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Am J Physiol Heart Circ Physiol 280: H2470-H2477, 2001;
0363-6135/01 $5.00
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Vol. 280, Issue 6, H2470-H2477, June 2001

SPECIAL TOPIC
Characterization of endothelium-derived hyperpolarizing factor in the human forearm microcirculation

Julian P. J. Halcox, Suresh Narayanan, Laura Cramer-Joyce, Rita Mincemoyer, and Arshed A. Quyyumi

Cardiology Branch, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, Maryland 20892


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

The identity of endothelium-dependent hyperpolarizing factor (EDHF) in the human circulation remains controversial. We investigated whether EDHF contributes to endothelium-dependent vasomotion in the forearm microvasculature by studying the effect of K+ and miconazole, an inhibitor of cytochrome P-450, on the response to bradykinin in healthy human subjects. Study drugs were infused intra-arterially, and forearm blood flow was measured using strain-gauge plethysmography. Infusion of KCl (0.33 mmol/min) into the brachial artery caused baseline vasodilation and inhibited the vasodilator response to bradykinin, but not to sodium nitroprusside. Thus the incremental vasodilation induced by bradykinin was reduced from 14.3 ± 2 to 7.1 ± 2 ml · min-1 · 100 g-1 (P < 0.001) after KCl infusion. A similar inhibition of the bradykinin (P = 0.014), but not the sodium nitroprusside (not significant), response was observed with KCl after the study was repeated during preconstriction with phenylephrine to restore resting blood flow to basal values after KCl. Miconazole (0.125 mg/min) did not inhibit endothelium-dependent or -independent responses to ACh and sodium nitroprusside, respectively. However, after inhibition of cyclooxygenase and nitric oxide synthase with aspirin and NG-monomethyl-L-arginine, the forearm blood flow response to bradykinin (P = 0.003), but not to sodium nitroprusside (not significant), was significantly suppressed by miconazole. Thus nitric oxide- and prostaglandin-independent, bradykinin-mediated forearm vasodilation is suppressed by high intravascular K+ concentrations, indicating a contribution of EDHF. In the human forearm microvasculature, EDHF appears to be a cytochrome P-450 derivative, possibly an epoxyeicosatrienoic acid.

vascular tone; endothelium; cytochrome P-450; miconazole; potassium; bradykinin; sodium nitroprusside


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

IN RESPONSE TO PHARMACOLOGICAL probes, such as ACh and bradykinin, or physiological increases in shear stress, endothelial cells release various relaxing factors, including nitric oxide (NO) and prostacyclin, that modulate vasodilator tone and function (13). Bradykinin and ACh also evoke endothelium-dependent hyperpolarization of vascular smooth muscle that leads to relaxation and appears to be mediated by a soluble transferable factor called endothelium-derived hyperpolarizing factor (EDHF) (11). Although NO and EDHF appear to be distinct entities, some reports suggest that NO may also hyperpolarize vascular smooth muscle by activating ATP-dependent K+ channels (1, 6, 14, 25). However, in the human microcirculation, combined inhibition of NO synthase and cyclooxygenase does not fully block endothelium-dependent vasodilation, suggesting that this residual vasodilation is dependent on the action of an EDHF distinct from NO (23, 24, 41).

Although the biochemical identity of EDHF remains a subject of intense controversy, endothelium-dependent hyperpolarization in human vascular tissue involves opening of K+ conductance channels (19, 24, 28). Because membrane potentials and K+ conductance cannot be measured in vivo, experimental studies have employed high extracellular concentrations of K+ (20-60 mmol/l) to "clamp" the smooth muscle membrane in a hyperpolarized or depolarized state (23, 41). By preventing any further cellular hyperpolarization, high extracellular K+ levels provide a means for nonspecifically blocking the action of EDHF. In this investigation, we infused KCl intra-arterially to achieve extracellular hyperkalemia in the human forearm.

Using specific antagonists of K+ conductance, several studies have shown that EDHF causes hyperpolarization of the human microcirculation via activation of Ca2+-activated K+ (KCa) channels (23, 24, 41), although other K+ channels may be involved in other species.

Evidence indicates that endothelial surface B2-receptor stimulation by bradykinin or muscarinic receptor activation by ACh and the resultant Ca2+ influx stimulate endothelial phospholipase A2 and NO synthase. Arachidonic acid released as a consequence of phospholipase A2 activation can be metabolized by cyclooxygenase, lipoxygenase, or cytochrome P-450 monooxygenase. Metabolites of cytochrome P-450 epoxygenase, such as epoxyeicosatrienoic acids (EETs) and their dihydroxyeicosatrienoic acid derivatives, are capable of activating KCa channels, resulting in hyperpolarization and relaxation of microvascular smooth muscle cells (7, 12, 26). Miconazole inhibits all cytochrome P-450 enzymes and impedes hyperpolarization of canine and human coronary microvessels. We therefore used miconazole in our study to investigate whether EDHF in the human forearm is a cytochrome P-450 metabolite.

Because EDHF-mediated vasodilation has been suggested to be enhanced or reduced in the setting of endothelial dysfunction and impaired NO bioavailability, it is crucial to demonstrate the presence of EDHF in the human circulation, so that strategies for improvement of endothelial function involving manipulation of EDHF can be explored. For this purpose, we investigated 1) whether EDHF contributes to endothelium-dependent vasomotion in the human forearm microvasculature by studying the effect of K+ on the response to bradykinin and 2) whether EDHF in the forearm microvasculature is a cytochrome P-450 metabolite that can be inhibited by miconazole.


    METHODS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Subjects

We studied 47 healthy volunteers. Each subject was screened by clinical history, physical examination, electrocardiogram, chest X-ray, and routine chemical analyses. All subjects were under 55 yr of age. None had evidence of present or past hypertension (blood pressure >140/90 mmHg) or hyperlipidemia (serum cholesterol >240 mg/dl) or clinical evidence of cardiovascular disease, diabetes, or any other systemic illness. None were smoking (within the past year) or taking medications at the time of the study. All participants gave written informed consent, and the study protocols were approved by the National Heart, Lung, and Blood Institute Investigational Review Board.

Study Protocols

All studies were performed in the morning in a quiet room with a temperature of 22°C. Participants fasted overnight and were asked to refrain from drinking alcohol or beverages containing caffeine for >= 24 h before the studies.

Each study consisted of the infusion of drugs into the brachial artery and measurement of the response of the forearm vasculature by means of strain-gauge venous occlusion plethysmography. All drugs were approved for human use by the Food and Drug Administration in the form of Investigational New Drugs and were prepared by the Pharmaceutical Development Service of the National Institutes of Health according to specific procedures to ensure accurate bioavailability and sterility of the solutions.

With the participants in the supine position, a 20-gauge polytetrafluoroethylene catheter (Arrow International) was inserted into the brachial artery of the nondominant arm. This arm was slightly elevated above the level of the right atrium, and a mercury-filled silicone-Elastomer strain gauge was placed on the widest part of the forearm (44). The strain gauge was connected to a plethysmograph (model EC-5R, D. E. Hokanson) that was calibrated to measure the percent change in volume and connected, in turn, to a chart recorder to record the flow measurements. For each measurement, a cuff placed around the upper arm was inflated to 40 mmHg with a rapid cuff inflator (model E-20, D. E. Hokanson) to occlude venous outflow from the extremity. A wrist cuff was inflated to suprasystolic pressures 1 min before each measurement to exclude the hand circulation (20). Flow measurements were recorded for 7 s every 15 s; seven readings were obtained for each mean value. Forearm vascular resistance (FVR) was calculated as mean arterial blood pressure divide  blood flow. Subjects were under continuous electrocardiographic monitoring, and systemic blood pressure was transduced directly from the arterial catheter (31).

In each of the following protocols, after arterial cannulation and application of the strain gauge, 5% dextrose solution was infused for 20 min to allow baseline conditions to stabilize before initiation of the protocol.

Protocol 1. Twelve subjects were given oral aspirin (1 g) 2 h before the study to inhibit cyclooxygenase-dependent production of vasoactive prostanoids. Bradykinin was initially infused at 400 ng/min for 5 min to test endothelium-dependent vasodilation after cyclooxygenase inhibition. NG-monomethyl-L-arginine (L-NMMA, 4 µmol/min) was then infused for 5 min to inhibit production of NO. During L-NMMA infusion, bradykinin was coinfused at 200 and 400 ng/min for 5 min each to test endothelium-dependent vasodilation independent of NO and prostanoids. After a 20-min recovery period, L-NMMA infusion was resumed and sodium nitroprusside was infused at 1.6 and 3.2 µg/min for 5 min each to test endothelium-independent vasodilation. After a 20-min recovery period, KCl solution (0.33 mmol/min) was infused, and dose-response curves were repeated with intra-arterial bradykinin and sodium nitroprusside during coadministration of KCl and L-NMMA. An 18-gauge cannula (Johnson and Johnson Medical) was inserted in an antecubital vein. Venous blood was drawn for measurement of serum K+ levels before and during KCl infusion.

Protocol 2. Nine subjects were given oral aspirin (1 g) 2 h before the study, and, as in protocol 1, dose-response curves to bradykinin and sodium nitroprusside were performed during coadministration of L-NMMA. KCl solution (0.33 mmol/min) was then coinfused for 5 min with the endothelium-independent vasoconstrictor phenylephrine to determine the dose of this agent (1-3 µg/min) that inhibited KCl-induced vasodilation. This dose of phenylephrine was then coadministered with KCl and L-NMMA, while the dose-response curves to bradykinin and sodium nitroprusside were repeated.

Protocol 3. In six subjects, ACh was administered at 15 and 30 µg/min for 5 min each to test endothelium-dependent vasodilation. After a 20-min recovery period, sodium nitroprusside was infused at 1.6 and 3.2 µg/min for 5 min each. Miconazole was then administered intra-arterially at 0.0125, 0.0375, and 0.125 mg/min for 10 min each. The dose-response curves to ACh and sodium nitroprusside were subsequently repeated during coadministration of miconazole at 0.125 mg/min.

Protocol 4. Fourteen subjects were given oral aspirin (1 g) 2 h before the study. Bradykinin was administered at 100, 200, and 400 ng/min for 5 min each during coadministration of L-NMMA (4 µmol/min). Miconazole was then infused at 0.125 mg/min for 15 min, and L-NMMA was coadministered during the last 5 min of the miconazole infusion. The dose-response curve to bradykinin was then repeated during coadministration of L-NMMA and miconazole. Sodium nitroprusside at 1.6, 3.2, and 6.4 µg/min for 5 min each was also administered to seven of these subjects before and after miconazole.

Reproducibility protocol. Reproducibility of the forearm blood flow responses to bradykinin and sodium nitroprusside was evaluated in six patients. Aspirin (1 g) was orally administered 2 h before initiation of the infusion protocol, and L-NMMA (4 µmol/min) was coadministered with bradykinin and sodium nitroprusside. Forearm blood flows with bradykinin (14.8 ± 2.3 and 14.2 ± 2.2 ml · min-1 · 100 ml-1 at 400 ng/min-1, r = 0.9) and sodium nitroprusside (9.8 ± 0.9 and 10.8 ± 1.7 ml · min-1 · 100 ml-1 at 3.2 µg/min, r = 0.8) were similar during repeat testing after an interval of 60 min.

Statistical Analysis

Values are means ± SE. The effect of KCl and miconazole on forearm blood flow and FVR was assessed by paired t-test. Dose-response curves with ACh, sodium nitroprusside, and bradykinin before and after KCl or miconazole were analyzed by two-way repeated-measures ANOVA (SAS, version 6.12, SAS Institute, Cary, NC). All calculated probability values are two tailed, and P < 0.05 was considered to indicate statistical significance.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

There were no changes in systemic arterial blood pressure or heart rate during the intra-arterial infusions in any of the studies.

Effect of KCl

Before administration of L-NMMA, bradykinin (400 ng/min) significantly increased forearm blood flow from 3.4 ± 0.3 to 25.6 ± 2.0 ml · min-1 · 100 ml-1 (P < 0.001). Administration of L-NMMA (4 µmol/min) caused significant vasoconstriction; forearm blood flow fell by 26% from 3.4 ± 0.3 to 2.5 ± 0.2 ml · min-1 · 100 ml-1 (P < 0.01). Furthermore, L-NMMA reduced the forearm blood flow response to bradykinin (400 ng/min) by 31%, from 25.6 ± 2 to 17.7 ± 2.1 ml · min-1 · 100 ml-1 (P < 0.001). This confirmed that NO contributes to the maintenance of basal vasodilator tone and to endothelium-dependent vasodilation with bradykinin. Nevertheless, significant microvascular dilation persisted after inhibition of NO synthase and cyclooxygenase.

Administration of KCl (0.33 mmol/min) increased forearm blood flow from 3.4 ± 0.3 to 8.5 ± 0.7 ml · min-1 · 100 ml-1 (P < 0.001). Venous serum K+ levels increased from 4.0 ± 0.1 to 7.5 ± 0.8 mmol/l (P < 0.01) during administration of KCl.

The forearm blood flow responses to bradykinin were similar before and during administration of KCl [P = not significant (NS) by ANOVA], whereas the response to sodium nitroprusside was greater during coadministration of KCl (P = 0.008 by ANOVA; Fig. 1). Thus, at the highest dose of bradykinin, the forearm blood flow was 17.7 ± 2.1 and 15.7 ± 2.0 ml · min-1 · 100 ml-1 before and during KCl, respectively (P = 0.31), and FVR was 6.24 ± 0.93 and 7.29 ± 0.94 mmHg · ml-1 · min · 100 ml-1 before and during KCl, respectively (P = 0.16).


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  		Fig. 1.   Effect of administration of KCl solution (0.33 mmol/l) on forearm blood flow responses (A and B) and forearm blood flow increment, calculated as the absolute increase in blood flow from baseline with or without KCl (C and D), in response to bradykinin and sodium nitroprusside, respectively, after inhibition of cyclooxygenase and nitric oxide synthase with aspirin and NG-monomethyl-L-arginine (L-NMMA). dagger P < 0.001, baseline flow pre- vs. post-KCl.

To account for the baseline vasodilation that was observed with KCl, we calculated the flow increment by subtracting the respective baseline value (at rest and after KCl) from the flow values obtained with each dose of bradykinin and sodium nitroprusside. Compared with control, the flow increment induced by bradykinin was lower during KCl (P = 0.004 by ANOVA) but that induced by sodium nitroprusside was unchanged (P = NS by ANOVA; Fig. 1).

The next study (protocol 2) was designed to preconstrict the forearm microvasculature with an alpha -adrenergic receptor agonist to restore blood flow to baseline levels after intra-arterial KCl. Coadministration of the endothelium-independent vasoconstrictor phenylephrine (1 µg/min in 4 patients and 3 µg/min in 4 patients) with KCl (0.33 mmol/min) significantly blunted vasodilation with KCl: 5.6 ± 1.6 and 3.0 ± 0.5 ml · min-1 · 100 ml-1 before and during KCl + phenylephrine, respectively (P = 0.11). The forearm blood flow response to bradykinin was attenuated by KCl + phenylephrine (P = 0.014 by ANOVA), whereas the response to sodium nitroprusside was unchanged (P = NS by ANOVA; Fig. 2). Thus, at the highest dose of bradykinin, blood flow was 21.4% lower (P < 0.001) and FVR was 39.5% higher (P < 0.001) after KCl.


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  		Fig. 2.   Effect of administration of KCl solution (0.33 mmol/l) during coadministration of phenylephrine (PE, 1-3 µg/min), at a dose that attenuated KCl-mediated vasodilation, on endothelium-dependent and -independent forearm blood flow responses with bradykinin (A) and sodium nitroprusside (B) after inhibition of cyclooxygenase and nitric oxide synthase with aspirin and L-NMMA. NS, not significant.

Effect of Miconazole on the Response to ACh

Miconazole did not affect basal vasomotor tone: forearm blood flow was 3.5 ± 0.5, 3.1 ± 0.4, 4.1 ± 0.6, and 4.1 ± 0.7 ml · min-1 · 100 ml-1 at rest and during infusion of miconazole at 0.0125, 0.0375, and 0.125 mg/min, respectively (P = NS by ANOVA). The dose-response curves with ACh (Fig. 3) and sodium nitroprusside (data not shown) were unchanged during coadministration of miconazole (both P = NS by ANOVA). Thus, at the highest dose of ACh, forearm blood flow was 21.0 ± 4.5 and 22.6 ± 4.0 ml · min-1 · 100 ml-1 before and during miconazole, respectively (P = 0.55), and FVR was 6.1 ± 1.4 and 5.2 ± 1.0 mmHg · ml-1 · min · 100 ml-1, respectively (P = 0.30).


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  		Fig. 3.   Effect of miconazole (0.125 mg/min) on endothelium-dependent forearm blood flow response to ACh.

Effect of Miconazole on the Response to Bradykinin After Aspirin and L-NMMA

Miconazole (0.125 mg/min) did not alter resting blood flow after blockade of NO synthase and cyclooxygenase: 2.05 ± 0.25 and 2.18 ± 0.25 ml · min-1 · 100 ml-1 before and after miconazole, respectively (P = 0.38). Vasodilation in response to bradykinin, but not to sodium nitroprusside, was inhibited by miconazole (Fig. 4). Thus, after miconazole, FVR was 27 ± 11% higher (from 9.3 ± 1 to 11.2 ± 1 mmHg · ml-1 · min · 100 ml-1, P = 0.02) at the peak dose of bradykinin but was unchanged during the highest dose of sodium nitroprusside (-4 ± 11%, P = NS).


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  		Fig. 4.   Effect of miconazole (0.125 mg/min) on percent increase in forearm blood flow (A and B) and percent reduction in forearm vascular resistance (C and D) in response to bradykinin and sodium nitroprusside, respectively, after inhibition of cyclooxygenase and nitric oxide synthase with aspirin and L-NMMA.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

We demonstrated that, in the healthy human forearm circulation, KCl attenuates NO- and prostaglandin-independent vasodilation in response to the endothelium-dependent agonist bradykinin without modulating the endothelium-independent response to sodium nitroprusside. Inhibition of cytochrome P-450 with miconazole does not affect basal forearm microvascular tone before or after inhibition of cyclooxygenase and NO synthase. In the presence of prostanoids and NO, miconazole does not inhibit the endothelium-dependent response to ACh. However, after inhibition of NO synthase and cyclooxygenase, miconazole selectively inhibited the response to bradykinin without affecting the response to sodium nitroprusside. Thus we believe that inhibition of endothelium-dependent hyperpolarization of the human forearm microcirculation in vivo can be demonstrated using high extracellular concentrations of K+. This EDHF is likely to be a cytochrome P-450 metabolite of arachidonic acid, probably an EET, that can be inhibited by miconazole.

As described previously, substantial bradykinin-mediated vasodilation of the forearm microcirculation persisted, despite inhibition of cyclooxygenase with aspirin and of NO with L-NMMA, revealing evidence for alternative vasodilator mechanisms such as EDHF release. There is broad consensus, at least in human blood vessels, that this vasodilation is due to activation of K+ channels (20, 25, 28).

We administered KCl solution at 0.33 mmol/min to raise the K+ concentration of the arterial serum to ~20 mmol/l. As previously reported, this dose of KCl provoked vasodilation of the forearm circulation, believed to be secondary to activation of the Na+-K+-ATPase and consequent hyperpolarization (8, 15, 22, 30, 34, 43). To overcome the impact of baseline vasodilation with KCl on interpretation of the subsequent dilation with endothelium-dependent and -independent agonists, we initially measured the flow increment in response to bradykinin and sodium nitroprusside. This analysis demonstrated that vasodilation with bradykinin was attenuated after KCl, whereas dilation with sodium nitroprusside remained unaffected. To confirm these findings, in protocol 2, we tested the response to bradykinin and sodium nitroprusside before and during administration of KCl after normalization of the resting blood flow by coadministration of the alpha -adrenoceptor agonist phenylephrine. The dose of phenylephrine required to reverse K+-mediated vasodilation (1-3 µg/min) was determined by individual titration during infusion of KCl. When KCl-mediated baseline vasodilation was "clamped" in this way, the response to bradykinin, but not to sodium nitroprusside, was attenuated by KCl. Because NO synthase and cyclooxygenase were inhibited in these experiments, it is likely that reduction of bradykinin-mediated hyperpolarization by high extracellular concentrations of K+ is the explanation for our findings, providing evidence for EDHF release in the healthy human peripheral microcirculation.

Whether K+ inhibits release of EDHF from endothelial cells or whether it inhibits further hyperpolarization of smooth muscle cells in response to EDHF by depolarizing cell membranes cannot be determined from our study. Furthermore, because inhibition of hyperpolarization occurs in the presence of NO synthase blockade, it is likely that EDHF in the human forearm microcirculation is not NO dependent. The concentration of K+ achieved in vivo in protocol 1 was lower than the levels traditionally used in experimental models for nonspecific inhibition of hyperpolarization (23, 41). Although we selected the dose of KCl to achieve an arterial K+ concentration of 20 mmol/l, with the assumption of resting forearm blood flow of 30 ml/min, the increase in blood flow during KCl infusion reduced the actual concentration achieved. Inhibition of KCl-mediated vasodilation with phenylephrine in protocol 2 is likely to have produced a higher local concentration of K+. Because most subjects experienced mild tingling in the hand with KCl and because there was a risk of cardiac toxicity, higher doses of KCl were not employed. Despite these limitations, inhibition of endothelium-dependent but not -independent vasodilation by K+ was observed and lends credence to the concept that hyperpolarization contributes to bradykinin-mediated vasodilation of the human forearm circulation. Because of the unavailability of specific inhibitors of KCa channels for parenteral use in humans, we were unable to determine whether hyperpolarization of the forearm microvessels with bradykinin is secondary to stimulation of the KCa channels, as shown in some in vitro studies. Previous studies in the human forearm circulation have demonstrated that inhibition of the ATP-dependent K+ channels with tolbutamide did not influence peak endothelium-dependent vasodilation (2), whereas nonspecific inhibition of Na+-K+-ATPase with ouabain attenuated residual bradykinin-mediated dilation after blockade of NO synthase (40).

Effect of Miconazole

With the observation that bradykinin-mediated forearm microvascular dilation is partly due to hyperpolarization that can be inhibited by high extracellular K+ concentrations, we examined whether EDHF in the human forearm microcirculation is a cytochrome P-450 metabolite. Studies have demonstrated EDHF to be a cytochrome P-450-derived arachidonic acid metabolite, probably an EET. Several members of the EET family, particularly 11,12-EET, are thought to be putative EDHFs, and their synthesis can be inhibited by miconazole (3, 7, 12, 39). The dose of miconazole required to inhibit EDHF-mediated vasodilation has been well established in vitro and in animal studies, and it has a documented safety profile in humans during parenteral administration. With an infusion rate of 0.125 mg/min, we aimed to produce an intra-arterial concentration of ~10 µmol/l, a level that effectively inhibits EDHF activity in vitro, without changing basal tone (7, 23). In our study, miconazole had no effect on resting forearm blood flow, suggesting that the cytochrome P-450 products do not influence basal microvascular tone in the healthy human forearm circulation, even after inhibition of cyclooxygenase and NO synthase. Furthermore, forearm blood flow responses to ACh and sodium nitroprusside were unchanged after miconazole, indicating that, in the presence of NO and prostaglandins, cytochrome P-450 inhibition did not influence EDHF-mediated vasodilation of the human forearm circulation. However, because NO inhibits production of EDHF in experimental studies (4, 5, 27) and EDHF activity in the human forearm circulation appears to be enhanced after NO synthase inhibition in healthy individuals (40), we tested whether miconazole inhibits endothelium-dependent vasodilation after inhibition of NO synthase and cyclooxygenase. Removing the inhibitory action of NO by pretreatment with L-NMMA attenuated the response to bradykinin, suggesting that miconazole, by antagonizing cytochrome P-450, blocks EDHF-mediated vasodilation.

Because a validated method for measurement of EETs or related cytochrome P-450 metabolites of arachidonic acid in human plasma has not yet been developed, the understanding of the mechanism underlying inhibition of the bradykinin response by miconazole is based on previous experimental work performed in vitro. Residual dilation after inhibition of NO and cyclooxygenase with arachidonic acid and bradykinin in human coronary microvessels has been unequivocally demonstrated to be due to cytochrome P-450 metabolites (23, 24), findings that are supported by observations in porcine, canine, bovine, and rat coronary arteries (17, 29, 35, 36, 38). We found that NO- and prostacyclin-independent, bradykinin-mediated vasodilation was 24% lower after miconazole.

There may be several reasons for the incomplete suppression of the bradykinin response after aspirin, L-NMMA, and miconazole in vivo. First, these are competitive antagonists and thus may not have completely inhibited cyclooxygenase, NO synthase, and cytochrome P-450 with the concentrations used. Second, bradykinin may stimulate release of other vasodilator molecules from the endothelium (e.g., carbon monoxide) (45) or from local nerves and mast cells (10, 42). Third, the nonspecific nature of the cytochrome P-450 enzyme inhibition with miconazole may be a factor; epoxygenation of arachidonic acid by this enzyme produces vasodilator EETs (11,12- and 14,15-EETs) that cause hyperpolarization, but hydroxylation of arachidonic acid produces vasoconstrictor substances, e.g., hydroxyeicosatetraenoic acids, that may offset activity of vasodilator EETs (9, 16, 18). These factors may have led to underestimation of the role of vasodilator EETs as EDHF in the human forearm circulation. Further study of this issue is required but is hindered by the lack of selective inhibitors of cytochrome P-450-mediated hydroxylation or epoxygenation that are approved for in vivo human use and the unavailability of validated assays capable of measuring stable EETs in human plasma.

Several other substances have been suggested as possible EDHFs with marked differences between species and vascular beds; however, few have been postulated as EDHFs in human vascular tissue. These include short-acting substances, such as carbon monoxide, hydroxyl radical, and hydrogen peroxide, that are capable of hyperpolarization. Others have suggested that K+ extruded from endothelial cells may itself mediate hyperpolarization of smooth muscle cells. These and other potential pathways also need to be rigorously studied in humans.

Limitations

Normalization of KCl-induced vasodilation with phenylephrine may have led to nonspecific inhibition of bradykinin-mediated dilation. This is unlikely, because sodium nitroprusside-induced vasodilation was unaffected, and previous studies have not demonstrated any action of alpha -adrenergic receptors on endothelium-dependent responses in the forearm (32).

It has been suggested that miconazole may directly inhibit KCa channels, and thus its inhibition of bradykinin responses may not necessarily be indicative of EETs as EDHF. Although this cannot be tested in humans in vivo, the attenuation of EDHF-mediated responses by cytochrome P-450 2C8/34 oligonucleotides in porcine coronary arteries strongly suggests that products of this enzyme constitute EDHF (12).

Conclusions and Implications

We previously demonstrated that the vascular effects of bradykinin in the human circulation are significantly divergent from those of ACh and other endothelium-dependent probes (37). Because bradykinin is tonically secreted from the vascular endothelium and appears to be partly responsible for NO and EDHF release, as shown in this study, it is important to determine how risk factors for atherosclerosis will impact on EDHF activity. This is particularly important in view of the fact that angiotensin-converting enzyme inhibitor-induced potentiation of bradykinin activity, which has been demonstrated to have a protective effect in patients with atherosclerosis, may act by improving bioavailability of NO and EDHF. Some studies have suggested that, in the presence of endothelial dysfunction and NO deficiency, EDHF activity may be upregulated (21, 33, 40), but other investigations have failed to confirm this observation (23, 24). Our finding that EDHF activity in vivo can be assessed using miconazole will facilitate these studies in human subjects.

In summary, we have demonstrated that bradykinin-mediated forearm microvascular vasodilation, in the presence of NO synthase and cyclooxygenase blockade, is inhibited by high extracellular concentrations of K+, providing indirect evidence for hyperpolarization. We have also provided evidence that miconazole inhibits bradykinin-mediated dilation, suggesting that EDHF in the human peripheral circulation is a cytochrome P-450 derivative, possibly an EET.


    ACKNOWLEDGEMENTS

We are grateful for the helpful advice provided by Dr. William B. Campbell regarding our studies with miconazole. We thank William H. Schenke and Gloria Zalos for technical assistance.


    FOOTNOTES

Address for reprint requests and other correspondence: A. A. Quyyumi, National Institutes of Health, Cardiology Branch, NHLBI, Bldg. 10, Rm. 7B15, 10 Center Dr., MSC 1650, Bethesda, MD 20892-1650 (E-mail: quyyumia{at}nih.gov).

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 26 December 2000; accepted in final form 29 January 2001.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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