Prostacyclin Formation Elicited by Endothelin-1 in Rat Aorta Is Mediated via Phospholipase D Activation and Not Phospholipase C or A2
Endothelin-1 (ET-1) is a potent vasoconstrictor peptide that also stimulates production of prostacyclin (PGI2) from arachidonic acid. The purpose of this study was to determine the contribution of phospholipases (PLs) A2, C, and/or D in ET-1–induced PGI2 formation in the rat aorta, measured as immunoreactive 6-ketoprostaglandin (PG) F1α. ET-1 increased 6-keto-PGF1α formation, which was not affected by a PLA2 inhibitor, 7,7-dimethyl eicosadienoic acid (DEDA). Furthermore, ET-1 failed to stimulate PLA2 activity measured in the cytosol (cPLA2), using phosphatidylcholine, l-a-1-palmitoyl-2-arachidonyl[14C] as a substrate. However, the adrenergic agonist norepinephrine increased 6-keto-PGF1α formation, which was attenuated by DEDA, and enhanced PLA2 activity. ET-1 enhanced PLC activity, as indicated by increased inositol phosphate production, which was prevented by a PLC inhibitor, U-73122. However, ET-1–induced 6-keto-PGF1α production was not altered by U-73122. An inhibitor of PLD activation, C2-ceramide, attenuated ET-1–induced PLD activity, as indicated by the production of phosphatidylethanol. Furthermore, ET-1–induced 6-keto-PGF1α formation was inhibited by C2-ceramide as well as by ethanol treatment. Moreover, inhibitors of phosphatidate phosphohydrolase (propranolol) and diacylglycerol lipase (RHC-80267), attenuated ET-1–induced 6-keto-PGF1α formation. Finally, ET-1–induced activation of PLD was not attenuated by a selective PKC inhibitor, bisindolylmaleimide I. These data suggest a novel pathway for ET-1–induced PGI2 formation in the rat aorta involving activation of PLD but not cPLA2 and independent of PLC or PKC activation.
Endothelin, isolated 8 years ago from porcine endothelial cells, is the most potent vasoconstrictor agent yet discovered.1 It has been implicated in a variety of cardiovascular diseases, such as atherosclerosis, hypertension, and renal failure.2 Previously, we have shown that ET-1 stimulates PGI2 formation in rat aorta via activation of ETA receptors in the smooth muscle layer. Moreover, activation of ETA receptors by ET-1 promotes influx of extracellular Ca2+ via voltage as well as receptor-operated Ca2+ channels, which in turn stimulates PGI2 production by a mechanism independent of PKC.3 PGI2 exerts a myriad of biological effects, which include inhibition of platelet aggregation,4 relaxation of vascular, gastric, and pulmonary smooth muscle, and a decrease in vascular reactivity to vasoconstrictor agents such as ET-1.5 PGI2 also inhibits the production and secretion of ET-1 from the endothelium,6 the major site of its synthesis in the vasculature.
PGI2 is synthesized from AA, which can be liberated from phospholipids via activation of PLs A2, C, and/or D.7 Activation of PLA2 is the most direct route for AA release in response to various stimuli from tissue lipids.8 A less common pathway for AA release is via PLC activation, which generates DAG that in turn is hydrolyzed by DAG lipase to form AA and monoacylglycerol.9 DAG may also be phosphorylated by DAG kinase to PA, which in turn can be hydrolyzed by PLA2 to generate AA and lysophosphatidic acid.10 Similarly, PLD activation could result in the release of AA from PA, either by PLA2 or after conversion by PP to DAG.11 Whether one or more of these pathways are involved in the rat aorta in the release of AA for PGI2 formation by ET-1 is not known. To test these hypotheses, we determined the effect of specific lipase inhibitors on ET-1–induced PGI2 formation in rat aortic rings. We also measured the activity of PLA2, PLC, and PLD, in the presence and absence of their respective inhibitors.
Materials and Methods
Preparation of Aortic Rings
The following protocol was reviewed and approved by our Institution Animal Care and Use Committee and conforms with the Guide for the Care and Use of Laboratory Animals (National Institutes of Health). The preparation of aortic rings was adapted from a previous method.12 Male Sprague-Dawley rats (300 to 350 g, Charles River, Wilmington, Mass) were anesthetized with pentobarbital (50 mg/kg IP) and the thoracic aortae removed and placed in a 37°C BSS containing the following (mmol/L): NaCl 116, KCl 5.4, MgCl2·6H2O 1.2, NaH2PO4.H2O 1.2, CaCl2.2H2O 1.8, glucose 5.5, and HEPES 25 (pH 7.4). Fat and connective tissue were carefully removed, as were the small blood vessels branching out of the aorta. The aortae were then cut into 0.2-cm-wide rings, unless otherwise indicated. For each experiment, aortae from two to three rats were pooled.
The first series of experiments was conducted to determine the role of different lipases in ET-1–induced 6-keto-PGF1α (the stable product of PGI2 hydrolysis) formation in rat aortic rings, using selective inhibitors. Separate groups of aortic rings were equilibrated in BSS overnight (with 95% O2/5% CO2) and then preincubated for 30 minutes with the following inhibitors (or vehicle): DEDA (for PLA2),13 U-73122 (for PLC),14 15 propranolol (for PP),16 or RHC 80267 (for DAG lipase).17 In an additional series of experiments, ET-1–induced 6-keto-PGF1α production was measured in aortic rings pretreated 16 hours with C2-ceramide, an inhibitor of PLD activation,18 19 or with ethanol for 10 minutes. PLD catalyzes a transphosphatidylation reaction in the presence of ethanol, resulting in the production of PEt and a consequent decrease in PA formation.20 The BSS was then removed and replaced with BSS containing ET-1 (10 nmol/L) or vehicle, with or without the respective inhibitors, for 20 minutes. In an additional experiment, the effect of NE (10 μmol/L) in the presence and absence of DEDA was determined. The rings were then removed and weighed, and the remaining buffer was assayed for 6-keto-PGF1α as described later.
This series of experiments was conducted to determine the effect of ET-1 and NE on PLA2 activity in rat aorta by use of an in vitro assay for PLA2.21 Aortae (one half aorta per experimental treatment) were equilibrated in BSS (with 95% O2/5% CO2) overnight and then treated with either ET-1 (10 nmol/L) or NE (10 μmol/L) for 10 minutes. Aortae were next frozen with liquid nitrogen and pulverized. All subsequent procedures were performed at 4°C. The powder was homogenized in a tissue-tearer homogenizer (Fisher) in 0.5 mL of buffer (pH 7.4) containing (mmol/L): HEPES 10, sucrose 340, EDTA 1, EGTA 1, PMSF 1, sodium fluoride 100, tetrasodium pyrophosphate 10, p-nitrophenyl phosphate 0.003, and sodium orthovanadate 0.2, as well as 25 μg/mL each of leupeptin and aprotinin. The homogenates were then centrifuged at 100 000g for 1 hour, the supernatant (cytosol) was removed and concentrated, and the protein concentration was determined using BSA as a standard (Bio-Rad). PLA2 activity in the cytosol was measured using phosphatidylcholine, l-a-1-palmitoyl-2-arachidonyl[14C] (57 mCi/mmol, American Radiolabeled Chemicals) as substrate (≈50 000 cpm per assay tube), cosonicated with 9 μmol/L dioleoylglycerol, 1 mg/mL BSA, 150 mmol/L NaCl, 5 mmol/L CaCl2, and 50 mmol/L Tris, pH 7.4. Twenty micrograms of cytosol protein was added and the reaction mixture incubated for 5 minutes at 37°C. The reaction was terminated with 2.5 mL of Dole reagent (2-propanol/heptane/0.5 mol/L H2SO4 [20:5:1, vol/vol/vol]), to which 1 mL of heptane and 1 mL of water (containing 20 μg of nonradiolabeled AA as carrier) was added. The tube was mixed and the heptane layer applied to a Sep-Pak 3-cc silica column (Waters) for separation of radiolabeled fatty acid. Each column was further washed with 1 mL of heptane, the eluates were air-dried, and radioactivity was measured by liquid scintillation spectroscopy. Data were expressed as the percentage above basal fractional release of [14C]AA.
This series of experiments was conducted to measure ET-1–induced PLC activity, measured as total IP production, in rat aortic rings in the absence and presence of a PLC inhibitor, U-73122. IP production was measured using a modification of a method previously described.22 Rat aortic rings were blotted and weighed and then incubated in 1 mL of BSS (with 95% O2/5% CO2) containing myo-[2-3H(N)]inositol (15 Ci/mmol, American Radiolabeled Chemicals), 5 μCi/mL per ring, for 24 hours. Labeled rings were then preincubated in BSS containing 20 mmol/L LiCl with or without U-73122 (10 μmol/L) or vehicle for 30 minutes. The rings were next incubated in BSS (with LiCl) containing ET-1 (10 nmol/L) alone, U-73122 alone, ET-1 and U-73122, or respective vehicles for 20 minutes. Incubations were terminated by the addition of 3 mL of ice-cold methanol/chloroform (2:1 [vol/vol]) containing 0.1% hydrochloric acid followed by 1 mL of chloroform. The solution containing the rings was sonicated for 1 hour in an ice bath and kept overnight at 4°C. Aqueous and lipid phases were separated by the addition of 2 mL of water. The upper phase was neutralized and applied to a Dowex-formate resin column (AG1-X8, Bio-Rad). The column was washed three times with 5 mL of water followed by three 5-mL aliquots of 60 mmol/L sodium formate with 5 mmol/L sodium tetraborate to elute glycerophosphoinositol. Total IPs were eluted by three 5-mL aliquots of 0.1 mol/L formic acid containing 1 mol/L ammonium formate. These eluates were collected in scintillation vials, and 5-mL aliquots of the eluate were combined with 2 mL of water and 10 mL of liquid scintillation fluid (Ultima Gold, Packard). Radioactivity was measured by scintillation spectroscopy. Data are expressed as counts per minute per milligram of tissue weight.
This series of experiments was conducted to determine the effect of ET-1 on PLD activity, measured as PEt production, in rat aortic rings in the absence and presence of C2-ceramide, or a PKC inhibitor, Bis I.23 PLD activity was measured by using a modification of methods previously described.24 25 Rat aortic rings (1 cm) were incubated overnight in 1 mL of BSS containing [3H]oleic acid (5 μCi/mL) (15 Ci/mmol, American Radiolabeled Chemicals) and 0.02% BSA, along with C2-ceramide (5, 10, or 50 μmol/L) when indicated. Labeled rings were then rinsed in BSS containing C2-ceramide, Bis I (20 nmol/L), or vehicle for 30 minutes. Before treatment with ET-1, the rings were incubated in BSS containing 108 mmol/L ethanol or none at all for 10 minutes. In the presence of ethanol, PLD transfers the phosphatidyl moiety of a phospholipid to ethanol to form PEt.20 Rings were incubated in BSS containing ET-1 (10 nmol/L), inhibitor alone, ET-1 and inhibitor, or respective vehicles for 10 minutes. The rings were then placed in tubes containing liquid nitrogen. Three milliliters of ice-cold methanol/chloroform/0.2 mol/L HCl (2:1:0.8 [vol/vol/vol]) was then added to each tube. The solutions containing rings were sonicated for 10 minutes in an ice bath, and then the rings were discarded. Thereafter, 1 mL each of chloroform and water was added to each tube, and the solution was mixed and then centrifuged for 10 minutes. The top aqueous layer was discarded, and 1.6 mL from the remaining bottom (2 mL) chloroform layer was transferred into a glass tube. From this 1.6 mL, an 80-μL aliquot was removed for scintillation spectroscopy to estimate radioactivity in the total lipid fraction. The remaining chloroform was dried under a nitrogen stream and the residue resuspended in 50 μL of methanol/chloroform (1:9 [vol/vol]) containing 10 μg of nonradiolabeled PEt standard (Biomol). Samples were spotted on a channeled silica gel thin-layer chromatography plate (Analtech). The plate was developed in a solvent system containing chloroform/methanol/acetic acid (65:15:2 [vol/vol/vol]) to separate PEt. After separation, the lipids were visualized in an iodine tank, and PEt (Rf=0.4) was identified by its comigration with standard. Lanes containing PEt were moistened with water and scraped into scintillation vials containing 10 mL of scintillation fluid. Radioactivity was measured by scintillation spectroscopy. Data were expressed as the fractional conversion to PEt of 3H label in total lipids×104.
Radioimmunoassay of 6-Keto-PGF1α
The content of 6-keto-PGF1α (the stable product of PGI2 hydrolysis) in the incubation buffer was determined by radioimmunoassay as previously described.26 Briefly, 50-μL samples were mixed with 3000 to 4000 cpm [3H]6-keto-PGF1α tracer (150 Ci/mmol, NEN) plus an appropriate concentration of antibody in polystyrene tubes. Tracer and antibody were prepared in buffer containing (g/L): NaN3 1.0, NaCl 9.0, KH2PO4 6.8, K2HPO4 26.1, and gelatin 2.0. Tubes were then vortexed and incubated overnight at 4°C. Dextran-coated charcoal (1 mL) was added to each tube to separate bound from free tracer, and radioactivity was determined by liquid scintillation spectroscopy. The antibody for 6-keto-PGF1α was kindly provided by Dr C. Leffler (University of Tennessee, Department of Physiology). Cross-reactivity of the 6-keto-PGF1α antibody was <0.1% with thromboxane B2, 13,14-dihydro-15-keto-PGF2, and PGI2 and <0.5% with PGE2 and PGF1α. Furthermore, none of the drugs used in this study were found to interfere with the radioimmunoassay.
The drugs used in this study were as follows: ET-1 from Peninsula Laboratories Inc; NE, propranolol, and timolol from Sigma Chemical Co; DEDA, U-73122, RHC 80267, C2-ceramide, wortmannin, and AG-126 from Biomol; and Bis I HCl, H-89, and ML-7 from Calbiochem. Stock solutions of DEDA, RHC 80267, U-73122, C2-ceramide, wortmannin, ML-7, AG-126, and H-89 were prepared in DMSO; NE, propranolol, timolol, and Bis I were prepared in double-distilled water. ET-1 was stored in water diluted with glacial acetic acid at −80°C until use. All of these compounds were diluted in BSS before their use. For all experiments, the effect of the appropriate vehicle was also determined.
Results are expressed as mean±SEM. Data were analyzed by one-way ANOVA; the unpaired Student's t test was applied to determine the difference between two groups. A value of P≤.05 was considered significant. Basal PGI2 output represents the amount of PGI2 in samples collected after removal of BSS and is expressed as picograms of immunoreactive 6-keto-PGF1α per milligram of tissue.
Effect of ET-1 and NE on 6-Keto-PGF1α Formation and PLA2 Activity in Rat Aortic Rings
ET-1 produces a consistent concentration-dependent increase in 6-keto-PGF1α production in rat aortic rings, which is maximal at 50 nmol/L.3 In these experiments, ET-1 (10 nmol/L)–induced 6-keto-PGF1α formation did not significantly differ between aortic rings pretreated (30 minutes) with DEDA (100 μmol/L) or vehicle. On the other hand, under identical conditions, NE (10 μmol/L)-induced 6-keto-PGF1α production was attenuated by DEDA (Table 1⇓). Homogenization of rat aorta in the presence of Ca2+ chelators results in the appearance of a PLA2 in the cytosol fraction (cPLA2).27 In cytosol fractions isolated from aorta treated with either NE (10 μmol/L) or ET-1 (10 nmol/L) for 10 minutes, NE but not ET-1 significantly increased cPLA2 activity (Fig 1⇓).
Effect of a PLC Inhibitor, U-73122, on ET-1–Stimulated 6-Keto-PGF1α Formation and PLC Activity in Rat Aortic Rings
ET-1 (10 nmol/L) caused a significant increase in PLC activity, as indicated by increased formation of IP. A PLC inhibitor, U-73122 (10 μmol/L), reduced basal as well as ET-1–induced production of IP. However, under identical conditions, U-73122 did not inhibit ET-1–induced 6-keto-PGF1α formation in rat aortic rings (Table 2⇓).
Effect of PLD, PP, and DAG Lipase Inhibitors on ET-1–Stimulated 6-Keto-PGF1α Formation in Rat Aortic Rings
Both C2-ceramide (Fig 2⇓, top), which inhibits PLD activation, and ethanol (Fig 2⇓, bottom), which decreases the formation of PA when PLD is activated, significantly attenuated ET-1–induced 6-keto-PGF1α formation in rat aortic rings. The conversion of exogenously added AA (10 μmol/L) to 6-keto-PGF1α (picograms of 6-keto-PGF1α synthesized per milligram of tissue) was not significantly altered in the presence of 50 μmol/L C2-ceramide (9943±1276 versus 9305±1240, AA versus AA+C2-ceramide, n=10) or 216 mmol/L ethanol (6561±969 versus 6028±580, AA versus AA+ethanol, n=6). A PP inhibitor, propranolol (Fig 3⇓, top), and a DAG lipase inhibitor, RHC 80267 (Fig 3⇓, bottom), significantly attenuated ET-1–induced 6-keto-PGF1α formation in rat aortic rings. Under identical conditions, timolol (200 μmol/L), like propranolol, a nonselective β-adrenergic receptor blocker, did not alter basal (165±48 versus 148±29, vehicle versus timolol) or ET-1 (10 nmol/L)–induced 6-keto-PGF1α production (picograms of 6-keto-PGF1α synthesized per milligram of tissue) in rat aortic rings (5829±1242 versus 5849±1549, ET-1 versus ET-1+timolol, n=6). Moreover, the conversion of exogenously added AA (10 μmol/L) to 6-keto-PGF1α (picograms of 6-keto-PGF1α synthesized per milligram of tissue) was not altered in the presence of 200 μmol/L propranolol or 50 μmol/L RHC 80267 (AA, 9239±1487; AA+propranolol, 9916±1009; AA+RHC 80267, 9322±645, n=4).
Effect of C2-Ceramide and Bis I on ET-1–Induced PLD Activity in Rat Aortic Rings
Treatment of rat aortic rings for 10 minutes with ET-1 (10 nmol/L) in the presence of ethanol (108 mmol/L) resulted in a significant increase in the production of PEt. This increase in PEt production was dose-dependently reduced by an inhibitor of PLD activation, C2-ceramide (Fig 4⇓, top). However, ET-1–induced activation of PLD in rat aortic rings was not inhibited by a selective PKC inhibitor, Bis I (20 nmol/L) (Fig 4⇓, bottom).
Our study demonstrates that in rat aortic rings ET-1–induced PGI2 formation is mediated through a novel pathway via activation of PLD, rather than the more commonly characterized route of PLA2 activation. This conclusion is based on our finding that ET-1 increased PLD activity (as indicated by an increase in PEt production) and that this increase in PLD activity, as well as the associated increase in 6-keto-PGF1α production, was attenuated by a cell-permeable inhibitor of PLD activation, C2-ceramide.18 19 Furthermore, ethanol, which decreases the amount of PA formed in favor of PEt during PLD activation, also inhibited ET-1–stimulated 6-keto-PGF1α formation in rat aortic rings. It is possible that ethanol may have nonspecific actions on the cell membrane. However, this is unlikely, since neither ethanol nor C2-ceramide altered cyclooxygenase activity, as indicated by their lack of effect on the conversion of exogenous AA to 6-keto-PGF1α. PLD activation leads to the production of PA from membrane phospholipids, which is then converted to DAG by PP. DAG is further metabolized by DAG lipase to release AA for PGI2 formation. Our finding that inhibitors of PP (propranolol) and DAG lipase (RHC 80267) also attenuated ET-1–induced 6-keto-PGF1α formation supports our contention that ET-1–induced activation of PLD initiates a pathway that releases AA for PGI2 formation in the rat aorta. The inhibitory effect of propranolol is not related to blockade of β-adrenergic receptors, because another nonselective β-adrenergic receptor blocker, timolol, did not alter 6-keto-PGF1α formation elicited by ET-1. Moreover, the effect of propranolol or RHC 80267 to attenuate ET-1–induced 6-keto-PGF1α formation was not due to a decrease in cyclooxygenase activity, because these agents did not reduce the conversion of exogenous AA to 6-keto-PGF1α. The conclusions reached regarding the contribution of PLD to ET-1–induced 6-keto-PGF1α production would be further substantiated if the mechanism whereby C2-ceramide inhibits activation of this enzyme were known or if more selective inhibitors of the lipase were available. One possibility is that C2-ceramide could activate a specific protein kinase or protein phosphatase that in turn inhibits the activation of PLD either directly or through another signaling molecule. Such a ceramide-activated protein kinase and phosphatase have been described.28 29 This, however, would not explain the long periods (hours) of incubation required for inhibition of PLD activation by C2-ceramide (ie, at least 4 hours).18 19 Therefore, an alternative possibility is that exogenously added ceramide might compete with the available pool of phosphocholine (ie, forming sphingomyelin) and thus decrease the synthesis of endogenous phosphatidylcholine, which is the substrate for PLD.
The predominant pathway for the release of AA for PG formation is considered to be activation of PLA2.7 8 Surprisingly, in the rat aortic rings, ET-1 failed to stimulate cPLA2 activity, and an inhibitor of PLA2s, DEDA, failed to alter ET-1–induced 6-keto-PGF1α formation. On the other hand, NE enhanced cPLA2 activity in the rat aorta, as well as 6-keto-PGF1α formation in rat aortic rings, and this was inhibited by DEDA. Furthermore, the ability of ET-1 to activate cPLA2 was not dose dependent, since concentrations of ET-1 as high as 50 nmol/L, which elicits maximal 6-keto-PGF1α production in the rat aorta,3 did not stimulate cPLA2 activity (data not shown). Therefore, these data suggest that the AA released for PGI2 formation in response to ET-1 stimulation in the rat aorta is not derived via cPLA2 activation.
ET-1–induced release of AA for PGI2 formation may also be derived via activation of PLC, which results in the formation of IP and DAG; the hydrolysis of the latter by DAG lipase may serve as a source of AA.9 In our study, ET-1 increased PLC activity, as indicated by an increase in IP production, and this action was blocked by the PLC inhibitor U-73122. However, U-73122 did not attenuate ET-1–induced 6-keto-PGF1α formation. The basal 6-keto-PGF1α formation was increased in the presence of U-73122. The reason for this increase, which could be stimulation of PLA2 and/or PLD, remains to be determined. Nevertheless, U-73122 did inhibit both basal and ET-1–induced PLC activity and did not significantly decrease ET-1–induced 6-keto-PGF1α formation compared with other inhibitors used in this study. Therefore, PLC does not appear to be involved in ET-1–stimulated PGI2 production in the rat aorta.
Having established that PLD is the primary phospholipase involved in ET-1–induced PGI2 production in rat aortic rings, we next investigated the possible contribution of PKC in this signaling pathway, since ET-1 has been reported to activate PKC,3 30 and PKC increases PLD activity in some cell systems.31 32 Our previous study3 showed that ET-1 increased PKC activity in rat aortic rings and that this action was blocked by two well-characterized PKC inhibitors, calphostin C and Bis I. However, neither calphostin C nor Bis I inhibited ET-1–induced 6-keto-PGF1α formation in rat aortic rings. In the present study, using a concentration of Bis I previously shown to block ET-1–induced PKC activation in rat aorta,3 we found that ET-1–stimulated PLD activity was likewise insensitive to Bis I treatment. Our results complement those of others23 24 who have shown PKC-independent regulation of PLD. Therefore, these data suggest that PKC does not mediate activation of PLD by ET-1 or PGI2 formation in the rat aorta.
In addition to PKC, we investigated the possible contribution of other protein kinases, ie, protein kinase A, tyrosine kinases, phosphatidylinositol 3 kinase, and myosin light chain kinase, on ET-1–induced 6-keto-PGF1α production in rat aortic rings by using their selective inhibitors, H-89, AG-126, wortmannin, and ML-7, respectively. None of these inhibitors altered ET-1–induced 6-keto-PGF1α formation in rat aortic rings (data not shown). It appears that these kinases do not mediate or modulate the effect of ET-1 to stimulate PGI2 production in the rat aorta. However, further work is required to exclude totally the contribution of one or more kinases in the activation of PLD, PP, and/or DAG lipase by ET-1 in the rat aorta.
In conclusion, our results suggest that ET-1 stimulates PLD, generating PA, which, after being converted to DAG, is hydrolyzed by DAG lipase to release AA for PGI2 formation. This signaling pathway does not require activation of cPLA2, PLC, or PKC.
Selected Abbreviations and Acronyms
|Bis I||=||bisindolylmaleimide I|
|BSS||=||balanced salt solution|
|DEDA||=||7,7-dimethyl eicosadienoic acid|
|PKC||=||protein kinase C|
This work was supported in part by National Institutes of Health (Heart, Lung, and Blood Institute) grant HL-19134-21. Dr Wright was supported in part by a National Research Service Award, Pre-Doctoral Research Training grant HL-07641-06. This work was done in partial fulfillment of requirements for the degree of Doctor of Philosophy by Dr Wright. We thank Anne Estes for her technical assistance.
- Received March 19, 1996.
- Accepted April 29, 1996.
Wright HM, Malik KU. Prostacyclin synthesis elicited by endothelin-1 in rat aorta is mediated by an ETA receptor via influx of calcium and is independent of protein kinase C. Hypertension. 1995;26:1035-1040.
De Nucci G, Thomas R, D'Orleans-Juste P, Antunes E, Walder C, Warner TD, Vane JR. Pressor effects of circulating endothelin are limited by its removal in the pulmonary circulation and by the release of prostacyclin and endothelium-derived relaxing factor. Proc Natl Acad Sci U S A. 1988;85:9797-9800.
Prins BA, Hu RM, Nazario B, Pedram A, Frank HJL, Weber MA, Levin ER. Prostaglandin E2 and prostacyclin inhibit the production and secretion of endothelin from cultured endothelial cells. J Biol Chem. 1994;269:11938-11944.
Gross RW. Myocardial phospholipases A2 and their membrane substrates. Trends Cardiovasc Med. 1992;2:115-121.
Dennis EA. Diversity of group types, regulation, and function of phospholipase A2. J Biol Chem. 1994;269:13057-13060.
Balsinde J, Diez E, Mollinedo F. Arachidonic acid release from diacylglycerol in human neutrophils. J Biol Chem. 1991;256:15638-15643.
Lapetina EG, Billah MM, Cuatrecasas P. The initial action of thrombin on platelets. J Biol Chem. 1981;256:5037-5040.
Lister MD, Glaser KB, Ulevitch RJ, Dennis EA. Inhibition studies on the membrane-associated phospholipase A2 in vitro and prostaglandin E2 production in vivo of the macrophage-like P388D1 cell. J Biol Chem. 1989;264:8520-8528.
Smith RJ, Sam LM, Justen JM, Bundy GL, Bala GA, Bleasdale JE. Receptor-coupled signal transduction in human polymorphonuclear neutrophils: effects of a novel inhibitor of phospholipase C–dependent processes on cell responsiveness. J Pharmacol Exp Ther. 1990;253:688-697.
Bleasdale JE, Thakur NR, Gremban RS, Bundy GL, Fitzpatrick FA, Smith RJ, Bunting S. Selective inhibition of receptor-coupled phospholipase C–dependent processes in human platelets and polymorphonuclear neutrophils. J Pharmacol Exp Ther. 1990;255:756-768.
Billah MM, Eckel S, Mullmann TJ, Egan RW, Siegel MI. Phosphatidylcholine hydrolysis by phospholipase D determines phosphatidate and diglyceride levels in chemotactic peptide–stimulated human neutrophils. J Biol Chem. 1989;264:17069-17077.
Sutherland CA, Amin D. Relative activities of rat and dog platelet phospholipase A2 and diglyceride lipase. J Biol Chem. 1982;257:14006-14010.
Gomez-Munoz A, Waggoner DW, O'Brien L, Brindley DN. Interaction of ceramides, sphingosine, sphingosine-1-phosphate in regulating DNA synthesis and phospholipase D activity. J Biol Chem. 1995;270:26318-26325.
Venable ME, Blobe GC, Obeid LM. Identification of a defect in the phospholipase D/diacylglycerol pathway in cellular senescence. J Biol Chem. 1994;269:26040-26044.
Bonser RW, Thompson NT, Randall RW, Garland LG. Phospholipase D activation is functionally linked to superoxide generation in the human neutrophil. Biochem J. 1989;264:617-620.
de Carvalho MGS, Carritano J, Leslie CC. Regulation of lysophospholipase activity of the 85-kDa phospholipase A2 and activation in mouse peritoneal macrophages. J Biol Chem. 1995;270:20439-20446.
Ohlstein EH, Horohonich S, Hay DWP. Cellular mechanisms of endothelin in rabbit aorta. J Pharmacol Exp Ther. 1989;250:548-555.
Toullec D, Pianetti P, Coste H, Bellevergue P, Grand-Perret T, Ajakane M, Baudet V, Boissin P, Boursier E, Loriolle F, Duhamel L, Charon D, Kerilovsky J. The bisindolylmaleimide GF 109203X is a potent and selective inhibitor of protein kinase C. J Biol Chem. 1991;266:15771-15781.
Liu Y, Geisbuhler B, Jones AW. Activation of multiple mechanisms including phospholipase D by endothelin-1 in rat aorta. Am J Physiol. 1992;262:941-949.
Llahi S, Fain JN. α1-Adrenergic receptor-mediated activation of phospholipase D in rat cerebral cortex. J Biol Chem. 1992;267:3679-3685.
Shaffer JE, Malik KU. Enhancement of prostaglandin output during activation of beta-1 adrenoceptors in the isolated rabbit heart. J Pharmacol Exp Ther. 1982;223:729-735.
Clark JD, Milona N, Knopf JL. Purification of a 110-kilodalton cytosolic phospholipase A2 from the human monocytic cell line U937. Proc Natl Acad Sci U S A. 1990;87:7708-7712.
Liu J, Mathias S, Yang Z, Kolesnick RN. Renaturation and tumor necrosis factor-α stimulation of a 97-kDa ceramide-activated protein kinase. J Biol Chem. 1994;269:3047-3052.
Hannun YA. The sphingomyelin cycle and the second messenger function of ceramide. J Biol Chem. 1994;269:3125-3128.
Xuan YT, Wang OL, Whorton AR. Regulation of endothelin-induced Ca2+ mobilization in smooth muscle cells by protein kinase C. Am J Physiol. 1994;266:C1560-C1567.
Geny B, Cockcroft S. Synergistic activation of phospholipase D by protein kinase C- and G-protein–mediated pathway in streptolysin O–permeabilized HL60 cells. Biochem J. 1992;284:531-538.
Gustavsson L, Moehren G, Torres-Marquez ME, Benistant C, Rubin R, Hoek JB. The role of cytosolic Ca2+, protein kinase C, and protein kinase A in hormonal stimulation of phospholipase D in rat hepatocytes. J Biol Chem. 1994;269:849-859.