Inhibition of Vasoconstriction by Angiotensin Receptor Antagonist GR117289C in Arterial Grafts

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1 Inhibition of Vasoconstriction by Angiotensin Receptor Antagonist GR117289C in Arterial Grafts Ming-Hui Liu, MD, H. Storm Floten, MD, Anthony P. Furnary, MD, Anthony P. C. Yim, MD, and Guo-Wei He, MD, PhD Cardiovascular Research, The Albert Starr Academic Center For Cardiac Surgery, Providence St. Vincent Hospital, Portland, Oregon, and Department of Surgery, The Chinese University of Hong Kong, Hong Kong SAR, China Background. Angiotensin II (AII) has been suggested to be one of the important factors for genesis of graft spasm in coronary artery bypass surgery. The aim of this work was to investigate the effects of the nonpeptide angiotensin receptor AT 1 antagonist GR117289C on the contraction induced by AII and other vasoconstrictors in isolated human internal mammary artery (IMA) preparations. Methods. Two hundred eight IMA rings taken from 64 patients undergoing coronary artery bypass grafting were studied in organ baths. The interaction between GR117289C and AII or the other vasoconstrictors (U46619, norepinephrine, endothelin-1, and potassium chloride) was investigated in two ways. Results. GR117289C induced near-maximal relaxation (94.5% 2.9%) in IMA rings precontracted by AII. In IMA rings incubated with 1 or 10 nmol/l GR117289C, contractile responses to AII were attenuated in a concentration-related manner, whereas the dose-response curve did not shift to the right when higher doses of AII were administered, suggesting that the AT 1 receptor blockade was noncompetitive in nature. Moreover, GR117289C also induced significant relaxation (82.9% 8.1%) in IMA rings precontracted by U46619, but no inhibitory responses to U46619 could be observed when IMA rings were incubated with GR117289C. GR117289C did not alter responses to potassium chloride, norepinephrine, and endothelin-1. Conclusions. These results indicate that GR117289C is a potent, selective, noncompetitive AT 1 receptor antagonist that may have a possible antagonistic effect on the thromboxane A 2 receptor. Because AII and thromboxane A 2 are important vasoconstrictors in the genesis of graft spasm, GR117289C may become an alternative treatment to relieve graft spasm. (Ann Thorac Surg 2000;70:2064 9) 2000 by The Society of Thoracic Surgeons The renin-angiotensin-aldosterone system is recognized to exert a vital physiopathologic role in blood pressure regulation and electrolyte balance [1]. The primary effector of this system is angiotensin II (AII), an octapeptide, which is responsible for a host of peripheral as well as central effects such as vasoconstriction, aldosterone biosynthesis and secretion, renal sodium reabsorption, catecholamine and vasopressin release, tissue remodeling, and an increase in drinking behavior [1]. These effects are elicited through the activation of specific AII receptors located predominantly on the cell surface of target organs. It has been revealed that at least two subtypes of AII receptors, the AT 1 and AT 2 receptors, exist [2]. AT 1 receptors appear to mediate most, if not all, of the established effects of AII, and the role, if any, of AT 2 receptors remains to be defined [3]. It has recently been reported that AT 2 receptors mediate vasodepressor responses in the rat [4]. Other evidence in the literature suggests that AT 2 receptors may play a role in fetal growth and development [5]. Accepted for publication April 19, Address reprint requests to Dr He, Division of Cardiothoracic Surgery, Department of Surgery, University of Hong Kong, 5A, Block B, Prince of Wales Hospital, Shatin, N.T., Hong Kong SAR, China; gwhe@cuhk.edu.hk. The therapeutic success of angiotensin-converting enzyme inhibitors, such as captopril and enalapril, in the treatment of hypertension and heart failure has confirmed the involvement of this system in the disease status. Despite their clinical success, the use of angiotensin-converting enzyme inhibitors is not without problems, dry cough, and a propensity to induce functional renal failure being among the most common [6]. Angioedema is a rare but potentially life-threatening side effect [7]. An alternative way of intervening in the reninangiotensin-aldosterone axis, which might avoid the side effects of angiotensin-converting enzyme inhibitors, is to prevent the action of AII at its sites of action. Losartan, a benzyl-substituted imidazole, is the first nonpeptide AII antagonist developed for clinical use [8]. GR117289C, a novel potent nonpeptide angiotensin AT 1 receptor antagonist, has been reported to exhibit potent AII antagonist activity in vitro and exert marked and prolonged antihypertensive activity after oral administration in renal hypertensive rats [9]. GR117289C has also been demonstrated to be a potent orally active AII receptor antagonist that resulted in a dose-dependent attenuation in AIIinduced vasoconstriction in the human forearm [10]. These compounds are being investigated as a potential novel treatment for hypertension and heart failure. Coronary artery bypass grafting (CABG) offers patients 2000 by The Society of Thoracic Surgeons /00/$20.00 Published by Elsevier Science Inc PII S (00)

2 Ann Thorac Surg LIU ET AL 2000;70: AT 1 ANTAGONIST IN IMA 2065 with ischemic heart disease significant improvement in quality of life and longevity. The internal mammary artery (IMA) is the most widely used arterial conduit for CABG. Spasm of arterial grafts has been well recognized. In addition, in the venous graft, smooth muscle cell proliferation deteriorates the graft function and results in recurrence of clinical symptoms. The mechanisms of these changes are complicated, but AII is suggested to be one of the important factors [10]. Angiotensin II is a potent endogenous vasoconstrictor. Elevated levels of AII and plasma renin have been found during and after CABG and have been implicated in the cause of postoperative hypertension, seen in 30% to 60% of patients after CABG [11, 12]. Other investigators [13] and we [14] have examined the vasoconstrictor effects of AII in the isolated human IMA, but the interaction between GR117289C and AII or other vasoconstrictors has not been studied. Therefore, in this study we have characterized the responses of the conduit arteries to GR117289C in the presence of AII and other vasoconstrictors, which may elicit graft spasm during and after CABG. Material and Methods General Human IMA rings were collected from 64 patients undergoing CABG. There were 51 male and 13 female patients with a mean age of years. Approval to use discarded IMA tissue was given by the Institutional Review Board of St. Vincent Hospital. Discarded IMA segments were collected and put in a container with oxygenated physiologic solution (Krebs ) maintained at 4 C and delivered to the laboratory. The Krebs solution has the following composition (in mmol/l): Na, 144; K, 5.9; Ca 2, 2.5; Mg 2, 1.2; Cl, 128.7; HCO 3, 25; SO 2 4, 1.2; H 2 PO 4, 1.2; and glucose, 11. Organ Bath Technique The IMA was placed in a glass dish with oxygenated Krebs solution, and the surrounding connective tissue was dissected out. The vessel was cut into 3-mm-long rings, and the number of rings taken from each patient varied from two to eight. Two hundred eight IMA rings were investigated in the present study. Artery rings were mounted on two parallel stainless steel wire hooks in 25-mL water-jacketed glass organ baths containing Krebs solution maintained at 37 C 0.1 C and continuously bubbled with a gas mixture of 95% O 2 and 5% CO 2. The lower hook was attached to a micrometeradjustable support leg and the upper to an isometric force transducer (model FT03, Grass Instruments, Astro- Med Inc, West Warwick, RI) to record changes in isometric force, which were amplified and recorded on a polygraph chart recorder (model 79, Grass Instruments). After a 60-minute equilibration period, a normalization technique was used to set the vascular rings at a pressure comparable with that of the in vivo situation. The details of this technique have been published previously [15]. Briefly, the rings were stretched in progressive steps to determine the length tension curve. A computer iterative fitting program (Vestand 2.1, Yang-Hui He, Princeton University, NJ) was used to determine the exponential curve, pressure, and the internal diameter. When the transmural pressure on the rings reached 100 mm Hg, determined from their own length tension curves, the stretching procedure was stopped, and the rings were released to 90% of their internal circumference at 100 mm Hg. This degree of passive tension was then maintained throughout the experiment. After the normalization procedure, the IMA rings were equilibrated for at least 45 minutes. The endothelium was intentionally preserved by cautiously dissecting and mounting the rings in our study as endothelium plays a modulation role in the contractility of the human IMA. We previously found that this technique allowed the experiment to be conducted with functionally intact endothelium, as determined by the relaxation response to acetylcholine in the isolated human IMA rings [16]. Protocol RELAXATION. GR117289C-induced relaxation was studied in IMA rings precontracted with AII (3 nmol/l; n 8), thromboxane A 2 (TXA 2 ) mimetic U46619 (10 nmol/l; n 8), norepinephrine (NE; 3 mol/l; n 8), endothelin-1 (ET-1; 10 nmol/l; n 8), and potassium chloride (K ;25 mmol/l; n 8). Another group of eight rings contracted by ET-1 (10 nmol/l) was studied as the time control, because in a previous study [17], we found that the contraction induced by ET-1 was not sustained. The concentrations of these vasoconstrictors were determined from the logistic curve-fitting equation. These concentrations are equal to the effective concentrations causing 50% to 80% response (EC 50 to EC 80 ) for the AII-, U46619-, NE-, ET-1-, and K -induced contraction in the human IMA from previous studies [15 17]. Cumulative concentration-relaxation curves to GR117289C were then established. Only one concentration-relaxation curve was obtained from each IMA ring. From eight rings (taken from 8 patients), a mean concentration-relaxation curve was constructed. The relaxation was expressed as percent reversal of the agonist-induced precontraction. DEPRESSION OF CONTRACTION BY PRETREATMENT WITH GR117289C. After equilibration of IMA rings for at least 45 minutes, 100 mmol/l K was added into the organ bath, and the contraction force was recorded. The ring was repeatedly washed with Krebs solution to restore the baseline pressure. To determine whether pretreatment with GR117289C would alter the contraction response to AII and other vasoconstrictors (U46619, NE, ET-1, and K ), cumulative concentration-contraction curves were constructed in IMA rings. Four rings from 1 patient were arranged for respective incubation for 30 minutes with 0.1, 1, or 10 nmol/l GR117289C, or with vehicle as control. The time for the pretreatment was decided by the average time (approximately 25 minutes) to reach a plateau for each dose of GR117289C in the relaxation experiment from the present study. The contraction was expressed as

3 2066 LIU ET AL Ann Thorac Surg AT 1 ANTAGONIST IN IMA 2000;70: percentage of the contraction force induced by 100 mmol/l K. Data Analysis The sensitivity of both vasoconstrictors and vasodilators was expressed as EC 50, the effective concentration that caused 50% of maximal contraction or relaxation. The EC 50 was determined from each concentrationcontraction (or relaxation) curve by a logistic curve-fitting equation: E MA p /(A p K p ), where E is response, M is maximal contraction (or relaxation), A is concentration, K is EC 50 concentration, and p is the slope parameter. A computerized program was used for the curve fitting. Statistical analysis was performed with SPSS software (SPSS Inc, Chicago, IL). All values were expressed as mean standard error of the mean. Statistical comparisons of the percentage relaxation or contraction under different treatments were performed by two-way analysis of variance (ANOVA) with repeated measures, followed by post hoc Bonferroni test to detect individual differences. The EC 50 values were compared by one-way ANOVA followed by post hoc Bonferroni test. A p value less than 0.05 was considered statistically significant. Materials Drugs used in this study and their sources were AII, ( )norepinephrine bitartrate, and potassium chloride (Sigma Chemical Company, St. Louis, MO); U46619 (Cayman Chemical, Ann Arbor, MI); and endothelin-1 (Peptides International, Louisville, KY). Stock solutions of AII and ET-1 were held frozen until required. GR117289C is a generous gift provided by Glaxo Research and Development Ltd., London, UK. GR117289C solution at 1 mmol/l was prepared by dissolving 1 mg of GR117289C in 50 L of sodium hydroxide (2 mol/l) and 50 L of ethanol, then diluting with distilled water. Results Resting Values of Internal Mammary Arterial Rings The internal diameter of the 208 IMA rings at an equivalent transmural pressure of 100 mm Hg was mm as determined from the normalization procedure. When the IMA rings were set at a resting diameter of 0.9 the diameter producing an equivalent transmural pressure of 100 mm Hg, the equivalent transmural pressure was mm Hg, and the resting force was g. Fig 1. Mean concentration (negative log molar [ LogM]) relaxation (percent reversal of the agonist-induced contraction) curves for GR117289C in human internal mammary arterial rings precontracted by angiotensin II (AII), U46619, norepinephrine (NE), endothelin-1 (ET-1), or potassium chloride (K). Number of internal mammary arterial rings in each group is eight (from 8 patients). Values are expressed as mean standard error of the mean. ( p less than versus angiotensin II group, p less than 0.05 versus U46619 group, two-way analysis of variance.) Relaxing Effect of GR117289C on Internal Mammary Arterial Rings Contracted by Angiotensin II, U46619, Norepinephrine, Endothelin-1, or Potassium Chloride The maximal relaxation caused by GR117289C was nearly complete in AII-precontracted IMA rings (94.5% 2.9%). GR117289C also markedly relaxed IMA rings precontracted by U46619 (82.9% 8.1%). For IMA rings precontracted by other vasoconstrictors, the maximal relaxation was 23.7% 9.0% for NE, 44.4% 10.5% for ET-1 (normalized by the time control), and 9.2% 2.5% for K. GR117289C-induced relaxation of AII or U46619 contractions was significantly greater than that for contractions produced by NE, ET-1, or K (p , two-way ANOVA; Fig 1). In IMA rings precontracted by vasoconstrictors, the EC 50 values for GR117289C were log molar for AII, log molar for U46619, log molar for NE, for ET-1, and log molar for K. In IMA rings precontracted by ET-1, the contraction was not sustained enough for the long period required for the cumulative dose-response curves, because the cumulative doses were given at 25-minute intervals (see Methods). The difference between the GR117289C-induced relaxation and the time control was not significant (p 0.08, two-way ANOVA; Fig 2). Depression of Contraction by Pretreatment With GR117289C on Internal Mammary Arterial Rings Cumulative addition of AII ( log molar) caused concentration-related contraction of human IMA preparations. After a 30-minute incubation period, GR117289C ( 10, 9, or 8 log molar) produced a concentration-dependent, insurmountable antagonism of AII (Fig 3). AII-induced contractions in IMA rings pretreated with 9 or 8 log molar GR117289C were significantly depressed (p 0.05 vs control). GR117289C at the concentration of 8 log molar caused approxi-

4 Ann Thorac Surg LIU ET AL 2000;70: AT 1 ANTAGONIST IN IMA 2067 Fig 2. Mean concentration (negative log molar [ LogM]) relaxation (percent reversal endothelin-1 [ET-1]-induced contraction) curves for GR117289C in human internal mammary arterial rings. Number of internal mammary arterial rings in each group is eight (from 8 patients). Values are expressed as mean standard error of the mean. (p 0.05, compared with the control group, two-way analysis of variance.) mately 80% depression of the maximal response to AII. Despite this profound, concentration-dependent depression of the maximal response to AII, GR117289C did not significantly change the EC 50 values of AII. The EC 50 values in the absence and presence of 10, 9, or 8 log molar GR117289C were , , , and log molar, respectively. In contrast, incubation with GR117289C for 30 minutes at concentrations of 10, 9, and 8 log molar had no significant effect on the contractile response to U46619, NE, ET-1, and K (p 0.05, two-way ANOVA; Fig 4). The EC 50 values for these vasoconstrictors also did not change significantly (p 0.05, one-way ANOVA; Fig 4). Comment Results of the present investigation demonstrated that the nonpeptide angiotensin AT 1 receptor antagonist GR117289C reduced contractile responses to AII in the human IMA. On the one hand, GR117289C caused a near-maximal relaxation (94.5% 2.9%; Fig 1) in the human IMA rings precontracted by AII. On the other hand, incubation with GR117289C attenuated the vasoconstriction evoked by AII in a concentration-dependent manner. In IMA rings incubated with 1 or 10 nmol/l GR117289C, the responses to AII were reduced markedly, but the dose-response curve did not shift to the right when higher doses of AII were administered, suggesting that the AT 1 receptor blockade was noncompetitive in Fig 3. Mean concentration (negative log molar [ LogM]) contraction (percent contraction induced by 100 mmol/l potassium chloride) curves to angiotensin II (AII) when vehicle (25 mol/l mixture of sodium hydroxide, ethanol, and distilled water; Control), 10 [GR ( 10)], 9 [GR ( 9)], or 8 [GR ( 8)] log molar GR117289C was added 30 minutes before the contraction started in human internal mammary arterial rings. Bar graph depicts the effective concentration causing 50% of the maximal response (EC 50 ) values for angiotensin II among the control (I) and GR-incubated groups at concentrations of 10 (II), 9 (III), and 8 (IV) log molar. Number of internal mammary arterial rings in each group is eight (from the same 8 patients). Values are expressed as mean standard error of the mean. ( p 0.02, p vs control group, two-way analysis of variance.) nature (Fig 3). Moreover, GR117289C induced marked relaxation (82.9% 8.1%; Fig 1) in the IMA rings precontracted by the TXA 2 mimetic U46619, but no inhibitory responses to U46619 could be observed when the IMA rings were incubated with 0.1, 1, or 10 nmol/l GR117289C (Fig 4A). GR117289C had no inhibitory effect on NE-, ET-1-, or K -induced vasoconstriction (Fig 4B, 4C, 4D). The IMA is widely used as a conduit for CABG. Numerous clinical studies have demonstrated the superiority of the IMA regarding long-term patency compared with saphenous vein. Vasospasm sometimes occurs during IMA grafting, and may limit graft function by reducing luminal blood flow. Spasm can result from endothelial injury during surgical preparation, which initiates the abnormal platelet-endothelium interaction [18]. The mechanism of graft spasm is not clear and may include mechanical, physical, and pharmacologic stimulation. Elevated plasma concentrations of AII and ET-1 have been observed during or after CABG [19]. These factors, either alone or in combination, may have implications in the genesis of graft spasm. Some of these changes may be mediated by AII. With its potent antagonistic effect to angiotensin AT 1 receptor and its high oral

5 2068 LIU ET AL Ann Thorac Surg AT 1 ANTAGONIST IN IMA 2000;70: Fig 4. Mean concentration (negative log molar [ LogM]) contraction (percent contraction induced by 100 mmol/l potassium chloride) curves to U46619 (A), norepinephrine (B), endothelin-1 (C) and potassium chloride (D) when vehicle (25 mol/l mixture of sodium hydroxide, ethanol, and distilled water; Control), 10 [GR ( 10)], 9 [GR ( 9)], or 8 [GR ( 8)] log molar GR117289C was added 30 minutes before the contraction started in human internal mammary arterial rings. Bar graph depicts the effective concentration causing 50% of the maximal response (EC 50 ) values for angiotensin II among the control (I) and GR-incubated groups at concentrations of 10 (II), 9 (III), and 8 (IV) log molar. Number of internal mammary arterial rings in each group is eight (from the same 8 patients). Values are expressed as mean standard error of the mean. (p 0.05, compared with the control group, two-way analysis of variance.) bioavailability, GR117289C may improve the graft longevity and function by relieving graft spasm and preventing smooth muscle cell proliferation stimulated by AII. Losartan, the first nonpeptide angiotensin receptor antagonist, has been available for clinical use since 1990 [8]. GR117289C (1-[[3-bromo-2-[2-(1H-tetrazol-5- yl)phenyl]-5-benzofuranyl]methyl]-2-butyl-4-chloro-1himidazole-5-carboxylic acid), a novel synthesized nonpeptide angiotensin receptor antagonist displaying high affinity for the AT 1 receptor, is a noncompetitive AT 1 receptor antagonist, whereas losartan is a competitive antagonist at the AT 1 receptor [20]. GR117289C has been shown to inhibit AII-induced aldosterone secretion and renal vasoconstriction in anesthetized dogs [21]. In addition, GR117289C has been shown to produce peripheral vasodilation in rats with renal hypertension after both systemic and oral administrations [22]. This novel AT 1 receptor antagonist has been shown to reduce the maximal contractile response to AII in rabbit aortic strips and to shift the dose-response curve for AII to the right in a nonparallel manner, providing support for the concept that GR117289C is a noncompetitive antagonist for the AT 1 receptor [20]. GR117289C has also been demonstrated to be a potent orally active AII receptor antagonist that results in a dose-dependent attenuation in AIIinduced vasoconstriction in human being in vivo [10]. The current study shows that GR117289C reduces the contractile response to AII in human IMA rings but does not shift the dose-response curve for AII, suggesting that GR117289C is also a noncompetitive antagonist for the AT 1 receptor in human IMA. A recent study by Li and colleagues [23] shows that losartan inhibits TXA 2 -induced contraction in canine coronary artery through TXA 2 /PGH 2 receptor antago-

6 Ann Thorac Surg LIU ET AL 2000;70: AT 1 ANTAGONIST IN IMA 2069 nism, and their study also indicates that the blocking actions of losartan on the TXA 2 receptor are quite specific, because administration of either the cyclooxygenase inhibitor, indomethacin, or a nitric oxide synthase inhibitor does not eliminate the antagonistic effect of losartan on the U46619-induced vasoconstriction. No reports have shown whether GR117289C also has an antagonistic effect for the TXA 2 /PGH 2 receptor. The present study shows that GR117289C induces near-maximal relaxation in human IMA rings precontracted by U46619 but does not attenuate the contractile response of AII in human IMA rings incubated with 10 nmol/l GR117289C for 30 minutes. It is unknown what accounts for the difference. Nevertheless, we have found, in a previous study [14], that some vasodilators such as nitroglycerin and aprikalim are potent in reversing existing vascular contraction but less effective if applied before the contraction. From this study, we may only speculate that GR117289C probably has an antagonistic effect on the TXA 2 /PGH 2 receptor in human IMA. In conclusion, the results of the present study show that GR117289C is a potent, selective, specific, noncompetitive AT 1 receptor antagonist, which produces concentration-dependent inhibition of AII-induced vasoconstriction in the human IMA. These data also indicate that GR117289C, which markedly reverses the vasoconstriction elicited by TXA 2 analog U46619, has a possible antagonistic effect on the TXA 2 /PGH 2 receptor. Because AII and TXA 2 are two important factors in spasm of coronary arterial grafts during or after CABG, GR117289C may become a valuable alternative therapy in CABG to relieve graft spasm and prevent smooth muscle cell proliferation of the graft. This study was supported by Providence St. Vincent Medical Foundation, and American Heart Association, Oregon affiliate, Portland, OR, Hong Kong Research Grants Council grants (CUHK7280/97 M and CUHK7246/99 M). The technical assistance of the surgical team and of Kay McCantz and other nurses in the Cardiac Operating Room, Providence St. Vincent Hospital, are also gratefully acknowledged. Doctor Liu is a Starr-He International Postdoctoral Fellow established by the Providence St. Vincent Medical Foundation. References 1. Vallotton MB. The renin angiotensin system. Trends Pharmacol Sci 1987;8: Bumpus FM, Catt KJ, Chiu AT, et al. 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