and TLC techniques with matrix-assisted laser desorption ionization-time-of-flight MS, we purified and identified sphingosine 1-phosphate (SPP) and sphingosylphosphocholine (SPC) as the plasma and serum factors responsible for activating the inwardly rectifying K+ channel (I(K)). With the use of MS the concentration of SPC was estimated at 50 nM in plasma and 130 nM in serum; those concentrations exceeded the 1.5 nM EC(50) measured in guinea-pig atrial myocytes. With the use of reverse-transcriptase-mediated PCR and/or Western blot analysis, we detected Edg1, Edg3, Edg5 and Edg8 as well as OGR1 sphingolipid receptor transcripts and/or proteins. In perfused guinea-pig hearts, SPC exerted a negative chronotropic effect with a threshold concentration of 1 microM. SPC was completely removed after perfusion through the coronary circulation at a concentration of 10 microM. On the basis of their constitutive presence in plasma, the expression of specific receptors, and a mechanism of ligand inactivation, we propose that SPP and SPC might have a physiologically relevant role in the regulation of the heart.

Phospholipase D activation by norepinephrine is mediated by 12(s)-, 15(s)-, and 20-hydroxyeicosatetraenoic acids generated by stimulation of cytosolic phospholipase a2. tyrosine phosphorylation of phospholipase d2 in response to norepinephrine.

Norepinephrine (NE) stimulates phospholipase D (PLD) through a Ras/MAPK pathway in rabbit vascular smooth muscle cells (VSMC). NE also activates calcium influx and calmodulin (CaM)-dependent protein kinase II-dependent cytosolic phospholipase A(2) (cPLA(2)). Arachidonic acid (AA) released by cPLA(2)-catalyzed phospholipid hydrolysis is then metabolized into hydroxyeicosatetraenoic acids (HETEs) through lipoxygenase and cytochrome P450 4A (CYP4A) pathways. HETEs, in turn, have been shown to stimulate Ras translocation and to increase MAPK activity in VSMC. This study was conducted to determine the contribution of cPLA(2)-derived AA and its metabolites (HETEs) to the activation of PLD. NE-induced PLD activation was reduced by two structurally distinct CaM antagonists, W-7 and calmidazolium, and by CaM-dependent protein kinase II inhibition. Blockade of cPLA(2) activity or protein depletion with selective cPLA(2) antisense oligonucleotides abolished NE-induced PLD activation. The increase in PLD activity elicited by NE was also blocked by inhibitors of lipoxygenases (baicalein) and CYP4A (17-octadecynoic acid), but not of cyclooxygenase (indomethacin). AA and its metabolites (12(S)-, 15(S)-, and 20-HETEs) increased PLD activity. PLD activation by AA and HETEs was reduced by inhibitors of Ras farnesyltransferase (farnesyl protein transferase III and BMS-191563) and MEK (U0126 and PD98059). These data suggest that HETEs are the mediators of cPLA(2)-dependent PLD activation by NE in VSMC. In addition to cPLA(2), PLD was also found to contribute to AA release for prostacyclin production via the phosphatidate phosphohydrolase/diacylglycerol lipase pathway. Finally, a catalytically inactive PLD(2) (but not PLD(1)) mutant inhibited NE-induced PLD activity, and PLD(2) was tyrosine-phosphorylated in response to NE by a MAPK-dependent pathway. We conclude that NE stimulates cPLA(2)-dependent PLD(2) through lipoxygenase- and CYP4A-derived HETEs via the Ras/ERK pathway by a mechanism involving tyrosine phosphorylation of PLD(2) in rabbit VSMC.

Angiotensin II-induced hypertension: contribution of Ras GTPase/Mitogen-activated protein kinase and cytochrome P450 metabolites.

We reported that norepinephrine and angiotensin II (Ang II) activate the Ras/mitogen-activated protein (MAP) kinase pathway primarily through the generation of cytochrome P450 (CYP450) metabolites. The purpose of the present study was to determine the contribution of Ras and CYP450 to Ang II-dependent hypertension in rats. Infusion of Ang II (350 ng/min for 6 days) elevated mean arterial blood pressure (MABP) (171+/-3 mm Hg for Ang II versus 94+/-5 for vehicle group, P<0.05). Ras is activated on farnesylation by farnesyl protein transferase (FPT). When Ang II was infused in combination with FPT inhibitor FPT III (232 ng/min) or BMS-191563 (578 ng/min), the development of hypertension was attenuated (171+/-3 mm Hg for Ang II plus vehicle versus 134+/-5 mm Hg for Ang II plus FPT III and 116+/-6 mm Hg for Ang II plus BMS-191563, P<0.05). Treatment with the MAP kinase kinase inhibitor PD-98059 (5 mg SC) reduced MABP. The CYP450 inhibitor aminobenzotriazole (50 mg/kg) also diminished the development of Ang II-induced hypertension to 113+/-8 mm Hg. The activities of Ras, MAP kinase, and CYP450 measured in the kidney were elevated in hypertensive animals. The infusion of FPT III, BMS-191563, or aminobenzotriazole reduced the elevation in Ras and MAP kinase activity. Morphological studies of the kidney showed that FPT III treatment ameliorated the arterial injury, vascular lesions, fibrinoid necrosis, focal hemorrhage, and hypertrophy of muscle walls observed in hypertensive animals. These data suggest that the activation of Ras and CYP450 contributes to the development of Ang II-dependent hypertension and associated vascular pathology.

Ras/mitogen-activated protein kinase mediates norepinephrine-induced phospholipase D activation in rabbit aortic smooth muscle cells by a phosphorylation-dependent mechanism.

Phospholipase D (PLD) activity is regulated by phosphatidylinositol 4,5-biphosphate, protein kinase C (PKC), ADP-ribosylation factor, and Rho. The present study was designed to investigate the mechanism of norepinephrine (NE)-mediated PLD activation in rabbit aortic vascular smooth muscle cells (VSMC). NE (10 microM) caused activation of PLD, as measured by the production of phosphatidylethanol in [(3)H]oleic acid-labeled cells. NE also increased PKC activity in VSMC. However, treatment of cells with bisindolylmaleimide, a PKC inhibitor, or long-term treatment with phorbol-12-myristate-13-acetate that depletes PKC did not decrease NE-induced activation of PLD. NE-stimulated PLD activity was attenuated by farnesyl transferase inhibitors (FPT III and SCH-56582), which reduce activation of both Ras and mitogen-activated protein (MAP) kinase. Moreover, transfection of VSMC with a dominant negative Ras resulted in inhibition of NE-stimulated MAP kinase and PLD activities. Treatment of cells with PD-98059, a MAP kinase kinase inhibitor, also reduced NE-stimulated PLD activity. These data suggest that NE-stimulated PLD activity is mediated via activation of Ras and MAP kinase in rabbit VSMC. To study the mechanism of activation of PLD by Ras/MAP kinase, NE-induced phosphorylation of PLD was examined. In VSMC, PLD of molecular mass 120 kDa was identified with polyclonal PLD antibody. Phosphorylation of PLD by NE, measured as (32)P incorporation into PLD, was inhibited by PD-98059. Moreover, PLD immunoprecipitated from VSMC lysates was phosphorylated in vitro by MAP kinase. Collectively, these results show a novel pathway for activation of PLD that appears to be mediated through Ras/MAP kinase pathway by a mechanism involving phosphorylation.

Contribution of Ras GTPase/MAP kinase and cytochrome P450 metabolites to deoxycorticosterone-salt-induced hypertension.

We recently reported that norepinephrine and angiotensin II activate the Ras/mitogen-activated protein (MAP) kinase pathway through generation of a cytochrome P450 (CYP450) and lipoxygenase metabolites. The purpose of this study was to determine the contribution of Ras/MAP kinase to deoxycorticosterone acetate (DOCA)-salt-induced hypertension in rats. Administration of DOCA and 1% saline drinking water to uninephrectomized rats for 6 weeks significantly elevated mean arterial blood pressure (MABP) (166+/-5 mm Hg, n=19) compared with that of normotensive controls (95+/-5 mm Hg, n=7) (P<0.05). The activity of Ras and MAP kinase measured in the heart was increased in DOCA-salt hypertensive rats. Infusion of the Ras farnesyl transferase inhibitors FPT III (138 ng/min) and BMS-191563 (694 ng/min) significantly (P<0.05) attenuated MABP to 139+/-4 mm Hg (n=14) and 126+/-1 mm Hg (n=4), respectively. Moreover, infusion of MAP kinase kinase inhibitor PD-98059 (694 ng/min) also reduced MABP in hypertensive rats. Morphological studies of the kidney showed that treatment of rats with FPT III, which reduced Ras activity, minimized the hyperplastic occlusive arteriosclerosis and fibrinoid vasculitis observed in untreated hypertensive rats. In addition, the rise in CYP450 activity and MABP in hypertensive rats was prevented by the CYP450 inhibitor aminobenzotriazole (50 mg/kg) and was associated with a decrease in Ras and MAP kinase activity in the heart. These data suggest that the Ras/MAP kinase pathway contributes to DOCA-salt-induced hypertension and associated vascular pathology consequent to activation of CYP450.

Activation of the L voltage-sensitive calcium channel by mitogen-activated protein (MAP) kinase following exposure of neuronal cells to beta-amyloid. MAP kinase mediates beta-amyloid-induced neurodegeneration.

Neuronal degeneration in Alzheimer's disease (AD) has been variously attributed to increases in cytosolic calcium, reactive oxygen species, and phosphorylated forms of the microtubule-associated protein tau. beta-Amyloid (betaA), which accumulates extracellularly in AD brain, induces calcium influx in culture via the L voltage-sensitive calcium channel. Since this channel is normally activated by protein kinase A-mediated phosphorylation, we examined kinase activities recruited following betaA treatment of cortical neurons and SH-SY-5Y neuroblastoma. betaA increased channel phosphorylation; this increase was unaffected by the protein kinase A inhibitor H89 but was reduced by the mitogen-activated protein (MAP) kinase inhibitor PD98059. Pharmacological and antisense oligonucleotide-mediated reduction of MAP kinase activity also reduced betaA-induced accumulation of calcium, reactive oxygen species, phospho-tau immunoreactivity, and apoptosis. These findings indicate that MAP kinase mediates multiple aspects of betaA-induced neurotoxicity and indicates that calcium influx initiates neurodegeneration in AD. betaA increased MAP kinase-mediated phosphorylation of membrane-associated proteins and reduced phosphorylation of cytosolic proteins without increasing overall MAP kinase activity. Increasing MAP kinase activity with epidermal growth factor did not increase channel phosphorylation. These findings indicate that redirection, rather than increased activation, of MAP kinase activity mediates betaA-induced neurotoxicity.

beta-hexosaminidase-induced activation of p44/42 mitogen-activated protein kinase is dependent on p21Ras and protein kinase C and mediates bovine airway smooth-muscle proliferation.

Late-phase and sustained activation of p44/42(MAPK) has been reported to be a critical factor in cell mitogenesis. We therefore hypothesized that p44/42(MAPK) is involved in mannosyl-rich glycoprotein-induced mitogenesis in bovine airway smooth-muscle cells (ASMC). Treatment of adherent ASMC with beta-hexosaminidase A (Hex A, 50 nM), an endogenous mannosyl-rich glycoprotein, resulted in a late-onset (30-min) activation of p44/42(MAPK) that lasted for 4 h. Activation of p44/42(MAPK) induced by Hex A was inhibited by an 18-mer phosphorothioate-derivatized antisense oligonucleotide (1-5 microM) directed to human p44(MAPK); the mitogen-activated protein kinase kinase (MEK1) inhibitor PD98059 (5 microM); the p42(MAPK) inhibitor Tyrphostin AG-126 (0.2 microM); the farnesyl transferase inhibitors SCH-56582 (10 microM) and FPT III (10 miroM), which inhibit p21Ras activation; and Calphostin C (0.2 microM), an inhibitor of protein kinase C. These agents also inhibited Hex A-induced cell proliferation in bovine ASMC. These data suggest that Hex A activates p44/42(MAPK) in a p21Ras- and PKC-dependent manner and that this activation mediates Hex A- induced mitogenesis in bovine ASMC.

20-Hydroxyeicosatetraenoic acid mediates calcium/calmodulin-dependent protein kinase II-induced mitogen-activated protein kinase activation in vascular smooth muscle cells.

Norepinephrine (NE) and angiotensin II (Ang II), by promoting extracellular Ca2+ influx, increase Ca2+/calmodulin-dependent kinase II (CaMKII) activity, leading to activation of mitogen-activated protein kinase (MAPK) and cytosolic phospholipase A2 (cPLA2), resulting in release of arachidonic acid (AA) for prostacyclin synthesis in rabbit vascular smooth muscle cells. However, the mechanism by which CaMKII activates MAPK is unclear. The present study was conducted to determine the contribution of AA and its metabolites as possible mediators of CaMKII-induced MAPK activation by NE, Ang II, and epidermal growth factor (EGF) in vascular smooth muscle cells. NE-, Ang II-, and EGF-stimulated MAPK and cPLA2 were reduced by inhibitors of cytochrome P450 (CYP450) and lipoxygenase but not by cyclooxygenase. NE-, Ang II-, and EGF-induced increases in Ras activity, measured by its translocation to plasma membrane, were abolished by CYP450, lipoxygenase, and farnesyltransferase inhibitors. An AA metabolite of CYP450, 20-hydroxyeicosatetraenoic acid (20-HETE), increased the activities of MAPK and cPLA2 and caused translocation of Ras. These data suggest that activation of MAPK by NE, Ang II, and EGF is mediated by a signaling mechanism involving 20-HETE, which is generated by stimulation of cPLA2 by CaMKII. Activation of Ras/MAPK by 20-HETE amplifies cPLA2 activity and releases additional AA by a positive feedback mechanism. This mechanism of Ras/MAPK activation by 20-HETE may play a central role in the regulation of other cellular signaling molecules involved in cell proliferation and growth.

Alpha-1A adrenergic receptor stimulation with phenylephrine promotes arachidonic acid release by activation of phospholipase D in rat-1 fibroblasts: inhibition by protein kinase A.

This study was conducted to determine the mechanism of arachidonic acid (AA) release elicited by phenylephrine (PHE) stimulation of alpha adrenergic receptor (AR), and its modulation by cyclic adenosine 3',5'-monophosphate (cAMP) in Rat-1 fibroblasts (R-1Fs) transfected with the alpha-1A, alpha-1B or alpha-1D AR. PHE increased AA release and also caused a marked accumulation of cAMP in R-1Fs expressing the alpha-1 AR subtypes, but not in those transfected with vector alone. PHE also enhanced phospholipase D (PLD), but not phospholipase A2 (PLA2) activity. The increase in PHE-induced AA release, PLD activity and cAMP accumulation differed among the various alpha AR subtypes with: alpha-1A > alpha-1B > alpha-1D AR. The effect of PHE to increase AA release was attenuated by C2-ceramide, an inhibitor of PLD; propranolol, a phosphatidate phosphohydrolase inhibitor; and RHC-80267, a diacylglycerol lipase inhibitor in R-1Fs expressing the alpha-1A AR. Forskolin, which activates adenylyl cyclase, increased cAMP accumulation and inhibited PHE-induced AA release and PLD activity in alpha-1A-AR-expressing R-1Fs. 8-(4-chlorophenyl-thio)-cAMP, a nonhydrolyzable analog of cAMP, also attenuated the rise in AA release and PLD activity elicited by PHE in these cells. In contrast, SQ 22536, an adenylyl cyclase inhibitor, and KT 5720, a protein kinase A inhibitor, increased PHE-induced AA release and PLD activity in R-1Fs expressing the alpha-1A AR. These data suggest that the alpha-1A, alpha-1B and alpha-1D ARs are coupled to PLD activation and cAMP accumulation. Moreover, PHE promotes AA release in R-1Fs expressing the alpha-1A AR through PLD activation. Furthermore, cAMP generated by alpha-1A AR stimulation acts as an inhibitory modulator of PLD activity and AA release via protein kinase A.

Cytochrome P-450 metabolites mediate norepinephrine-induced mitogenic signaling.

Norepinephrine (NE) stimulates release of arachidonic acid (AA) from tissue lipids in blood vessels, which is metabolized via cyclooxygenase, lipoxygenase (LO), and cytochrome P-450 (CYP-450) pathways to biologically active products. Moreover, NE and AA have been shown to stimulate proliferation of vascular smooth muscle cells (VSMCs) of rat aorta. The purpose of this study was to determine the possible contribution of AA and its metabolites to NE-induced mitogenesis in VSMCs of rat aorta and the underlying mechanism of their actions. NE (0.1 to 10 micromol/L) increased DNA synthesis as measured by [3H]thymidine incorporation in VSMCs, and this effect was attenuated by inhibitors of CYP-450 (17-octadecynoic acid, 5 micromol/L; 12-diabromododec-11-enoic acid, 10 micromol/L; and dibromo-dodecenyl-methylsulfimide, 10 micromol/L) and by the LO inhibitor (baicalein, 20 micromol/L), but not by the cyclooxygenase inhibitor (indomethacin, 5 micromol/L). CYP-450 and LO metabolites of AA, 20-hydroxyeicosatetraenoic acid (HETE) (0.1 to 0.5 micromol/L) and 12(S)-HETE, respectively, increased [3H]thymidine incorporation in VSMCs. Both NE and 20-HETE increased mitogen activated protein (MAP) kinase activity as measured by the in-gel kinase assay. The inhibitor of MAP kinase kinase, PD-98059 (50 micromol/L), attenuated NE as well as 20-HETE induced [3H]thymidine incorporation and MAP kinase activation in VSMCs. These data suggest that products of AA formed via CYP-450, most likely 20-HETE, and via LO mediate NE induced mitogenesis in VSMCs.

Signal transduction mechanisms involved in angiotensin-(1-7)-stimulated arachidonic acid release and prostanoid synthesis in rabbit aortic smooth muscle cells.

This study investigated the signal transduction mechanisms of angiotensin-(1-7) [Ang-(1-7)]- and Ang II-stimulated arachidonic acid (AA) release for prostaglandin (PG) production in rabbit aortic vascular smooth muscle cells. Ang II and Ang-(1-7) enhanced AA release in cells prelabeled with [3H]AA. However, 6-keto-PGF1 alpha synthesis produced by Ang II was much less than that caused by Ang-(1-7). In the presence of the lipoxygenase inhibitor baicalein, Ang II enhanced production of 6-keto-PGF1 alpha to a greater degree than Ang-(1-7). Angiotensin type (AT)1 receptor antagonist DUP-753 inhibited only Ang II-induced [3H]AA release, whereas the AT2 receptor antagonist PD-123319 inhibited both Ang II- and Ang-(1-7)-induced [3H]AA release. Ang-(1-7), receptor antagonist D-Ala7-Ang-(1-7) inhibited the effect of Ang-(1-7), but not of Ang II. In cells transiently transfected with cytosolic phospholipase A2 (cPLA2), mitogen-activated protein (MAP) kinase or Ca(++)-/cal-modulin-dependent protein (CAM) kinase II antisense oligonucleotides, Ang-(1-7)- and Ang II-induced [3H]AA release was attenuated. The CaM kinase II inhibitor KN-93 and the MAP kinase kinase inhibitor PD-98059 attenuated both Ang-(1-7)- and Ang II-induced cPLA2 activity and [3H]AA release. Ang-(1-7) and Ang II also increased CaM kinase II and MAP kinase activities. Although KN-93 attenuated MAP kinase activity, PD-98059 did not affect CaM kinase II activity. Both Ang II and Ang-(1-7) caused translocation of cytosolic PLA2 to the nuclear envelope. These data show that Ang-(1-7) and Ang II stimulate AA release and prostacyclin synthesis via activation of distinct types of AT receptors. Both peptides appear to stimulate CaM kinase II, which in turn, via MAP kinase activation, enhances cPLA2 activity and release of AA for PG synthesis.

Types of purinoceptors and phospholipase A2 involved in the activation of the platelet-activating factor-dependent transacetylase activity and arachidonate release by ATP in endothelial cells.

Acyl analogs of PAF are the major products synthesized during agonist stimulation of endothelial cells. We have previously shown that PAF: 1-acyl-2-lyso-sn-glycero-3-phosphocholine transacetylase in calf pulmonary artery endothelial cells is activated by ATP through protein phosphorylation, and the increase in transacetylase activity by ATP contributes to the biosynthesis of acyl analogs of PAF (J. Biol. Chem. 272, 17431-17437, 1997). To understand the mechanisms(s) by which ATP stimulates acyl analogs of PAF production, we have identified the subtypes of the purinergic receptor that are linked to the activation of two enzymes involved in the generation of acyl analogs of PAF, namely, transacetylase and phospholipase A2. Experiments with transient transfection of the cells with antisense and sense thio-oligonucleotide to cytosolic phospholipase A2 (cPLA2) were also performed to evaluate whether downstream activation of cPLA2 is involved in ATP-receptor mediated induction of arachidonate release and synthesis of radylacetyl-GPC. We found that the P2u/P2Y2 receptor, which recognizes a pyrimidine nucleotide, UTP, as well as purine nucleotides, shows a potency profile of UTP > ATP = ATP gamma S > 2-methylthio-ATP in mediating the activation of PAF: lysophospholipid transacetylase. On the other hand, ADP beta S and 2-methylthio-ATP have similar potencies as ATP but have lower potencies than UTP and ATP gamma S in stimulating the release of arachidonate. These results suggest that both P2u/P2Y2 and P2y/P2Y1 receptor subtypes promote arachidonate release. In addition, transient transfection of endothelial cells with cPLA2 antisense but not the sense thio-oligonucleotide inhibited the stimulation of arachidonate release and [3H]acetate incorporation into radyl[3H]acetyl-GPC. Thus, our data suggest that a receptor-mediated process is involved in the activation of transacetylase for the induced synthesis of acyl analogs of PAF in endothelial cells. Furthermore, it is likely that cPLA2 supplies the lysophospholipids as substrates for the transacetylation reaction.

Signal transduction mechanism(s) involved in prostacyclin production elicited by acetylcholine in coronary endothelial cells of rabbit heart.

The purpose of this study was to elucidate the mechanism by which acetylcholine (ACh) promotes prostacyclin (PGI2) production in cultured coronary endothelial cells (CEC) of the rabbit heart. ACh-induced production of PGI2, measured as immunoreactive 6-keto-PGF1alpha, was enhanced by increasing the extracellular calcium (Ca++) concentration and reduced by Ca++ depletion. The receptor-operated Ca++ channel blocker SK&F96365, but not the voltage-dependent Ca++ channel blockers verapamil or nifedipine, attenuated ACh-induced 6-keto-PGF1alpha production and the associated rise in cytosolic Ca++. Thapsigargin, which depleted Ca++ accumulation from the intracellular Ca++ store, did not prevent the ACh-induced rise in cytosolic Ca++. In the absence of extracellular Ca++, ACh and ATP increased cytosolic Ca++ but did not alter 6-keto-PGF1alpha production. In permeabilized CEC, guanosine 5'-O-(3-thiotriphosphate) (GTP-gamma-S) but not ACh enhanced 6-keto-PGF1alpha synthesis. ACh increased 6-keto-PGF1alpha production in the presence of GTP-gamma-S. These effects of GTP-gamma-S were attenuated by guanosine 5'-O-(2-thiotriphosphate). In the absence of extracellular Ca++, ACh or ATP increased cytosolic Ca++ in cells permeabilized with beta-escin and loaded with GTP-gamma-S; this effect was attenuated by guanosine 5'-O-(2-thiotriphosphate). The effect of ATP but not ACh to mobilize intracellular Ca++ or increase 6-keto-PGF1alpha was inhibited by pertussis toxin. The phospholipase C inhibitor D609, which attenuated ACh- and ATP-induced mobilization of intracellular Ca++, did not alter 6-keto-PGF1alpha production. The NO synthase inhibitor N-monomethyl-arginine also failed to alter ACh-induced 6-keto-PGF1alpha synthesis. These data suggest that, in CEC of the rabbit heart, ACh stimulates prostacyclin production via a pertussis toxin-insensitive G protein and by increasing the influx of extracellular Ca++ through a G protein-independent receptor-operated Ca++ channel.

Beta adrenergic receptor stimulated prostacyclin synthesis in rabbit coronary endothelial cells is mediated by selective activation of phospholipase D: inhibition by adenosine 3'5'-cyclic monophosphate.

Activation of beta adrenergic receptors in the isolated rabbit heart by catecholamines stimulates prostacyclin (PGI2) synthesis, which is inhibited by adenosine 3'5'-cyclic monophosphate (cAMP). The purpose of this study was to determine if activation of beta adrenergic receptors in cultured coronary endothelial cells (CEC) of rabbit heart with isoproterenol (ISOP) stimulates PGI2 synthesis and if cAMP inhibits the synthesis of this prostanoid and to investigate the underlying mechanism. Incubation of CEC with ISOP increased production of cAMP and PGI2, measured as immunoreactive cAMP and 6-keto-prostaglandin F1alpha, (6-keto-PGF1alpha), respectively. Forskolin, an activator of adenylyl cyclase, increased cAMP accumulation and inhibited ISOP-stimulated 6-keto-PGF1alpha synthesis. 8-(4-chlorophenyl-thio) cAMP also inhibited ISOP-induced 6-keto-PGF1alpha production. However, miconazole, an inhibitor of adenylyl cyclase, reduced cAMP accumulation and enhanced ISOP-stimulated 6-keto-PGF1alpha synthesis in CEC. ISOP-induced 6-keto-PGF1alpha synthesis was attenuated by C2-ceramide, an inhibitor of phospholipase D (PLD) by propranolol, a beta-AR antagonist that also inhibits phosphatidate phosphohydrolase and by the diacylglycerol lipase inhibitor 1,6-bis-(cyclohexyloximinocarbonylamino)-hexane (RHC 80267). Acetylcholine (ACh) induced 6-keto-PGF1alpha synthesis was also inhibited by these agents. Both ISOP and ACh increased PLD activity, which was inhibited by C2-ceramide but not by RHC 80267 or propranolol. ACh but not ISOP increased phospholipase A2 activity in CEC. ISOP- but not ACh-induced increase in PLD activity was attenuated by forskolin and 8-(4-chlorophenyl-thio)-adenosine 3'-5'-cyclic monophosphate and augmented by miconazole. These data suggest that beta adrenergic receptors activation promotes PGI2 synthesis in the CEC by selective activation of PLD and that cAMP decreases PGI2 synthesis by decreasing PLD activity. Moreover, beta adrenergic receptors activated PLD appears to be distinct from that stimulated by ACh.