Nitric Oxide–Independent Dilation of Conductance Coronary Arteries to Acetylcholine in Conscious Dogs
Abstract NO and prostacyclin formation cannot entirely account for receptor-operated endothelium-dependent dilation of coronary vessels, since vasodilator responses are not completely suppressed by inhibitors of these agents. Therefore, we considered that another factor, such as an endothelium-derived hyperpolarizing factor described in vitro, may participate in NO- and prostacyclin-independent coronary dilator responses. In conscious instrumented dogs, intracoronary acetylcholine (ACh, 30.0 ng · kg−1 · min−1) increased the external epicardial coronary diameter (CD) by 0.18±0.03 mm (from 3.44±0.11 mm) when increases in coronary blood flow (CBF) were prevented and increased the CD by 0.20±0.05 when CBF was allowed to increase. After the administration of intracoronary Nω-nitro-l-arginine methyl ester (L-NAME), CBF responses to ACh were abolished, but CD responses (0.23±0.05 from 3.22±0.09 mm) were maintained. Blockade of NO formation was confirmed by reduced CD baselines and blunted flow-dependent CD responses caused by adenosine and transient coronary artery occlusions after L-NAME administration. ACh-induced CD increases resistant to L-NAME and indomethacin were reduced after the administration of intracoronary quinacrine, an inhibitor of phospholipase A2, or proadifen, an inhibitor of cytochrome P-450. Quinacrine or proadifen alone (without L-NAME) did not alter CD responses to ACh, but L-NAME given after proadifen blunted ACh-induced increases in CD. The increases in CD caused by arachidonic acid given after L-NAME+indomethacin were antagonized by proadifen but not altered by quinacrine. Thus, a cytochrome P-450 metabolite of arachidonic acid accounts for L-NAME–resistant and indomethacin-resistant dilation of large epicardial coronary arteries to ACh. Conversely, NO formation is the dominant mechanism of ACh-induced dilation after blockade of the cytochrome P-450 pathway.
In vivo, NO formation from l-arginine is apparently the major pathway involved in the dilation of canine large epicardial coronary arteries caused by elevated shear stress and by ACh, the archetype of receptor-operated endothelium-dependent agents. This conclusion is supported by smaller ACh1–6 and flow-dependent responses after blockade of NO formation with arginine analogues.1–4,7 The residual dilation to ACh commonly observed after arginine analogues suggests that NO may not be the sole intermediate involved in vivo. Aside from an incomplete blockade of NO formation, the residual receptor-operated dilation after arginine analogues may involve the release of an EDHF.8–15 This EDHF may be a cytochrome P-450 monooxygenase metabolite of arachidonic acid, such as one of the EETs synthesized by isolated vessels.16–19 Direct evidence for the involvement of a factor distinct from NO or a cyclooxygenase-derived metabolite of arachidonic acid in receptor-operated dilation is lacking in vivo.
Conceivably, the relative contribution of NO and EDHF may differ according to the stimulus (ie, flow-dependent or receptor-operated) used to elicit vascular relaxation. In that eventuality, the importance of the dilation remaining after blockade of NO and prostacyclin (PGI2) formation may depend on the strategy used to trigger vascular relaxation.
Therefore, we compared the extent to which L-NAME, a blocker of NO formation, antagonized flow-dependent dilation of large epicardial coronary arteries caused by either brief coronary artery occlusions or intracoronary infusions of adenosine and the receptor-operated dilation elicited by intracoronary ACh infusions. We also examined the effects of quinacrine, an inhibitor of phospholipase A2,20 or proadifen, an inhibitor of cytochrome P-450,21 on ACh-induced dilation resistant to the combined blockades of NO and PGI2 formation.
Materials and Methods
After general anesthesia with sodium pentobarbital (30 mg/kg IV) and under sterile conditions, 29 mongrel dogs (30±1 kg) underwent a left thoracotomy at the fifth intercostal space under artificial ventilation. Instrumentation was implanted as previously described.22,23 Briefly, a catheter implanted in the thoracic aorta and an external transducer (model 800, Bentley Trantec) were used to monitor MAP. A Konigsberg pressure transducer was used to record LVP and to obtain its first derivative (LV dP/dt). The pressure gauge was cross-calibrated against measurements made with a catheter also implanted in the LV cavity. CBF was monitored using a 10-MHz pulsed Doppler flowmeter24 with a probe implanted around the circumflex coronary artery. The vessel cross-sectional area was measured at necropsy and used to calculate a calibration factor (in mL · min−1 · kHz−1). A Silastic (Dow Corning Co) catheter was implanted in the proximal circumflex coronary artery using the approach described by Gwirtz.25 Distal to the Doppler flow probe and before the first marginal coronary artery, a pair of miniature ultrasonic crystals sutured on opposite sides of the artery were used with an ultrasonic sonomicrometer (model 120.2, Triton Technology) to measure external CD. A hydraulic occluder was placed around the coronary artery distal to the dimension crystals before the first marginal coronary artery in 6 dogs. The chest was closed in layers, and analgesia was provided after surgery with 0.3 mg IM buprenorphine (Temgesic, Reckitt and Colman Pharmaceuticals). Prophylactic procaine penicillin G (300 000 U IM) and benzathine penicillin G (300 000 U IM) were administered for 10 days after the surgery. Enteric-coated aspirin (650 mg/d PO) was begun the day after surgery and given on a daily basis thereafter.
Hemodynamic variables were recorded on a VHS tape using a PCM recording adaptor (model 4000A, A.R. Vetter Co) and monitored on a direct ink-writing strip-chart recorder (model 2800s, Gould).
Flow-Dependent Versus Receptor-Operated Dilation After L-NAME
Experiments were initiated 2 to 4 weeks after surgery in 6 conscious healthy dogs lying quietly on their right sides. While HR, LVP, LV dP/dt, MAP, phasic and mean CBF, and CD were monitored, intracoronary infusions of 10.0 and 30.0 ng · kg−1 · min−1 ACh chloride (Sigma Chemical Co) were performed until a steady-state increase in CD was achieved, ie, 4 to 6 minutes after the beginning of the infusion. Starting at the initial rise in CBF, the hydraulic occluder was immediately inflated in order to prevent any increase in CBF and to maintain CBF near preinfusion baseline level until the infusion was completed. ACh (30.0 ng · kg−1 · min−1) was also infused without controlling CBF. The effects of intracoronary infusions of 500 ng · kg−1 · min−1 adenosine (Sigma Chemical Co) were examined under normal and controlled CBF. Flow-dependent dilation of large epicardial coronary arteries was created by the release of transient coronary arterial occlusions lasting 5, 10, and 15 seconds. In 3 dogs, a complete suppression of CBF could not be achieved during coronary artery occlusion because of small arterial branches located between the occluder and the Doppler flow probe. In these dogs, substantial but presumably submaximal increases in CBF following the release of the occlusion were created. Care was taken to ensure that CBF fell to the same level after coronary artery occlusions of various durations.
Drugs were freshly dissolved in warm saline (38.9°C) and infused at a rate of 0.8 mL/min. A continuous intracoronary infusion of warm saline (0.8 mL/min) was used to ensure the patency of the intracoronary catheter throughout these experiments. The sequence of drug administration or coronary artery occlusions was randomly selected. Five to 8 minutes was allowed between infusions or occlusions for the return of hemodynamics to a steady-state baseline.
The same procedure was repeated after intracoronary administration of L-NAME at 50.0 μg · kg−1 · min−1 for 12 minutes (Sigma), delivered in 0.5 mL/min of saline. At least 10 minutes was allowed after the completion of L-NAME delivery to reach a steady-state baseline CD level. CBF was not controlled during ACh and adenosine infusions after L-NAME.
L-NAME–Resistant Dilation to ACh
Quinacrine. In 6 dogs pretreated with 5.0 mg/kg IV indomethacin (Sigma), the effects of an inhibitor of phospholipase A2, quinacrine (6.7 μg · kg−1 · min−1 for 30 minutes followed by 0.67 μg · kg−1 · min−1 IC for the duration of the experiments, Sigma), on ACh responses were examined after intracoronary L-NAME. Quinacrine was dissolved in saline and infused at a rate of 0.8 mL/min. Intracoronary infusions of ACh (30.0 ng · kg−1 · min−1) and intracoronary bolus injections of 50.0 ng/kg NTG (Parke-Davis) were made before L-NAME, after L-NAME, and 60 minutes after the onset of quinacrine delivery. Adenosine (500 ng · kg−1 · min−1 IC) was given before and after L-NAME to confirm the adequacy of the blockade of NO formation in large epicardial coronary vessels. At least 48 hours later, responses to ACh and NTG were examined before and after quinacrine alone in the same animals.
Proadifen. In 6 dogs pretreated with indomethacin, quinacrine was replaced by intracoronary proadifen (20.0 μg · kg−1 · min−1 for 20 minutes followed by 2.0 μg · kg−1 · min−1 for the duration of the experiments, Sigma), an inhibitor of the cytochrome P-450 monooxygenase, and the same protocols described for quinacrine were repeated. In 6 dogs, ACh-induced responses were examined before proadifen, after proadifen, and after proadifen+L-NAME.
L-NAME–Resistant Dilation to Arachidonic Acid
In 6 additional dogs, the effects of an intracoronary infusion of arachidonic acid (30.0 μg · kg−1 · min−1 for 10 minutes) were examined after L-NAME+indomethacin and after L-NAME+indomethacin+proadifen. On a different day, the effects of quinacrine on L-NAME–resistant and indomethacin-resistant dilation to arachidonic acid were examined in 5 of these dogs. Arachidonic acid was dissolved in saline under a nitrogen stream, and aliquots of 20 mg/mL were frozen at −70°C. Immediately before each infusion, a sample of the stock solution of arachidonic acid was thawed and dissolved in saline for infusion at a rate of 0.8 mL/min.
L-NAME–Resistant Dilation to ACh in Dogs Not Treated With Aspirin
In 5 additional dogs never exposed to aspirin, the effects of intracoronary ACh (30 ng · kg−1 · min−1), adenosine (500 ng · kg−1 · min−1), and NTG (50 ng/kg) were examined before L-NAME, after intracoronary L-NAME, and after L-NAME+proadifen, as previously described. These animals were instrumented as de-scribed above and studied at least 2 weeks after surgery. Dogs were pretreated with intravenous indomethacin (5.0 mg/kg) before the experiments.
Hemodynamic data were read directly from the strip charts under baseline and steady-state conditions during the intracoronary administration of ACh and adenosine. Data for reactive hyperemic and NTG responses were read at peak increases in CBF and CD. The volume of the blood flow deficit during coronary arterial occlusions (ie, the flow debt) and the excess of blood flow that followed the release of the occlusion (ie, the flow repayment) were obtained by planimetry and reported in milliliters. The duration of the debt and repayment were directly measured on the strip charts. Data are reported as mean±SEM throughout.
Comparisons of relationships between peak CBF, the volume of flow repayment, and the duration of hyperemic responses (as covariates) before and after L-NAME to changes in CD were performed by ANCOVA.26 Simultaneous comparisons of baseline CBF, CD, LVP, LV dP/dt, MAP, and HR before L-NAME, after L-NAME, and after L-NAME+quinacrine/proadifen were made by ANOVA for repeated measurements followed by Bonferroni’s t test.26,27 CBF, CD, LVP, LV dP/dt, MAP, and HR at steady state during ACh and adenosine administration were compared with baselines values by paired t test. A one-way ANOVA followed by Bonferroni’s t test to isolate specific contrasts was used for simultaneous comparisons of baselines and responses at peak CBF and CD when hyperemic and NTG responses were analyzed. CBF and CD responses to ACh (10.0 and 30.0 ng · kg−1 · min−1) and transient arterial occlusions before and after L-NAME were simultaneously compared by ANOVA for repeated measurements. CBF and CD responses before L-NAME, after L-NAME, and after L-NAME+quinacrine/proadifen were compared by one-way ANOVA for repeated measurements followed by Bonferroni’s t test. Baselines and CBF and CD responses to ACh and NTG before and after quinacrine or proadifen were compared by paired t test. ANOVA was used to compare baselines and responses before proadifen, after proadifen, and after proadifen+L-NAME. Paired t tests were used to compare baselines and responses to arachidonic acid after indomethacin+L-NAME with and without proadifen or quinacrine.
Statistical significance was reached at P<.05 in all cases.
All experimental procedures were approved by an ethical committee on animal care and performed in accordance with Guide to the Care and Use of Experimental Animals (Canadian Council on Animal Care, publication No. [ISBN] 0 to 919087-18-3, Ottawa, 1993).
Flow-Dependent Versus Receptor-Operated Dilation After L-NAME
CD responses to ACh and adenosine under normal and controlled CBF and reactive hyperemic responses were examined in the same 6 dogs before and after L-NAME. Overall comparisons of baseline hemodynamics revealed significant decreases in CD and HR after L-NAME, as reported in Table 1⇓. Baseline LVP, LV dP/dt, MAP, and CBF were not significantly altered after L-NAME.
CD Responses to ACh With or Without Controlled CBF
A recording of responses elicited by intracoronary infusions of ACh (30.0 ng · kg−1 · min−1) with controlled CBF before L-NAME and with normal CBF after L-NAME is displayed in Fig 1⇓.
ACh (30.0 ng · kg−1 · min−1 IC) increased (P<.05) CBF by 16±5% from 53±5 mL/min and CD by 0.20±0.05 from 3.45±0.11 mm. When CBF increases were prevented, ACh-induced increases in CD averaged 0.18±0.03 from 3.44±0.11 mm, not different from those observed under normal CBF.
After L-NAME, baseline CD fell (P<.01) to 3.22±0.09 mm, but CBF was not altered (56±3 mL/min). In spite of the failure of ACh to increase CBF after L-NAME, CD increases averaged 0.23±0.05 mm, not different from responses observed before L-NAME with and without controlled CBF. CD responses to ACh with and without controlled CBF before and after L-NAME are reported in Fig 2⇓.
Except for CBF and CD, ACh had no other significant hemodynamic effects.
CD Responses to Adenosine With and Without Controlled CBF
Adenosine (500 ng · kg−1 · min−1 IC) increased (P<.01) CBF by 75±17% from 52±5 mL/min and CD by 0.20±0.04 from 3.44±0.11 mm. When CBF increases were prevented (5±2% from 50±3 mL/min), adenosine failed to increase CD (−0.02±0.01 from 3.43±0.11 mm), consistent with the involvement of a flow-dependent mechanism.
After L-NAME, adenosine still increased CBF substantially by 49±10% from 56±5 mL/min, although less (P<.05) than before L-NAME. In contrast, CD increases were nearly abolished (0.03±0.01 from 3.24±0.09 mm) and did not differ from responses elicited under controlled CBF before L-NAME.
Except for CBF and CD, adenosine had no other significant hemodynamic effects.
CD Responses to Transient Coronary Artery Occlusions
The release of transient coronary artery occlusions lasting 5, 10, and 15 seconds resulted in graded increases in CD, in peak CBF, and in the volume and duration of flow repayment.
After L-NAME, peak increases in CBF after transient coronary artery occlusions were not significantly reduced. However, the volume and the duration of flow repayment were smaller after L-NAME. CD increases were dramatically reduced after L-NAME. The relationships between changes in peak CBF or the volume and duration of flow repayment and CD responses revealed significant downward shifts after L-NAME; ie, for any given increase in peak, volume, or duration of CBF responses, CD increases were disproportionately smaller after L-NAME. These data are reported in Fig 3⇓.
Except for CBF and CD increases, transient coronary artery occlusions had no other significant hemodynamic effects.
L-NAME–Resistant Dilation to ACh
Except for changes in CBF and CD, ACh, adenosine, and NTG had no other significant effects on hemodynamics under the various experimental conditions.
In 6 dogs pretreated with indomethacin, the effects of quinacrine on L-NAME–resistant CD responses to ACh (30.0 ng · kg−1 · min−1 IC) were examined.
Consistent with the data reported above, L-NAME did not prevent CD responses elicited by ACh but abolished CBF increases. Adequacy of blockade of NO formation by L-NAME was further demonstrated by the complete suppression of CD responses to adenosine (0.22±0.06 from 3.09±0.07 mm before L-NAME to 0.00±0.01 from 2.86±0.06 mm after L-NAME). In contrast, CBF responses to adenosine were only partially inhibited (98±14% before L-NAME to 64±7% after L-NAME).
Except for a decrease in LV dP/dt, the addition of quinacrine after L-NAME had no influence on baseline CBF and CD or other hemodynamic variables (Table 2⇓). In this situation, ACh-induced increases in CD were significantly reduced, as reported in Fig 4⇓. ACh failed to increase CBF after quinacrine, as we observed after L-NAME alone.
Bolus injections of NTG (50.0 ng/kg) caused similar peak increases in CBF before L-NAME (62±11%), after L-NAME (67±10%), and after L-NAME+quinacrine (57±7%). CD responses to NTG were augmented (P<.05) after L-NAME, and quinacrine did not further alter CD responses (Fig 4⇑).
On a different day, ACh and NTG responses were examined before and after quinacrine alone in the same animals pretreated with indomethacin.
Intracoronary quinacrine alone had no significant effects on baseline CBF and CD or other hemodynamic variables. ACh induced similar increases in CBF before (17±4%) and after (18±7%) quinacrine. CD responses to ACh were also similar before and after quinacrine (Fig 4⇑). Quinacrine did not alter CBF responses to NTG (57±6% before and 70±6% after quinacrine), but CD responses were increased after quinacrine (Fig 4⇑).
In 6 dogs pretreated with indomethacin, proadifen given after L-NAME did not further influence baseline CBF and CD or other hemodynamic variables (Table 3⇓). The L-NAME–resistant ACh-induced increases in CD were reduced after the addition of proadifen, as illustrated in Fig 5⇓ and summarized in Fig 6⇓. CBF increases caused by ACh were blunted after L-NAME, and pro- adifen had no further effects. Adequacy of blockade of NO formation was demonstrated by blunted CD responses to adenosine after L-NAME.
NTG-induced increases in CBF did not differ before L-NAME (55±5%), after L-NAME (65±5%), and after L-NAME+proadifen (64±10%). In contrast, CD increases caused by NTG were augmented after L-NAME and not further altered by the addition of proadifen (Fig 6⇑).
On a different day, ACh and NTG responses were examined before and after proadifen alone in the same animals pretreated with indomethacin. Proadifen alone increased baseline MAP (92±3 to 105±6 mm Hg, P<.05) and LVP (112±3 to 129±4 mm Hg, P<.01) but did not alter CBF, CD, LV dP/dt, and HR.
CBF responses to ACh did not differ before (19±7%) and after (25±5%) proadifen. CD responses to ACh were similar before and after proadifen (Fig 6⇑). Proadifen did not alter CBF responses to NTG (77±12% before and 77±9% after proadifen) or CD responses (Fig 6⇑).
In 6 additional dogs, L-NAME given after proadifen blunted CD responses (Fig 7⇓). Increases in CBF were similar before and after proadifen and blocked by the addition of L-NAME. CD responses to NTG were maintained under these conditions.
L-NAME–Resistant and Indomethacin-Resistant Dilation to Arachidonic Acid
Arachidonic acid (30.0 μg · kg−1 · min−1) injected after indomethacin+L-NAME increased CD without altering baseline CBF. Other hemodynamic effects of arachidonic acid were slight and did not reach statistical significance. CD increases to arachidonic acid were blunted by proadifen (n=6) but maintained after quinacrine (n=5, Fig 8⇓).
L-NAME–Resistant Dilation to ACh in Dogs Not Treated With Aspirin
In 5 additional dogs, ACh (30 ng · kg−1 · min−1) increased (P<.01) CBF by 20±3% from 49±6 mL/min and CD by 0.23±0.02 from 3.04±0.13 mm before L-NAME. After L-NAME, increases in CBF caused by ACh were abolished (−11±5% from 43±6 mL/min), but CD increases persisted (Fig 9⇓). After L-NAME+proadifen, ACh caused smaller (P<.01) increases in CD compared with L-NAME alone. Except for a decrease in CD and HR, L-NAME had no other significant effects on baseline hemodynamics. The addition of proadifen had no further effects (Table 4⇓).
Before L-NAME, adenosine (500 ng · kg−1 · min−1) increased CD by 0.26±0.05 from 3.07±0.13 mm and CBF by 98±31% from 47±6 mL/min. After L-NAME, CD increases were abolished, and changes in CBF (68±11% from 43±6 mL/min) did not statistically differ from responses before L-NAME.
NTG-induced increases in CBF did not differ before L-NAME (61±6%), after L-NAME (82±17%), and after L-NAME+proadifen (80±14%). In contrast, CD increases caused by NTG were augmented after L-NAME and not further altered by the addition of proadifen (Fig 9⇑).
The present study demonstrates that blockade of receptor-operated dilation of large epicardial coronary arteries to ACh requires the simultaneous blockade of NO formation and the phospholipase A2/cytochrome P-450 pathway. Consequently, NO formation cannot entirely account for receptor-operated dilation of conductance coronary vessels to ACh. After adequate blockade of NO formation by L-NAME, which was demonstrated by blunted flow-dependent dilations, ACh-induced dilation of large epicardial coronary arteries persisted. These L-NAME–resistant dilations to ACh were sensitive to quinacrine, an inhibitor of phospholipase A2,20 and to proadifen, an inhibitor of cytochrome P-450 monooxygenase.21 To our knowledge, these data are the first to show the involvement of a cytochrome P-450–derived metabolite of arachidonic acid in ACh-induced dilation of large epicardial coronary arteries in vivo. Our data showing a proadifen-sensitive and quinacrine-insensitive dilation of large epicardial coronary arteries to arachidonic acid after L-NAME and indomethacin provide direct evidence for the involvement of a cytochrome P-450 metabolite (presumably EDHF) as a key intermediate in the regulation of large epicardial coronary arteries in vivo.
Flow-dependent dilation involves NO formation as the major intermediate responsible for vascular relaxation. Consistent with this conclusion, reactive dilations caused by transient coronary arterial occlusions, a flow-dependent phenomenon,28 were dramatically reduced after analogues of l-arginine.1–4,7,23,29 In the present experiments, relationships between peak CBF, the volume of flow repayment, and the duration of hyperemic responses to changes in CD were shifted downward after L-NAME. For a given increase in CBF, changes in CD were disproportionately smaller after L-NAME. Thus, substantial blockade of NO formation was achieved under the present experiments. We further considered that sustained flow-dependent responses, in contrast to transient flow-dependent dilations, may be differentially altered by L-NAME. Therefore, adenosine was used to elicit sustained flow-dependent increases in CD, which were abolished by preventing the rise in CBF. Our data are in agreement with those of Holtz et al,30 who determined that the effects of adenosine on CD were completely flow dependent. A direct effect of adenosine in addition to flow-dependent effects was apparently observed by Hintze and Vatner.31 The extent to which hemodynamic changes associated with intravenous adenosine influenced CD responses in these earlier studies was not completely accounted for. In our experiments, the flow-dependent responses caused by intracoronary adenosine were prevented by L-NAME in the face of smaller but still substantial increases in CBF, consistent with adequate blockade of NO formation in large epicardial coronary arteries. This agrees with our earlier data22 and with data of Huckstorf et al,29 who reported that the effects of adenosine on CD were blunted after L-NAME. In contrast, Canty and Schwartz3 reported that dilations to intracoronary infusions of adenosine delivered distally to the site of CD measurements were entirely flow dependent and resistant to L-NAME. Why our data differed is not apparent.
A receptor-operated process was primarily involved in ACh-induced increases in CD, because CD responses to ACh were similar whether CBF was allowed to increase or was maintained near baseline before L-NAME. The small amplitude of CBF increases caused by ACh before L-NAME may explain why a contribution of a flow-dependent component to increases in CD was not apparent. After L-NAME, ACh-induced increases in CD were maintained in spite of adequate blockade of NO formation.
The present findings are in general agreement with reports suggesting the involvement of a vasorelaxant factor other than NO or PGI2 in ACh-induced dilation. In vitro, ACh and bradykinin cause hyperpolarization and relaxation of the underlying smooth muscle cells through the release of an EDHF(s).8–17,32,33 This hyperpolarizing effect may account for the L-NAME–resistant and indomethacin-resistant dilation to endothelium- dependent agonists. The chemical identity of EDHF has been inferred on the basis of the blockade of vascular relaxation and/or hyperpolarization by inhibitors of phospholipase A2 or cytochrome P-450 monooxygenases in some16–18,33,34 but not all35 earlier studies. Other substances have also been suggested to act as EDHF.13 Recently, Campbell et al18 showed that EDHF closely resembles EETs, which are cytochrome P-450 metabolites of arachidonic acid. In fact, the cytochrome P-450 inhibitors, proadifen and miconazole, attenuated both the relaxation and hyperpolarization caused by methacholine in bovine isolated coronary arteries.18 Direct evidence for the production of EETs by isolated vessels and for their relaxing and hyperpolarizing effects on vascular smooth muscle cells through opening of Ca2+-activated K+ channels further supports the possibility that EETs are, in fact, EDHF.18 Our data indicate that the factor responsible for L-NAME–resistant and indomethacin-resistant dilation to ACh displays characteristics also shared by EDHF, such as the insensitivity to blockade of NO and PGI2 formation and the vulnerability to inhibitors of phospholipase A2 or cytochrome P-450. Although our approach cannot allow us to directly demonstrate the involvement of membrane hyperpolarization in ACh-induced dilation, our data support the possibility that EETs and presumably EDHF intervened.
We were concerned about the possibility that quinacrine and proadifen blocked L-NAME–resistant and indomethacin-resistant dilation to ACh through nonspecific effects.36–40 To address that issue, ACh-induced responses were examined before and after quinacrine or proadifen alone. No significant effects of either drug on baseline CBF or CD could be demonstrated when given alone or even after L-NAME. Furthermore, neither quinacrine nor proadifen alone interfered with ACh-induced increases in CBF and CD. NTG responses were not decreased after quinacrine or proadifen given alone or after L-NAME, consistent with a maintained coronary reactivity after these inhibitors. Thus, in our hands, nonspecific effects of quinacrine and proadifen were not apparent. Blockade of NO formation was essential for demonstrating a proadifen- and quinacrine-sensitive component to ACh-induced increases in CD. When NO formation was blocked, EDHF became the dominant factor in ACh-induced dilation of large epicardial coronary arteries. Conversely, when proadifen was given first to block EDHF formation, ACh-induced increases in CD were sensitive to the blockade of NO formation. Taken together, these data highlight the cross talk between NO and EDHF whereby the specific contribution of each factor to vasodilator responses largely depends on the status of the alternate pathway. In this connection, NO may normally have an inhibitory effect on the cytochrome P-450 pathway, which becomes the dominant mechanism of dilation when NO formation is impaired.
Resistance coronary vessels differed from those large-conductance vessels, since L-NAME-resistant dilation to ACh infusions was not apparent. Our data are in general agreement with most of earlier studies showing that arginine analogues blunt ACh-induced increases in CBF.4–6,22,29,41–43 In contrast to the present findings, blockade of NO formation has been reported to partially prevent ACh-induced dilation of conductance coronary arteries.1–6 One noticeable difference between our approach and the one used by others is the intracoronary delivery of agonists and blockers of NO formation. In this connection, when a similar approach was used in previous studies in conscious dogs, little blockade of CD responses to ACh could be demonstrated with L-NAME in spite of the blockade of CD responses caused by adenosine or transient coronary artery occlusions.22,29 Conceivably, the chronic use of aspirin, which targeted the reduction of platelet aggregation for maintaining the patency of the intracoronary catheter, and the blockade of the cyclooxygenase to prevent PGI2 synthesis may have accounted for the failure of L-NAME to block ACh-induced dilation of large epicardial coronary vessels in our dogs. An augmented NO production caused by the blockade of PGI2 formation44 could conceivably override the effects of L-NAME and increase the residual dilation to ACh. To directly address that issue, additional experiments were performed in 5 dogs not treated with aspirin. In these animals, ACh-induced dilation of large epicardial coronary arteries was also resistant to L-NAME+indomethacin and sensitive to proadifen, as we observed in dogs treated with aspirin. In addition, adenosine-induced CD dilation was blunted by L-NAME, consistent with adequate blockade of NO formation. Thus, the chronic use of aspirin could not have accounted for the L-NAME–resistant dilation to ACh in our experiments. A factor distinct from NO and PGI2 intervened, since quinacrine and proadifen selectively antagonized the L-NAME–resistant and indomethacin-resistant dilation to ACh. This conclusion agrees with most of earlier in vitro and in vivo studies in which aspirin was not used and in which an L-NAME–resistant dilation of large epicardial coronary arteries to ACh was apparent.
Neither quinacrine nor proadifen in the presence or absence of L-NAME had significant influence on baseline CD and CBF. Thus, EDHF derived from the phospholipase A2/cytochrome P-450 pathway was not an important determinant of baseline vascular tone in the present experiments. This agrees with earlier reports in which quinacrine given alone or after NG-nitro-l-arginine failed to influence coronary perfusion pressure in isolated rat hearts.16,34 NO apparently had a greater influence than did EDHF on baseline vascular tone in large coronary arteries, as suggested by the constriction elicited by L-NAME.
Our data suggest that a sequential action of phospholipase A2 and cytochrome P-450 monooxygenase may be involved in ACh-induced dilation resistant to L-NAME and indomethacin. Consistent with this hypothesis, exogenous arachidonic acid given after L-NAME and indomethacin caused a proadifen-sensitive but quinacrine-insensitive dilation of large coronary arteries.
In conclusion, ACh-induced receptor-operated dilation of large epicardial coronary arteries resistant to blockade of NO and PGI2 synthesis is antagonized by inhibitors of phospholipase A2 or cytochrome P-450 monooxygenases. Thus, a cytochrome P-450 metabolite of arachidonic acid may be the intermediate involved in L-NAME–resistant and indomethacin-resistant dilation to ACh of large epicardial coronary arteries in conscious dogs.
Selected Abbreviations and Acronyms
|CBF||=||coronary blood flow|
|EDHF||=||endothelium-derived hyperpolarizing factor|
|L-NAME||=||Nω-nitro-l-arginine methyl ester|
|LVP||=||LV systolic pressure|
|MAP||=||mean arterial pressure|
This study was supported through grants from the Medical Research Council of Canada, Canadian Heart and Stroke Foundation, Fonds de la Recherche en Santé du Québec, and Fonds de la Recherche de l’Institut de Cardiologie de Montréal. The authors thank Claude Mousseau, Jean-Pierre Turcotte, and Jhésabelle Voyer for skillful assistance in conducting these studies.
- Received March 21, 1997.
- Accepted September 11, 1997.
- © 1997 American Heart Association, Inc.
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