This Article Abstract Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Weintraub, N. L. Articles by Lonigro, A. J. Search for Related Content PubMed PubMed Citation Articles by Weintraub, N. L. Articles by Lonigro, A. J.

(Hypertension. 1995;26:684-690.)
© 1995 American Heart Association, Inc.

Articles

Relationship of Arachidonic Acid Release to Porcine Coronary Artery Relaxation

Neal L. Weintraub; Alan H. Stephenson; Randy S. Sprague; Lorraine McMurdo; Andrew J. Lonigro

From the Departments of Internal Medicine (N.L.W., R.S.S., L.M., A.J.L.) and Pharmacological and Physiological Science (A.H.S., A.J.L.), St Louis (Mo) University School of Medicine.

Correspondence to Neal L. Weintraub, MD, St Louis University School of Medicine, Division of Clinical Pharmacology, Room M205, 1402 S Grand Blvd, St Louis, MO 63104.

	Abstract

Top Abstract Introduction Methods Results Discussion References

 
Abstract In porcine coronary artery endothelium-dependent relaxation to bradykinin is in part attributed to a chemically unidentified factor, termed endothelium-derived hyperpolarizing factor (EDHF). We hypothesize that arachidonic acid, acting through a cyclooxygenase-independent mechanism, is responsible for EDHF production. To define the relationship between EDHF production and arachidonic acid release, we investigated the role of phospholipase C in bradykinin-induced relaxation and prostaglandin I₂ production (an index of arachidonic acid release) in porcine coronary artery. The phospholipase C inhibitor U73122 (1 µmol/L) abolished bradykinin-induced, nitric oxide–mediated relaxation but did not inhibit either bradykinin-induced, EDHF-mediated relaxation or prostaglandin I₂ production. However, when given at a larger dose (20 µmol/L) U73122 abolished both bradykinin-induced, EDHF-mediated relaxation and prostaglandin I₂ production. Similarly, the calcium-ATPase inhibitor thapsigargin, given at a dose (1 µmol/L) that abolished bradykinin-induced increases in intracellular calcium concentration in cultured porcine coronary artery endothelial cells, eliminated both bradykinin-induced, EDHF-mediated relaxation and prostaglandin I₂ production. Although thapsigargin abolished bradykinin-induced prostaglandin I₂ production, the basal production of prostaglandin I₂ was enhanced, and contraction of endothelium-intact rings was attenuated. These latter responses are most likely related to enhanced basal arachidonic acid release and associated EDHF production. These observations suggest that phospholipase C activation and increased intracellular calcium concentration are required for both bradykinin-induced arachidonic acid release and EDHF production in porcine coronary artery. Moreover, EDHF production in porcine coronary artery appears to be closely associated with arachidonic acid release, thus supporting the hypothesis that arachidonic acid, acting through a cyclooxygenase-independent mechanism, is responsible for EDHF production in porcine coronary artery.

Key Words: endothelium-derived factors • epoprostenol • nitric oxide • phospholipase C • arachidonic acid • arteries • phospholipases A

	Introduction

Top Abstract Introduction Methods Results Discussion References

 
The vascular endothelium produces relaxation of vascular smooth muscle via release of at least two well-characterized autacoids, that is, NO, synthesized from L-arginine by the action of NO synthase,^{1 2 3} and PGI₂, a product of cyclooxygenase-mediated arachidonic acid metabolism.⁴ In the PCA, however, endothelium-dependent relaxation to agonists such as bradykinin is also mediated by an as yet chemically unidentified factor termed EDHF.^{5 6 7 8} This factor is believed to effect relaxation of vascular smooth muscle by opening potassium channels in the smooth muscle membrane, resulting in hyperpolarization and consequently reduced calcium entry through voltage-dependent calcium channels.^{6 7 8} Several endothelium-derived products of arachidonic acid metabolism have been suggested to promote vasorelaxation by such a mechanism.^{9 10 11 12 13}

Previously, we reported that in the PCA arachidonic acid as well as bradykinin in the presence of cyclooxygenase and NO synthase inhibition produced endothelium-dependent relaxations that were abolished by depolarizing concentrations of KCl.¹⁴ In contrast, although the bradykinin-induced, EDHF-mediated relaxations of the PCA were blocked by the nonselective phospholipase inhibitors quinacrine and 4-bromophenacyl bromide, those relaxations elicited by arachidonic acid were not.¹⁴ These observations suggested that PCA relaxations attributed to EDHF might be mediated by a noncyclooxygenase product of arachidonic acid metabolism. However, neither arachidonic acid– nor bradykinin-induced relaxations were abolished by inhibitors of the three known pathways of arachidonic acid metabolism, that is, cyclooxygenase, lipoxygenase, or cytochrome P-450 monooxygenase pathways.^{14 15} Thus, if relaxation responses attributed to EDHF in the PCA were indeed mediated by arachidonic acid, then the responses might have resulted from the production of a heretofore unknown arachidonic acid metabolite or participation of arachidonic acid in a second messenger pathway that influences vascular tone. If, however, the inhibition of bradykinin-induced relaxations by quinacrine and 4-bromophenacyl bromide was related to actions other than blocking of arachidonic acid liberation from tissue stores, then the relationship of arachidonic acid to EDHF production would be unclear.

In the present study, to define the role of arachidonic acid and/or its products in mediating the relaxation response to bradykinin in PCA, we investigated the role of PLC in bradykinin-induced arachidonic acid release (estimated by PGI₂ synthesis) and EDHF-mediated relaxations in PCA rings.

	Methods

Top Abstract Introduction Methods Results Discussion References

 
Measurement of Relaxation Responses
Right coronary arteries were dissected from pig hearts immediately after removal at a local slaughterhouse. The arteries were placed in ice-cold modified KRB solution (composition in mmol/L: NaCl 118.3, KCl 4.7, CaCl₂ 2.5, MgSO₄ 1.2, KH₂PO₄ 1.2, NaHCO₃ 25.0, NaEDTA 0.026, and glucose 11.1) and transported to the laboratory. The arteries, stored at 4°C for a maximum of 3 days, were cut into rings (3 to 5 mm in width) immediately before use. Care was taken not to disturb the endothelium. The rings were mounted onto stainless steel triangles, which were attached by thread to isometric force transducers (model FT-03, Grass Instrument Co) coupled to a polygraph (model 7, Grass) for continuous recording of ring tension. Each ring was affixed to a stationary post and suspended in a water-jacketed (37°C) organ bath (Radnoti Glass Technology, Inc) containing 10 or 20 mL KRB solution that was continuously aerated with 95% O₂/5% CO₂. Basal ring tension was gradually adjusted to 10 g, a value previously shown to yield a maximal contraction to KCl (60 mmol/L).¹⁴ Each ring was repetitively contracted with KCl (60 mmol/L) until a stable tension was achieved. Indomethacin (10 µmol/L) was present in the KRB solution throughout all experiments, except for those in which PGI₂ was used as an estimate of arachidonic acid release from tissue stores. When used, L-NAME (100 µmol/L) was introduced into the bath 45 minutes before the initial contraction and was thereafter included for the duration of the experiment.

Each ring was contracted with either PGF₂(15 to 75 µmol/L), U46619 (a thromboxane mimetic; 7 to 50 nmol/L), or KCl (25 to 35 mmol/L) to achieve 40% to 80% of the maximal contraction obtained with KCl (60 mmol/L). When contraction was stable, the ring was relaxed in a cumulative-dose fashion with bradykinin (0.3 to 100 nmol/L). The bath was rinsed, and after a 30-minute stabilization period the ring was exposed to the PLC inhibitor U73122 (1 to 20 µmol/L)¹⁶ for 20 minutes or the Ca²⁺-ATPase inhibitor thapsigargin (1 µmol/L)¹⁷ for 10 minutes. The contractile agent was then readministered, with the concentration adjusted to achieve a tension similar in degree to that achieved in the previous contraction. Relaxation responses were then repeated. In some experiments control relaxation responses to nitroglycerin (0.03 to 100 µmol/L), arachidonic acid (0.3 to 100 µmol/L), or cromokalim (30 to 1000 nmol/L) (which relaxes smooth muscle by activating potassium channels¹⁸ ) were determined before and after treatment with U73122 or thapsigargin. In some experiments mechanical disruption of the endothelium was achieved by rubbing the rings with a wooden toothpick. Rings were considered denuded of endothelium when maximal relaxation to bradykinin (100 nmol/L) in U46619- or PGF₂-contracted rings was less than 10%.

Relaxation responses were expressed as the percent decrease from the U46619-, PGF₂-, or KCl-induced tension.

Estimation of PGI₂ Production
In separate experiments PCA rings were equilibrated in KRB solution at 10 g basal tension and repetitively contracted with KCl (60 mmol/L) as described above. Indomethacin was not included in the KRB solution for these experiments. The rings were then contracted with U46619 and relaxed with bradykinin (100 nmol/L) to document adequate endothelium-dependent relaxation; rings that relaxed less than 50% were discarded. The baths were rinsed, and 30 minutes later U73122, thapsigargin, or vehicle (DMSO) was introduced. After incubation (20 minutes for U73122, 10 minutes for thapsigargin, and 10 or 20 minutes for DMSO) the KRB solution was replaced with fresh solution containing the same concentration of U73122, thapsigargin, or DMSO. Ten minutes later a 500-µL aliquot of KRB solution was withdrawn from the bath for estimation of basal PGI₂ production, measured as 6-keto-PGF₁, the nonenzymatic hydrolysis product of PGI₂. Bradykinin (100 nmol/L), vehicle (distilled water; control in Figs 1B and 4), or arachidonic acid (10 µmol/L) was then introduced into the bath, and 10 minutes later a second 500-µL aliquot of KRB solution was withdrawn for estimation of bradykinin-induced or control PGI₂ production.

View larger version (34K): [in this window] [in a new window]   Figure 1. A, Line graph shows effects of the PLC inhibitor U73122 on bradykinin-induced, NO-mediated relaxation of PCA rings. Rings were contracted with KCl in the presence of indomethacin (10 µmol/L), and bradykinin-induced relaxations were determined before (control) and after incubation with U73122 (1 µmol/L). B, Bar graph shows effects of U73122 on bradykinin-induced PGI2 release from PCA rings. Rings were pretreated with either vehicle (DMSO), 1 µmol/L U73122, or 20 µmol/L U73122. Bradykinin (100 nmol/L) or its vehicle (control) was then administered. After 10 minutes an aliquot of Krebs' solution was removed for estimation of PGI2 production, measured as 6-keto-PGF1, by enzyme immunoassay. Results were expressed as nanograms 6-keto-PGF1 per gram tissue, corrected for bath volume. All rings were maintained at a resting tension of 10 g. Results are expressed as mean±SEM. *P<.05 vs relaxation before U73122 (A) and vs control PGI2 production (B); P<.05 vs bradykinin-induced PGI2 production from vehicle- and 1 µmol/L U73122–pretreated rings. n=4 (A) and n=5-7 (B).

View larger version (19K): [in this window] [in a new window]   Figure 4. Bar graph shows effects of the Ca2+-ATPase inhibitor thapsigargin on bradykinin-induced PGI2 release from PCA rings. Refer to Fig 1B for details. Because thapsigargin enhanced the basal release of PGI2 (see text), results are expressed as percent increase over basal values (mean±SEM). *P<.05 vs PGI2 release from vehicle-pretreated rings; n=5-7.

Immediately after withdrawal from the bath the aliquot of KRB solution was placed in a 2-mL vial containing 50 µL of a solution of indomethacin (100 mg/mL) and EDTA (0.5 mg/mL) in 0.1 mol/L Na₂CO₃ (pH 7.0). The vial was vortexed and stored at -20°C for analysis. Concentrations of 6-keto-PGF₁ were determined by means of an enzyme-linked immunoassay as previously described.^{15 19} The values were corrected for the volume of solution in the bath and ring wet weight. The effects of U73122 and thapsigargin on basal PGI₂ production were determined by comparing basal 6-keto-PGF₁values among U73122-, thapsigargin-, and vehicle (DMSO)-pretreated rings. The effects of the inhibitors on bradykinin-induced PGI₂ production were determined in U73122-, thapsigargin-, and vehicle (DMSO)-pretreated rings by comparing bradykinin-induced 6-keto-PGF₁ values to vehicle (control)-induced 6-keto-PGF₁ values.

Estimation of [Ca²⁺]_i
Right coronary arteries from pigs were placed in sterile Dulbecco's phosphate-buffered saline containing 2 mL of a solution of penicillin (5000 U/mL), streptomycin (5 mg/mL), and neomycin (10 mg/mL) per 100 mL phosphate-buffered saline and transported to the laboratory. In a laminar flow hood the arteries were incised longitudinally and pinned, intimal surface up, to a Styrofoam board. The intimal surface was scraped gently with a scalpel, which was then immersed in a tube containing Dulbecco's minimum essential medium with 20% fetal calf serum and vigorously agitated. The medium was repetitively pipetted and plated onto 6x24-mm coverslips. The cells were maintained at 37°C in a 5% CO₂ atmosphere and provided with fresh medium every 2 to 3 days. The cells exhibited contact inhibition and stained positively for membrane low density lipoprotein receptors (Biomedical Technologies, Inc).

On reaching confluence the cells were loaded with fura 2-AM (10 µmol/L) in medium containing 10% fetal calf serum and probenecid (5 mmol/L, to prevent fura 2 secretion²⁰ ) at 37°C for 2 to 4 hours. The coverslips were then placed in HEPES-buffered Krebs' solution (composition in mmol/L: NaCl 118, KCl 4.8, MgSO₄ 1, NaHCO₃ 2.4, dextrose 11, CaCl₂ 2.5, and HEPES 10, pH 7.4 with NaOH) for 20 minutes and transferred to a quartz cuvette containing 3 mL fresh Krebs' solution. The cuvette was placed into an Aminco-Bowman series 2 luminescence spectrometer at a 30° angle to the excitation beam, stirred continuously with a magnetic bar, and maintained at 32°C. Fluorescence emission was monitored at 510 nm during dual-wavelength excitation (340 and 380 nm) alternating at 0.5-second intervals. The ratio (R) of fluorescence emission during excitation at 340 and 380 nm was used to estimate [Ca²⁺]_i with the equation of Grynkiewicz et al²¹ : [Ca²⁺]_i=K_d · [(R-R_min)/(R_max-R)] · (S_f2/S_b2), where K_d is the dissociation constant for the fura 2–Ca²⁺ complex; R_min and R_max are the fluorescence ratios obtained in the absence of Ca²⁺ (0 mmol) and in the presence of saturating concentrations (2 mmol) of Ca²⁺, respectively; and S_f2/S_b2 is the fluorescence emission ratio during excitation at 380 nm in 0 versus 2 mmol Ca²⁺. K_d was assumed to be 224 nmol/L²⁰ ; R_min, R_max, and S_f2/S_b2 were determined with a method described by Dolor et al.²²

Chemicals
Bradykinin (acetate salt), arachidonic acid (sodium salt), U46619, PGF₂, indomethacin, L-NAME, cromokalim, thapsigargin, and probenecid were purchased from Sigma Chemical Co. Nitroglycerin was purchased from American Regent Laboratories, Inc; U73122 from Biomol, Inc; and fura 2-AM from Calbiochem, Inc. Indomethacin, PGF₂, and cromokalim were dissolved in ethanol; U46619 in methanol; and U73122, thapsigargin, and fura 2-AM in DMSO. Final bath concentrations of ethanol and DMSO did not exceed 0.1%. All other compounds were dissolved in distilled water.

Statistical Analyses
All data are expressed as mean±SEM. Differences between means were analyzed by Student's t tests for paired or unpaired data, as appropriate. Differences between means of multiple groups were analyzed by ANOVA, with a least significant difference test applied if the F ratio was significant. Probability values of .05 or less were considered to be statistically significant.

	Results

Top Abstract Introduction Methods Results Discussion References

 
Effects of U73122
Bradykinin-induced relaxations of KCl-contracted PCA rings are mediated by NO,^{6 7 14 23 24} the synthesis of which depends on PLC activation (for review see References 25 through 27^{25 26 27} ). Hence, to establish U73122 concentrations that effectively inhibited bradykinin-induced PLC activation, bradykinin-induced relaxations of PCA rings contracted with KCl (25 to 35 mmol/L) were determined before and after treatment with various U73122 concentrations. In KCl-contracted rings exposure to U73122 at 1 µmol/L or greater abolished relaxations produced by bradykinin (0.3 to 100 nmol/L, Fig 1A). In contrast, U73122 concentrations up to 20 µmol/L did not abolish relaxations to nitroglycerin or attenuate contractions to KCl (data not shown).

Basal PGI₂ production from rings pretreated with vehicle (72.9±18.8 ng/g) did not differ from basal PGI₂ production from rings pretreated with U73122 at either 1 or 20 µmol/L (63.5±13.3 and 46.1±9.5 ng/g PGI₂, respectively) (data not shown in figure). In vehicle-pretreated rings a 10-minute exposure to bradykinin (100 nmol/L) resulted in a significant increase in PGI₂ production compared with control values (Fig 1B). This increased PGI₂ production in response to bradykinin was unaffected by pretreatment with 1 µmol/L U73122 (Fig 1B); however, pretreatment with 20 µmol/L U73122 abolished the bradykinin-induced increased PGI₂ production (Fig 1B). In contrast, 20 µmol/L U73122 did not inhibit basal PGI₂ production and did not affect the increased PGI₂ production resulting from arachidonic acid administration (10 µmol/L) (487.6±102.3 [U73122] versus 479.3±78.9 ng/g [vehicle]).

Bradykinin-induced relaxations of PGF₂- or U46619-contracted PCA rings pretreated with NO synthase and cyclooxygenase inhibitors are mediated by EDHF.^{6 7} Hence, to investigate the effects of U73122 on relaxations mediated by EDHF, we determined responses to bradykinin before and after administration of U73122 in PCA rings precontracted with PGF₂ and pretreated with the NO synthase inhibitor L-NAME (100 µmol/L). Bradykinin-induced, EDHF-mediated relaxations were not inhibited by treatment with 1 µmol/L U73122 (Fig 2A). However, exposure of rings to 20 µmol/L U73122 abolished bradykinin-induced (Fig 2B) but not nitroglycerin- (data not shown) or arachidonic acid-induced (Fig 2C) relaxations. Contractions to PGF₂ were not attenuated by 20 µmol/L U73122 (data not shown).

View larger version (16K): [in this window] [in a new window]   Figure 2. Line graphs show effects of U73122 on bradykinin-induced, EDHF-mediated relaxations of PCA rings. Rings were contracted with PGF2 in the presence of L-NAME (100 µmol/L) and indomethacin (10 µmol/L) and relaxed with bradykinin before (control) and after incubation with 1 µmol/L (A) or 20 µmol/L (B) U73122. In C, arachidonic acid–induced relaxations of PCA rings contracted with PGF2 are shown in the presence of L-NAME and indomethacin before (control) and after U73122 (20 µmol/L). Results are expressed as mean±SEM. *P<.05 vs relaxation before U73122. n=4 for all experiments.

Effects of Thapsigargin
The administration of bradykinin (100 nmol/L) to cultured PCA endothelial cells grown to confluence on coverslips and loaded with fura 2-AM resulted in a rapid increase in [Ca²⁺]_i (Fig 3A). The administration of thapsigargin (1 µmol/L) produced a slower increase in [Ca²⁺]_i, which remained persistently elevated after 10 minutes (Fig 3B). The bradykinin-induced increase in [Ca²⁺]_i was abolished by pretreatment with thapsigargin (1 µmol/L) for 10 minutes (Fig 3B).

View larger version (12K): [in this window] [in a new window]   Figure 3. Representative tracings show [Ca2+]i measurements obtained from PCA endothelial cells grown on coverslips and loaded with fura 2-AM. Bradykinin (BK, 100 nmol/L) produced a rapid increase in [Ca2+]i (A) that was abolished by pretreatment with thapsigargin (TG, 1 µmol/L) (B). Thapsigargin produced a slower rise in [Ca2+]i, which remained elevated 10 minutes after administration.

Basal PGI₂ production from thapsigargin-pretreated rings was increased approximately threefold compared with basal production from vehicle-pretreated rings (241.1±51.4 versus 72.9±18.8 ng/g, P<.05). Thus, in rings pretreated with thapsigargin PGI₂ production in response to bradykinin was expressed as the percent increase over basal values.

In vehicle-pretreated rings a 10-minute exposure to bradykinin (100 nmol/L) resulted in increased PGI₂ production compared with control values (Fig 4). This increased PGI₂ production in response to bradykinin was abolished by pretreatment with 1 µmol/L thapsigargin (Fig 4). In contrast, 1 µmol/L thapsigargin did not inhibit PGI₂ production in response to arachidonic acid (10 µmol/L) (670.0±82.5 [thapsigargin] versus 479.3±78.9 ng/g [vehicle]).

In PCA rings pretreated with L-NAME (100 µmol/L), exposure to 1 µmol/L thapsigargin resulted in attenuation of the contractile responses to U46619 (Fig 5A), such that higher concentrations of U46619 were required to produce tensions similar to the prethapsigargin values. This thapsigargin-induced attenuation of contraction in response to U46619 was not observed in endothelium-denuded rings (Fig 5A).

View larger version (16K): [in this window] [in a new window]   Figure 5. A, Bar graph shows contractile responses to U46619 in endothelium (Endo)-intact and endothelium-denuded PCA rings expressed as percent maximal tension (to 60 mmol/L KCl) divided by U46619 concentration. Contractile responses to U46619 were determined before (control) and after a 10-minute incubation with 1 µmol/L thapsigargin. L-NAME and indomethacin were included in all experiments with endothelium-intact but not endothelium-denuded rings. B, Line graph shows effects of thapsigargin on bradykinin-induced, EDHF-mediated relaxations of PCA rings. Rings were contracted with U46619 in the presence of L-NAME and indomethacin and relaxed with bradykinin before (control) and after a 10-minute exposure to thapsigargin (1 µmol/L). C, Line graph shows cromokalim-induced relaxations of endothelium-denuded rings contracted with PGF2 before (control) and after 1 µmol/L thapsigargin. Results are expressed as mean±SEM. *P<.05 vs prethapsigargin concentration of U46619 (A) and vs relaxation before thapsigargin (B). n=4 for all experiments.

After treatment with 1 µmol/L thapsigargin, endothelium-intact, L-NAME–pretreated rings contracted with U46619 failed to relax to bradykinin (Fig 5B), whereas each ring relaxed fully to nitroglycerin and cromokalim (data not shown). Likewise, neither nitroglycerin-induced (data not shown) nor cromokalim-induced (Fig 5C) relaxations of endothelium-denuded rings contracted with U46619 were inhibited by 1 µmol/L thapsigargin.

	Discussion

Top Abstract Introduction Methods Results Discussion References

 
We undertook the present study to investigate the hypothesis that relaxations attributed to EDHF in the PCA are mediated by arachidonic acid or an as yet unidentified product of arachidonic acid metabolism. If this hypothesis is correct, then inhibition of the phospholipase or phospholipases responsible for the bradykinin-induced release of arachidonic acid should also result in inhibition of PGI₂ synthesis as well as inhibition of relaxations mediated by EDHF. Bradykinin can activate PLA₂ and PLC, either or both of which can release arachidonic acid from phospholipid stores.²⁸ It has been reported that in vascular endothelial cells bradykinin-induced stimulation of arachidonic acid release is, like bradykinin-induced stimulation of NO production, dependent on activation of the PLC-phosphoinositol pathway.^{29 30} Activation of PLC results in the release of diacylglycerol, which can liberate arachidonic acid by the action of diacylglycerol lipase.³¹ The increase in [Ca²⁺]_i and activation of protein kinase C, which occur secondary to PLC activation, may activate PLA₂, thereby producing some if not most of the bradykinin-induced arachidonic acid release.^{32 33} However, arachidonic acid release may be independent of PLC activity, resulting from the direct activation of PLA₂ by bradykinin, most likely via direct coupling of receptor–G protein complexes with PLA₂ (for reviews see References 34 through 36^{34 35 36} ). Indeed, in cultured porcine aortic endothelial cells bradykinin-induced activation of PLA₂ was demonstrated to occur independently of PLC activation.³⁷ In the present study incubation with the PLC inhibitor U73122 (20 µmol/L) or the Ca²⁺-ATPase inhibitor thapsigargin (1 µmol/L) prevented the bradykinin-induced production of PGI₂ from PCA rings. Since neither compound inhibited PGI₂ production resulting from the administration of arachidonic acid, the prevention of bradykinin-induced PGI₂ production by U73122 and thapsigargin most likely resulted from inhibition of bradykinin-induced arachidonic acid release. Accordingly, these observations suggest that in the PCA, PLC activation and intracellular Ca²⁺ mobilization are required for the bradykinin-induced release of arachidonic acid.

Incubation of PCA rings with U73122 (20 µmol/L) or thapsigargin (1 µmol/L) also abolished bradykinin-induced relaxation mediated by EDHF. Since neither U73122 (20 µmol/L) nor thapsigargin (1 µmol/L) prevented relaxation to nitroglycerin, the inhibition of bradykinin-induced relaxation most likely did not result from nonspecific interference with vascular smooth muscle relaxation. Moreover, neither U73122 (20 µmol/L) nor thapsigargin (1 µmol/L) inhibited relaxation produced by arachidonic acid or cromokalim, respectively. Since both arachidonic acid and cromokalim produce relaxation through potassium-sensitive mechanisms,^{14 18} these observations suggest that the inhibitors did not abolish bradykinin-induced EDHF-mediated relaxation by blocking potassium channels. Thus, the most likely explanation for the abolition of bradykinin-induced EDHF-mediated relaxation by U73122 and thapsigargin is that these compounds inhibited EDHF production. Accordingly, these observations suggest that PLC activation and intracellular Ca²⁺ mobilization are required for bradykinin-induced EDHF production. The signal transduction mechanisms leading to EDHF production have not been defined; however, a role for Ca²⁺ has been suggested. In rabbit carotid artery, endothelium-dependent hyperpolarization elicited by ATP was partially inhibited by removal of Ca²⁺ from the Krebs' solution.³⁸ In PCA, the Ca²⁺ ionophore A23187 produced endothelium-dependent hyperpolarization of vascular smooth muscle cells,⁷ and in canine coronary artery, endothelium-dependent hyperpolarization and nitro-L-arginine–resistant relaxation produced by bradykinin were attenuated by calmodulin inhibitors.^{39 40} These findings suggest that increases in [Ca²⁺]_i stimulate EDHF production. However, the mechanisms by which increases in [Ca²⁺]_i stimulate EDHF production are unknown.

Thapsigargin not only abolished the production of PGI₂ and the relaxation mediated by EDHF in response to bradykinin, it also stimulated an increase in the basal production of PGI₂. Furthermore, thapsigargin administration in the presence of L-NAME and indomethacin attenuated the contractile response to U46619. Since attenuation of contraction did not occur in endothelium-denuded rings, the most likely explanation for this effect is that thapsigargin stimulated an increase in basal EDHF production. Although this study did not address the mechanisms responsible for the thapsigargin-induced stimulation of PGI₂ and EDHF production, a likely explanation is the sustained increase in [Ca²⁺]_i (Fig 4) induced by thapsigargin.^{22 30} Thapsigargin induces a two-component increase in [Ca²⁺]_i, with the early component resulting from Ca²⁺ mobilization from intracellular stores and the late component resulting from enhanced Ca²⁺ influx, apparently consequent to depletion of intracellular Ca²⁺ stores.²² Thapsigargin concentrations similar to that used in this study have been shown to prevent refilling of the Ca²⁺ stores, thereby resulting in the sustained enhancement of Ca²⁺ influx²² and an increase in the basal production of NO and PGI₂.³⁰ It is likely that this sustained Ca²⁺ influx was also responsible for the enhanced production of both PGI₂ and EDHF observed in the present study. These observations confirm the importance of Ca²⁺ in the production of both PGI₂ and EDHF in the PCA.

In the present study U73122 (1 µmol/L) abolished bradykinin-induced relaxation of PCA rings contracted with KCl in the presence of indomethacin. Since under these experimental conditions bradykinin-induced relaxation is mediated entirely by NO,^{6 7 14 23 24} this observation suggests that U73122 (1 µmol/L) inhibited the production and/or interfered with the action of NO. Considering that U73122 concentrations of up to 20 µmol/L did not abolish nitroglycerin-induced relaxation of KCl-contracted rings, the compound most likely abolished bradykinin-induced relaxation at least in part by inhibiting NO production. In contrast, U73122 (1 µmol/L) did not inhibit either bradykinin-induced PGI₂ production or EDHF-mediated relaxation, whereas a higher concentration of the compound (20 µmol/L) abolished both. The reason why a higher concentration of U73122 was required to abolish bradykinin-induced PGI₂ production and EDHF-mediated relaxation than was required to abolish bradykinin-induced, NO-mediated relaxation is not clear. It is possible that differences in experimental conditions could have accounted for these observations. Alternatively, it is possible that the differential inhibition observed with U73122 resulted from the participation of more than one PLC isoform in the bradykinin-induced production of NO, PGI₂, and EDHF in PCA. Indeed, the existence of multiple structurally and functionally distinct PLC isoforms within other cell types has been reported.^{41 42} Since all PLC isoforms are not equally inhibitable by U73122,⁴³ it is possible that the PLC isoform or isoforms responsible for bradykinin-induced NO production are more sensitive to U73122 inhibition than the isoform or isoforms responsible for bradykinin-induced PGI₂ and EDHF production. Finally, the higher U73122 concentration could have abolished bradykinin-induced PGI₂ production and EDHF-mediated relaxation through mechanisms distinct from phospholipase inhibition and release of arachidonic acid (ie, nonspecific effects). Indeed, U73122 concentrations of greater than 1 µmol/L were reported to inhibit GTPase activity of polymorphonuclear neutrophil membranes.⁴⁴ However, the observations that U73122 (20 µmol/L) did not inhibit arachidonic acid–induced PGI₂ production or arachidonic acid–induced relaxation of PCA rings suggest that such nonspecific effects were not likely to have accounted for the observed inhibition.

In summary, we found that bradykinin-induced production of PGI₂ and relaxation mediated by EDHF in the PCA were abolished by the PLC inhibitor U73122 and the Ca²⁺-ATPase inhibitor thapsigargin. These observations suggest that both arachidonic acid release and EDHF production in the PCA depend on PLC activation and intracellular Ca²⁺ mobilization. These findings support the hypothesis that relaxations attributed to EDHF in the PCA are mediated by arachidonic acid or an as yet unidentified arachidonic acid metabolite.

	Selected Abbreviations and Acronyms

 

[Ca2+]i = intracellular Ca2+ concentration DMSO = dimethyl sulfoxide EDHF = endothelium-derived hyperpolarizing factor KRB = Krebs-Ringer bicarbonate L-NAME = N-nitro-L-arginine methyl ester NO = nitric oxide PCA = porcine coronary artery PGI2, PGF2, PGF1 = prostaglandin I2, F2, F1 PLC, PLA2 = phospholipase C, A2

	Acknowledgments

 
This work was supported in part by National Heart, Lung, and Blood Institute Specialized Center of Research grant HL-30572. N.L.W. was supported by a National Research Service Award Post-Doctoral Research Training grant (2 T 32 HL-07050-18). The authors gratefully acknowledge the invaluable assistance of Jo Schreiweis in carrying out the experiments.

Received April 12, 1995; first decision May 15, 1995; accepted July 10, 1995.

	References

Top Abstract Introduction Methods Results Discussion References

 
1. Furchgott RF, Zawadzki JV. The obligatory role of endothelial cells in the relaxation of arterial smooth muscle by acetylcholine. Nature. 1980;288:373-376. [Medline] [Order article via Infotrieve]

2. Palmer R, Ferrige A, Moncada S. Nitric oxide release accounts for the biological activity of endothelium-derived relaxing factor. Nature. 1987;327:524-526. [Medline] [Order article via Infotrieve]

3. Ignarro LJ, Byrns RE, Buga GM, Woods KS. Endothelium-derived relaxing factor from pulmonary artery and vein possesses pharmacologic and chemical properties identical to those of nitric oxide radical. Circ Res. 1987;61:866-879. [Abstract/Free Full Text]

4. Moncada S, Gryglewski R, Bunting S, Vane JR. An enzyme isolated from arteries transforms prostaglandin endoperoxides into an unstable substance that inhibits platelet aggregation. Nature. 1976;263:663-665. [Medline] [Order article via Infotrieve]

5. Beny J-L, Brunet PC. Neither nitric oxide nor nitroglycerin accounts for all the characteristics of endothelially mediated vasodilation of pig coronary arteries. Blood Vessels. 1988;25:308-311. [Medline] [Order article via Infotrieve]

6. Cowan CL, Cohen RA. Two mechanisms mediate relaxation by bradykinin of pig coronary artery: NO-dependent and -independent responses. Am J Physiol. 1991;261:H830-H835. [Abstract/Free Full Text]

7. Nagao T, Vanhoutte PM. Hyperpolarization as a mechanism for endothelium-dependent relaxations of the porcine coronary artery. J Physiol (Lond). 1992;445:355-367. [Abstract/Free Full Text]

8. Von der Weid P-Y, Beny J-L. Effect of Ca²⁺ ionophores on membrane potential of pig coronary artery endothelial cells. Am J Physiol. 1992;262:H1823-H1831. [Abstract/Free Full Text]

9. Pinto A, Abraham NG, Mullane KM. Arachidonic acid-induced endothelial-dependent relaxations of canine coronary arteries: contribution of a cytochrome P-450-dependent pathway. J Pharmacol Exp Ther. 1987;240:856-863. [Abstract/Free Full Text]

10. Pfister SL, Campbell WB. Arachidonic acid– and acetylcholine-induced relaxations of rabbit aorta. Hypertension. 1992;20:682-689. [Abstract/Free Full Text]

11. Rosolowsky M, Campbell WB. Role of PGI₂ and epoxyeicosatrienoic acids in relaxation of bovine coronary arteries to arachidonic acid. Am J Physiol. 1993;264:H327-H335. [Abstract/Free Full Text]

12. Gebremedhin D, Ma Y-H, Falck JR, Roman RJ, VanRollins M, Harder DR. Mechanism of action of cerebral epoxyeicosatrienoic acids on cerebral arterial smooth muscle. Am J Physiol. 1992;263:H519-H525. [Abstract/Free Full Text]

13. Jackson WF, Konig A, Dambacher T, Busse R. Prostacyclin-induced vasodilation in rabbit heart is mediated by ATP-sensitive potassium channels. Am J Physiol. 1993;264:H238-H243. [Abstract/Free Full Text]

14. Weintraub NL, Joshi SN, Branch CA, Stephenson AH, Sprague RS, Lonigro AJ. Relaxation of porcine coronary artery to bradykinin: role of arachidonic acid. Hypertension. 1994;23(part 2):976-981.

15. Lonigro AJ, Weintraub NL, Branch CA, Stephenson AH, McMurdo L, Sprague RS. Endothelium-dependent relaxation to arachidonic acid in porcine coronary artery: is there a fourth pathway? Pol J Pharmacol.. 1994;46:567-577. [Medline] [Order article via Infotrieve]

16. Bleasdale JE, Bundy GL, Bunting S, Fitzpatrick FA, Huff RM, Sun FF, Pike JE. Inhibition of phospholipase C dependent processes by U-73,122. In: Samuelsson P, Wong PY-K, Sun FF, eds. Advances in Prostaglandin, Thromboxane, and Leukotriene Research. New York, NY: Raven Press Publishers; 1989:590-593.

17. Thastrup O, Cullen PJ, Drobak BK, Hanley MR, Dawson AP. Thapsigargin, a tumor promoter, discharges intracellular Ca²⁺ stores by specific inhibition of the endoplasmic reticulum Ca²⁺-ATPase. Proc Natl Acad Sci U S A.. 1990;87:2466-2470. [Abstract/Free Full Text]

18. Weston AH. Smooth muscle K⁺ channel openers: their pharmacology and clinical potential. Pflugers Arch. 1989;414(suppl 1):S99-S105.

19. Pradelles P, Grassi J, Maclouf J. Enzyme immunoassays of eicosanoids using acetylcholine esterase as label: an alternative to radioimmunoassay. Anal Chem. 1985;57:1170-1173. [Medline] [Order article via Infotrieve]

20. Di Virgilio F, Steinberg TH, Silverstein SC. Inhibition of fura-2 sequestration and secretion with organic anion transport blockers. Cell Calcium. 1990;11:57-62. [Medline] [Order article via Infotrieve]

21. Grynkiewicz G, Poenie M, Tsien RY. A new generation of Ca²⁺_i indicators with greatly improved fluorescence properties. J Biol Chem.. 1985;260:3440-3450. [Abstract/Free Full Text]

22. Dolor RJ, Hurwitz LM, Mizra Z, Strauss HC, Whorton AR. Regulation of extracellular Ca²⁺ entry in endothelial cells: role of intracellular Ca²⁺ pool. Am J Physiol. 1992;262:C171-C181. [Abstract/Free Full Text]

23. Kilpatrick EV, Cocks TM. Evidence for differential roles of nitric oxide (NO) and hyperpolarization in endothelium-dependent relaxation of pig isolated coronary artery. Br J Pharmacol. 1994;112:557-565. [Medline] [Order article via Infotrieve]

24. Holzmann S, Kukovetz WR, Windischhofer W, Paschke E, Graier WF. Pharmacologic differentiation between endothelium-dependent relaxations sensitive and resistant to nitro-L-arginine in coronary arteries. J Cardiovasc Pharmacol. 1994;23:747-756. [Medline] [Order article via Infotrieve]

25. Dinerman JL, Lowenstein CJ, Snyder SH. Molecular mechanisms of nitric oxide regulation: potential relevance to cardiovascular disease. Circ Res. 1993;73:217-222. [Free Full Text]

26. Busse R, Mulsch A, Fleming I, Hecker M. Mechanisms of nitric oxide release from the vascular endothelium. Circulation. 1993;87(suppl V):V-18-V-25.

27. Himmel HM, Whorton AR, Strauss HC. Intracellular calcium, currents, and stimulus-response coupling in endothelial cells. Hypertension. 1993;21:112-127. [Abstract/Free Full Text]

28. Irvine RF. How is the level of free arachidonic acid controlled in mammalian cells? Biochem J. 1982;204:3-16. [Medline] [Order article via Infotrieve]

29. De Nucci G, Gryglewski RJ, Warner TD, Vane JR. Receptor-mediated release of endothelium-derived relaxing factor and prostacyclin from bovine aortic endothelial cells is coupled. Proc Natl Acad Sci U S A. 1988;85:2334-2338. [Abstract/Free Full Text]

30. Macarthur H, Hecker M, Busse R, Vane JR. Selective inhibition of agonist-induced but not shear stress-dependent release of endothelial autacoids by thapsigargin. Br J Pharmacol. 1993;108:100-105. [Medline] [Order article via Infotrieve]

31. Bell RL, Kennerly DA, Stanford N, Majerus PW. Diglyceride lipase: a pathway for arachidonic acid release from human platelets. Proc Natl Acad Sci U S A. 1979;76:3238-3241. [Abstract/Free Full Text]

32. Parker J, Daniel LW, Waite M. Evidence of protein kinase C involvement in phorbol diester-stimulated arachidonic acid release and prostaglandin synthesis. J Biol Chem. 1987;262:5385-5393. [Abstract/Free Full Text]

33. Rittenhouse SE. Activation of human platelet phospholipase C by ionophore A23187 is totally dependent upon cyclo-oxygenase products and ATP. Biochem J. 1984;222:103-110. [Medline] [Order article via Infotrieve]

34. Axelrod J. Receptor-mediated activation of phospholipase A₂ and arachidonic acid release in signal transduction. Biochem Soc Trans. 1990;18:503-507. [Medline] [Order article via Infotrieve]

35. Bereziat G, Etienne J, Kokkinidis M, Olivier JL, Pernas P. New trends in mammalian non-pancreatic phospholipase A₂ research. J Lipid Mediat. 1990;2:159-172. [Medline] [Order article via Infotrieve]

36. Cockcroft S, Nielson CP, Stutchfield J. Is phospholipase A₂ activation regulated by G-proteins? Biochem Soc Trans. 1991;19:333-336. [Medline] [Order article via Infotrieve]

37. Kaya H, Patton GM, Hong SL. Bradykinin-induced activation of phospholipase A₂ is independent of the activation of polyphosphoinositide-hydrolyzing phospholipase C. J Biol Chem. 1989;264:4972-4977. [Abstract/Free Full Text]

38. Chen G, Suzuki H. Endothelium-dependent hyperpolarization elicited by adenine compounds in rabbit carotid artery. Am J Physiol.. 1991;260:H1037-H1042. [Abstract/Free Full Text]

39. Nagao T, Illiano S, Vanhoutte PM. Calmodulin antagonists inhibit endothelium-dependent hyperpolarization in the canine coronary artery. Br J Pharmacol. 1992;107:382-386. [Medline] [Order article via Infotrieve]

40. Illiano S, Nagao T, Vanhoutte PM. Calmidazolium, a calmodulin inhibitor, inhibits endothelium-dependent relaxations resistant to nitro-L-arginine in the canine coronary artery. Br J Pharmacol. 1992;107:387-392. [Medline] [Order article via Infotrieve]

41. Rhee SG, Choi KD. Multiple forms of phospholipase C isozymes and their activation mechanisms. In: Putney JW Jr, ed. Advances in Second Messenger and Phosphoprotein Research. New York, NY: Raven Press Publishers; 1992;26:35-60.

42. Majerus PW, Ross TS, Cunningham TW, Caldwell KK, Bennett Jefferson A, Banasl VS. Recent insights in phosphatidylinositol signaling. Cell. 1990;63:459-465. [Medline] [Order article via Infotrieve]

43. 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. [Abstract/Free Full Text]

44. 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.[Abstract/Free Full Text]

This article has been cited by other articles:

				M. A. Carroll, A. B. Doumad, J. Li, M. K. Cheng, J. R. Falck, and J. C. McGiff Adenosine2A receptor vasodilation of rat preglomerular microvessels is mediated by EETs that activate the cAMP/PKA pathway Am J Physiol Renal Physiol, July 1, 2006; 291(1): F155 - F161. [Abstract] [Full Text] [PDF]

				L. L. Stoll, G. M. Denning, W.-G. Li, J. B. Rice, A. L. Harrelson, S. A. Romig, S. T. Gunnlaugsson, F. J. Miller Jr, and N. L. Weintraub Regulation of Endotoxin-Induced Proinflammatory Activation in Human Coronary Artery Cells: Expression of Functional Membrane-Bound CD14 by Human Coronary Artery Smooth Muscle Cells J. Immunol., July 15, 2004; 173(2): 1336 - 1343. [Abstract] [Full Text] [PDF]

				Y. Zhang, T. Tazzeo, S. Hirota, and L. J. Janssen Vasodilatory and Electrophysiological Actions of 8-iso-Prostaglandin E2 in Porcine Coronary Artery J. Pharmacol. Exp. Ther., June 1, 2003; 305(3): 1054 - 1060. [Abstract] [Full Text] [PDF]

				S. P. Marrelli Mechanisms of endothelial P2Y1- and P2Y2-mediated vasodilatation involve differential [Ca2+]i responses Am J Physiol Heart Circ Physiol, October 1, 2001; 281(4): H1759 - H1766. [Abstract] [Full Text] [PDF]

				M. H. Zink, C. L. Oltman, T. Lu, P. V. G. Katakam, T. L. Kaduce, H.-C. Lee, K. C. Dellsperger, A. A. Spector, P. R. Myers, and N. L. Weintraub 12-Lipoxygenase in porcine coronary microcirculation: implications for coronary vasoregulation Am J Physiol Heart Circ Physiol, February 1, 2001; 280(2): H693 - H704. [Abstract] [Full Text] [PDF]

				Y. Qiu and J. Quilley Vascular effects of arachidonic acid in the rat perfused heart: role of the endothelium, cyclooxygenase, cytochrome P450, and K+ channels J. Lipid Res., December 1, 1999; 40(12): 2177 - 2184. [Abstract] [Full Text] [PDF]

				N. L. Weintraub, X. Fang, T. L. Kaduce, M. VanRollins, P. Chatterjee, and A. A. Spector Epoxide hydrolases regulate epoxyeicosatrienoic acid incorporation into coronary endothelial phospholipids Am J Physiol Heart Circ Physiol, November 1, 1999; 277(5): H2098 - H2108. [Abstract] [Full Text] [PDF]

				S. G. Clark and L. C. Fuchs Role of Nitric Oxide and Ca++-Dependent K+ Channels in Mediating Heterogeneous Microvascular Responses to Acetylcholine in Different Vascular Beds J. Pharmacol. Exp. Ther., September 1, 1997; 282(3): 1473 - 1479. [Abstract] [Full Text]

				N. L. Weintraub, X. Fang, T. L. Kaduce, M. VanRollins, P. Chatterjee, and A. A. Spector Potentiation of Endothelium-Dependent Relaxation by Epoxyeicosatrienoic Acids Circ. Res., August 19, 1997; 81(2): 258 - 267. [Abstract] [Full Text]

				J. Bauersachs, R. Popp, M. Hecker, E. Sauer, I. Fleming, and R. Busse Nitric Oxide Attenuates the Release of Endothelium-Derived Hyperpolarizing Factor Circulation, December 15, 1996; 94(12): 3341 - 3347. [Abstract] [Full Text]

				S. V. Smirnov and P. I. Aaronson Modulatory Effects of Arachidonic Acid on the Delayed Rectifier K+ Current in Rat Pulmonary Arterial Myocytes: Structural Aspects and Involvement of Protein Kinase C Circ. Res., July 1, 1996; 79(1): 20 - 31. [Abstract] [Full Text]

				K. B. Brosnihan, P. Li, and C. M. Ferrario Angiotensin-(1-7) Dilates Canine Coronary Arteries Through Kinins and Nitric Oxide Hypertension, March 1, 1996; 27(3): 523 - 528. [Abstract] [Full Text]

This Article Abstract Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Weintraub, N. L. Articles by Lonigro, A. J. Search for Related Content PubMed PubMed Citation Articles by Weintraub, N. L. Articles by Lonigro, A. J.