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Am J Physiol Heart Circ Physiol 281: H1553-H1560, 2001;
0363-6135/01 $5.00
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Vol. 281, Issue 4, H1553-H1560, October 2001

Endothelium-derived hyperpolarizing factor in coronary microcirculation: responses to arachidonic acid

Christine L. Oltman1, Neal L. Kane1,2, Jonathon L. Fudge2, Neal L. Weintraub2, and Kevin C. Dellsperger1,2

1 Department of Veterans Affairs, Iowa City, 52246; and 2 Department of Internal Medicine and the Cardiovascular Center, University of Iowa, Iowa City, Iowa 52242


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

In coronary resistance vessels, endothelium-derived hyperpolarizing factor (EDHF) plays an important role in endothelium-dependent vasodilation. EDHF has been proposed to be formed through cytochrome P-450 monooxygenase metabolism of arachidonic acid (AA). Our hypothesis was that AA-induced coronary microvascular dilation is mediated in part through a cytochrome P-450 pathway. The canine coronary microcirculation was studied in vivo (beating heart preparation) and in vitro (isolated microvessels). Nitric oxide synthase (NOS) (Nomega -nitro-L-arginine, 100 µM) and cyclooxygenase (indomethacin, 10 µM) or cytochrome P-450 (clotrimazole, 2 µM) inhibition did not alter AA-induced dilation. However, when a Ca2+-activated K+ channel channel or cytochrome P-450 antagonist was used in combination with NOS and cyclooxygenase inhibitors, AA-induced dilation was attenuated. We also show a negative feedback by NO on NOS-cyclooxygenase-resistant AA-induced dilation. We conclude that AA-induced dilation is attenuated by cytochrome P-450 inhibitors, but only when combined with inhibitors of cyclooxygenase and NOS. Therefore, redundant pathways appear to mediate the AA response in the canine coronary microcirculation.

coronary circulation; coronary microcirculation; intravital microscopy


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

PREVIOUS STUDIES in the coronary circulation of several species, including humans, showed that responses to endothelium-dependent agonists persist despite inhibition of nitric oxide (NO) synthase (NOS) and cyclooxygenase (11, 20, 21). These NOS- and cyclooxygenase-independent relaxations are mediated by hyperpolarization of smooth muscle membranes and have been attributed to endothelium-derived hyperpolarizing factor (EDHF). EDHF-mediated relaxations appear to be especially prominent in resistance arterioles, suggesting an important role in the physiological regulation of blood flow (7, 15). In pathological states, where the bioactivity of NO is impaired, EDHF has been suggested as a backup mechanism for the maintenance of endothelium-dependent vasodilation (7). Also EDHF may be feedback inhibited by NO (4, 16). Elucidating the contribution of EDHF to endothelium-dependent relaxations under physiological conditions is more complicated than previously appreciated. Whereas the chemical identity of EDHF remains in question, a number of recent investigations suggest that it may be a noncyclooxygenase metabolite(s) of arachidonic acid (AA) (5, 9, 10, 12, 14, 22).

One pathway that is putatively responsible for endothelium-dependent hyperpolarization and vasodilation is cytochrome P-450 metabolism of AA. In particular, the cytochrome P-450-derived epoxyeicosatrienoic acids and dihydroxyeicosatrienoic acids have been shown to potently dilate canine coronary microvessels (18). Therefore, we tested the hypothesis that AA-induced coronary microvascular dilation is mediated in part through a cytochrome P-450 pathway. To investigate this hypothesis, we performed comprehensive in vivo and in vitro coronary microvascular techniques, which permitted a complimentary evaluation of the potential mechanisms of relaxation. In vivo coronary microvascular responses were evaluated in the beating canine left ventricle. In vitro coronary microvascular responses were studied in isolated microvascular baths to provide a focused evaluation of the vasculature independent of confounding influences present in the intact animal.


    METHODS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

All protocols were approved by the University of Iowa Animal Care and Use Committee and the Veteran Affairs Research Committee and conform to the Guide For The Care And Use Of Laboratory Animals published by the National Institutes of Health (NIH Publication No. 85-23, Revised 1985).

In Vivo Study: General Preparation

Mongrel dogs (n = 25, 4-8 kg body wt) of either sex were sedated with ketamine (20 mg/kg sc) and acepromazine (0.2 mg/kg sc) and anesthetized with alpha -chloralose (60 mg/kg iv), urethane (150 mg/kg iv), and sodium borate (25 mg/kg iv). Additional doses of anesthetic were administered throughout the experiment to maintain the depth of anesthesia. A polyethylene catheter (PE-150) was inserted into the external jugular vein for administration of drugs and fluids. Another catheter (PE-205) was inserted into the common carotid artery for monitoring arterial pressure and measurement of arterial blood gases. A cuffed endotracheal tube was inserted into the trachea. To minimize respiration-induced cardiac motion, dogs were ventilated with a high-frequency jet ventilator synchronized to the cardiac cycle as previously described (6, 8). Positive end-expiratory pressure (3-5 cmH2O) was applied to prevent atelectasis.

A left thoracotomy was performed in the fifth intercostal space, the fourth and fifth ribs were resected, and a left lower lobectomy was performed to create an adequate thoracic window. The pericardium was incised to suspend the heart in a cradle. A catheter (PE-150) was inserted into the left atrium via the left atrial appendage to administer fluorescein-labeled dextran. A 5-Fr catheter (Millar Instruments; Houston, TX) was inserted into the left ventricle via the left atrial appendage for measurement of left ventricular pressure and the first derivative of pressure with respect to time (dP/dt). Snares were placed around the descending thoracic aorta and the inferior vena cava to control arterial pressure. The epicardial surface was kept moist by superfusion with warmed (37°C) Krebs solution bubbled with 20% O2-5% CO2-75% N2 at the rate of 2 ml/min. The Krebs solution contained (in mM) 118.3 NaCl, 4.7 KCl, 1.2 CaCl2, 1.2 MgPO4, 25.0 NaHCO3, and 1.2 KH2PO4; pH 7.4 at 37°C. Core temperature was maintained at 37°C with a servo-controlled thermal blanket. Arterial blood gases were maintained within the physiological range throughout the protocols (pH: 7.39 ± 0.02; PCO2: 34 ± 2 mmHg; PO2: 95 ± 10 mmHg).

Microscope and Video System

Measurements of coronary microvessels were obtained using intravital microscopy (Zeiss), with epiillumination of the cardiac surface by a computer-controlled strobe (Chadwick Helmuth; Almonte, CA) as described previously (21). The strobe was triggered by the left ventricular dP/dt signal causing a flash once per cardiac cycle in late diastole. Fluorescein isothiocyanate dextran (molecular weight 487,000, Sigma Chemical; St. Louis, MO) was injected into the left atrium to illuminate the internal microvascular diameter and to differentiate arterioles from venules by sequence of illumination. A Zeiss Neufluora (×6.3, numerical aperature = 0.02) objective, when coupled with a ×6.3 relay lens, measured microvascular diameters with 2.5-µm resolution. Images were transmitted to a video camera (General Electronic; Owensboro, KY) via a ×1.0 or ×6.3 relay lens. Digital data images were selected and stored in a computer (IBM 486). Images were later recalled on a high-resolution monitor, and the microvascular diameters were measured using a digitizing tablet and a computer to calculate the vessel diameter in microns. All vessel measurements represent the mean of up to three images at each experimental condition.

Protocols

After the general surgical preparation, at least 30 min was allowed for stabilization of monitored variables. Baseline measurements of hemodynamics, hematocrit, microvascular diameters, and arterial blood gases were performed. After this stabilization period, sodium nitroprusside (SNP, 10 µM) was applied topically to determine the viability of the preparation. Vessels dilating <30% in response to 10 µM SNP were excluded from analysis. The preparation was allowed to recover for 30 min after SNP. Baseline images were again acquired, AA (1, 5, and 10 µM topically) was then given, and coronary microvascular diameters were measured 1, 5, and 10 min following the initiation of the topical suffusion. The time course of AA-induced vasodilation was examined because vasodilation attributed to EDHF has been reported to be transient and, in some preparations, may not be present after steady state has been achieved (10 min) (1, 3). The timing for images began as the drug reached the myocardial surface. Because the measurements were time-course measurements, only one microscopic field could be monitored per animal. Blood gases were measured before and after completion of each protocol. Hemodynamics were continuously monitored and were unaffected by the topical suffusion of AA or SNP.

After the protocols were completed, the dogs were euthanized with an overdose of anesthetic, followed by saturated potassium chloride (10 ml).

Protocol 1 examined repeated concentration responses to AA to determine whether tachyphylaxis was present. Four dogs served as the control group. Inhibitors [indomethacin, Nomega -nitro-L-arginine (L-NNA), or clotrimazole] were not administered in this group. The percent change from baseline in response to topically applied AA (1, 5, and 10 µM) was determined. The vessels were allowed to return to resting diameters, and dose responses to AA were again determined.

The following protocols used combinations of inhibitors of cyclooxygenase (indomethacin), NOS (L-NNA), and cytochrome P-450 (clotrimazole).

Protocol 2 evaluated the role of cyclooxygenase in AA-induced coronary microvascular dilation. The percent change from baseline in response to topically applied AA (1, 5, and 10 µM) was determined. A single dose of indomethacin (5 mg/kg iv) was given to the dogs 30-60 min before the AA concentration-response curve was repeated.

Protocol 3 evaluated the role of cytochrome P-450 in the presence of cyclooxygenase inhibition (indomethacin) on AA-induced coronary microvascular dilation. All dogs in this group were treated with indomethacin (5 mg/kg iv) before surgery, and an AA concentration-response curve was performed. Dogs were then treated with clotrimazole (1.6 µM topically) for 30 min, and AA concentration-response curves were repeated.

Protocol 4 was designed to determine the role of cytochrome P-450 in the presence of combined cyclooxygenase plus NOS inhibition on AA-induced coronary microvascular dilation. Indomethacin was administered to all dogs. L-NNA (70 µM) was continuously applied topically to the heart. AA concentration-response curves were performed. After clotrimazole (1.6 µM) was given topically for 30 min, a second AA concentration-response curve was evaluated.

In Vitro Study

Microvessel procurement. Thirty-nine adult mongrel dogs of either sex (3-8 kg) were euthanized with an overdose of Pentothal Sodium (50 mg/kg). The hearts were quickly harvested and immediately placed in cold (4°C), oxygenated Krebs bicarbonate buffer solution for dissection.

Isolated microvessels. A standard in vitro pressurized arteriole preparation was used to study coronary microvessels (17, 18). Ventricular microvessels (75-175 µm intraluminal diameter and ~1 mm in length) were carefully removed from the myocardium, cleaned with the aid of a dissecting microscope, and placed in an organ chamber. Each end of the microvessel was cannulated with a glass micropipette and secured with 10-0 ophthalmic suture. The organ chamber was placed on the stage of an inverted microscope. Attached to the microscope were a video camera, a video monitor, and a calibrated video caliper. The organ chamber was connected to a rotary pump that continuously circulated oxygenated Krebs buffer. Krebs-Henseleit solution contained (in mM) 120.0 NaCl, 4.7 KCl, 2.5 CaCl2, 1.2 MgSO4, 23.0 NaHCO3, 1.2 KH2PO4, 11.0 glucose, and 0.025 EDTA. Solutions were aerated with 20% O2-5% CO2-75% N2 and maintained at 37°C with pH at 7.4. An image of the microvessel was displayed on the video monitor, and intraluminal diameters were measured by manually adjusting the video micrometer. The resolution of the system allowed measurement of very small (1-2 µm) changes in vessel diameter.

Microvessels were allowed to equilibrate for 30 min at a hydrostatic distending pressure of 20 mmHg under conditions of no flow. KCl (50 mM) was added to the bath to test constrictor capacity. After being washed with fresh Krebs buffer, vessel diameter returned to baseline. Endothelin-1 (0.2-0.8 nM) was used to constrict the microvessels to 30-60% of their resting diameter. Cumulative concentration-response relationships were evaluated for AA (10-10 to 10-5 M) and SNP (10-10 to 10-4 M) by adding the drug directly to the organ bath. A single dose of SNP (10-4 M) or papaverine (10-4 M) was given at the end of each experiment to determine maximal diameter.

To examine the mechanisms responsible for AA-induced dilation in isolated canine coronary arterioles, several combinations of inhibitors were utilized. Microvessels were incubated in the presence of inhibitors for 30 min before the concentration-response curve was performed. Inhibitors included indomethacin (10 µM) to investigate the role of cyclooxygenase, L-NNA (100 µM) to examine the role of NOS, cinnamyl-3,4-dihydroxy-alpha -cyanocinnamate (CDC, 1 µM) to examine the role of the lipoxygenase pathway, and clotrimazole (2 µM) to examine the role of the cytochrome P-450 pathway. To determine the role of potassium channels in AA-induced dilation, arterioles were treated with an isotonic solution of KCl to produce 30-60% constriction of their resting diameter before administration of AA. Vessels were also incubated with several K+ channel antagonists, including tetrabutylammonium chloride (TBA, 1 mM), tetraethylammonium chloride (TEA, 1 mM), charbydotoxin (50 nM), and glibenclamide (1 µM), or combinations of L-NNA and indomethacin and a K+ channel antagonist. To determine whether NO can modulate AA-induced vasodilation through a feedback inhibition mechanism, as was suggested by Bauersachs et al. (4) and Nishikawa et al. (16), responses to AA were evaluated in the presence of indomethacin, L-NNA, and an NO donor (SNP, 1 nM). This dose of SNP was selected because it had little (<= 25%) vasodilator effect. We also used papaverine (1 nM), a non-NO donor, in the presence of indomethacin and L-NNA, to evaluate AA-induced relaxation.

The following criteria were required for an acceptable microvessel experiment. First, microvessels could not demonstrate obvious leaks. Second, microvessels had to constrict >30% to 50 mM KCl and >30% to endothelin. Finally, microvessels had to dilate by >80% to 10-4 M SNP or 10-4 M papaverine.

Solutions and Drugs

AA (sodium salt) was obtained from Nu-Chek-Prep, Endothelin-1 was purchased from Phoenix Peninsula Laboratories (San Carlos, CA). L-NNA, clotrimazole, SNP, TBA, TEA, papaverine, glibenclamide, charybdotoxin, and indomethacin were purchased from Sigma Chemical. CDC was obtained from Biomol. All solutions and vasoactive agents were prepared fresh on the day of the experiment. AA and SNP were dissolved in Krebs-Henseleit buffer, whereas indomethacin, L-NNA, and clotrimazole were dissolved in saline. Agents for topical application were added to the epicardial perfusate at 10 times their final concentration using a Harvard infusion pump at a rate of 0.2 ml/min. For topical application, clotrimazole was initially dissolved in 100% ethanol to provide an initial stock solution of 10-2 M and was subsequently dissolved into the Krebs perfusate to provide a final concentration of 1.6 µM. Vehicle studies demonstrated that this concentration of ethanol did not affect AA-induced vasodilation.

Statistical Analysis

Data are means ± SE. Sigmastat software (Jandel Scientific) was used for statistical analyses. For in vivo studies, a one-way analysis of variance (ANOVA) with repeated measures was used to evaluate the changes in hemodynamic variables and microvascular diameters during each protocol. Blood gas data within groups were analyzed by Student's t-tests. Baseline diameters for each treatment group were compared with baseline diameters from the control group using an unpaired Student's t-test and the Bonferroni correction. All concentration-response curves were evaluated for differences using two-way repeated-measures ANOVA, followed by the Fisher least-significant difference correction for multiple comparisons. Differences with P <=  0.05 were considered significant.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Coronary Microvascular Dilation to AA In Vivo

To determine whether tachyphylaxis to AA was present in vivo, repeated concentration-response curves to AA were performed. Application of AA resulted in concentration-dependent vasodilation, which was unchanged on repeat determination (Fig. 1). Therefore, further studies were performed as sequential measurements before and after application of inhibitors, which allowed the same vessels to be studied.


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  		Fig. 1.   Percent change in diameter of canine coronary microvessels in response to increasing concentrations of arachidonic acid (AA) (1, 5, and 10 µM topically). Responses were generated using intravital microscopy in a beating canine heart. There were no differences in the concentration-response curves to AA between run 1 and run 2. Values are means ± SE.

To investigate the role of cyclooxygenase in AA-induced relaxation, AA concentration-response curves were performed before and after treatment with indomethacin (5 mg/kg iv). Indomethacin did not alter the vasodilatory response to AA in vivo (Fig. 2A), suggesting that AA-induced dilation is resistant to inhibition of cyclooxygenase, at least when other vasodilatory pathways are intact.


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  		Fig. 2.   Role of cyclooxygenase and cytochrome P-450 pathways on AA-induced vasodilation in canine coronary microvessels. A: in vivo response where cyclooxygenase inhibition alone did not affect AA-induced vasodilation. Indo, indomethacin. B: in vivo dilation to AA in the absence and presence of clotrimazole (CLO, 1.6 µM topically). All dogs received Indo (5 mg/kg iv). A reduction in vasodilation to the highest concentration of AA was observed; nevertheless, the amount of dilatation in response to AA was substantial. *P <=  0.05 without vs. with CLO.

To examine the role of the cytochrome P-450 pathway in AA-induced microvascular dilation, responses to AA were determined in vivo before and after treatment with clotrimazole (1.6 µM topically). We previously showed that this dose of clotrimazole selectively inhibited acetylcholine-induced coronary vasodilation in vivo in the presence of NOS and cyclooxygenase inhibitors, suggesting functional inhibition of the cytochrome P-450 pathway (21). All dogs in this group were treated with indomethacin (5 mg/kg iv) before surgery. Under these conditions, clotrimazole slightly attenuated AA-induced microvascular dilation in vivo (Fig. 2B), suggesting a role for cytochrome P-450 metabolism in mediating dilation to AA in the presence of cyclooxygenase inhibition.

We then studied the effects of clotrimazole in the presence of indomethacin and L-NNA on AA-induced coronary microvascular dilation in vivo. Baseline heart rate, mean arterial blood pressure, and blood gas measurements are shown in Table 1 and were similar among all groups. There were no significant changes in these parameters during each protocol. When NOS and cyclooxygenase inhibitors were applied, AA (10 µM) produced 28 ± 5% dilation. However, in the presence of cyclooxygenase, NOS, and cytochrome P-450 inhibitors, AA-induced microvascular dilation was markedly attenuated (5.8 ± 2.5%; Fig. 3). These data suggest that the cytochrome P-450 pathway plays a prominent role in AA-induced dilation in vivo when both NOS and cyclooxygenase are blocked.

                              
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  		Table 1.   Resting diameter, heart rate, mean arterial pressure, and blood gases



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  		Fig. 3.   Role of cytochrome P-450 pathway to nitric oxide synthase (NOS)-cyclooxygenase-resistant portion of AA-induced dilation. AA-induced coronary microvascular vasodilation in vivo in the presence of combined cyclooxygenase inhibition (Indo, 5 mg/kg iv) and NOS inhibition [NG-nitro-L-arginine (L-NNA), 70 µM topically] with and without cytochrome P-450 inhibition CLO (1.6 µM topically). There was significant inhibition of AA-induced vasodilation at all three concentrations of AA. n = 6, *P <=  0.05. These results demonstrate that AA-induced vasodilation resistant to the combined inhibition of cyclooxygenase and NOS is mediated by cytochrome P-450 metabolites of AA.

Coronary Microvascular Dilation to AA In Vitro

We examined the potential contribution of cyclooxygenase, NOS, and K+ channels to AA-induced coronary microvascular dilation in vitro. In isolated vessels, baseline diameter was 88.1 ± 11.0, 120.3 ± 13.1, and 122.4 ± 12.9 µm (P = not significant) for microvessels in the control, indomethacin + L-NNA, and KCl groups, respectively. Microvessels were constricted with endothelin to 37 ± 3% and 48 ± 4% and with KCl to 41 ± 6% resting diameter in the control, indomethacin + L-NNA, and KCl groups, respectively. Baseline diameters and the magnitude of constriction were not different between control groups and any of the treatment groups. AA produced concentration-dependent vasodilation in isolated canine coronary microvessels (Fig. 4A). The highest concentration of AA (10 µM) produced nearly complete relaxation (96 ± 4%) of endothelin-preconstricted vessels. Inhibitors of both the NOS and cyclooxygenase pathways did not alter AA-induced vasodilation of coronary microvessels (maximal relaxation = 86 ± 6%). However, preconstriction with isotonic KCl solution (32 ± 2 mM) virtually abolished dilation to AA (maximal relaxation = 12 ± 9%, Fig. 4A). This concentration of KCl produced essentially the same degree of preconstriction as endothelin. These results suggest that AA-induced vasodilation of isolated coronary microvessels is resistant to inhibitors of the NOS and cyclooxygenase pathways, and that hyperpolarization likely plays an important role in AA-induced vasodilation in coronary microvessels in vitro.


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  		Fig. 4.   Role of combined cyclooxygenase and NOS and cytochrome P-450 inhibition on AA-induced vasodilation in isolated coronary microvessels. A: relaxation to AA was not altered by the combination of Indo (10 µM) and L-NNA (100 µM) in endothelin-constricted vessels, however, was blocked by KCl preconstriction. B: AA-induced relaxation in isolated microvessels in the presence of CLO (2 µM) and Indo (10 µM) or L-NNA (100 µM). Values are means ± SE; n = 5 or 6 animals in each group. *P <=  0.05.

To evaluate the role of the cytochrome P-450 pathway in AA-induced dilation in vitro, microvessels were treated with clotrimazole and either indomethacin or L-NNA before performing the AA concentration-response curve. Combinations of clotrimazole and either indomethacin or L-NNA did not significantly alter AA-induced dilation (max dilation: indomethacin and clotrimazole: 86 ± 8%, L-NNA and clotrimazole: 97 ± 7%, Fig. 4B).

To evaluate the cytochrome P-450 contribution of the NOS-cyclooxygenase-resistant portion of AA-induced dilation, studies were performed in the presence of indomethacin, L-NNA, and clotrimazole. Treatment of coronary microvessels with clotrimazole, in the presence of L-NNA and indomethacin, inhibited AA-induced dilation (max relaxation: 27 ± 9%, Fig. 5A). Relaxation was only observed with the highest concentration of AA. In contrast, concentration-response curves to the direct smooth muscle vasodilator SNP were not altered in microvessels that had been incubated with L-NNA, indomethacin, and clotrimazole (Fig. 5B). Maximal SNP-induced relaxation was 94 ± 2% and 98 ± 3% in coronary microvessels from control and L-NNA and indomethacin and clotrimazole groups, respectively. That selective inhibition of AA-induced dilation required the presence of inhibitors of all three pathways suggests redundant vasodilator mechanisms may be involved.


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  		Fig. 5.   AA- and SNP-induced vasodilation in isolated coronary microvessels. A: AA-induced dilation of endothelin-constricted vessels in control and NOS (L-NNA, 100 µM), cyclooxygenase (Indo, 10 µM), and cytochrome P-450 inhibition (CLO, 2 µM). n = 5 or 6 animals. *P <=  0.05. B: SNP-induced complete relaxation of endothelin-induced constriction and dilation was not altered by this combination of inhibitors. n = 4 animals, P = not signifcant.

Because preconstriction with KCl abolished AA-induced dilation (Fig. 4A), further in vitro studies were performed to determine which K+ channel(s) mediates the response. Inhibitors of K+ channels known to be present in the coronary circulation were tested. TBA (1 mM), a nonspecific calcium-sensitive K+ channel inhibitor, significantly attenuated AA-induced dilation (Fig. 6). Charybdotoxin (50 nM), a large-conductance Ca2+-activated K+ channel inhibitor, slightly inhibited dilation induced by submaximal doses of AA. However, TEA (1 mM), which predominately blocks Ca2+-activated K+ channel, did not alter AA-induced dilation. Intermediate conductance calcium-activated K+ channels may be involved because both TBA and charybdotoxin may block these channels. There is no known direct inhibitor of these channels.


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  		Fig. 6.   Role of K+ channels in AA-induced dilation in vitro. AA-induced dilation was attenuated in the presence of tetrabutylammonium chloride (TBA, 1 mM) and slightly inhibited by charybdotoxin (50 nM). Tetraethylammonium chloride (TEA, 1 mM) did not alter AA-induced dilation. n = 4-6 animals. *P <=  0.05, control vs. TBA, star P <=  0.05, control vs. charybdotoxin.

To examine the effects of inhibitors of K+ channels on the NOS- and cyclooxygenase-resistant dilation in response to AA, K+ channel inhibitors were utilized along with L-NNA and indomethacin (Fig. 7). The combination of L-NNA, indomethacin, and glibenclamide did not alter AA-induced dilation. However, the Ca2+-activated K+ channel inhibitors charybdotoxin or TEA significantly attenuated the NOS- and cyclooxygenase-resistant AA-induced vasodilation. In the presence of L-NNA, indomethacin, and TBA (nonspecific calcium-sensitive K+ channel inhibitor), AA-induced relaxation was completely inhibited. Thus Ca2+-activated K+ channels appear to be important in AA-induced coronary microvascular dilation, particularly when the NOS and cyclooxygenase pathways are blocked.


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  		Fig. 7.   Contribution of K+ channels in the presence of NOS and cyclooxygenase inhibition in vitro. In the presence of L-NNA (100 µM) and Indo (10 µM), glibenclamide did not alter AA-induced relaxation. Both charybdotoxin and TEA attenuated the NOS-PGI2-independent response. TBA completely inhibited AA-induced dilation. n = 5 animals in each group. cross P <=  0.05, L-NNA + Indo vs. L-NNA, Indo and charybdotoxin, *P <=  0.05, L-NNA + Indo vs. L-NNA, Indo + TEA, star P <=  0.05, L-NNA + Indo vs. L-NNA, Indo, and TBA.

To assess the role of the lipoxygenase pathway on the NOS-cyclooxygenase-resistant portion of AA-induced dilation, studies were performed in the presence of indomethacin, L-NNA, and CDC. CDC did not alter AA-induced vasodilation (maximal dilation with CDC: 68 ± 12%, n = 4 vs. L-NNA + Indo alone: 86 ± 6%, P = NS). Thus the lipoxygenase pathway most likely does not participate in AA-induced relaxation of coronary microvessels.

Interaction Between NO and EDHF

Because a recent report (16) suggested that NO may feedback inhibit EDHF-dependent dilation, we sought to determine whether exposure to NO would block AA-induced relaxation in vitro. Vasodilation was evaluated in the presence of indomethacin, L-NNA, and a NO donor (SNP, 1 nM). This dose of SNP produced <25% dilation of preconstricted arterioles. AA-induced dilation was virtually abolished in the presence of indomethacin, L-NNA, and SNP (Fig. 8), suggesting NO may inhibit EDHF-mediated coronary microvascular vasodilation in vitro.


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  		Fig. 8.   NO inhibits endothelium-derived hyperpolarizing factor (EDHF)-mediated dilation in vitro. AA-induced residual dilation is blocked by the additional administration of sodium nitroprusside (SNP). In the presence of 1 nM SNP, the NOS-PGI2-independent AA-induced is inhibited. This response was not observed in the presence of papaverine. n = 4-6 animals. *P <=  0.05, L-NNA + Indo vs. L-NNA, Indo + SNP.

To determine whether a non-NO donor vasodilating substance is associated with the observed inhibition of the EDHF response, AA-induced vasodilation was performed in the presence of indomethacin, L-NNA, and papaverine (1 nM). This concentration of papaverine produces similar relaxation as 1 nM SNP and did not alter AA-induced dilation.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

The most significant new findings of this study are 1) blockade of all three traditional dilator pathways (cyclooxygenase, NOS, and cytochrome P-450) abolishes dilation to AA, whereas cyclooxygenase, NOS, or cytochrome P-450 inhibition alone does not inhibit AA-induced dilation; 2) AA-induced dilation is in part mediated by K+ channels; and 3) there is negative feedback by NO on NOS-cyclooxygenase-resistant AA-induced vasodilation. We conclude that AA-induced canine coronary microvascular dilation is mediated by redundant mechanisms that include stimulation of NO production and metabolism of AA by cytochrome P-450.

In our study we used two different but complementary methods to evaluate AA-induced dilation in the coronary microcirculation. The in vivo beating heart preparation allows us to investigate all influences controlling coronary microvascular dilation. To complement this technique, a standard in vitro preparation was also used to study isolated canine ventricular microvessels. An advantage of the in vitro technique is that it provides a milieu for focused evaluation of the contractile properties of coronary arteries independent of confounding (neural, humoral, metabolic, and physical) influences present in the intact animal.

AA is a ubiquitous fatty acid in membrane phospholipids that can be converted to vasoactive metabolites through several enzymatic pathways, including the lipoxygenase, cyclooxygenase, and cytochrome P-450 pathways. Several of these substances can induce vascular relaxation/dilatation by hyperpolarizing vascular smooth muscle (5, 19, 22). Thus metabolites of AA are candidates for EDHF, although the chemical nature of EDHF remains elusive.

Recently, Widmann et al. (21) showed in the coronary microcirculation of the dog that acetylcholine-induced dilation is only blocked by inhibiting the cyclooxygenase, NOS, and cytochrome P-450 pathways, whereas in larger vessels acetylcholine responses are predominately NOS mediated (13). In resistance arterioles of different vascular beds, EDHF has been shown to contribute substantially to endothelium-dependent vasodilation (7, 15), particularly when the production or bioactivity of NO is diminished in disease states such as atherosclerosis. Thus, in the microcirculation, EDHF may play a major role in the physiological or pathophysiological regulation of blood flow, whereas in large arteries, NO appears to play a dominant role.

Evaluation of AA-induced dilation in the canine coronary microcirculation involved studies designed to investigate the role of each endothelial-dependent pathway separately and in combination with other pathways. Studies looking at each pathway separately did not exclude any of the pathways tested. Alone, the cyclooxygenase pathway does not appear to play a major role in AA-induced dilation. In vivo, indomethacin had no effect on relaxation, and in vitro, the combination of indomethacin and L-NNA did not alter AA-induced dilation. The lipoxygenase pathway most likely does not participate in AA-induced dilation, because the NOS-cyclooxygenase-resistant vasodilation was not blocked by CDC.

To investigate the hypothesis that NOS-cyclooxygenase-resistant vasodilation is mediated by the cytochrome P-450 pathway, AA-induced relaxation was evaluated in the presence of indomethacin, L-NNA, and clotrimazole. Both in vivo and in vitro, this combination of inhibitors blocked AA-induced dilation. Our results also showed that K+ channels mediate AA-induced vasodilation. High extracellular K+ concentrations, which prevent membrane hyperpolarization by depolarization of smooth muscle, blocked AA-induced dilation. The nonspecific calcium-activated K+ channel inhibitor attenuated the AA-induced response, whereas specific inhibitors of calcium-activated K channels channels failed to demonstrate significant effects. Intermediate conductance calcium-activated K channels may be involved. In addition, the NOS-cyclooxygenase-resistant vasodilation was inhibited by both charybdotoxin and TEA and completely blocked by TBA, suggesting that EDHF-induced dilation is mediated through calcium-activated K channels.

Bauersachs et al. (4) showed the production of EDHF is damped by NO as bradykinin and acetylcholine relaxation was attenuated in the presence of an NO donor in rabbit carotid and porcine conduit coronary arteries. Nishikawa et al. (16) showed canine coronary arteriole dilation to bradykinin was abolished in the presence of an NO donor (SNP), NOS inhibitor (L-NMMA), and cyclooxygenase inhibitor (indomethacin). These studies suggest that NO inhibits or provides a negative feedback to EDHF-induced dilation. In our laboratory, we were able to completely block AA-induced vasodilation in isolated coronary microvessels in the presence of SNP (a NO donor) (Fig. 8).

Together, these observations lead one to speculate that AA-induced vasodilation is mediated in part through activation of calcium-activated K channels channels, particularly in the presence of inhibitors of cyclooxygenase and NOS. This NOS-cyclooxygenase-resistant dilation, likely mediated through EDHF, is also greatly affected by the presence of NO. This negative feedback, while important in the normal circulation, may be interrupted during times of disease processes such as diabetes and atherosclerosis (2). Without the tonic inhibition, a predominant regulatory pathway in disease could become the EDHF pathway. Thus chemical identification of EDHF is an important future direction for work in this area.

In conclusion, AA-induced coronary microvascular dilation is significant and mediated through potentially redundant pathways, including the cyclooxygenase, NOS, and cytochrome P-450 enzyme systems. The final dilatory effect appears to be mediated through a hyperpolarizing K+ channel.


    ACKNOWLEDGEMENTS

The authors thank Len Brooks for technical assistance.


    FOOTNOTES

The support for this study included Veterans Affairs (VA)/JDF Diabetes Research Center (to K. C. Dellsperger), VA Merit Review (to K. C. Dellsperger), American Heart Association (AHA) Heartland Affiliate Beginning Grant-in-Aid (to C. L. Oltman), and National Heart, Lung, and Blood Institute Grants HL-49264 and HL-62984 (to N. L. Weintraub). N. L. Weintraub is a Clinician-Scientist awardee of the AHA.

Address for reprint requests and other correspondence: C. L. Oltman, Cardiovascular Research (151), VA Medical Center, Highway 6 West, Iowa City, IA 52246 (E-mail: christine-oltman{at}uiowa.edu).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received 2 December 2000; accepted in final form 18 January 2001.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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Am J Physiol Heart Circ Physiol 281(4):H1553-H1560


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