Nitric oxide (NO)-induced coronary vasodilation is mediated through production of cyclic guanosine monophosphate (cGMP) and through inhibition of the endothelin-1 (ET) system. We previously demonstrated that phosphodiesterase-5 (PDE5)-mediated cGMP breakdown and ET each exert a vasoconstrictor influence on coronary resistance vessels. However, little is known about the integrated control of coronary resistance vessel tone by these two vasoconstrictor mechanisms. In the present study, we investigated the contribution of PDE5 and ET to the regulation of coronary resistance vessel tone in swine both in vivo, at rest and during graded treadmill exercise, and in vitro. ETA/ETB receptor blockade with tezosentan (3 mg/kg iv) and PDE5 inhibition with EMD360527 (300 μg·min−1·kg−1 iv) each produced coronary vasodilation at rest and during exercise as well as in preconstricted isolated coronary small arteries. In contrast, tezosentan failed to produce further coronary vasodilation in the presence of EMD360527, both in vivo and in vitro. Importantly, EMD360527 (3 μM) and cGMP analog 8-Br-cGMP (100 μM) had no significant effects on ET-induced contractions of isolated porcine coronary small arteries, suggesting unperturbed ET receptor responsiveness. In contrast, PDE5 inhibition and cGMP blunted the contractions produced by the ET precursor Big ET, but only in vessels with intact endothelium, suggesting that PDE5 inhibition limited ET production in the endothelium of small coronary arteries. In conclusion, PDE5 activity exerts a vasoconstrictor influence on coronary resistance vessels that is mediated, in part, via an increase in endothelial ET production.
- PDE5 inhibition
- ETA/ETB blockade
- Big ET
- coronary vasomotor tone
coronary blood flow is tightly coupled to myocardial oxygen demand to maintain a consistently high level of myocardial oxygen extraction (10, 16, 26). This tight coupling has been proposed to depend on a myriad of vasodilators and vasoconstrictors, including nitric oxide (NO) and endothelin-1 (ET). NO causes coronary vasodilation by stimulating guanylyl cyclase in vascular smooth muscle to generate cyclic guanosine monophosphate (cGMP), which activates cGMP-dependent protein kinase G (PKG) (32). Levels of cGMP in vascular smooth muscle are tightly regulated by several cyclic nucleotide phosphodiesterases (PDEs) that hydrolyze cGMP and terminate its vasodilator effect (2). PDE5 is present in vascular smooth muscle cells in the coronary vascular bed and thus has the potential to regulate coronary blood flow by exerting a vasoconstrictor influence. Indeed, PDE5 inhibition resulted in relaxation of coronary conduit arteries in swine in vitro (45) and in vivo (1), and produced coronary resistance vessel dilation, thereby increasing coronary blood flow in humans (24) and swine (31).
NO not only induces coronary vasodilation through cGMP-mediated activation of vascular smooth muscle K+ channels (26) but also produces vasodilation by inhibiting the coronary vasoconstrictor influence of ET during exercise (30). We recently observed that this inhibition of ET by NO, which is likely cGMP-mediated (5, 14, 25), occurs principally through a reduced production of ET from Big ET (7). Consequently, we hypothesized that PDE5 exerts a vasoconstrictor influence in the coronary microcirculation, in part, by abrogating the cGMP-mediated inhibition of ET. To test this hypothesis, we investigated the coronary vasodilator effects of ET-receptor blockade in the absence and presence of PDE5 inhibition in awake swine at rest and during graded treadmill exercise. Since we observed that the vasodilation produced by ET receptor blockade was lost in the presence of PDE5-inhibition, we further evaluated the interaction between the two vasoconstrictor mechanisms in isolated coronary small arteries.
Studies were performed in accordance with the “Guiding Principles in the Care and Use of Laboratory Animals” as approved by the Council of the American Physiological Society, and with approval of the Animal Care Committee at Erasmus University Medical Center Rotterdam. Sixteen cross-bred Yorkshire Landrace swine (2–3 mo old; 22 ± 1 kg at the time of surgery) of either sex entered the in vivo study. Daily adaptation of animals to laboratory conditions started 1 wk before surgery and continued during the first week after surgery. In vitro studies were performed in coronary small arteries isolated from the hearts of 29 slaughterhouse pigs.
Swine were sedated with ketamine (20 mg/kg iv) and midazolam (1 mg/kg im), anaesthetized with thiopental (10 mg/kg iv), intubated and ventilated with a mixture of O2 and N2 (1:2), to which 0.2–1% (vol/vol) isoflurane was added (12, 29). Anesthesia was maintained with midazolam (2 mg/kg iv) and fentanyl (10 μg·kg−1·h−1 iv). Under sterile conditions, the chest was opened via the fourth left intercostal space. A fluid-filled polyvinylchloride catheter was inserted into the aortic arch for measurement of aortic pressure and blood sampling for determination of Po2, Pco2, and pH (ABL-600, Radiometer), and O2 saturation and hemoglobin concentration (OSM3, Radiometer). A microtipped, high-fidelity pressure-transducer (P4.5, Konigsberg Instruments) was inserted into the left ventricle (LV) via the apex for measurement of LV pressure and its first derivative LV dP/dt. Polyvinylchloride catheters were also inserted into the left ventricle to calibrate the Konigsberg transducer left ventricular pressure signal and into the pulmonary artery for administration of drugs. A Transonic flow probe was placed around the proximal left anterior descending coronary artery (LAD) for measurement of coronary blood flow. Finally, two small angiocatheters were inserted into the anterior inter-ventricular vein for coronary venous blood sampling (11). Electrical wires and catheters were tunneled subcutaneously to exit at the back. Then, the chest was closed, and animals were allowed to recover, receiving analgesia (0.3 mg buprenorphine im) and a slow-release fentanyl patch (12 μg/h) for 3 days, and antibiotic prophylaxis (25 mg/kg amoxicillin and 5 mg/kg gentamycin iv) for 5 days. All catheters were flushed daily with heparinized saline (1,000–5,000 IU/ml saline) to prevent the formation of blood clots and to secure catheter patency.
Studies were performed 1–2 wk (11 ± 1 days) after surgery, with animals exercising on a motor-driven treadmill. Swine (n = 16) were subjected to two different experimental protocols. In the first group, nine swine were subjected to 1) control exercise and 2) exercise in the presence of ETA/ETB receptor antagonist tezosentan (a gift from Actelion Pharmaceuticals, Allschwil, Switzerland). In the second group, eight animals (one of which was subjected to both protocols) were subjected to 1) control exercise, 2) exercise in the presence of PDE5 inhibitor EMD360527 (a gift from Merck, Darmstadt, Germany), and 3) exercise in the presence of combined EMD360527 and tezosentan. We have previously observed excellent reproducibility of consecutive control exercise protocols (12).
Effects of ETA/ETB receptor blockade.
With swine lying quietly on the treadmill, resting hemodynamic measurements were obtained, and arterial and coronary venous blood samples were collected. Hemodynamic measurements were repeated, and rectal temperature was measured with animals standing on the treadmill. Subsequently, a five-stage exercise protocol (1–5 km/h) was begun, with each stage lasting 3 min. Hemodynamic variables were continuously recorded, and blood samples were collected during the last 30 s of each exercise stage at a time when hemodynamics had reached a steady state. At the conclusion of the exercise trial, animals were allowed to rest for 90 min, resulting in a complete return of hemodynamic variables to baseline values. Subsequently, tezosentan was infused intravenously over 10 min in a dose of 3 mg/kg, followed by a continuous intravenous infusion of 6 mg·kg−1·h−1 (29, 30), and the five-stage exercise trial was repeated. Tezosentan is a highly selective and potent ET receptor antagonist, with a Ki of 10 nM for ETB and a Ki of 20 nM for ETA (6). We have previously shown that that the dosage of tezosentan used in the present study virtually abolished the systemic pressor responses elicited by intravenous ET in doses up to 100 ng·kg−1·min−1 iv, indicating highly effective ET-receptor blockade (29).
Effects of PDE5 inhibition and ETA/ETB receptor blockade.
Ninety minutes after completing a control exercise trial as described above, swine received a continuous intravenous infusion of EMD360527 in a dose of 300 μg·kg−1·min−1. Ten minutes after the infusion was started, the five-stage exercise trial was repeated, while infusion of EMD360527 was continued (23, 31). Upon completion of the exercise trial, EMD360527 infusion was halted, and animals were allowed to rest for another 90 min. Then, the EMD360527 infusion was continued while an intravenous infusion of tezosentan was started in dose-regimen identical as described above for the individual infusions, and the five-stage exercise protocol was repeated. EMD360527 is a highly selective and potent PDE5 inhibitor, demonstrating at least 45-fold selectivity for PDE5 (IC50=0.007 mM) compared with PDE6 (IC50 = 0.32 mM), 94-fold selectivity for PDE1 (IC50 = 0.66 mM), 137-fold selectivity for PDE10 (IC50 = 0.96 mM), and >1,400-fold selectivity for PDE2, PDE3, PDE4, and PDE7 (all IC50 > 10 mM). EMD360527 in a dose of 300 μg·kg−1·min−1 iv results in plasma drug concentrations of 15.4 μM (31), indicating that this dose reaches concentrations that are well above the IC50 for PDE5 and are at least an order of magnitude below the IC50 of other PDE isoforms. Selectivity for PDE5 inhibition is also supported by our observation that EMD360527 has negligible effects on LV dP/dtmax compared with the PDE3 inhibitor pimobendan (31).
Myograph Studies of Isolated Coronary Small Arteries
Swine hearts (n = 29) were collected at a local slaughterhouse. Coronary small arteries (diameter of ∼250 μm) were dissected out and stored overnight in cold Krebs bicarbonate solution aerated with 95% O2/5% CO2 of the following composition (in mM): 118 NaCl, 4.7 KCl, 2.5 CaCl2, 1.2 MgSO4, 1.2 KH2PO4, 25 NaHCO3, and 8.3 glucose; pH 7.4 (42). The next day, coronary arteries were cut into segments of ∼2-mm length and mounted in microvascular myographs (Danish Myo Technology) with separated 6-ml organ baths containing Krebs bicarbonate solution aerated with 95% O2/5% CO2 and maintained at 37°C. Changes in contractile force were recorded with a Harvard isometric transducer. Following a 30-min stabilization period, the internal diameter was set to a tension equivalent to 0.9 times the estimated diameter at 100 mmHg effective transmural pressure. The vessels were then exposed to 30 mM KCl twice. Endothelial integrity was verified by observing dilation to 10 nM substance P after preconstriction with 100 nM of the stable thromboxane-A2 analog 9,11-dideoxy-11α,9α epoxymethanoprostaglandin F2α (U46619). Then, vessels were subjected to 100 mM KCl to determine the maximal vascular contraction. Thereafter, vessels were allowed to equilibrate in fresh Krebs solution for 30 min before different experimental protocols were initiated (4, 46). Only one protocol was executed per vessel, and vessels within one experimental protocol were obtained from different animals.
Effects of PDE5 inhibition and ETA/ETB blockade.
To study the interaction between PDE5 and ET receptors more directly, i.e., without the influence of perturbations in hemodynamics or myocardial metabolism, dose responses to EMD360527, tezosentan, and their combination were studied in isolated coronary small arteries. For this purpose, vessels were preconstricted with U46619, and concentration-response curves of EMD360527 (10−9 to 3 × 10−6 M) (n = 6), tezosentan (10−9 to 3 × 10−6 M) (n = 6), and combined EMD360527 and tezosentan (n = 6) were studied.
Effects of PDE5 inhibition and cGMP on ET receptor sensitivity and ET production.
To study the mechanism of interaction between PDE5 and ET, vessels were exposed to ET (10−9 to 10−7 M) and Big ET (10−8 to 10−6 M) in the absence and presence of EMD360527 (3 μM) (n = 15 vessels for ET; n = 16 vessels for Big ET), and in the absence and presence of 8-Br-cGMP (100 μM) (n = 6 in each group). In a separate group of vessels, the endothelium was mechanically removed, and vessels were subsequently exposed to ET (10−9 to 10−7 M) (n = 6) and to Big ET (10−8 to 10−6 M) in the absence (n = 7) and presence (n = 6) of 8-Br-cGMP (100 μM). Based on in vivo (7) and in vitro (35, 43) observations, we selected a higher dose range of Big ET than ET to achieve similar vasoconstrictor responses. Big ET has no direct vasomotor effect (34), so that the Big ET-induced vasoconstriction reflects the conversion from Big ET to vasoactive ET (7).
Data Analysis and Statistics
Digital recording and offline analysis of hemodynamic data and computation of myocardial O2 consumption (MV̇o2) have been described in detail elsewhere (12, 29). Myocardial O2 delivery (MDo2) was computed as the product of LAD coronary blood flow and arterial blood O2 content. MV̇o2 in the region of myocardium perfused by the LAD was calculated as the product of coronary blood flow and the difference in O2 content between arterial and coronary venous blood. Myocardial oxygen extraction (MEo2) was computed as the ratio of MV̇o2 and MDo2.
In vitro vascular contraction responses to ET and Big ET were normalized to 100 mM KCl. Vascular relaxation responses to EMD360527, tezosentan, and combined EMD360527 and tezosentan were expressed as a percentage of contraction to U46619.
Statistical analysis of hemodynamic data was performed using two-way ANOVA for repeated measures followed by post hoc test using Scheffe's test for the effect of exercise and paired t-testing with modified Bonferroni correction (22) for the effect of drug interventions. To test the effects of drug treatment on the relation between MV̇o2 [or the product of heart rate and LV systolic pressure, i.e., the rate pressure product (RPP)] and MEo2, coronary venous O2 tension (cvPo2), and coronary venous O2 saturation (cvSo2), regression analysis was performed using drug treatment and MV̇o2 or RPP, as well as their interaction as independent variables, and by assigning a dummy variable to each animal to account for inter-animal variability. Linearity of the relations was confirmed by plotting the predicted value of the dependent variable against the residual value (measured value-predicted value). In all cases, residuals were scattered around zero over the entire range of predicted values. Statistical analysis of the effects of EMD360527, tezosentan, as well as combined EMD360527 and tezosentan on preconstricted isolated coronary small arteries, and the effects of EMD360527 and 8-Br-cGMP on ET and Big ET responses in isolated coronary small arteries was performed using two-way ANOVA. Statistical significance was accepted when P < 0.05 (two-tailed). Data are presented as means ± SE.
Integrated Effects of EMD360527 and tezosentan in the Coronary Circulation
Graded treadmill exercise had minimal effects on mean aortic blood pressure but produced marked increases in heart rate, LV systolic pressure, LV dP/dtmax and RPP (Table 1). The consequent increase in MV̇o2 was met by a commensurate increase in coronary blood flow and MDo2 (Table 2) so that MEo2 and hence cvSo2 and cvPo2 remained relatively constant during exercise (Figs. 1 and 2).
Intravenous infusion of the ETA/ETB receptor blocker tezosentan produced a 5- to 10-mmHg decrease in mean aortic pressure, reaching statistical significance during exercise, with no significant effect on heart rate (Table 1). In contrast, EMD360527 produced a 10- to 15-mmHg decrease in blood pressure that was accompanied by, presumably baroreceptor reflex-mediated, increases in heart rate. Combined infusion of EMD360527 and tezosentan produced a further decrease in mean aortic pressure compared with EMD360527 alone, which was similar to that produced by tezosentan alone (Table 1).
Tezosentan tended to increase both coronary blood flow and hemoglobin (Table 2), resulting in an upward shift of the relation between MDo2 and MV̇o2, thereby allowing a small but significant decrease in MEo2 that resulted in a small increase in cvPo2 and cvSo2, both at rest and during exercise (Figs. 1 and 2). EMD360527 resulted in a significant increase in coronary blood flow while hemoglobin was reduced (Table 2). The increase in coronary blood flow outweighed the decrease in hemoglobin, resulting in an overall increase in MDo2, thereby allowing a decrease in MEo2, resulting in a marked increase in cvPo2 as well as cvSo2 (Figs. 1 and 2). Strikingly, in the presence of EMD360527, the effects of tezosentan on MEo2, cvSo2, and cvPo2 were no longer observed, indicating that the vasoconstrictor influence of endogenous ET was abolished by PDE5 inhibition (Figs. 1 and 2).
Integrated Effects of EMD360527 and tezosentan in isolated Coronary Small Arteries
Concentration responses to tezosentan, EMD360527, and combined tezosentan and EMD360527 were studied in isolated porcine coronary small arteries preconstricted with U46619. Both tezosentan and EMD360527 produced dose-dependent vascular relaxation (Fig. 3). Similar to the observations in vivo, we observed not only that the vasorelaxation responses to tezosentan in vitro were smaller than the responses to EMD360527 but also that the vasorelaxation responses to tezosentan were lost in the presence of EMD360527. These in vitro findings suggest that the in vivo observations were not due to systemic hemodynamic changes resulting from intravenous administration of tezosentan and EMD360527, and hence suggest a local interaction between PDE5 and ET.
Effects of EMD360527 and 8-Br-cGMP on ET Receptor Sensitivity and ET Production
To further study the local vascular interaction between PDE5 and ET, we conducted experiments in isolated coronary small arteries exposed to either ET or Big ET, both in the absence and presence of EMD360527 and 8-Br-cGMP. Although EMD360527 had no significant effect on the concentration-dependent contractions produced by ET, it attenuated vascular contraction by Big ET (Fig. 4). Similarly, 8-Br-cGMP significantly attenuated Big ET-induced, but not ET-induced, vascular contraction (Fig. 4). These findings suggest that ET production from Big ET, rather than ET receptor sensitivity, was modulated by PDE5 activity and cGMP signaling.
To further investigate the site of interaction between PDE5 and ET production, we performed studies in denuded coronary small arteries. Denudation enhanced ET-induced vascular contraction (Fig. 5), likely due to unopposed vascular smooth muscle ET receptor activation following loss of endothelial ETB receptor-mediated vasodilator influence. In contrast, denudation had no effect on Big ET-induced vascular contraction (Fig. 5). In view of the enhanced responsiveness to ET in denuded vessels, the lack of increase in contraction produced by Big ET implies a reduced ET production following denudation. These findings indicate that a significant part of the ET production occurs in the endothelium. Finally, the attenuation by 8-Br-cGMP of Big ET-induced vascular contraction, which was observed in intact vessels, was no longer detected in denuded vessels (Fig. 6), suggesting that the attenuation of ET production by cGMP principally occurs in endothelial cells.
The main findings of the present study in the porcine coronary circulation were that 1) PDE5 inhibition with EMD360527 and ET receptor blockade with tezosentan each produced coronary vasodilation, with vasodilation by PDE5 inhibition being significantly greater than that produced by ET blockade; 2) in the presence of PDE5 inhibition, ET receptor blockade failed to produce additional coronary vasodilation; 3) both PDE5 inhibition and cGMP significantly attenuated Big ET but not ET-induced contraction in isolated coronary small arteries; 4) denudation of coronary small arteries enhanced ET-induced contraction but had no effect on Big ET-induced contraction; and 5) cGMP failed to blunt Big ET-induced contractions in denuded vessels. The implication of these findings will be discussed below.
Intravenous administration of blockers of vasoactive substances not only results in changes in coronary resistance vessel tone but can also produce pronounced changes in systemic hemodynamics. In the present study, PDE5 inhibition, ETA/ETB blockade, and their combination resulted in decreases in mean aortic pressure, which are the result of systemic vasodilation (23, 29, 31) and which require autoregulatory adjustments in coronary vascular resistance. In addition, the baroreceptor reflex-mediated increases in heart rate and contractility, together with the decrease in aortic blood pressure, will result in changes in myocardial metabolism, requiring adjustments of myocardial oxygen supply and hence coronary blood flow. Finally, ET receptor blockade tended to increase hemoglobin levels at rest [possibly due to an indirect reflex-activation of α-adrenergic receptors on the splenic capsule (26)], whereas PDE5 inhibition produced a slight reduction in hemoglobin during exercise [possibly due to a direct cGMP-mediated relaxing action on the splenic capsule (26)], which requires adjustments in coronary blood flow to maintain myocardial oxygen delivery. Consequently, changes in coronary blood flow and/or coronary vascular resistance in response to PDE5 inhibition and ET blockade in vivo not only reflect direct effects of these pharmacological interventions on coronary resistance vessel tone but are also influenced by their effects on perfusion pressure, myocardial metabolic demand, and/or the blood's oxygen carrying capacity. Hence, interpretation of changes in coronary blood flow and coronary vascular resistance data is difficult when the effects of pharmacological interventions on coronary resistance vessel tone are investigated in awake animals over a wide range of myocardial metabolic activities.
A sensitive way to study the regulation of coronary resistance vessel tone during exercise is to examine the relation between coronary venous oxygen levels and MV̇o2 (10, 38). Thus a direct decrease in vasomotor tone by PDE5 inhibition, ETA/ETB blockade, or their combination will increase MDo2 at a given level of MV̇o2. This increase in MDo2 relative to MV̇o2 will allow a decrease in MEo2, thereby leading to increases in cvPo2 and cvSo2 and hence in an upward shift of the relation between MV̇o2 and coronary venous oxygen levels. The coronary venous oxygen level thus represents a sensitive index of myocardial tissue oxygenation (i.e., the balance between MDo2 and MV̇o2) and is therefore commonly used to reflect coronary resistance vessel tone (10, 38).
Although we (10) and other coronary physiologists (3, 9, 15, 21, 33, 37, 39, 44) often employ the myocardial oxygen balance to study the regulation of coronary resistance vessel tone, potential limitations of this approach have been pointed out (20). For example, plotting MV̇o2 vs. cvPo2 or cvSo2 can be considered inappropriate because cvPo2 and cvSo2 are part of the formula to calculate MV̇o2, so that MV̇o2 is not an independent variable. For this purpose, we also plotted RPP vs. MEo2, cvPo2, and cvSo2, and found that substituting RPP for MV̇o2 yielded virtually identical results (see Fig. 2).
Another potential concern is that the oxygen dissociation curve is influenced by various factors, including pH, Pco2, and NO, which could affect computation of MV̇o2, particularly when cvSo2 is estimated from cvPo2 using an assumed oxygen dissociation curve (3, 13). In the present study, MV̇o2 was determined using highly accurate and direct measurements of not only cvPo2 but also of Hb concentration and cvSo2, thereby accounting for any potential changes in the oxygen dissociation curve. Finally, a linear relation between MV̇o2 and cvPo2 or cvSo2 is often assumed, even when this is not apparent from visual inspection (18). In the present study, we validated the use of linear regression analysis by statistically confirming linearity of all relations between MV̇o2 and MEo2 as well as coronary venous oxygen levels.
Integrated Control of Coronary Resistance Vessel Tone in Swine by ET and PDE5
Regulation of coronary resistance vessel tone is the result of interplay of a myriad of not only vasodilator but also vasoconstrictor influences (10, 26). Many of the coronary vascular control mechanisms are highly similar across species (26), but some interspecies differences exist, including a lack of significant α-adrenergic constriction of coronary resistance vessels in swine (20). Importantly, coronary vasoconstrictor influences of ET and PDE5 have been demonstrated across a variety of species, including swine (8, 29–31), dogs (17, 23a), and humans (24, 27).
In accordance with previous studies from our laboratory, we observed in the present study that ET receptor blockade (29, 30) and PDE5 inhibition (31), each resulted in coronary vasodilation, as evidenced by the decreased MEo2 and increased cvPo2 and cvSo2. The vasodilator effect produced by PDE5 inhibition was greater than that produced by ET blockade both in vivo and in vitro, indicating that the vasoconstrictor influence of PDE5 in coronary resistance vessels is greater than that of endogenous ET. Importantly, in the presence of PDE5 inhibition with EMD360527, ETA/ETB blockade with tezosentan failed to produce additional vasodilation of coronary resistance vessels beyond that produced by PDE5-inhibition alone. This observation is highly suggestive of a direct interaction between the NO-cGMP and ET systems. Indeed, we have previously shown that NO elicits coronary vasodilation in part via inhibition of ET-mediated vasoconstriction (30), which was likely mediated through reduced production of ET from Big ET (7). Experiments in the porcine aorta (5), the rat heart (14), and the cultured pulmonary arterial endothelial cells (25) have shown that not only NO but also the non-hydrolysable cGMP analog 8-Br-cGMP can suppress ET production and release. Interestingly, we found that both PDE5 inhibition and 8-Br-cGMP significantly attenuated the vasoconstrictor response of isolated coronary small arteries to Big ET, but not to ET, indicating that cGMP suppressed ET production rather than ET receptor sensitivity.
To investigate the site of interaction between PDE5 and ET production in more detail, we performed studies in denuded coronary small arteries. Denudation enhanced ET-induced vascular contraction, likely due to unopposed vascular smooth muscle ET receptor activation following loss of an endothelial ETB-receptor vasodilator influence (29). In contrast, denudation had no effect on Big ET-induced vascular contraction. In view of the enhanced responsiveness to ET in denuded vessels, the lack of increase in contraction produced by Big ET implies a reduced ET production following denudation. Together, these observations suggest that a significant part, but not all, of the ET production occurs in the endothelium of porcine coronary small arteries. Interestingly, the attenuation by 8-Br-cGMP of Big ET-induced vascular contraction, which was observed in vessels with intact endothelium, was no longer observed in endothelium-denuded vessels. These latter findings suggest that, similar to what has been reported in pulmonary endothelial cells (25), the cGMP-mediated blunting of ET production principally occurs in endothelial cells of coronary small arteries in swine.
Insight into the mechanisms regulating coronary microvascular tone is of critical importance in understanding the regulation of coronary blood flow under physiological conditions, such as during exercise, but also in understanding the mechanisms of microvascular dysfunction and its contribution to perturbations in myocardial oxygen supply in coronary vascular disease states. This is especially true in light of increasing evidence that coronary microvascular dysfunction is present in a variety of coronary vascular disease states (19), with evidence of reduced NO/cGMP signaling (40) and increased ET signaling (36). Those observations have fueled the concept that microvascular dysfunction is an important component of, and contributor to, myocardial ischemia in patients with coronary artery disease (28). The present study shows that loss of NO/cGMP signaling and enhanced ET signaling could both result from an increased PDE5 activity. Future studies should therefore be aimed at investigating the role of enhanced PDE5 activity, and its interaction with NO/cGMP and ET, in coronary microvascular dysfunction in animal models and patients with ischemic heart disease.
In conclusion, the present study demonstrated that PDE5 inhibition blunted the vasoconstrictor influence of ET in porcine coronary resistance vessels, which appeared to be principally the result of inhibition of ET production in the endothelium, with no change in ET responsiveness. These findings indicate that PDE5 exerts a vasoconstrictor influence in the porcine coronary microcirculation that is mediated in part via an enhanced ET production.
This study was supported by The Netherlands Heart Foundation (2000T042 to D. Merkus), The European Commission (FP7-Health-2010; MEDIA-261409 to Dirk J. Duncker and D. Merkus), The China Scholarship Council (2009624027 to Z. Zhou), the National Institutes of Health (HL-36088 to M. H. Laughlin and T32-AR-048523 to S. B. Bender), and the Department of Veteran's Affairs BLR&D CDA-2 (002030). This work was also partially supported with resources and the use of facilities at the Harry S. Truman Memorial Veteran's Hospital in Columbia, MO.
No conflicts of interest, financial or otherwise, are declared by the author(s).
Author contributions: Z.Z., V.J.d.B., S.B.B., and D.M. performed experiments; Z.Z. and V.J.d.B. analyzed data; Z.Z., V.J.d.B., A.J.D., D.M., M.H.L., and D.J.D. interpreted results of experiments; Z.Z. prepared figures; Z.Z., D.M., and D.J.D. drafted manuscript; Z.Z., V.J.d.B., S.B.B., A.J.D., D.M., M.H.L., and D.J.D. edited and revised manuscript; Z.Z., V.J.d.B., S.B.B., A.J.D., D.M., M.H.L., and D.J.D. approved final version of manuscript; D.M. and D.J.D. conception and design of research.
The authors gratefully acknowledge the expert technical assistance of Annemarie Verzijl.
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