A significant endothelium-dependent vasodilation persists after inhibition of nitric oxide synthase (NOS) and cyclooxygenase (COX) in the coronary vasculature, which has been linked to the activation of cytochrome P-450 (CYP) epoxygenases expressed in endothelial cells and subsequent generation of vasodilator epoxyeicosatrienoic acids. Here, we investigated the contribution of CYP 2C9 metabolites to regulation of porcine coronary vasomotor tone in vivo and in vitro. Twenty-six swine were chronically instrumented. Inhibition of CYP 2C9 with sulfaphenazole (5 mg/kg iv) alone had no effect on bradykinin-induced endothelium-dependent coronary vasodilation in vivo but slightly attenuated bradykinin-induced vasodilation in the presence of combined NOS/COX blockade with Nω-nitro-l-arginine (20 mg/kg iv) and indomethacin (10 mg/kg iv). Sulfaphenazole had minimal effects on coronary resistance vessel tone at rest or during exercise. Surprisingly, in the presence of combined NOS/COX blockade, a significant coronary vasodilator response to sulfaphenzole became apparent, both at rest and during exercise. Subsequently, we investigated in isolated porcine coronary small arteries (∼250 μm) the possible involvement of reactive oxygen species (ROS) in the paradoxical vasoconstrictor influence of CYP 2C9 activity. The vasodilation by bradykinin in vitro in the presence of NOS/COX blockade was markedly potentiated by sulfaphenazole under control conditions but not in the presence of the ROS scavenger N-(2-mercaptoproprionyl)-glycine. In conclusion, CYP 2C9 can produce both vasoconstrictor and vasodilator metabolites. Production of these metabolites is enhanced by combined NOS/COX blockade and is critically dependent on the experimental conditions. Thus production of vasoconstrictors slightly outweighed the production of vasodilators at rest and during exercise. Pharmacological stimulation with bradykinin resulted in vasodilator CYP 2C9 metabolite production when administered in vivo, whereas vasoconstrictor CYP 2C9 metabolites, most likely ROS, were dominant when administered in vitro.
- reactive oxygen species
- coronary blood flow
- coronary vasomotor tone
coronary blood flow is tightly regulated to maintain a consistently high level of myocardial oxygen extraction over a wide range of myocardial oxygen demands (8, 9, 15). This tight regulation is dependent on a myriad of vasoconstrictor and vasodilator influences, exerted by the autonomic nervous system, myocardium, endothelium, and blood (8, 9). Nitric oxide (NO) and prostacyclin (PGI2) are well-known endothelium-derived vasodilators that exert significant vasodilator influences in the coronary microcirculation of swine (36) and that are in part mediated via inhibition of endothelin, particularly during exercise (37). In addition to NO and PGI2, there is a variety of vasodilators, including S-nitrosothiols, K+, H2O2, and cytochrome P-450 (CYP) metabolites (2, 14, 20, 32), that produce vasodilation via opening of vascular smooth muscle K+ channels and subsequent membrane hyperpolarization (7, 19). The contribution of these so-called endothelium-derived hyperpolarizing factors (EDHFs; Refs. 5, 17, 41) to endothelium-dependent vasodilation varies, depending on the species and vascular bed studied (4, 7), as well as on the agonist used to stimulate the endothelium (7, 42).
One group of EDHFs is produced by CYP isoenzymes (23, 33, 39), of which there are two classes: 1) epoxygenases that catalyze the formation of epoxyeicosatrienoic acids (EETs), and 2) ω-hydroxylases that produce 20-hydroxyeicosatetraenoic acid (23, 27). Endothelial cells are capable of synthesizing the four regioisomeric EETs, 14,15-, 11,12-, 8,9-, and 5,6-EETs, with 11,12-EETs being the major metabolite produced by CYP 2C9 epoxygenase (13, 18, 20, 40). A CYP 2C epoxygenase homologous to CYP 2C8/9 has been identified as a putative coronary EDHF synthase in porcine coronary vasculature (21) and was shown to mediate the bradykinin-induced endothelium-dependent hyperpolarization of vascular smooth muscle cells, in the presence of NO synthase (NOS) and cyclooxygenase (COX) blockade, in isolated porcine coronary large arteries (19). In contrast, another study (2) failed to observe an effect of CYP 2C9 inhibition on the bradykinin-mediated vasodilation of porcine coronary small arteries (∼300 μm) in vitro. However, most of the coronary vascular resistance resides in vessels <200 μm in diameter (45). Furthermore, the role of CYP 2C9 metabolites in porcine coronary resistance vessel dilation produced by bradykinin in vivo has not been investigated to date. Consequently, the first aim of the present study was to study the contribution of CYP 2C9 metabolites to the coronary vasodilation produced by bradykinin in awake resting swine in the absence and presence of inhibition of NOS and COX.
The mechanisms that mediate exercise hyperemia in the heart remain incompletely understood (8, 9). Since there is evidence that CYP 2C9 metabolites contribute to skeletal muscle exercise hyperemia and enhance oxygen uptake in the human leg (24), we hypothesized that CYP 2C9 metabolites play a similar role in exercise hyperemia in the heart. Consequently, the second aim of the present study was to determine the contribution of endogenous CYP 2C9 metabolites in the control of coronary resistance vessel tone in swine at rest and during exercise.
The results of these in vivo studies showed that, whereas CYP 2C9 metabolites contributed to bradykinin-induced coronary vasodilation, CYP 2C9 metabolites exerted a basal coronary vasoconstrictor influence. Since CYP 2C9 has been shown to produce reactive oxygen species (ROS; Refs. 20, 22), the third aim of our study was to further explore the contribution of CYP 2C9 metabolites to bradykinin-induced coronary vasodilation. Specifically, we tested in vitro the hypothesis that ROS scavenging would enhance the CYP 2C9-mediated vasodilation by bradykinin.
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 Medical Center Rotterdam. Twenty-six crossbred Yorkshire x Landrace swine (2- to 3-mo-old and 22 ± 1 kg at the time of surgery) of either sex entered the study. Daily adaptation of animals to laboratory conditions started 1 wk before surgery and continued during the first week after surgery.
Swine were sedated with ketamine (30 mg/kg iv), anesthetized 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 (10, 11, 43). 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 and a fluid-filled polyvinylchloride catheter was inserted into the aortic arch for the measurement of mean aortic pressure and blood sampling for the determination of Po2, Pco2, pH, O2 saturation, and hemoglobin concentration. A microtipped high fidelity pressure-transducer (P4.5; Konigsberg Instruments) was inserted into the left ventricle via the apex. A polyvinylchloride catheter was also inserted into the left ventricle to calibrate the Konigsberg transducer left ventricle pressure signal. Other polyvinylchloride catheters were inserted into the left atrium to measure pressure and into the pulmonary artery for the systemic administration of drugs. A Transonic flow probe was placed around the proximal left anterior descending coronary artery (LAD) for the measurement of coronary blood flow (35). Finally, small angiocatheters were inserted into the anterior interventricular vein for coronary venous blood sampling (11) and into the left anterior descending coronary artery for intracoronary infusion of bradykinin. Electrical wires and catheters were tunneled subcutaneously to the back. Then, the chest was closed and animals were allowed to recover, receiving analgesia (0.3 mg buprenorphine im) for 2 days and antibiotic prophylaxis (25 mg/kg amoxicillin and 5 mg/kg gentamycin iv) for 5 days.
In Vivo Experimental Protocols
In vivo studies were performed 1–3 wk after surgery. In the first series of experiments, we investigated the contribution of CYP 2C9 metabolites to bradykinin-induced coronary vasodilation in resting swine, while in the second series of experiments, we investigated the contribution of CYP 2C9 metabolites to the coronary hyperemia during exercise. Since both NO and PGI2 can act on CYP 2C9 enzymes and inhibit the formation of EDHFs (3, 29), studies were performed in the absence and presence of combined NOS and COX inhibition. All four protocols were performed on different days and in random order.
Contribution of CYP 2C9 metabolites to bradykinin-induced coronary vasodilation.
Hemodynamic measurements, consisting of left atrial and aortic blood pressures, heart rate, and coronary blood flow, were obtained in nine swine, while animals were resting quietly. Subsequently, the coronary blood flow responses to bradykinin were determined. Bradykinin was infused into the coronary artery in consecutive doses of 0.03, 0.1, 0.3, 1.0, and 3.0 μg/min, with each infusion step lasting 3 min. Hemodynamic measurements were recorded at baseline and throughout the entire infusion protocol. Then, the infusion was stopped and coronary blood flow was allowed to return to baseline values. Thirty minutes later, the selective CYP 2C9 inhibitor sulfaphenazole [inhibition constant (Ki) value of 0.3 ± 0.1 μM for CYP 2C9], which results in >100-fold less inhibition of CYP 2C8 (Ki of 63 μM) or 2C18 (Ki of 29 μM) and no inhibition of CYP 1A1, 1A2, 3A4, and 2C19 (20, 31), was infused intravenously in a dose of 5 mg/kg over 10 min. Ten minutes after completion of administration, the bradykinin dose-response protocol was repeated.
On a different day, four resting swine received systemic administration of the endothelial NOS inhibitor Nω-nitro-l-arginine (NLA) in a dose of 20 mg/kg followed by systemic administration of the COX inhibitor indomethacin in a dose of 10 mg/kg (25). Ten minutes later, the intracoronary bradykinin dose-response protocol, as described above, was performed. Thirty minutes after completion of bradykinin infusion, 5 mg/kg indomethacin was intravenously infused (25). Then, sulfaphenazole was infused intravenously, in a dose of 5 mg/kg, and 10 min later the bradykinin dose response protocol was repeated.
Contribution of CYP 2C9 metabolites to coronary resistance vessel tone at rest and during exercise.
With fourteen 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 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 protocol, animals were allowed to rest for 90 min, resulting in a complete return of hemodynamic variables to baseline values. Subsequently, sulfaphenazole was intravenously administered in a dose of 5 mg/kg, and 10 min after completion of administration, the exercise protocol was repeated. We (11) have previously observed excellent reproducibility of two consecutive exercise protocols.
On a different day, the NLA (20 mg/kg) and indomethacin (10 mg/kg) were infused intravenously in 10 swine (25), and the exercise protocol as described above was performed. Ninety minutes later, indomethacin was administered in a dose of 5 mg/kg (which, in combination with the long-lasting effect of NLA, results in similar hemodynamic conditions as observed after administration of NLA and indomethacin before the first exercise protocol; Ref. 25), followed by intravenous infusion of sulfaphenazole (5 mg/kg) and the exercise protocol was repeated.
In Vitro Experimental Protocols
Swine hearts (n = 16) were collected at a local slaughterhouse. Coronary small arteries (diameter ≈250 μm) were dissected out and stored overnight in cold, oxygenated Krebs bicarbonate solution of the following composition (mM): 118 NaCl, 4.7 KCl, 2.5 CaCl2, 1.2 MgSO4, 1.2 KH2PO4, 25 NaHCO3, 8.3 and glucose pH 7.4. The next day, coronary arteries were cut into segments of ≈2 mm in 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 U46619. Then, vessels were subjected to 100 mM KCl to determine the maximal vascular contraction. Thereafter, vessels were allowed to equilibrate in fresh organ bath fluid for 30 min before initiating different experimental protocols (2). Only one protocol was executed per vessel, and each experimental protocol was performed in five to eight vessels, each obtained from different animals.
Contribution of CYP 2C9 metabolites to bradykinin-induced coronary vasodilation in vitro.
Following preconstriction with U46619 (100 nM), concentration-responses to bradykinin (10−10-10−7 M) were conducted in control vessels and in vessels incubated with sulfaphenazole (10−5 M), N-(2-mercaptoproprionyl)-glycine (MPG; 3 × 10−4 M) and combined sulfaphenazole and MPG. All experiments were performed in the absence and presence of Nω-nitro-l-arginine methyl ester HCl (l-NAME; 10−4 M) and indomethacin (10−5 M), respectively.
Digital recording and offline analysis of hemodynamic data and computation of myocardial O2 consumption (MV̇o2) have been described in detail elsewhere (11, 12, 34). Coronary vascular conductance was calculated as coronary blood flow divided by mean aortic pressure minus left atrial pressure. 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. Vascular relaxation responses to bradykinin were expressed as percentage of contraction to U46619.
Statistical analysis of in vivo hemodynamic data was performed using two-way ANOVA for repeated measures, followed by post hoc analysis using Scheffé's test (for the effects of bradykinin and exercise) and t-test (for the effects of drugs NLA + Indo and Sulfa) when appropriate. To test the effects of drug treatment on the relation between MV̇o2 and coronary venous O2 tension (cvPo2) and coronary venous O2 saturation (cvSo2), regression analysis was performed using drug treatment and MV̇o2, as well as their interaction as independent variables, and by assigning a dummy variable to each animal. The effects of drug treatment on the bradykinin responses in vitro were analyzed using two-way (bradykinin × drug) ANOVA. Statistical significance was accepted when P < 0.05. Data are presented as means ± SE.
Contribution of CYP 2C9 Metabolites to Bradykinin-Induced Coronary Vasodilation
Intracoronary infusion of bradykinin resulted in dose-dependent increases in coronary blood flow and in coronary vascular conductance up to 180% (Table 1; Fig. 1). Inhibition of CYP 2C9 with sulfaphenazole had no effect on bradykinin-induced coronary vasodilation.
After blockade of NOS/COX pathways by NLA and indomethacin, the coronary blood flow increments in response to bradykinin were blunted at lower dosages (0.03, 0.1, and 0.3 μg/min) but enhanced at higher dosages (1 and 3 μg/min; Table 1). The latter was likely due to an increase in coronary perfusion pressure as aortic blood pressure increased from ∼80 to ∼140 mmHg following NLA and indomethacin. Consequently, at a lower dosage of bradykinin the increase in coronary vascular conductance was attenuated in the presence of blockade of NOS/COX pathways, whereas at higher dosages the bradykinin-mediated increments in coronary conductance were comparable to those observed in the absence of NOS/COX blockade (Fig. 1). These findings suggest that bradykinin-induced coronary vasodilation is principally dependent on NO and PGI2 at lower dosages but at higher dosages appears independent of NO and PGI2.
In the presence of NOS/COX inhibition, sulfaphenazole attenuated the bradykinin-mediated increases in coronary blood flow (Table 1) and coronary vascular conductance (Table 1 and Fig. 1), particularly at higher dosages of bradykinin (both P < 0.01 for bradykinin × sulfaphenazole). These findings indicate that CYP 2C9 metabolites contribute to bradykinin-induced coronary resistance vessel dilation, but only when NOS/COX pathways are blocked.
Contribution of CYP 2C9 Metabolites to Coronary Resistance Vessel Tone at Rest and During Exercise
Exercise produced graded increase in heart rate, left ventricular systolic pressure, and the maximum rate of rise in left ventricular pressure with minimal effects on mean aortic blood pressure (Table 2). The consequent increase in MV̇o2 during exercise was met by a similar increase in coronary blood flow and myocardial oxygen supply, so that oxygen extraction, cvPo2, and cvSo2 were maintained relatively constant during exercise (Table 3 and Fig. 2).
Intravenous infusion of sulfaphenazole had no systemic hemodynamic effects (Table 2) but resulted in a slight increase in coronary vascular conductance (Table 3, P < 0.05 by ANOVA) leading to an increase in myocardial O2 supply that exceeded the O2 demand, thereby resulting in small increases in cvPo2 and cvSo2 both at rest and during exercise (Fig. 2). The small increases in cvPo2 (overall 0.9 ± 0.2 mmHg) were, however, highly consistent and hence reached statistical significance (P < 0.05), indicating that sulfaphenazole produced a small degree of coronary vasodilation that was similar at rest and during exercise.
In accordance with previous studies from our laboratory (36), administration of NLA and indomethacin resulted in a pronounced increase in aortic pressure that was accompanied by a, probably baroreflex-mediated, decrease in heart rate; these effects waned with incremental exercise levels (Table 2). NLA and indomethacin also produced coronary vasoconstriction, as reflected in the marked reductions in cvPo2 and cvSo2, that was well maintained during exercise (Fig. 2). Subsequent intravenous infusion of sulfaphenazole resulted in increases in cvPo2 and cvSo2 at rest that were maintained during exercise (Fig. 2), reflecting coronary vasodilation. Importantly, the increases cvPo2 and cvSo2 by sulfaphenazole were enhanced by pretreatment with NOS/COX inhibition (Fig. 2). Taken together, these findings suggest that endogenous CYP 2C9 metabolites exert a net vasoconstrictor influence on the coronary vasculature, which is blunted by NO and PGI2.
Contribution of ROS to the CYP 2C9-Mediated Vasoconstriction
To investigate the role of ROS in opposing the bradykinin-induced vasodilation, we studied the effect of ROS scavenging with MPG on the vasodilator response to bradykinin in isolated coronary small arteries in vitro, both in the absence and presence of NOS and COX inhibition and/or CYP 2C9 inhibition. Bradykinin resulted in dose-dependent small artery relaxation (Fig. 3). MPG shifted the bradykinin concentration-response curve to the left, indicating that bradykinin-induced vasorelaxation was limited by simultaneous ROS production. However, sulfaphenazole had no effect on bradykinin-induced relaxation either in the presence or absence of MPG (Fig. 3), indicating that, similar to our in vivo observations, CYP 2C9 does not contribute to bradykinin-induced vasorelaxation in vitro. Blockade of NOS and COX by N-nitro-l-arginine methyl ester and indomethacin significantly attenuated bradykinin-induced vasorelaxation. The effect of subsequent ROS scavenging with MPG was enhanced, indicating that ROS production was increased following inhibition of NOS and COX. In contrast to our in vivo observations, administration of sulfaphenazole in the presence of N-nitro-l-arginine methyl ester and indomethacin enhanced the bradykinin-induced vasorelaxation in vitro (Fig. 3), indicating that production of vasoconstrictor metabolites by CYP 2C9 predominated in vitro. The observation that this effect of sulfaphenazole was no longer present in the presence of MPG suggests that indeed CYP 2C9 is a functionally significant source of ROS in isolated porcine coronary small arteries, but only when NOS and/or COX are blocked.
The present study is the first to investigate the contribution of CYP 2C9 metabolites to the bradykinin-induced vasodilation in the coronary microcirculation in vivo and the contribution of CYP 2C9 metabolites to exercise hyperemia. The main findings were that 1) CYP 2C9 inhibition by sulfaphenazole did not affect bradykinin-induced coronary vasodilation under control conditions either in vivo or in vitro, but 2) in the presence of combined NOS/COX blockade, CYP 2C9 inhibition blunted the bradykinin-induced coronary vasodilation in vivo, whereas 3) it enhanced the bradykinin-induced coronary vasodilation in vitro; 4) the potentiation by CYP 2C9 inhibition of bradykinin-induced vasodilation in vitro was abolished following ROS scavenging with MPG; 5) inhibition of CYP 2C9 produced slight but consistent coronary vasodilation in vivo at rest that was maintained during exercise; and 6) the vasodilation by CYP 2C9 inhibition was enhanced in the presence of NOS/COX blockade. The implications of these findings will be discussed below.
Contribution of CYP 2C9 Metabolites to Bradykinin-Induced Coronary Vasodilation
The endothelium plays a critical role in regulation of coronary vasomotor tone, not only by production of well-known factors like NO and PGI2 but also through production of other factors that influence the membrane-potential of the vascular smooth muscle cells (16). One of these EDHFs is 11,12-EET, a metabolite of CYP 2C9. The present study investigated the contributions of NO, prostanoids, and CYP 2C9 metabolites on the bradykinin-induced coronary resistance vessel dilation in swine in vivo. We found that bradykinin-induced vasodilation of the coronary microcirculation was not altered by CYP 2C9 inhibition with sulfaphenazole, whereas inhibition of NOS and COX attenuated the vasodilator response to bradykinin. Subsequent administration of sulfaphenazole further attenuated the vasodilator response to bradykinin. Similarly, in vitro studies (1, 2, 6, 20, 28), in isolated porcine coronary large and small arteries, indicate that endothelium-dependent vasodilation produced by bradykinin is principally NO mediated, while CYP 2C9 metabolites only contribute to bradykinin-induced endothelium-dependent coronary vasodilation in the absence of NO and PGI2 (6, 19, 28). In contrast, in the in vivo canine heart, bradykinin-induced dilation of both large (100–200 μm) and small (<100 μm) coronary arterioles remained unaffected by NOS and COX inhibition but was abolished by subsequent CYP 2C9 inhibition (38), suggesting an important role for CYP 2C9 vasodilator metabolites in the vasodilation produced by bradykinin, particularly in vivo.
Recent studies (30) indicate that NO, PGI2, and CYP 2C9 metabolites are not the only substances produced in response to bradykinin and indicate a role for hydrogen peroxide in bradykinin-induced vasodilation of coronary arterioles. In contrast, we found in the present study that ROS scavenging with MPG enhanced the bradykinin-induced vasodilation of isolated coronary small arteries in vitro, suggesting release of vasoconstrictor ROS in response to bradykinin that limits the bradykinin induced vasodilation. To our surprise we observed that, following NOS and COX inhibition, sulfaphenazole enhanced, rather than reduced, bradykinin-induced vasodilation in vitro. Moreover, in the presence of MPG, this effect of sulfphenazole was no longer observed, suggesting that CYP 2C9 was at least in part responsible for the bradykinin-induced ROS production. This notion is in accordance with previous studies (20, 22) showing that CYP 2C9 can be a physiologically relevant source of ROS in the coronary vasculature. The divergent findings that, in response to bradykinin in vivo, CYP 2C9 exerts principally a vasodilator influence, whereas a vasoconstrictor influence predominates in vitro, are difficult to explain but illustrate that the balance between CYP 2C9 derived vasodilator and vasoconstrictor metabolites depends on the experimental conditions. Finally, our findings in vivo as well as in vitro both support the concept that CYP 2C9 activity is suppressed under physiological conditions by NO and PGI2 (3, 29, 38). This likely involves a direct effect on CYP 2C9 or may be due to membrane depolarization following loss of NO and PGI2, leading to a membrane potential that is more susceptible to the actions of EETs and ROS.
Contribution of CYP 2C9 Metabolites to Exercise Hyperemia in the Heart
Observations in the human leg indicate that CYP 2C9 metabolites contribute to exercise hyperemia when NOS is inhibited (24), suggesting that CYP 2C9 can also be activated by physiological stimuli such as exercise. We therefore investigated in our study whether CYP 2C9 metabolites also contribute to coronary hyperemia during exercise. Since intravenous administration of inhibitors of vasomotor pathways often results in systemic hemodynamic changes, and hence myocardial metabolism, changes in coronary vasomotor tone during exercise cannot simply be assessed by changes in coronary blood flow or coronary vascular conductance. 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 myocardial oxygen consumption (9, 44). Thus an increase in vasomotor tone, produced by blockade of an endogenous vasodilator pathway, will result in a decrease in myocardial oxygen delivery at a given level of myocardial oxygen consumption, forcing the heart to increase its oxygen extraction to fulfill its oxygen requirement, thereby resulting in a decrease in coronary venous oxygen level. The coronary venous O2 level therefore represents an index of myocardial tissue oxygenation, i.e., the balance between O2 delivery and O2 consumption that is determined by coronary vasomotor tone (9, 44).
Using the myocardial O2 balance as an index of coronary vasomotor tone, we (34, 36, 37) have previously shown that alterations in the balance between endothelial vasodilators, including NO and prostanoids, and endothelial vasoconstrictors contribute to exercise hyperemia in the porcine coronary vasculature. In the present study, we investigated the vasomotor influence of CYP 2C9 activity in exercise hyperemia. Surprisingly, inhibition of CYP 2C9 with the selective inhibitor sulfaphenazole (20, 31) resulted in a parallel upward shift of the relations between myocardial O2 consumption and cvPo2 as well as cvSo2, indicating that inhibition of CYP 2C9 resulted in a small degree of vasodilation both at rest and during exercise. These findings show that CYP 2C9 metabolites exerted a net vasoconstrictor influence rather than, or in excess of, a vasodilator influence. The coronary vasodilator effect of sulfaphenazole was enhanced by prior inhibition of NOS and COX, consistent with previous observations that CYP 2C9 activity is blunted by NO and PGI2 (3, 29, 38).
The exact identity of the vasoconstrictor(s) produced by CYP 2C9 under basal conditions remains to be elucidated. However, the present study, as well as previous studies (20, 22), has shown that CYP 2C9 can be a physiologically relevant source of ROS in the porcine coronary vascular bed. Apparently, the production of ROS, which induce vasoconstriction, can occur simultaneously with the production of vasodilator EETs and can also be suppressed by sulfaphenazole (22). The observation that CYP 2C9 inhibition reduced the vasodilator response to bradykinin in vivo, but enhanced it in vitro, while it also resulted in vasodilation at rest and during exercise, suggests that the balance of vasodilators and vasoconstrictors produced by CYP 2C9 varies based on the experimental conditions. To our knowledge, it is unknown which factors affect this balance. However, there may be a role for the endogenous antioxidants and hence the “redox” status of the vasculature. Thus, under basal conditions, and particularly in situations where production of ROS is increased (such as during exercise or in the presence of endothelial dysfunction), when antioxidants are decreased (endothelial dysfunction) or in the presence of increased oxidative stress (high oxygen concentrations in vitro), the vasoconstriction due to production of ROS may predominate, whereas upon infusion of a NO-dependent vasodilator like bradykinin or acetylcholine, NO, ROS, and EETs all increase. In this situation, part of the NO may be used to scavenge ROS, allowing the vasodilator influence of EETs to dominate. In accordance with this concept, sulfaphenazole enhances the NO-mediated component of bradykinin-induced vasodilation in healthy vessels (22); improved endothelium-dependent, NO-mediated, vasodilation to acetylcholine in the forearm of patients with coronary artery disease (18); and restored postischemia vascular dysfunction in rats (26). In all cases, administration of sulfaphenazole was accompanied by a decrease in oxidative stress (18, 22, 26).
The present study demonstrates that CYP 2C9 can produce both vasoconstrictor and vasodilator metabolites. Production of these metabolites is enhanced by combined NOS/COX blockade and is critically dependent on the experimental conditions. Thus production of vasoconstrictor CYP 2C9 metabolites was dominant over production of vasodilator metabolites in swine at rest and during treadmill exercise. In contrast, pharmacological stimulation with bradykinin resulted in vasodilator CYP 2C9 metabolite production when studied in the intact coronary circulation in vivo, whereas vasoconstrictor CYP 2C9 metabolites, most likely ROS, were dominant when bradykinin was studied in isolated coronary resistance arteries in vitro. Future studies are warranted to further investigate the mechanisms that underlie these divergent influences of CYP 2C9 on coronary resistance vessel tone.
This study was supported by The Netherlands Heart Foundation (2000T042) and China Scholarship Council (2009624027).
No conflicts of interest, financial or otherwise, are declared by the author(s).
Author contributions: Z.Z., V.H., V.J.d.B., F.G., M.H., and D.M. performed experiments; Z.Z., V.H., V.J.d.B., and M.H. analyzed data; Z.Z., V.H., V.J.d.B., F.G., D.M., and D.J.D. interpreted results of experiments; Z.Z. and V.H. prepared figures; Z.Z. and V.H. drafted manuscript; V.J.d.B., D.M., and D.J.D. edited and revised manuscript; D.M. and D.J.D. conception and design of research; D.M. and D.J.D. approved final version of manuscript.
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