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Am J Physiol Heart Circ Physiol 290: H37-H45, 2006. First published July 15, 2005; doi:10.1152/ajpheart.00491.2005
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Role of phospholipase C and diacylglyceride lipase pathway in arachidonic acid release and acetylcholine-induced vascular relaxation in rabbit aorta

Xin Tang,1 Erik M. Edwards,1 Blythe B. Holmes,1 John R. Falck,2 and William B. Campbell1

1Department of Pharmacology and Toxicology, Medical College of Wisconsin, Milwaukee, Wisconsin; and 2Department of Biochemistry, University of Texas Southwestern Medical Center, Dallas, Texas

Submitted 12 May 2005 ; accepted in final form 11 July 2005


   ABSTRACT
TOP
 ABSTRACT
METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
ACh stimulates arachidonic acid (AA) release from membrane phospholipids of vascular endothelial cells (ECs). In rabbit aorta, AA is metabolized through the 15-lipoxygenase pathway to form vasodilatory eicosanoids 15-hydroxy-11,12-epoxyeicosatrienoic acid (HEETA) and 11,12,15-trihydroxyeicosatrienoic acid (THETA). AA is released from phosphatidylcholine (PC) and phosphatidylethanolamine (PE) by phospholipase A2 (PLA2), or from phosphatidylinositol (PI) by phospholipase C (PLC) pathway. The diacylglycerol (DAG) lipase can convert DAG into 2-arachidonoylglycerol from which free AA can be released by monoacylglycerol (MAG) lipase or fatty acid amidohydrolase (FAAH). We used specific inhibitors to determine the involvement of the PLC pathway in ACh-induced AA release. In rabbit aortic rings precontracted by phenylephrine, ACh induced relaxation in the presence of indomethacin and N{omega}-nitro-L-arginine (L-NNA). These relaxations were blocked by the PLC inhibitor U-73122, DAG lipase inhibitor RHC-80267, and MAG lipase/FAAH inhibitor URB-532. Cultured rabbit aortic ECs were labeled with [14C]AA and stimulated with methacholine (10–5 M). Free [14C]AA was released by methacholine. Methacholine decreased the [14C]AA content of PI, DAG, and MAG fractions but not PC or PE fractions. Methacholine-induced release of [14C]AA was blocked by U-73122, RHC-80267, and URB-532 but not by U-73343, an inactive analog of U-73122. The data suggested that ACh activates PLC, DAG lipase, and MAG lipase pathway to release AA from membrane lipids. This pathway is important in regulating vasodilatory eicosanoid synthesis and vascular relaxation in rabbit aorta.

endothelium-dependent hyperpolarizing factor; phospholipid; monoacylglycerol lipase; trihydroxyeicosatrienoic acid; hydroxyepoxyeicosatrienoic acid

VASCULAR ENDOTHELIAL CELLS (ECs) regulate vascular tone by synthesis and release of many bioactive compounds. Ligands such as hormones, ACh, bradykinin, and cytokines stimulate release of arachidonic acid (AA) from cellular membranes (6, 15, 26, 43, 55), and AA induces endothelium-dependent vasodilation in isolated rabbit aorta preconstricted with phenylephrine, which is not blocked by nitric oxide (NO) synthase (NOS) and cyclooxygenase (COX) inhibitors (19, 39, 50). The phospholipase inhibitors mepacrine and dimethyl-eicosadienoic acid (DEDA) decrease relaxation to ACh but not AA, suggesting AA or a metabolite may be involved in the relaxations (11). AA is metabolized by COX, lipoxygenase (LO), and cytochrome P-450 pathways into bioactive eicosanoids (10, 30, 40, 48). Using specific inhibitors in rabbit aorta, Singer and Peach (51) first concluded that a LO metabolite mediates the NO- and PG-resistant relaxation to ACh (51). The LO inhibitors cinnamyl-3,4-dihydroxy-{alpha}-cyanocinnamate, nordihydroguaiaretic acid, and ebselen block ACh and AA-induced vasodilation in rabbit aorta (11, 39, 42). Previous studies in our lab identified the 15-LO metabolites of AA in rabbit aorta. AA is oxidized by 15-LO to 15-hydroperoxyeicosatetraenoic acid (15-HPETE), which is converted to hydroxyepoxyeicosatrienoic acid (HEETA) and trihydroxyeicosatrienoic acid (THETA) (11, 42). 15-Hydroxy-11,12-epoxyeicosatrienoic acid (15-H-11,12-EETA) and 11,12,15-THETA relax the aorta, an effect blocked by high potassium, or by apamin, an inhibitor of small-conductance, calcium-activated potassium channels (11, 23, 42). Therefore, in rabbit aorta, 15-LO metabolites of AA represent a new group of endothelium-dependent hyperpolarizing factors (EDHF) that are involved in regulation of vascular tone.

The generation of free AA from membrane lipids is essential to the production of eicosanoids. In ECs, ACh may stimulate AA release through phospholipase A2 (PLA2) and phospholipase C (PLC) pathways. Mammalian PLA2s hydrolyze the sn-2 fatty-acyl bond of phospholipids, preferentially phosphatidylcholine (PC) and phosphatidylethanolamine (PE) (3, 52). Cytosolic PLA2 (cPLA2) is the main regulator of AA mobilization and eicosanoid synthesis in human umbilical vein EC (14, 24) and bovine aortic ECs (49). Alternatively, PLCs metabolize phosphatidylinositol (PI) and phosphatidylinositol phosphate to inositol phosphates and diacylglycerol (DAG) (45). DAG is metabolized by DAG lipase to 2-arachidonylglycerol (2-AG) (12, 34). AA can be released from 2-AG by monoacylglycerol (MAG) lipase or fatty acid amidohydrolase (FAAH)(12). This PLC and DAG lipase pathway mediates AA release in human platelets (5) and bovine pulmonary artery ECs (56). Inhibition of PLC pathway by the PLC inhibitor U-73122 and the DAG lipase inhibitor RHC-80267 blocked ACh-induced vasorelaxation and AA release in porcine coronary arteries and guinea pig carotid arteries (44). In this study, we measured the effects of several PLC pathway inhibitors on vascular activity of rabbit aorta and AA release in cultured rabbit aorta endothelial cells (RA-ECs). The data suggest that ACh activates PLC and DAG lipase to metabolize membrane lipids and liberate AA, which is converted to vasodilatory eicosanoids.


   METHODS
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 ABSTRACT
 METHODS
RESULTS
 DISCUSSION
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Western immunoblotting assay. Primary antibodies (goat anti-human cPLA2 antibody, mouse anti-human sPLA2 II antibody, rabbit anti-human PLC{gamma}1 antibody, rabbit anti-human PLC{gamma}2 antibody, rabbit anti-bovine PLC{beta}1 antibody, and rabbit anti-human PLC{beta}2 antibody) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Human type II sPLA2 standard was purchased from Cayman Chemical (Ann Harbor, MI). Secondary antibodies [rabbit anti-goat IgG and goat anti-rabbit IgG horseradish peroxidase (HRP)-conjugated antibody] were from Calbiochem (San Diego, CA). Goat anti-mouse IgG HRP-conjugated antibody was from Jackson ImmunoResearch (West Grove, PA). Protein concentration was measured using Bio-Rad protein assay system (Bio-Rad, Hercules, CA). Fifty micrograms of protein were loaded in each lane and separated by SDS-PAGE by using a 10% resolving gel and 4% stacking gel. Protein were then transferred to nitrocellulose membranes. Nonspecific binding was blocked by incubating the membrane with Tris buffered saline (TBS) buffer containing 20 mM Tris-base, 150 mM NaCl, 0.1% sodium azide, and 3% BSA overnight at 4°C. Membranes were exposed to primary antibody (dilution 1:500) in TBS blocking buffer for 1 h at room temperature and rinsed with TBS buffer containing 0.1% Tween-20. Membrane was then incubated with appropriate secondary antibodies (1:5,000) for 1 h at room temperature. Excessive secondary antibody was then removed by rinsing with TBS buffer. Immunoreactive bands were identified using the Renaissance chemiluminescence detection kit (NEN, Boston, MA) and exposed to Kodak BioMax ML film.

Vascular activity. Animal protocols were approved by the Animal Care Committee of the Medical College of Wisconsin, and procedures were performed in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals (1996). Isolated thoracic aortic rings from the 4- to 6-wk-old New Zealand White rabbits were suspended in a tissue bath with 6 ml of Krebs bicarbonate solution (in mM: 119 NaCl, 4.8 KCl, 24 NaHCO3, 1.2 KH2PO4, 1.2 MgSO4, 11 glucose, 0.02 EDTA, and 3.2 CaCl2) as previously described (11, 39, 41). The buffer was maintained at 37°C and bubbled with 95% O2-5% CO2. Isometric tension was measured with Grass FT-03 force-displacement transducers connected to ETH-400 bridge amplifier and recorded by a MacLab 8e analog-to-digital converter, MacLab software V 4.1.1 (AD Instruments, MA) and a Macintosh Computer. Vessels were gradually adjusted to basal tension of 1.75 g, allowed to equilibrate for 1 h, and then contracted with 40 mM KCl. After the vessels reached peak contraction, tissue baths were rinsed, and vessels returned to resting tension. Vessels were then pretreated with L-NNA (3 x 10–5 M) and indomethacin (10–5 M) for 10 min and then contracted by 10–7–10–6 M of phenylephrine to 50–80% of the maximal KCl contraction. When the contraction was stabilized, cumulative concentrations of AA (10–9–10–4 M) or ACh (10–9–10–5 M) were added to the bath, and changes in isometric tension were measured. Similar experiments were performed in vessels pretreated for 10 min with phospholipase pathway inhibitors. Vasorelaxation was expressed as percentage of maximum precontraction. The vascular activity data are expressed as means ± SE. One-way ANOVA was performed between each group (P < 0.05 is considered significant).

RA-EC culture and [U-14C]AA uptake. ECs were cultured from thoracic aortas of 6-wk-old rabbits as previously described (41). Cells were cultured in 60-mm plastic petri dishes at 37°C in an atmosphere of 5% CO2 in air using Minimum Essential Medium (GIBCO-BRL, Carlsbad, CA) containing 10% rabbit serum, 10% fetal bovine serum (Nova-Tech, San Diego, CA), 1% L-glutamine, 1% antibiotic/antimycotic, and 1% ampicillin sodium (Sigma, St. Louis, MO) until 80% confluent. In experiments involving AA uptake, cells were washed with serum-free media twice, and 5 ml of serum free media with 0.1 mCi [U-14C]AA (specific activity = 900 mCi/mmol, NEN, Beverly, MA) was added to each dish. The cells were then incubated at 37°C for 18 h.

Assay of methacholine-induced AA release from membrane lipids. After incubation, the cells were washed with HEPES buffer containing (in mM) 10 HEPES, 150 NaCl, 5 KCl, 2 CaCl2, 1 MgCl2, 6 glucose, pH = 7.4, with 0.2% BSA. Five milliliters of HEPES buffer were added in each dish with 10–5 M of methacholine, inhibitors, or vehicle. The inhibitors were dissolved in DMSO; 5 µl of stock solution were added in each sample. The final concentration of DMSO was 0.1%. The inhibitors and their final concentrations were 10 µM for U-73122 [1-(6-((17{beta}-3-methoxyestra-1,3,5,10 trien-17-yl)amino)hexyl)-1H-pyrrole-2,5-dione] and 10 µM for U-73343 [1-(6((17{beta}-3-methoxyestra-1,3,5,10 trien-17-yl)amino)hexyl)-2,5-pyrrolidine-dione]; both U-73122 and U-73343 were purchased from Biomol (Plymouth Meeting, PA). Diazoarachidonyl ketone (DAK; 10 µM) and URB-532 (10 µM) were synthesized as previously described (16, 28). RHC-80267 [1,6-bis(cyclohexyloximinocarbonylamino)-hexane; 10 µM in DMSO] was purchased from Caymen Chemical. Incubations were carried out from 5 to 30 min at 37°C. After incubation, the buffer was removed, and 1 ml ice-cold methanol was added to the cells to stop the reactions. To analyze [3H]myoinositol incorporation in membrane lipids, cells were washed twice with HEPES buffer containing 0.2% BSA. HEPES buffer (1 ml) was added in each dish with 20 µCi of [3H]myoinositol (370–740 GBq/mmol, 10–20 Ci/mmol, 37 MBq/ml, 1 mCi/ml, Amersham Bioscience, UK) and 10–5 M of methacholine and vehicle or RHC-80267. The buffer was removed, and 1 ml ice-cold methanol was added to stop the reactions. The cells were scraped from the dishes with a rubber policeman. Distilled water (1 ml) was added to each dish, and the mixture was transferred to a 15-ml glass conical tube. The membrane lipids were extracted by adding 4 volumes of chloroform and methanol (CHCl3/CH3OH = 1:2) (8, 47). The tubes were mixed and allowed to stand for 30 min. Then 1.2 volumes of water and 1.2 volumes of CHCl3 were added to each tube. The extraction tubes were mixed again and centrifuged at 900 g for 3 min. The water phase was removed and organic phase was dried under a gentle stream of nitrogen, reconstituted in 200 µl of CHCl3, and known phospholipid and neutral lipid standards (15 µg each) were added. The samples were spotted on Whatman LD5 channeled silica gel G TLC plates (Whatman, UK) using an automatic spotter. The extracts were subjected to TLC in solvent system I (CHCl3/CH3OH/acetic acid/H2O = 50:30:8:1.5) for separation of phospholipids or solvent system II (heptane:diethyl ether:acetic acid = 100:20:4) for separation of neutral lipids (47). The plates were allowed to air dry, and autoradiographic images of labeled lipids were acquired with a Packard Instant Imager (Packard Instrument, Meriden, CT). Radioactive bands were scraped from the plates, and radioactivity was determined by liquid scintillation counting. Data are expressed as means ± SE. One-way ANOVA was performed between each group (P < 0.05 is considered significant).

Metabolism of [14C]AA. Isolated aortic rings were placed into 5 ml HEPES buffer. Vessels were incubated at 37°C with or without inhibitors in the presence of 10–5 M indomethacin for 10 min, and then [U-14C]AA (0.05 µCi) was added to final concentration of 10–7 M. After 5 min, calcium ionophore A-23187 (10–5 M) was added. After an additional 10 min, the reaction was stopped by adding ethanol (15% final concentration). The samples were then stored at –40°C until analyzed. The buffer was acidified to pH 3.5 with glacial acetic acid and extracted using Bond Elute octadecylsilyl columns (Varian, Palo Alto). The extracted lipid metabolites were analyzed by reverse-phase high-pressure liquid chromatography (HPLC) using solvent system III and a Nucleosil C18 (5 µm, 4.6 x 250 mm) column (42). A 40-min linear gradient from 50% solvent B (acetonitrile with 0.1% glacial acetic acid) in solvent A (deionized water) to 100% solvent B was used. The flow rate was 1 ml/min. The column effluent was collected in 0.2-ml fractions, and the radioactivity was determined by scintillation counting.


   RESULTS
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 ABSTRACT
 METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Effects of PLC pathway inhibitors on vascular activity in rabbit aorta. In phenylephrine-precontracted rabbit aorta, ACh induced a concentration-dependent vasorelaxation in the presence of L-NNA (30 µM) and indomethacin (10 µM) (Fig. 1). The maximal relaxation to ACh was ~44.0 ± 2.0% at 3 x 10–7 M. The PLC inhibitor U-73122 (10 µM) completely abolished the relaxations to ACh (maximum relaxation was –1.5 ± 0.8%, Fig. 1A). U-73343 (10 µM), an inactive analog of U-73122, shifted the relaxation curve to the right without affecting maximal relaxation to ACh (Fig. 1C). The DAG lipase inhibitor RHC-80267 (10 µM) and MAG lipase/FAAH inhibitors URB-532 (10 µM) and DAK (10 µM) also reduced maximal ACh relaxations to 9.0 ± 4.0% (Fig. 1E), 14.3 ± 4.0% (Fig. 1G), and 31 ± 4.4% (Fig. 1I), respectively. These results indicate that PLC, DAG lipase, and MAG lipase/FAAH inhibitors block the EDHF-mediated vasorelaxation induced by ACh in rabbit aorta. AA (10–7–10–4 M) elicits relaxations in indomethacin-treated rabbit aorta precontracted with phenylephrine (Fig. 1B). U-73122, RHC-80267, and URB-532 did not significantly affect these relaxations (Fig. 1, B, D, F, H). U-73343 shifted the concentration-response curves to AA slightly to the right. Surprisingly, DAK blocked AA-induced relaxation (Fig. 1J). These data indicate that AA acts distal to PLC, DAG lipase, and MAG lipase.



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  		Fig. 1. Effect of U-73122 (10 µM) (A and B), U-73343 (10 µM) (C and D), RHC-80267 (10 µM) (E and F), URB-532 (10 µM) (G and H), and diazoarachidonyl ketone (DAK; 10 µM) (I and J) on vascular activity of rabbit aortas. Aortic rings were pretreated for 10 min with indomethacin and N{omega}-nitro-L-arginine (L-NNA). Vessels were precontracted with 10–7 M of phenylephrine, and relaxation response to ACh (A, C, E, G) and arachidonic acid (AA; B, D, F, H) were determined, Control (filled symbol) and inhibitor-treated (open symbol) data are expressed as percent relaxations, and each value represents mean ± SE (n = 4–64). *P < 0.05, **P < 0.01, ***P < 0.001.

 
The inhibitors were tested for nonspecific effects on AA metabolism in rabbit aorta. [14C]AA was metabolized to 14C-labeled metabolites that comigrated with THETA, HEETA, and 15-hydroxyeicosatetraenoic acid (15-HETE). U-73343 partially inhibited AA metabolism in rabbit aorta. It decreased production of 15-HETE and vasodilatory THETA and HEETA (Fig. 2A), which explains the slight reduction in relaxations to ACh with U-73343. DAK inhibits FAAH, MAG lipase, and DAG lipase. DAK markedly inhibited [14C]AA metabolism. At 10 µM, it decreased 15-HETE productions by 90% (Fig. 2B). This explains DAK blockade of ACh- and AA-induced relaxations in rabbit aortas (Fig. 1, I and J). Two other MAG lipase inhibitors, methyl arachidonyl fluorophosphonate (10 µM) and 3-octylthio-1,1,1,-trifluoro-2-propanone (10 µM), also inhibited the 15-LO metabolism of [14C]AA (data not shown). Because of the effect of these inhibitors on [14C]AA metabolism, they were not used in these studies. In contrast, URB-532 did not affect [14C]AA metabolism by aortic rings (Fig. 2C).



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  		Fig. 2. Effects of U-73343, DAK, and URB-532 on [14C]AA metabolism by rabbit aorta. Aortic rings with intact endothelium were pretreated with 10 µM of U-73343 (A), DAK (B), or URB-532 (C). The rings were then incubated with [14C]AA in the presence of indomethacin. Samples were extracted, and the eicosanoids were resolved by HPLC. Migration times of known standards are shown above the chromatograms. HEETA, 15-hydroxy-11,12-epoxyeicosatrienoic acid; THETA, 11,12,15-trihydroxyeicosatrienoic acid; HETE, hydroxyeicosatetraenoic acid.

 
Methacholine-induced [14C]AA release from membrane lipids of cultured RA-ECs. We used cultured RA-ECs to study AA release from membrane lipids in response to agonist stimulation. The membrane lipids of RA-ECs were prelabeled by incubating the cells with [14C]AA for 18 h. [14C]AA was incorporated in major lipid fractions in the following proportions (n = 26): PC (22.3 ± 4.9%), PE (23.5 ± 6.5%), PI (12.8 ± 3.0%), MAG (14.2 ± 4.3%), DAG (13.6 ± 3.1%), and triacylglycerol (TAG, 19.7 ± 3.7%). When cells were stimulated by 10–5 M of methacholine from 0 to 30 min, free [14C]AA was released from RA-ECs into the buffer (Fig. 3A). [14C]AA and its 14C-labeled metabolites in the incubation buffer increased within 5 min after methacholine stimulation. At 10 min, the amount of [14C]AA metabolites in the buffer reached a plateau that lasted for 20 min. The cellular lipids were extracted and resolved by TLC. Figure 3 shows that the release of [14C]AA corresponded with the decrease of radioactivity in the membrane lipids. Methacholine reduced [14C]AA in PI, DAG, and MAG but not from PC, PE, or TAG (Fig. 3).



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  		Fig. 3. Time course of AA release from membrane lipids of cultured rabbit aorta endothelial cells (RA-EC). Cells were labeled with [14C]AA for 18 h, washed, and then treated with 10–5 M methacholine. The buffer was collected, and radioactivity was measured. A: release of [14C]AA at 5–30 min of incubation. Cells were collected. The membrane lipids were extracted and resolved by thin-layer chromatography (TLC). The bands comigrating with known lipid standards were scraped, and radioactivity was determined. The major lipid fractions measured are phosphatidylcholine (PC; B), phosphatidylethanolamine (PE; C), phosphatidylinositol (PI; D), monoacylglycerol (MAG; E), diacylglycerol (DAG; F), and triacylglycerol (TAG; G). They are plotted as percentage of total lipids at each time point. Each value represents mean ± SE (n = 6–7). *P < 0.05, **P < 0.01.

 
We studied the effects of the PLC inhibitor U-73122 (10 µM) and its inactive analog U-73343 (10 µM) on methacholine-induced [14C]AA release from RA-ECs (Fig. 4). Cells were pretreated with vehicle (DMSO) or inhibitors followed by 5 min of stimulation with 10–5 M methacholine. Vehicle did not affect [14C]AA release from membrane lipids (data not shown). U-73122, but not U-73343, completely blocked methacholine-induced [14C]AA release from RA-ECs into the buffer (Fig. 4A). With methacholine, there was no significant change in the radioactivity in the PC, PE, or TAG (Fig. 4, B, C, and G). The proportion of radioactivity in PI decreased from 12.4 ± 3.0% to 8.1 ± 1.5% (Fig. 4D), MAG decreased from 15.2 ± 4.6% to 7.4 ± 2.1% (Fig. 4E), and DAG from 13.3 ± 3.4% to 7.9 ± 1.4% (Fig. 4F). U-73122 blocked the methacholine-induced decrease in [14C]AA from PI, DAG, and MAG. In comparison, U-73343 had no effect on [14C]AA release from these lipids. These data suggest that PLC is involved in AA release from PI in RA-ECs.



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  		Fig. 4. Effects of the phospholipase C (PLC) inhibitor U-73122 and its inactive analog U-73343 on [14C]AA release and membrane lipids of cultured RA-ECs. Cells were labeled with [14C]AA for 18 h, then treated with U-73122 (10 µM), U-73343 (10 µM), 10–5 M methacholine (Mech) alone, or methacholine with U-73122 or U-73343. The buffer was collected, and radioactivity was measured (A). Cells were extracted, radioactivity in the major lipid fractions were separated, and radioactivity was measured in PC (B), PE (C), PI (D), MAG (E), DAG (F), or TAG (G). Data were expressed as percentage of total lipids for each group. Each value represents mean ± SE (n = 6–13). *P < 0.05, **P < 0.01, ***P < 0.001.

 
Next, we tested the effect of the DAG lipase inhibitor RHC-80267 (10 µM) (Fig. 5). The [14C]AA in membrane lipids was not affected by RHC-80267 alone. Figure 5A shows that RHC-80267 inhibited methacholine-induced [14C]AA release from RA-ECs. The [14C]AA in the PC and PE fractions was not significantly different compared with control groups (Fig. 5, B and C). [14C]AA in PI, MAG, and DAG were reduced after 5 min incubation with methacholine (Fig. 5,D–F). RHC-80267 completely blocked the reduction in DAG (Fig. 5F) and partially inhibited the reduction in MAG (Fig. 5E). Surprisingly, RHC-80267 completely reversed the decrease in [14C]AA in PI by methacholine (Fig. 5D). We therefore investigated this blockade by examining PI synthesis from [3H]inositol. The incorporation of [3H]inositol into PI was not affected by methacholine stimulation. However, in the presence of RHC-80267 alone, the incorporation of [3H]inositol in PI was increased. This incorporation was greater in the presence of both methacholine and RHC-80267 (Table 1). Thus, when RHC-80267 blocks the degradation of DAG to MAG and AA, a portion of the accumulated DAG is used to synthesize PI. This is consistent with the failure of DAG to increase with RHC-80267 and with RHC-80267 inhibition of methacholine-induced decrease in [14C]AA-labeled PI.



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  		Fig. 5. Effect of the DAG lipase inhibitor RHC-80267 on [14C]AA release and membrane lipids of cultured RA-ECs. Cells were labeled with [14C]AA for 18 h, washed, and then treated with RHC-80267 (10 µM) alone, 10–5 M methacholine alone, or methacholine with RHC-80267 (RHC + Mech). The buffer was collected, and radioactivity was measured (A). The major lipid fractions from the cells are separated as PC (B), PE (C), PI (D), MAG (E), DAG (F), or TAG (G) and are plotted as percentage of total lipids for each group. Each value represents mean ± SE (n = 6–13). **P < 0.01, ***P < 0.001.

 

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  		Table 1. Incorporation of [3H]myoinositol in membrane phosphatidylinositol

 
Figure 6 shows effects of the MAG lipase inhibitor URB-532 (10 µM) on [14C]AA release from RA-ECs. Methacholine increased [14C]AA release into the media (Fig. 6A). It was blocked by URB-532. PC, PE, and TAG were not affected by URB-532 alone or methacholine stimulation (Fig. 6, B, C, and G). URB-532 partially blocked methacholine-induced decrease in [14C]DAG degradation (Fig. 6F) and abolished the decrease in [14C]MAG (Fig. 6E). Interestingly, URB-532 inhibited the methacholine-induced decrease in [14C]PI (Fig. 6D). It is assumed that this is related to DAG conversion to PI as with RHC-80267 (Table 1). These data indicate that in RA-ECs, methacholine releases AA from PI by activating the PLC pathway. This is blocked by PLC, DAG lipase, and MAG lipase/FAAH inhibitors.



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  		Fig. 6. Effect of the MAG lipase/fatty acid amidohydrolase (FAAH) inhibitor URB-532 on AA release and membrane lipids of cultured RA-ECs. Cells were labeled with [14C]AA for 18 h, then treated with URB-532 (10 µM) alone, 10–5 M methacholine alone, or methacholine with URB-532 (URB). The buffer was collected, and radioactivity was measured (A). The major lipid fractions from the cells were collected and shown as PC (B), PE (C), PI (D), MAG (E), DAG (F), or TAG (G) and plotted as percentage of total lipids for each group. Each value represents mean ± SE (n = 6–13). ***P < 0.001.

 
Expression of PLA2 and PLC isoforms in RA-ECs and smooth muscle cells. Specific antibodies were used to identify major PLA2 and PLC isoforms expressed in RA-ECs and RA-smooth muscle cells (RA-SMCs) by immunoblotting (Fig. 7). In both RA-ECs and RA-SMCs, an antibody against human cPLA2 detected a 110-kDa protein, the same molecular weight reported for cPLA2 (14, 24). cPLA2 was expressed at similar amounts in both cell types (Fig. 7A). sPLA2II was not detected in either RA-ECs or RA-SMCs (Fig. 7B). PLC{gamma}1 is universally expressed in most tissues (46), whereas PLC{gamma}2 is an inducible isoform (35). Strong expression of PLC{gamma}1 (140 kDa) appeared in RA-ECs and a much weaker signal was detected in RA-SMCs (Fig. 7C). An antibody against human PLC{gamma}2 did not detect any PLC{gamma}2 expression in either RA-ECs or RA-SMCs (Fig. 7D). The PLC{beta} isoforms, PLC{beta}1 and PLC{beta}2, were both expressed in RA-ECs but not in RA-SMCs (Fig. 7, E and F). These data show the cell-specific expression of some phospholipase isoforms in RA-ECs and RA-SMCs and suggest that PLC{beta} and/or PLC{gamma} are involved in lipid metabolism of the vascular endothelium.



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  		Fig. 7. Expression of PLA2s and PLCs in RA-ECs and rabbit aorta smooth muscle cells (SMCs). Western immunoblots of EC and SMC proteins are shown for cPLA2 (110 kDa; A), sPLA2 II (14 kDa with human sPLA2 II as positive standard; B), PLC{gamma}1 (140 kDa; C), PLC{gamma}2 (150 kDa with rat spleen lysates as positive standard; D), PLC{beta}1 (110 kDa; E), and PLC{beta}2 (110 kDa; F).

 
In summary, our results support the hypothesis that PLC, DAG lipase, and the MAG lipase/FAAH pathway mediate AA release induced by stimulation of muscarinic receptors in RA-ECs.


   DISCUSSION
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 ABSTRACT
 METHODS
 RESULTS
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GRANTS
 REFERENCES
 
In 1980, Furchgott and Zawadzki (21) first described that ACh induced endothelium-dependent relaxation of the rabbit aorta. After 25 years of study, the physiological importance of endothelial cholinergic receptors is not completely understood. Blood vessels receive cholinergic innervation (38), and ECs have cholinergic receptors (2, 53). The density of innervation is greater in large arteries than small arteries (38), However, because the cholinergic nerve terminals are located in the adventitial-medial junction, it is doubtful that released ACh will penetrate the media and reach the endothelium in large arteries. Thus cholinergic nerves are probably not the source of ACh responsible for endothelium-dependent dilation in arteries such as the aorta and other large arteries. However, in other vascular beds such as the adrenal gland, nerve stimulation increases adrenal blood flow, and this increase is blocked by cholinergic antagonists (9). Thus neurally released ACh can induce vasodilation in the adrenal gland. Alternatively, the artery may be the source of ACh. ECs contain choline acetyltransferase and synthesize and release ACh (29, 36). However, the factors that regulate endothelial ACh release are not known, and a role for endogenous endothelial ACh has not been demonstrated. Thus the source of acetylcholine to cause endothelium-dependent relaxation of arteries is not clear.

ACh caused endothelium-dependent relaxation of the rabbit aorta through a NO-mediated mechanism and a mechanism independent of NO and PGs (11, 13, 21). The relaxation to ACh is reduced but not blocked by the NOS and PG synthesis inhibitors, L-NNA and indomethacin (11, 13, 39). In rabbit aortas, ACh elicits synthesis of prostaglandins (26) and other AA metabolites (40) by activating muscarinic receptors (25). We reported that AA metabolites of 15-LO pathways, specifically 11,12,15-THETA and 15-H-11,12-EETA, mediate these L-NNA- and indomethacin-resistant vasorelaxations through opening apamin-sensitive K+ channels (11, 23, 42). The studies suggest that THETA and HEETA serve as EDHFs in rabbit aorta. Free AA from membrane lipids is the substrate for vasodilator eicosanoid synthesis. In many tissues or cells, free AA is released by PLA2 cleavage of the sn-2 bond of PC and PE or by PLC metabolism of PI and subsequent hydrolysis of DAG and MAG (3, 5, 14, 15, 27). In vascular tissues, both PLA2 and PLC pathways are implicated in ACh-induced eicosanoid synthesis. In previous studies, we have shown that PLA2 inhibitors mepacrine and DEDA decreased L-NNA- and indomethacin-resistant relaxations to ACh, but not to AA in rabbit aorta (11). This suggests that PLA2 is involved in AA release in aortic endothelial cells. There is evidence that AA may also be released through the PLC pathway. In guinea pig carotid and rat mesentery arteries, ACh-induced hyperpolarizations were inhibited by PLC inhibitors but not PLA2 inhibitors (20, 44). The PLC-DAG lipase-MAG lipase pathway was also reported to be a source of AA in rat brain (33), murine macrophages and fibrosarcoma cells (4, 31), and human platelets (5). We postulated that this pathway is stimulated by ACh in rabbit aorta and provides AA as a source of vasodilatory eicosanoids. In the present study, we focused on whether the PLC pathway contributes to AA release and vasorelaxation in rabbit aorta on ACh stimulation. Our data indicate that the PLC, DAG-lipase dependent pathway is very important in this process.

The PLC blocker U-73122 completely blocked ACh-induced relaxation. Similar inhibition was seen with the DAG lipase inhibitor RHC-80267 and the MAG lipase/FAAH inhibitor URB-532. AA-induced relaxations were not affected by these inhibitors. These data suggest that PLC, DAG lipase, and MAG lipase inhibitors act downstream of muscarinic receptor activation and upstream of AA metabolism in aortic endothelium. U-73343, an inactive analog of U-73122, did not block AA release by the PLC pathway but decreased AA metabolism by 15-LO in rabbit aorta. Thus both ACh- and AA-induced relaxations were reduced by U-73343. This is a previously unrecognized activity of U-73343 and may limit its usefulness as a control compound in some studies.

To detect AA release from membrane lipids, we labeled the membrane lipids of cultured RA-ECs with [14C]AA. [14C]AA or its 14C-labeled metabolites were released into the media with methacholine treatment. This release was associated with a decrease in radioactivity in PI, DAG, and MAG fractions of RA-ECs. The radioactivity in the PC and PE fractions was not significantly altered by methacholine stimulation. It is reported that PLA2, especially an AA specific cPLA2, releases AA from PC and PE (17, 57). This does not seem to occur in rabbit aorta. PLCs are more specific for PI (14, 27). These data suggest that AA is released from PI by the sequential action of PLC, DAG lipase, and MAG lipase/FAAH. Further experiments show that both [14C]AA release and reductions in [14C]PI, [14C]DAG, and [14C]MAG caused by methacholine stimulation were inhibited by PLC pathway blockers. All these data indicate that the PLC, DAG lipase pathway contributes to AA release and thus vasodilatory eicosanoid synthesis and relaxation to ACh.

There is evidence that stimulation of muscarinic receptors increases PI turnover (32). The DAG formed by PLC activation can be degraded by DAG lipase or phosphorylated by DAG kinase and used to regenerate PI (37). When RHC-80267 was used to block DAG lipase in the presence of methacholine, the accumulated DAG increased PI synthesis. Thus the synthesis of PI is regulated by the cellular concentration of DAG.

Although phenylephrine was used to precontract the aortic rings, we did not investigate its effect on AA release from rabbit aortic ECs because several studies show adrenergic agonists do not alter AA metabolism. In previous studies, norepinephrine did not affect endothelial prostacyclin release (1). Similarly, stimulation of adrenergic receptors increased AA metabolism in rabbit aorta; however, this release was not affected by removal of the endothelium (26). Thus adrenergic agonists increase AA metabolism in SMCs and not ECs. The endothelial {alpha}-adrenergic receptors that mediate endothelium-dependent relaxations are of the {alpha}2-subtype (2). However, the presence of {alpha}2-adrenergic receptors on the aortic endothelium has been questioned (53). Phenylephrine is an {alpha}1-selective adrenergic agonist and will not activate endothelial {alpha}2-adrenergic receptors. Thus it is unlikely that phenylephrine will affect AA release from aortic ECs.

Western immunoblotting with specific antibodies demonstrated the expression of PLC and PLA2 isoforms in rabbit aortic cells. Interestingly, our data indicate different expression patterns of PLA2s and PLCs in the vascular wall. cPLA2 expression was reported in RA-SMCs (18). Our data suggest cPLA2 is expressed in both ECs and SMCs in equal amount. In comparison, PLC{beta}1,2 and PLC{gamma}1 exist mainly in ECs with very little expression in SMCs. This suggests that PLCs may be more important in the rabbit aorta endothelium, while cPLA2s are less tissue specific. In contrast to our findings, PLC{beta} expression has been reported in vascular SMC of the pig, human, and rat (7). PLC{beta} is coupled to many receptors including the muscarinic receptor in a Gq-dependent manner (46). The PLC{gamma} family is activated through the protein kinase pathway (22, 27). DAG lipase and MAG lipase are still not characterized in the rabbit. URB-532 inhibits both MAG lipase and FAAH, so which enzyme contributes to AA release is unknown (54). More research is needed to define the various components of this pathway.

In conclusion, we demonstrated that a PLC-, DAG lipase-, and MAG lipase-dependent pathway mediates the release of free AA from membrane lipids and plays an important regulatory role in ACh-induced vasodilatory eicosanoid synthesis and vasorelaxation.


   GRANTS
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
These studies were supported by grants from the National Heart, Lung, and Blood Institute (HL-37981), the National Institute of General Medical Sciences (GM-31278), and the Robert A. Welch Foundation.


   ACKNOWLEDGMENTS
 
We thank Gretchen Barg for secretarial assistance.


   FOOTNOTES
 

Address for reprint requests and other correspondence: W. B. Campbell, Dept. of Pharmacology and Toxicology, Medical College of Wisconsin, 8701 Watertown Plank Rd., Milwaukee, WI 53226 (e-mail: wbcamp{at}mcw.edu)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


   REFERENCES
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 

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