Ann Thorac Surg 2000;70:1327-1331
© 2000 The Society of Thoracic Surgeons
Original articles: cardiovascular
Relaxation induced by cGMP phosphodiesterase inhibitors sildenafil and zaprinast in human vessels
Pascual Medina, PhDa,
Gloria Segarra, BSca,
Juan B. Martínez-León, MDb,
José M. Vila, PhDa,
Martín Aldasoro, MDa,
Eduardo Otero, MDb,
Salvador Lluch, MDb
a Department of Physiology, University of Valencia, Valencia, Spain
b Department of Surgery, University of Valencia, Valencia, Spain
Address reprint requests to Dr Lluch, Department of Physiology, Facultad de Medicina y Odontología, Avda Blasco Ibáñez 17, 46010 Valencia, Spain
e-mail: medinap{at}post.uv.es
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Abstract
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Background. Sildenafil is currently used in the treatment of erectile dysfunction. However, assessment of direct effects of sildenafil on coronary arteries and on arteries used as coronary grafts is unknown. This study was designed to investigate the effects of sildenafil on contracted human coronary, internal mammary, and radial arteries obtained from multiorgan donors. The observations were extended to forearm veins. Zaprinast was included in this study for comparison.
Methods. Segments of left coronary, internal mammary, and radial arteries, and forearm veins were obtained from 16 multiorgan donors. Vascular rings were suspended in organ bath chambers and isometric tension was recorded. Then the effects of sildenafil, zaprinast, and sodium nitroprusside on precontracted vessels were studied.
Results. Sildenafil (10-8 - 3 x 10-5 mol/L) caused concentration-dependent relaxation in the internal mammary arteries, radial arteries, and forearm veins. In the coronary arteries, sildenafil had a modest relaxant effect. In addition, sildenafil amplified the relaxation induced by sodium nitroprusside in all four vessels. Relaxation was unaffected by the inhibitor of nitric oxide synthase NG-monomethyl-L-arginine (10-4 mol/L). Compared with zaprinast, sildenafil was eight to ten times more potent in terms of EC50 values.
Conclusions. The direct relaxant effects of sildenafil together with its synergistic interaction with nitric oxide donors should be considered in patients undergoing coronary bypass surgery, patients with low blood pressure, and patients receiving antihypertensive regimes.
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Introduction
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Sildenafil, a selective inhibitor of type 5 cyclic guanosine monophosphate (cGMP) phosphodiesterase (PDE) [1–3] has proved to be effective in the treatment of erectile dysfunction after oral administration in men [1, 4, 5]. By inhibiting the hydrolytic breakdown of cGMP, sildenafil increases the levels of cGMP. This provides the signal for smooth muscle relaxation of corpora cavernosa [3, 6]. Erectile dysfunction and cardiovascular disease share several risk factors, including hypertension, diabetes and hyperlipidemia [7]. Moreover, there is a close correlation between the severity of ischemic heart disease and erectile dysfunction [8]. Thus patients with cardiovascular disease may also receive treatment for erectile dysfunction. Although several reports indicate that sildenafil is well tolerated in these patients [9, 10], assessment of direct effects on coronary arteries and on arteries used as coronary grafts is unknown. Agents that inhibit cGMP hydrolysis such as sildenafil may enhance the relaxation induced by nitrovasodilators (nitric oxide donors). Nitric oxide (NO) donors release NO within smooth muscle and, as a result, increase the level of cGMP [11, 12]. The synergistic action between sildenafil and sodium nitroprusside in blood vessels has been recently observed in human penile arteries and veins [13]. In addition, to enhance the NO-stimulated cGMP signal, sildenafil may cause relaxation of penile vessels through mechanisms independent of NO formation (in the absence of NO formation). Therefore, this study was designed to investigate the effects of sildenafil given alone or in combination with the NO donor, sodium nitroprusside, on contracted human coronary, internal mammary, and radial arteries obtained from multiorgan donors. We extended some of these observations to forearm veins. The PDE 5 inhibitor zaprinast [14] was included in this study for comparison.
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Material and methods
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Collection of samples
Portions of the anterior descending branch of the left coronary, internal mammary, and radial arteries, and forearm veins (cephalic) were obtained from 16 multiorgan donors (10 men and 6 women [age range, 16 to 60 years]) during procurement of organs for transplantation. This study was approved by the ethical committee of the University of Valencia, Valencia, Spain. The vessels were immediately placed in cold (4°C) modified Krebs solution (see Organ Bath Experiments for composition) and aerated with 95% oxygen and 5% carbon dioxide until they were used. Experiments started within 2 hours, or not more than 8 hours after organs were removed. When used within 2 to 8 hours, the vessels were stored in the modified Krebs solution, which was found not to alter either the maximum effect of PDE inhibitors or the contractile response to potassium chloride (KCl) or U-46619.
Epicardial coronary arteries were obtained from 7 donors (4 men and 3 women [age range 31 to 50 years]). Five of these donors were victims of traumas caused by motor vehicle collisions; their hearts were not used for transplantation because of myocardial contusion (n = 4) or hemopericardium (n = 1). One heart was obtained from a donor who had congenital heart disease. Another heart was obtained from a 40-year-old-man who died of intracerebral hemorrhage associated with hypertension. The branches of the coronary arteries used in these experiments showed no signs of atheroma on gross examination and under a dissection microscope.
Internal mammary and radial arteries, and forearm veins (cephalic) were obtained from 9 multiorgan donors (6 men and 3 women [age range, 16 to 60 years]). Five of these donors died of myocardial infarction, and 4 were victims of motor vehicle collisions.
Organ bath experiments
The vessels were cleaned of adherent connective tissue and cut into rings (3 mm in length) under a dissection microscope. Each ring was suspended between two stainless-steel L-shaped pins in 4 ml organ baths containing modified Krebs solution of the following composition (mmol/L): NaCl, 115; KCl, 4.6; MgSO4, 1.2; CaCl2, 2.5; NaHCO3, 25; KH2PO4, 1.2; glucose, 11.1 and disodium ethylenediaminetetraacetic acid, 0.01. The solution was equilibrated with 95% oxygen (O2) and 5% carbon dioxide (CO2) to give a pH of 7.3 to 7.4. The temperature was held at 37°C. One pin was fixed to the organ bath wall while the other was connected to a strain gauge (model Grass FT03; Grass Instruments Division of Astro-Med, Inc, West Warwick, RI). Resting tension was set to the optimal level for development of active force in response to 100 mmol/L KCl. The optimal resting tensions were 3 g for coronary and internal mammary arteries, 4 g for radial arteries and 2 g for cephalic veins. The rings were allowed to attain a steady level of tension during a 2 to 3 hour accommodation period before testing. Changes in isometric force were recorded on a Macintosh computer (Apple Computers, Cupertino, CA) by use of Chart Version 3.4/s software and a MacLab/8e data acquisition system (ADInstruments, East Sussex, UK). Functional integrity of the endothelium was confirmed routinely by the presence of relaxation induced by bradykinin (10-7 - 3 x 10-7 mol/L) during contraction obtained with thromboxane A2 receptor agonist U-46619 (1 - 3 x 10-7 mol/L).
The effects of sildenafil, zaprinast, and sodium nitroprusside, and the combination of either sildenafil or zaprinast with sodium nitroprusside were studied in vessels contracted with 10-9 - 10-8 mol/L of U-46619. After a stable contraction was obtained, concentration-response curves were recorded. In some experiments the relaxant effects of sildenafil were determined in vessels preincubated with N6-monomethyl-L-arginine, 10-4 mol/L (L-NMMA (10-4 mol/L)) for 20 minutes.
Chemicals
Sildenafil was synthesized by Pfizer Ltd, Sandwich, Kent, UK. All other chemicals were purchased from Sigma Chemical, St. Louis, MO.
Data analysis
All values are expressed as the mean ± the standard error of the mean. In each experimental group, n indicates the number of donors. The relaxations are expressed as a percentage decrease in tension of that contraction induced by U-46619. The negative logarithm of the molar concentration at which half-maximum relaxation occurs (pEC50) was determined from individual concentration-response curves by a logistic, curve-fitting equation: 
where E is response, M is maximal relaxation, A is concentration, K is EC50 concentration, and P is the slope parameter. The pEC50 values were compared by an unpaired t test and an analysis of variance with Scheffe’s test as a post hoc test. Statistical significance was accepted at p less than 0.05.
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Results
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Sildenafil and zaprinast, at low concentrations (10-7 - 3 x 10-6 mol/L), had a small relaxant effect on U-46619 precontracted coronary arteries (Fig 1). Relaxation at the highest concentrations (3 x 10-5 mol/L for sildenafil, 10-4 mol/L for zaprinast) was approximately 50% for each. The NO donor, sodium nitroprusside, induced concentration-dependent relaxations of the coronary artery (Fig 1) that were enhanced about eight times in the presence of 10-6 mol/L sildenafil and four times in the presence of 10-5 mol/L zaprinast (Table 1). Higher concentrations of sildenafil (10-5 mol/L) and zaprinast (3 x 10-5 mol/L) did not further potentiate the relaxation induced by sodium nitroprusside.
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Fig 1. Concentration-response curves to sildenafil, zaprinast, and sodium nitroprusside in the absence and in the presence of either sildenafil (10-6 mol/L) or zaprinast (10-5 mol/L) in coronary and internal mammary arteries. Each point represents the mean ± the standard error of the mean. (n = number of donors [6 to 10 rings from each donor]).
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Table 1. Relaxant Effects of Sildenafil, Zaprinast, Sodium Nitroprusside and Sodium Nitropruside in the Presence of Either 10-6 mol/L Sildenafil or 10-5 mol/L Zaprinast on U-46619 Precontracted Vessels
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Sildenafil relaxed precontracted internal mammary arteries in a concentration-dependent manner. Zaprinast was approximately six-fold less potent than sildenafil (Table 1). Sodium nitroprusside induced concentration-dependent relaxation of the internal mammary arteries (Fig 1) that were enhanced approximately eight times by 10-6 mol/L sildenafil and by 10-5 mol/L zaprinast.
The relaxant effects of sildenafil and zaprinast in radial arteries and forearm veins are shown in Figure 2 and Table 1. Both sildenafil (10-6 mol/L) and zaprinast (10-5 mol/L) potentiated the relaxation induced by sodium nitroprusside.
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Fig 2. Concentration-response curves to sildenafil, zaprinast, and sodium nitroprusside in the absence and in the presence of either sildenafil (10-6 mol/L) or zaprinast (10-5 mol/L) in radial arteries and forearm veins. Each point represents the mean ± the standard error of the mean. (n = number of donor [6 to 10 rings from each donor]).
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The relaxations induced by sildenafil or zaprinast in the four vessels studied were not affected by previous treatment with the inhibitor of NO synthase L-NMMA (10-4 mol/L) (n = 4 for each compound).
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Comment
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Our in vitro experiments demonstrate that sildenafil causes significant relaxation in the human internal mammary artery, radial artery, and forearm vein. In the coronary artery, sildenafil had a modest relaxant effect only at the highest concentration. Sildenafil amplified the relaxation induced by the NO donor, sodium nitroprusside, in the four vessels. It is assumed that sildenafil elicits these effects as a consequence of enhancing cGMP levels [6, 15]. In vascular smooth muscle, cGMP has been shown to reduce the intracellular free Ca2+ levels by stimulating Ca2+-pumping adenosine triphosphatase (ATPase), or by opening Ca2+-activated K+ channels leading to hyperpolarization and inhibition of voltage-sensitive Ca2+ channels [16, 17]. Another mechanism that could contribute to the effect of cGMP on intracellular calcium levels is the inhibition of phospholipase phospholipase C activation [18]. Whatever mechanism was involved to cause this effect of sildenafil, the results suggest that relaxation does not involve the intervention of the L-arginine-NO pathway because L-NMMA, an inhibitor of nitric oxide synthase [19], did not modify this effect. Similar findings have been recently observed in human penile arteries and veins from multiorgan donors [13]. Compared with zaprinast, sildenafil was approximately 10-fold more potent in amplifying relaxations induced by sodium nitroprusside.
The relaxation induced by sildenafil in the internal mammary artery, radial artery, and forearm vein does not support the proposal that the action of sildenafil is only dependent on preexisting activation of the NO- cGMP levels. This proposal is based on experiments showing that sildenafil had little relaxation effect on human and rabbit corpus cavernosum strips contracted with norepinephrine [3, 6]. We observed that the relaxation induced by sildenafil was evident in the absence of an NO donor. However, in the coronary artery, sildenafil had little relaxant effects but enhanced the relaxation by sodium nitroprusside, thus indicating that the action of sildenafil in this particular artery is dependent on the preexisting activation of the NO-cGMP system; this finding is similar to that observed in isolated strips of rabbit corpus cavernosum [6] and aortic rings [15]. The maximal amplifying effects of sildenafil on relaxation induced by sodium nitroprusside occurred at a concentration of 10-6 mol/L, which is ten times greater than the concentration of sildenafil required to induce maximum increase in cGMP levels in canine coronary artery smooth muscle [15].
Unlike cAMP-specific PDE 3 inhibitors, sildenafil has not been found to have direct inotropic effects [15], which is consistent with the absence of PDE 5 in human myocardium [15, 20]. In contrast, relatively high levels of PDE 5 are found in most vascular and nonvascular smooth muscle preparations [1, 15, 20]; this distribution appears to be consistent with the observed vascular effects of sildenafil. Because sildenafil and NO elevate smooth muscle cGMP levels, it is expected that a synergistic action between sildenafil and the NO donor, sodium nitroprusside, must exist. Our data indicate that relatively low concentrations of sildenafil will potentiate the relaxation to sodium NO donors in all the vessels tested. This interaction between sildenafil and NO donors has been considered as a risk of developing potentially life-threatening hypotension [21, 22]. Our data from isolated human vessels give experimental support to recent guidelines from the American College of Cardiology and the American Heart Association reiterating the need for caution in the use of sildenafil in certain high risk patients, even in the absence of nitrate use [22].
Of particular interest are the results in the forearm vein. The venous system contains the major part of the total blood volume and is therefore an important determinant for the regulation of cardiac output [23]. In a pilot study of men with ischemic heart disease modest reductions in pulmonary arterial pressure (-27%) and cardiac output (-7%) were observed after 40 mg intravenous doses of sildenafil [10]. These changes, together with the marked relaxant effects of sildenafil on human forearm veins observed in our study, reinforce the limitations recently described [22] in relation to the use of sildenafil in patients with heart failure and borderline low blood pressure, and those receiving antihypertensive regimes.
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Acknowledgments
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This study was supported by the Comisión Interministerial de Ciencia y Tecnología, Ministerio de Sanidad and Generalitat Valenciana. Gloria Segarra was the recipient of a Fellowship of the Instituto de Salud Carlos III (99/9016).
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Accepted for publication June 5, 2000.
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Abstract
Introduction
