Bovine coronary arteries relax in response to bradykinin, methacholine, sodium nitroprusside, isoproterenol, and arachidonic acid in a concentration-dependent manner. The relaxations to methacholine, bradykinin, and arachidonic acid are lost when endothelium is removed. Indomethacin, a cyclooxygenase inhibitor, attenuated the relaxations to methacholine, bradykinin, and arachidonic acid and shifted the EC50 (control versus indomethacin) to each (1×10−7 versus 3×10−7 mol/L, 3×10−10 versus 2×10−9 mol/L, and 3×10−7 versus 2×10−6 mol/L, respectively). Nitro-l-arginine, a nitric oxide synthase inhibitor, also attenuated the relaxations to methacholine, bradykinin, and arachidonic acid and shifted the EC50 (control versus nitro-l-arginine) to each (1×10−7 versus 3×10−7 mol/L, 3×10−10 versus >10−9 mol/L, and 3×10−7 versus >10−6 mol/L, respectively). The combination of indomethacin and nitro-l-arginine blunted the relaxations to these agents and also shifted the EC50 values (control versus indomethacin plus nitro-l-arginine) to each (1×10−7 versus 5×10−7 mol/L, 3×10−10 versus >10−9 mol/L, and 3×10−7 versus >10−6 mol/L, respectively). Methacholine, bradykinin, and arachidonic acid stimulated the release of prostaglandin I2, measured as 6-keto-PGF1α. Indomethacin, but not nitro-l-arginine, inhibited arachidonic acid–induced release of 6-keto-PGF1α. Vascular cGMP content was unchanged by arachidonic acid but was significantly elevated by bradykinin. Relaxations to prostaglandin I2 and sodium nitroprusside, but not 8,9-epoxyeicosatrienoic acid or isoproterenol, were inhibited by nitro-l-arginine. We conclude that the endothelium-dependent relaxations to methacholine, bradykinin, and arachidonic acid are partly due to prostaglandin I2 release. The remainder of the responses to these agents is due to the release of other relaxing factor or factors. Since bradykinin increased cGMP and nitro-l-arginine partially inhibited its relaxant effects, nitric oxide also appears to participate in the bradykinin-induced effect. Since the combination of indomethacin and nitro-l-arginine failed to completely block the relaxations to methacholine, bradykinin, and arachidonic acid, another endothelial factor must contribute to their vascular effects. Surprisingly, nitro-l-arginine attenuated the relaxations to arachidonic acid; however, l-arginine failed to reverse the effects of nitro-l-arginine on arachidonic acid–induced relaxations. In addition, arachidonic acid failed to increase cGMP. Nitro-l-arginine also reduced the responses to prostaglandin I2 and sodium nitroprusside. These data indicate that these arginine analogues may have effects other than competitive inhibition of nitric oxide synthase.
Substances such as acetylcholine, bradykinin, ATP, substance P, and the calcium ionophore A23187 have been shown to relax blood vessels through the release of EDRFs.1 The importance of the endothelium in regulating vascular tone as well as its role in regulating hemostasis and inflammation has generated a great deal of interest in determining the exact mechanisms involved in this regulation. Both PGI2, a cyclooxygenase metabolite of arachidonic acid, and NO represent endothelium-derived vasodilators that mediate agonist-induced dilation.2 3 4 5 6 7 8 However, there is debate as to the possible existence of other EDRFs.9 10 11 12 13
The control of coronary vascular tone and the role of the endothelium in this regulation is generally examined with the use of isolated rings or strips of vessel.14 15 The participation of the endothelium in agonist-induced relaxation is determined by removal of the endothelium physically or chemically. Thus, agents may be classified as being either endothelium-dependent or -independent. With the discovery that l-arginine is the precursor of NO,16 several l-arginine analogues have been found to be competitive inhibitors of NO synthase.17 These l-arginine analogues are used for determination of the involvement of NO in many agonist-induced vasorelaxant effects; however, some reports have indicated the apparent nonspecificity of these agents.18 19 20
Arachidonic acid also causes an endothelium-dependent relaxation of coronary arteries.21 Previous reports indicate that PGI2 and a cytochrome P-450 metabolite or metabolites mediate these relaxations.12 The latter metabolite(s) appear to be EETs. While examining arachidonic acid–induced relaxations, we found that L-NA was capable of attenuating these relaxations. This would suggest a possible involvement of NO in mediating this effect. Alternatively, it could also suggest a role for NO in modulating the synthesis and/or release of PGI2 or the EETs. Therefore, in the present study we examined further the mediators of arachidonic acid–induced relaxation in bovine coronary arteries. Since agents such as methacholine and bradykinin can exert some of their effects by the release of arachidonic acid and its subsequent conversion to vasoactive metabolites, we compared the relaxations to arachidonic acid with the relaxations in response to these agonists. Our results fail to show a contribution by NO to the relaxations induced by arachidonic acid, but we did observe a contribution by PGI2 and some other endothelial factor. Although L-NA was effective in attenuating the relaxations to arachidonic acid, sodium nitroprusside, and to some extent PGI2, it did not affect relaxations to isoproterenol. This attenuation may reflect some as yet unidentified effect of L-NA.
Bovine hearts (2 to 4 kg) were obtained in saline at ambient temperature from a local slaughterhouse. The epicardial left anterior descending coronary artery was dissected, cleaned of adhering fat and connective tissue, and placed in a Krebs' bicarbonate solution containing (mmol/L) NaCl 119, KCl 5, NaHCO3 24, KH2PO4 1.2, MgSO4 1.2, glucose 11, EDTA 0.02, and CaCl2 3.2. The vessels were cut into rings, with care taken not to damage the endothelium. In some vessels, the endothelium was deliberately removed by gentle rubbing of the lumen with forceps. The rings were suspended on a pair of stainless steel hooks in a 15-mL water-jacketed organ chamber. One hook was anchored to a steel rod and the other was attached to a force transducer (model FT-03C, Grass Instrument Co). Tension was recorded on a polygraph (model 7D, Grass). The organ chamber was filled with Krebs' bicarbonate solution that was bubbled with 95% O2/5% CO2 and maintained at 37°C. The vessels were challenged with repeated exposures to 20 mmol/L KCl and progressive increases in basal tension for determination of optimal resting tension. This tension was found to be 8 g for vessels 4 mm in diameter. After the vessels equilibrated for 1.5 hours, KCl (40 mmol/L) was added until reproducible contractions were obtained. The thromboxane mimetic U46619 (2×10−8 mol/L) was then administered to increase basal tone to approximately 50% of maximum. U46619 was selected as the precontracting agent because it produced reproducible, sustained contractions that did not fade with time. Cumulative additions of methacholine (10−9 to 10−5 mol/L), bradykinin (10−12 to 5×10−9 mol/L), arachidonic acid (10−9 to 10−5 mol/L), PGI2 (10−9 to 10−5 mol/L), 8,9-EET (10−9 to 10−5 mol/L), isoproterenol (10−9 to 10−5 mol/L), or sodium nitroprusside (10−9 to 10−5 mol/L) were made. To establish the mechanisms of relaxation, we treated the same vessels with indomethacin (1×10−5 mol/L), L-NA (3×10−5 mol/L), or a combination of L-NA and indomethacin, and repeated the concentration-response curves to the agonists. To examine the specificity of L-NA on arachidonic acid–induced relaxations, we pretreated vessels with either vehicle or l-arginine (1×10−4 mol/L) for 45 minutes and added either vehicle or L-NA (3×10−5 mol/L) for 10 minutes before the addition of U46619. Results are expressed as percent relaxation (relative to the U46619 contraction), with 100% relaxation representing the basal, pre-U46619 tension, which was 8 g. Occasionally, the tension of the vessel fell below basal tension after treatment with high concentrations of bradykinin. Therefore, maximal relaxation was greater than 100%.
Determination of cGMP in Bovine Coronary Arteries
Rings of bovine coronary arteries were mounted in organ chambers as described above. After the vessels were precontracted with U46619, they were treated with either vehicle, bradykinin (10−9 mol/L), arachidonic acid (10−5 mol/L), or isoproterenol (10−7 mol/L). At the point of maximal relaxation, typically between 1 minute for bradykinin and isoproterenol and 2 minutes for arachidonic acid, the organ chambers were lowered and the rings freeze-clamped. cGMP content was measured by the method of Katsuki and Murad.22 Radioimmunoassays for cGMP were performed with a commercial kit purchased from Advanced Magnetics Inc.
Metabolism of [14C]Arachidonic Acid by Bovine Coronary Arteries
Vessels were dissected from bovine hearts as described above and cut into rings. Rings were placed into 1 mL of a HEPES buffer containing (mmol/L) HEPES 10, NaCl 149, KCl 5, CaCl2 1.8, MgCl2 1.0, and glucose 5.5 and pretreated for 15 minutes with L-NA (3×10−5 mol/L), indomethacin (10−5 mol/L), a combination of both inhibitors, or vehicle. Then, 0.5 μCi (10−7 mol/L) of [14C(U)]arachidonic acid (1 Ci/mmol; New England Nuclear) and 2×10−8 mol/L U46619 were added, and the incubation was continued for 10 minutes. At the end of the incubation period, the incubation medium was decanted and extracted (see below).
Extraction and Chromatographic Methods
For isolation of the metabolites of [14C]arachidonic acid that were synthesized by the blood vessel, incubation medium was acidified to pH 3.0 with glacial acetic acid and treated with ethanol to a final concentration of 15%. The solution was extracted over an octadecylsilyl extraction column (Analytichem), and the metabolites were eluted with ethyl acetate, as previously described.23 The organic phases were pooled and dried under a stream of nitrogen.
Extracts were dissolved in acetonitrile and chromatographed on a reversed-phase high-performance liquid chromatographic system with a Nucleosil C18 column (5 μm, 4.6×250 mm, Phenomenex). The solvent program consisted of solvent A, which was distilled water, and solvent B, which contained acetonitrile/glacial acetic acid (999:1). A linear gradient from 50% solvent B in solvent A to 100% solvent B over 40 minutes was used at a flow rate of 1 mL/min. The effluent was collected in 0.2-mL fractions and mixed with scintillation fluid, and radioactivity was measured by liquid scintillation spectrometry to obtain a profile of radioactive metabolites.
In some experiments, aliquots of the buffer were removed from the organ chamber before and after administration of methacholine or bradykinin for measurement of the release of PGI2 by bovine coronary artery rings. Radioimmunoassays for 6-keto-PGF1α were performed on the buffer as previously described with the use of antibodies produced in our laboratory.24
Bovine coronary artery endothelial cells were cultured onto six-well plates as previously described.23 The cells were washed with HEPES buffer and then incubated in 3 mL buffer containing L-NA (3×10−5 mol/L) or vehicle for 10 minutes at 37°C. Then, either bradykinin (10−6 mol/L) or arachidonic acid (10−5 mol/L) was added, and the incubation was continued for 2 hours. After incubation, the supernatant was removed for nitrite measurement, and the cells were frozen for protein determination. Nitrite was measured by the Greiss reaction with a multichannel flow injection analyzer (Lachat Instruments, Inc) that included an in-line cadmium column for reduction of nitrate to nitrite.25 26 27 Since nitrate was converted to nitrite, the results are expressed as total nitrite (picomoles per milligram protein). Protein was measured by the method of Lowry et al.28
Determination of EC50
EC50 values were determined graphically with the maximal relaxation for each agonist expressed as 100%. With arachidonic acid, the highest concentration could not exceed 10−5 mol/L because of the detergent-like effects of this agent.
Statistical analysis was performed by an ANOVA for determination of significant differences among groups followed by Dunnett's multiple comparison test for determination of differences between groups. A value of P<.05 was considered statistically significant.
PGI2 was purchased from Caymen Chemicals Co and L-NA from Eastman Kodak. 8,9-EET was prepared as previously described.12 Methacholine, bradykinin, isoproterenol, sodium nitroprusside, and most of the other chemicals were purchased from Sigma Chemical Co. [3H]6-Keto-PGF1α and [14C(U)]arachidonic acid were obtained from New England Nuclear. Advanced Magnetics Inc was the source of the cGMP radioimmunoassay kit.
Bovine coronary arteries relaxed to both methacholine and bradykinin in a concentration-dependent manner (Fig 1⇓). Maximal relaxations to methacholine and bradykinin occurred at 1×10−6 and 5×10−9 mol/L, respectively. The EC50 values for methacholine and bradykinin were 1×10−7 and 3×10−10 mol/L, respectively. Endothelium removal eliminated these relaxation responses (data not shown). Although pretreatment with indomethacin had no effect on basal tone, we frequently saw increases in basal tone with L-NA pretreatment (40±9%). Pretreatment with L-NA (3×10−5 mol/L) or indomethacin (1×10−5 mol/L) significantly attenuated the relaxations to methacholine and bradykinin (Fig 1⇓). The combination of indomethacin and L-NA further blocked the relaxations to methacholine but produced no greater effect than either inhibitor alone with bradykinin.
Arachidonic acid also relaxed bovine coronary arteries in a concentration-dependent manner (Fig 2⇓). Maximal relaxations to arachidonic acid occurred at 1×10−5 mol/L, with an EC50 of 3×10−7 mol/L. These relaxations were significantly attenuated after pretreatment with L-NA, and the concentration-response curve shifted to the right (Fig 2A⇓). Endothelium removal eliminated the arachidonic acid–induced relaxations (data not shown). Indomethacin also blocked the arachidonic acid–induced relaxations, whereas combinations of L-NA and indomethacin caused no further attenuation of the arachidonic acid–induced relaxations. If L-NA reduced arachidonic acid–induced relaxations by blocking NO synthesis, the addition of an excess of the NO precursor l-arginine should overcome the L-NA blockade. The relaxations to arachidonic acid were slightly greater in the presence of l-arginine alone; however, this was not statistically significant (Fig 2B⇓). The inhibition of arachidonic acid–induced relaxation by L-NA was not altered by l-arginine treatment.
Both PGI2 and 8,9-EET relaxed coronary arteries (Fig 3⇓). Maximal relaxation occurred at 1×10−6 mol/L PGI2, with an EC50 of 10−7 mol/L, and 10−5 mol/L 8,9-EET, with an EC50 of 3×10−6 mol/L. L-NA significantly attenuated the relaxations to PGI2 (P<.05 at 3×10−8, 10−7, and 3×10−7 mol/L) but not 8,9-EET. Isoproterenol and sodium nitroprusside also relaxed coronary arteries (Fig 3⇓). The relaxations to sodium nitroprusside (EC50 of 2×10−8 mol/L) were markedly attenuated by pretreatment with L-NA (EC50 of 2×10−7 mol/L), whereas isoproterenol-induced relaxations (EC50 of 8×10−8 mol/L) were unaltered by pretreatment with the drug (EC50 of 2.5×10−8 mol/L).
Since indomethacin blocked a significant portion of the relaxation to methacholine and bradykinin, aliquots were removed from the organ baths and assayed for PGI2 release. As shown in the Table⇓, 6-keto-PGF1α synthesis increased significantly with methacholine, and this increase was related to concentration. Similarly, bradykinin stimulated 6-keto-PGF1α synthesis. Pretreatment with indomethacin completely inhibited the methacholine- or bradykinin-induced PGI2 synthesis. Bovine coronary artery strips treated with arachidonic acid (10−6 mol/L) in the presence and absence of indomethacin displayed the same pattern of 6-keto-PGF1α synthesis and inhibition as the vessels treated with methacholine or bradykinin. Specifically, arachidonic acid increased the concentration of 6-keto-PGF1α to 5807±836 pg/mL, compared with basal concentrations of 1180±45 pg/mL. Pretreatment with indomethacin (10−5 mol/L), L-NA (3×10−5 mol/L), or a combination of indomethacin and L-NA inhibited arachidonic acid–induced 6-keto-PGF1α release by 95% (P<.05) (238±60 pg/mL), 22% (4545±870 pg/mL), and 96% (P<.05) (223±21 pg/mL), respectively. The change in 6-keto-PGF1α release with L-NA was not statistically significant.
Because L-NA was effective at blocking the relaxations to methacholine, bradykinin, and arachidonic acid, we measured cGMP content in vessels treated with these agonists (Fig 4A⇓). cGMP content was increased from 8.7±2.0 to 28.5±4.4 pmol cGMP/g tissue (P<.05) when the vessels were stimulated with 10−9 mol/L bradykinin. In contrast, arachidonic acid had no effect on cGMP content compared with control (8.8±0.9 versus 8.7±2.0 pmol cGMP/g tissue). Furthermore, nitrite production by bovine coronary artery endothelial cells stimulated with bradykinin (10−6 mol/L) increased significantly. This stimulation was attenuated by pretreatment with L-NA (Fig 4B⇓). Arachidonic acid (10−5 mol/L) failed to significantly change the nitrite production from control.
Because arachidonic acid failed to increase vascular cGMP content as well as nitrite production by endothelial cells and because L-NA was effective at attenuating a portion of the arachidonic acid–induced relaxations, we examined the profile of radioactive metabolites formed on incubation of [14C]arachidonic acid in the presence and absence of L-NA. Coronary vessels converted arachidonic acid to products comigrating with the prostaglandins, hydroxyeicosatetraenoic acids (HETEs), and EETs (Fig 5⇓). In the presence of L-NA, the same products were produced, and the radioactive peaks comigrating with the 15-HETE and 14,15-EET standards increased slightly. Indomethacin predominantly decreased the formation of the prostaglandins and to a lesser extent the HETEs and EETs.
The control of vascular tone by bradykinin, ATP, substance P, acetylcholine, and the calcium ionophore A23187 has been shown to be related to the release of an EDRF or EDRFs.6 7 8 Cholinergic agonists have also been shown to control vascular tone by the release of PGI2 and EDRF in vivo and in vitro, although differences in vascular beds and species can lead to confusing and often contradicting results.29 30 31 Although it is now recognized that EDRF is NO or an NO-like compound,6 7 8 the existence of several other EDRFs has been suggested.9 10 11
The effects of local mediators on the coronary circulation is a subject of intense investigation and is extremely important in disease states such as myocardial ischemia, congestive heart failure, and hypertension. We chose to examine arachidonic acid–induced relaxations of bovine coronary arteries precontracted with the thromboxane mimetic U46619. Our goal was to determine the possible mediators involved in these relaxations. Since both NO and PGI2 were candidates for the mediation of these effects, we began by examining the effects of indomethacin and the l-arginine analogue L-NA on arachidonic acid–induced relaxations. We found that indomethacin was effective in blocking the relaxations to methacholine, bradykinin, and arachidonic acid. Also, these agents increased 6-keto-PGF1α release, an effect inhibited by indomethacin. These findings indicated that PGI2 participated in the relaxant effects of these three agonists.
In agreement with previous reports,17 the relaxations to methacholine and bradykinin were also attenuated by the inhibitor of NO synthase L-NA. Bradykinin also increased the cGMP content of coronary vessels, an effect blocked by L-NA. Since cGMP mediates the vasorelaxant effects of NO,32 33 these studies indicate that NO also participates in the relaxations to bradykinin and methacholine. Indomethacin and L-NA only partially inhibited bradykinin- and methacholine-induced relaxations. These findings indicate that some other endothelial factor must mediate a portion of the bradykinin-induced relaxation because a major portion of the relaxations was not blocked by inhibition of both cyclooxygenase and NO synthase. The contribution of this factor or another factor to methacholine-induced relaxations must be small because a majority of the response was blocked by this combination.
L-NA was effective alone and in combination with indomethacin at attenuating the arachidonic acid–induced relaxations. This would suggest that NO release accounts for a portion of the relaxant effects of the fatty acid. However, arachidonic acid failed to increase the vascular content of cGMP or the endothelial production of nitrate/nitrite, indexes of NO formation. Moreover, addition of l-arginine, the precursor to NO, to L-NA–treated vessels failed to reverse the inhibitory effects of L-NA on arachidonic acid–induced relaxations. These findings fail to substantiate a role for NO in arachidonic acid–induced relaxations. We have previously shown that arachidonic acid–induced relaxations are mediated by a cyclooxygenase metabolite, PGI2, and a cytochrome P-450 metabolite, EET.12 Thus, it is possible that L-NA also inhibits the actions of these metabolites on vascular tone. In support of this, L-NA inhibited the relaxations to PGI2. Surprisingly, L-NA attenuated the relaxations to sodium nitroprusside, an endothelium-independent agent. Whether this inhibition reflects a chemical inactivation of sodium nitroprusside or some direct effect of L-NA on vascular smooth muscle remains to be determined.
Given these experimental findings, it would seem that L-NA has actions other than the inhibition of NO synthase. Indeed, Peterson et al18 have suggested that these inhibitors may have effects on iron-containing enzyme systems apart from NO synthase. They reported that the NO synthase inhibitors NG-nitro-l-arginine methyl ester and NG-monomethyl-l-arginine inhibited the reduction of ferric cytochrome c by ferrous iron. Along these lines, Koller et al19 have suggested that l-arginine analogues may blunt prostaglandin-related dilation of rat arterioles. The inhibitory effects of L-NA on the relaxation responses to a variety of agonists, arachidonic acid, PGI2, and sodium nitroprusside, suggest that they exert their effects at some common mechanism involved in the regulation of smooth muscle contractility. This action remains to be determined.
In addition, since arginine analogues appear to have vascular effects other than inhibition of NO synthase, caution must be used in assessing the contribution of NO to the regulation of vascular tone with the use of these drugs. This raises the possibility that the present and previous studies may have overestimated the contribution of NO in the vascular response to bradykinin, methacholine, and other agonists. This consideration further raises the possibility that endothelial cell factors other than NO and PGI2 may participate in the vascular effects of a number of agonists.
Finally, these findings indicate that PGI2, NO, and some other factor or factors mediate the relaxant effects of methacholine and bradykinin. Arachidonic acid–induced relaxations are mediated by PGI2 and some other metabolite. Previous studies suggested that this metabolite is probably an EET.12 NO does not appear to mediate the relaxant effects of arachidonic acid; rather, L-NA may have actions other than inhibition of NO synthase.
Selected Abbreviations and Acronyms
|[14C(U)]arachidonic acid||=||uniformly labeled [14C]arachidonic acid|
|EDRF||=||endothelium-derived relaxing factor|
These studies were supported by grants from the National Heart, Lung, and Blood Institute (HL-17669 and HL-51055). The authors express their appreciation to Martha Williams for her technical assistance and Gretchen Barg for her secretarial assistance.
Reprint requests to William B. Campbell, PhD, Department of Pharmacology and Toxicology, Medical College of Wisconsin, 8701 Watertown Plank Rd, Milwaukee, WI 53226.
- Received February 2, 1996.
- Revision received March 12, 1996.
Furchgott RF. Role of endothelium in responses of vascular smooth muscle. Circ Res. 1983;53:557-573.
Raz A, Isakson PC, Minkes MS, Needleman P. Characterization of a novel metabolic pathway of arachidonate in coronary arteries which generates a potent endogenous coronary vasodilator. J Biol Chem. 1977;252:1123-1126.
Ignarro LJ, Byrns RE, Buga GM, Wood 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.
Rubanyi GM, Vanhoutte PM. Nature of endothelium-derived relaxing factor: are there two relaxing mediators? Circ Res. 1987;61(suppl II):II-61-II-67.
Angus JA, Cocks TM. Endothelium-derived relaxing factor. J Pharmacol Exp Ther. 1989;41:303-351.
Rosolowsky M, Campbell WB. Role of PGI2 and EETs in the relaxation of bovine coronary arteries to arachidonic acid. Am J Physiol. 1993;264:H327-H335.
Pfister SL, Campbell WB. Arachidonic acid- and acetylcholine-induced relaxations of rabbit aorta. Hypertension. 1992;20:682-689.
Holzmann S. Endothelium-induced relaxation by acetylcholine associated with layer rises in cyclic GMP in coronary arterial strips. J Cyclic Nucl Res. 1982;8:409-419.
Koller A, Sun D, Messina EJ, Kaley G. L-arginine analogues blunt prostaglandin-related dilation of arterioles. Am J Physiol. 1993;264:141194-141199.
Archer SL, Hampl V. NGmonomethyl L-arginine causes nitric oxide synthesis in isolated arterial rings: trouble in paradise. Biochem Biophys Res Commun. 1992;188:59.
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.
Katsuki S, Murad F. Regulation of adenosine cyclic 3′,5′-monophosphate levels and contractility in bovine tracheal smooth muscle. Mol Pharmacol. 1977;13:330-341.
Revtyak GE, Johnson AR, Campbell WB. Cultured bovine coronary arterial endothelial cells synthesize HETEs and prostacyclin. Am J Physiol. 1988;254:C8-C19.
Ibe BO, Falck JR, Johnson AR, Campbell WB. Regulation of synthesis of prostacyclin and HETEs in human endothelial cells. Am J Physiol. 1989;256:C1168-C1175.
Lowry OH, Rosebrough NJ, Farr AL, Randall RJ. Protein measurement with the Folin phenol reagent. J Biol Chem. 1951;193:265-275.
Toda N. Isolated human coronary arteries in response to vasoconstrictor substances. Am J Physiol. 1983;245:H937-H941.
Förstermann U, Mugge A, Alheid U, Haverich A, Frolich JC. Selective attenuation of endothelium-mediated vasodilation in atherosclerotic human coronary arteries. Circ Res. 1988;62:185-190.
Ignarro LJ, Harbison RG, Wood KS, Kadowitz PJ. Dissimilarities between methylene blue and cyanide on relaxation and cyclic GMP formation in endothelium-intact intrapulmonary artery caused by nitrogen oxide-containing vasodilators and acetylcholine. J Pharmacol Exp Ther. 1986;236:30-36.
Ignarro LJ, Harbison RG, Wood KS, Kadowitz PJ. Activation of purified soluble guanylate cyclase by endothelium-derived relaxing factor from intrapulmonary artery and vein: stimulation by acetylcholine, bradykinin and arachidonic acid. J Pharmacol Exp Ther. 1986;237:893-900.