(Circulation. 2001;103:1702.)
© 2001 American Heart Association, Inc.
Basic Science Reports |
Endothelium-Derived Hyperpolarizing Factor
Identification and Mechanisms of Action in Human Subcutaneous Resistance Arteries
From the Department of Biological and Biomedical Sciences, Glasgow Caledonian University (P.C., F.J., C.H.), and the Department of Medicine and Therapeutics, Western Infirmary (J.M., J.J.V.M), Glasgow, Scotland.
Correspondence to Dr Paul Coats, School of Biological and Biomedical Sciences, Glasgow Caledonian University, Cowcaddens Road, Glasgow, Scotland, UK. E-mail p.coats{at}gcal.ac.uk
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Abstract |
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Background—Both a vascular endothelial cytochrome P450 (CYP450) product of arachidonic acid metabolism and the potassium ion (K+) have been identified as endothelium-derived hyperpolarizing factors (EDHFs) in animal vascular tissues. We studied the relative importance of EDHF, nitric oxide (NO), and prostacyclin (PGI2) as vasodilators in human subcutaneous arteries. We also examined the mechanisms underlying the vasodilator action of EDHF to elucidate its identity.
Methods and
Results—Subcutaneous resistance arteries were
obtained from 41 healthy volunteers. The contribution of EDHF to the
vasodilation induced by acetylcholine was assessed by inhibiting
production of NO, PGI2, and membrane
hyperpolarization. The mechanisms underlying the
relaxation evoked by K+ and EDHF were
elucidated. EDHF was found to account for 
80% of
acetylcholine-mediated vasorelaxation. Its action was insensitive to
the combination of barium and ouabain, whereas barium and ouabain
reversed K+-mediated vasorelaxation.
EDHF-mediated vasorelaxation, however, was sensitive to the
phospholipase A2 inhibitor
oleyloxyethyl phosphorylcholine and the CYP450 inhibitor
ketoconazole.
Conclusions—EDHF is the major contributor to endothelium-dependent vasorelaxation in human subcutaneous resistance arteries. A product of phospholipase A2/CYP450–dependent metabolism of arachidonic acid and not K+ is the likely identity of EDHF in human subcutaneous resistance arteries.
Key Words: endothelium-derived factors • nitric oxide
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Introduction |
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Endothelial dysfunction is known to occur in a number of cardiovascular diseases, including atherosclerosis, hypertension, and heart failure.1 2 3 4 5 6 Therefore, considerable interest is attached to understanding the mechanisms underlying endothelium-dependent vasorelaxation. It is hoped that therapeutic strategies may be developed to counter vascular endothelial dysfunction in these and other diseases.7 The vascular endothelium modulates local blood flow via the dynamic release of numerous vasoactive factors. Receptor-dependent agonists, such as acetylcholine, bradykinin, and substance P, indirectly relax vascular smooth muscle by inducing the release of the known endothelium-derived relaxing factors (EDRFs) nitric oxide (NO), prostacyclin (PGI2), and endothelium-derived hyperpolarizing factor (EDHF).8 9 10 11 At present, there is no clear consensus on the identity of EDHF or the exact mechanisms by which EDHF relaxes vascular smooth muscle. Some evidence, however, demonstrates that the action of EDHF involves calcium-sensitive K+ channels (KCa), which are sensitive to the combined effects of the toxins apamin and charybdotoxin.12 13 14 15
To date, numerous studies have aimed to identify the nature of EDHF and its mechanisms of action. Experimental evidence suggests that the most likely candidates are the P450 metabolite of arachidonic acid metabolism, 11,12-epoxyeicosatrienoic acid (11,12-EET), and the potassium ion (K+).15 16 17 18 19 K+ has long been known to possess vasodilator properties.20 21 22 Recently, Edwards and colleagues15 proposed that K+, as an EDHF, was extruded from the endothelium into the myoendothelial gap, leading to activation of vascular smooth muscle inwardly rectifying potassium channels and Na2+,K+-ATPase pumps. This in turn resulted in efflux of K+ from the vascular smooth muscle, inducing hyperpolarization and vasorelaxation. Other recent and compelling evidence, however, suggests that the CYP450 enzyme product 11,12-EET, derived from the endothelium-dependent metabolism of arachidonic acid, is an EDHF in porcine coronary arteries and hamster gracilis muscle artery.19 23 The evidence that K+ or a P450 enzyme product is an EDHF, however, remains controversial.24 25 26 27 28 29 Therefore, real uncertainty exists regarding the identification of EDHF.
There have been few studies of EDHF in human arteries. Nonetheless, it appears that an endothelium-dependent non-NO, nonprostanoid mechanism of vasorelaxation predominates in these arteries.30 31 32 33 34
To date, there has been no specific study of EDHF in human subcutaneous resistance arteries. Therefore, in this study we aimed to identify the mechanisms of action and the likely identity of EDHF in human small subcutaneous resistance arteries.
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Methods |
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Patients and Vessel Preparation
The hospital ethics review committee approved the study, and each subject gave informed written consent. Clinical characteristics of the volunteers are given in Table 1
. Healthy volunteers with no history of
vascular disease, diabetes, hypertension, or renal impairment attended
the Clinical Investigations Research Unit at the Western Infirmary,
Glasgow. Subcutaneous gluteal fat biopsies (1.5x1.5x0.5 cm) were
excised under local anesthesia with 1% lidocaine and
immediately transferred to cold physiological
saline solution (PSS). Forty-six subcutaneous resistance-size arteries
(lumen diameter 123±3 µm) were isolated from 41 biopsies and cleaned
of any adherent tissue under a dissection microscope (Zeiss Stemi 2000,
magnification x6 to x45).
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Isolated arteries were mounted on a pressure myograph (Danish MyoTech P100) and studied pharmacologically at an intraluminal pressure of 40 mm Hg in a no-flow state by methods described previously.35 Briefly, PSS was gassed with 95% O2/5% CO2, with pH maintained at 7.4 at 37°C. Functional viability was assessed by maximum vasoconstriction to 60 mmol/L K+ PSS and norepinephrine (10 µmol/L) and vasorelaxation (>80%) to acetylcholine. All arteries fulfilled these criteria, and none were discarded.
Pharmacological Protocols
All studies were carried out in arteries
preconstricted with norepinephrine to 80% of maximum
response. In experiments in which
K+-modified PSS was used, the concentration
of norepinephrine was reduced to maintain a preconstriction
diameter similar to previous cumulative concentration-response curves
(CCRCs).
Relative Importance of EDHF
CCRCs were constructed with acetylcholine alone and
after cumulative incubation with 100 µmol/L
NG-nitro-L-arginine
(L-NOARG) for 1 hour, 30 µmol/L indomethacin
for 30 minutes, and 25 mmol/L K+
PSS.36 37 38
Identification of K+
Channels
All subsequent acetylcholine experiments were carried
out with arteries preincubated with 100 µmol/L L-NOARG+30 µmol/L
indomethacin. CCRCs were then constructed after
intraluminal incubation with either (1) glibenclamide (10 µmol/L);
(2) apamin (100 nmol/L); (3) charybdotoxin (100 nmol/L); (4)
apamin+charybdotoxin; (5) apamin+iberiotoxin; (6) barium
(Ba2+, 30 µmol/L);(7) ouabain
(1 mmol/L); and (8)
Ba2++ouabain.
Role of Arachidonic
Acid
Acetylcholine CCRCs were constructed after
intraluminal incubation with oleyloxyethyl phosphorylcholine (OOPC) and
ketoconazole. Endothelial specificity of these
inhibitors was assessed by repeating CCRCs with sodium
nitroprusside, pinacidil, and 1-ethyl-2-benzimidazoline (EBIO) in the
presence of maximal concentrations of OOPC and
ketoconazole.
Mechanisms of
K+-Mediated Vasorelaxation
In standard PSS, the extracellular
K+ concentration
([KCl]o) was elevated from 4.6 to 20
mmol/L in 2-mmol/L steps. This protocol was then repeated in the
presence of Ba2+ or ouabain and
Ba2++ouabain.
Determination of cGMP Content
Human subcutaneous artery segments (3
mm) were isolated from 3 subcutaneous biopsies and incubated in PSS in
the presence of either (1) norepinephrine (1
µmol/L), (2) norepinephrine+acetylcholine (10
µmol/L), (3) norepinephrine+acetylcholine+L-NOARG (100
µmol/L), or (4)
norepinephrine+acetylcholine+oxadiazoloquinoxalin (ODQ, 10
µmol/L). All experiments were in the presence of
isobutylmethylxanthine (10 µmol/L). At the
end of the incubation period, the tissues were immersed in liquid
nitrogen, homogenized in 95% ethanol, and
centrifuged at 3000 rpm for 15 minutes. The supernatant was
extracted and cGMP determined by
radioimmunoassay.39 The
amount of protein in the centrifuged pellet was determined by
Bradford’s
assay.40
Drugs and Solutions
Acetylcholine,
isobutylmethylxanthine,
indomethacin, L-NOARG, norepinephrine,
pinacidil, EBIO, and sodium nitroprusside were purchased from Sigma
Chemical Co; apamin from Calbiochem; charybdotoxin from Bachem;
ketoconazole from Tocris Cookson Ltd; and OOPC from Affinity Research.
Anti-cGMP was a gift from Dr David Bunton (Glasgow Caledonian
University, UK). PSS composition (in mmol) was NaCl 119, KCl 4.5,
NaHCO3 25,
KH2PO4 1.0,
MgSO4 · 7H2O 1.0,
glucose 11.0, and CaCl2 2.5.
K+ PSS composition (25 and 60 mmol/L)
was equimolar substitution of NaCl with KCl.
Data and Statistical Analysis
Relaxation data are represented as
relaxation relative to the preconstricted diameter of the artery.
Values are presented as mean±SEM. Statistical comparisons of
pEC50 (concentration required to produce 50% of
the maximum response) and maximum response were performed with
Student’s paired t test
followed by multiple comparisons by Bonferroni’s test where
appropriate. Not calculable (NC) appears where the
pEC50 could not be determined. Comparison of
CCRCs was by one-way ANOVA for repeated measures. Statistical
significance was assumed at a value of
P<0.05.
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Results |
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The effects of L-NOARG, indomethacin, and 25 mmol/L K+ PSS on acetylcholine-mediated vasorelaxation are shown in Figure 1
. Incubation with L-NOARG significantly reduced both
the maximum relaxation and pEC50 (137±24 versus
72±14 nmol/L, P<0.05) to
acetylcholine. Subsequent incubation with indomethacin
failed to modify this response further. Exchange of PSS with 25
mmol/L K+ PSS, however, abolished the
relaxation response to acetylcholine
(Figure 1).
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Figure 2 summarizes the results of the cGMP
radioimmunoassay. As expected, acetylcholine substantially increased
cGMP generation. Incubation with L-NOARG or ODQ abolished the
acetylcholine-dependent liberation of cGMP. Both L-NOARG (100 µmol/L)
and ODQ (10 µmol/L) were equipotent at inhibiting the generation of
cGMP.
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Incubation with 10 µmol/L glibenclamide had no effect on
the acetylcholine-dependent response
(Figure 3). Apamin or charybdotoxin alone also had little
effect on the response to acetylcholine
(Figure 4). Incubation with the combination of apamin and
charybdotoxin, however, abolished the relaxation to acetylcholine.
Substitution of charybdotoxin with iberiotoxin had no effect on the
relaxation response
(Figure 5).
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Cumulative addition of KCl on preconstricted arteries
initially produced a concentration-dependent relaxation (4.6 to 14
mmol/L). This was reversed at higher concentrations (16 to 20
mmol/L) to a vasoconstriction response
(Figure 6). Ba2+ (30 µmol/L) or
ouabain (1 mmol/L) alone was unable to significantly modify the
K+-mediated responses (data not
presented). When combined, however,
Ba2+ and ouabain reversed
K+-induced vasorelaxation, resulting in
vasoconstriction at all concentrations of KCl
(Figure 6). In contrast, Ba2+ and
ouabain either alone (data not shown) or combined, at concentrations
that reversed K+-mediated vasorelaxation,
had no effect on acetylcholine-mediated vasorelaxation
(Figure 7).
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The phospholipase A2
inhibitor OOPC reduced both sensitivity and maximum
relaxation responses to the
L-NOARG/indomethacin-insensitive component of
acetylcholine-mediated vasorelaxation in a concentration-dependent
manner
(Figure 8; EC50: ACh, 0.2±0.1
µmol/L; +30 µmol/L OOPC, 1.0±0.6 µmol/L; +100 µmol/L OOPC, NC;
P<0.05, 30 µmol/L OOPC
versus ACh). OOPC had no effect on the sensitivity to
norepinephrine, because the concentration used for
preconstriction remained relatively constant. Furthermore, OOPC failed
to inhibit the response to cumulative addition of sodium nitroprusside
(Table 2). Incubation with the highest OOPC concentration
(100 µmol/L) failed to modify the relaxation response to the
ATP-sensitive K+ channel opener pinacidil
and the Ca2+-sensitive
K+ channel opener EBIO
(Table 2).
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The cytochrome P450 inhibitor ketoconazole also
resulted in a concentration-dependent inhibition of the
L-NOARG/ indomethacin-insensitive relaxation to
acetylcholine
(Figure 9; EC50: ACh 0.3±0.1
µmol/L; +1 µmol/L ketoconazole, 1.0±0.6 µmol/L; +10 µmol/L
ketoconazole, 3.5±1.7 µmol/L; +30 µmol/L ketoconazole, NC; +100
µmol/L ketoconazole, NC;
P<0.05, 1 µmol/L
ketoconazole versus ACh and 10 µmol/L ketoconazole versus 1 µmol/L
ketoconazole). Again, ketoconazole did not modify the sensitivity of
the tissue to the preconstricting agonist norepinephrine.
Also, sodium nitroprusside–, EBIO-, and pinacidil-dependent responses
were unaffected by the highest concentration of ketoconazole (100
µmol/L,
Table 3).
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Discussion |
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This study has 2 important findings. First, it demonstrates that EDHF in human subcutaneous resistance arteries is the major component of endothelium-dependent vasorelaxation. Second, a product of arachidonic acid metabolism, probably a cytochrome P450 product rather than the potassium ion, is likely to be an EDHF in these arteries.
A number of studies have suggested that a substance
independent of NO and PGI2 is the major EDRF(s)
in resistance arteries. Our results demonstrate that the
L-NOARG/indomethacin-insensitive component of
acetylcholine-mediated relaxation is the major component of
endothelium-dependent relaxation in human subcutaneous
resistance arteries. The NO component, ie, the L-NOARG–sensitive
component, accounted for only 20% of the maximum relaxation
response to acetylcholine. Furthermore, indomethacin
had no effect on the endothelium-dependent relaxation,
indicating that PGI2 plays no role in the
relaxation response in these ex vivo conditions. In contrast, the major
component of the endothelium-dependent relaxation was
sensitive to 25 mmol/L K+ PSS. This
sensitivity to high concentrations of K+
PSS, which has been shown to be a characteristic of EDHF, accounted for
75% of the maximum response to
acetylcholine.15 36 38 41 42
This study confirms the relative importance of EDHF and that NO and
PGI2 play relatively minor roles in
endothelium-dependent relaxation of human resistance
arteries.11 34 43 44
In vessels in which NO synthase and cyclooxygenase-1 are inhibited, glibenclamide, a KATP channel inhibitor previously shown to inhibit EDHF-mediated vasorelaxation in rabbit cerebral arteries, had no measurable effect on EDHF-mediated relaxation to acetylcholine in human subcutaneous resistance arteries.45 This indicates that KATP channels have no role in this response.
One of the few points of consensus to emerge in EDHF research has been that preincubation of arteries with either apamin, an inhibitor of large-conductance calcium-sensitive K+ channels (BKCa), or charybdotoxin, an inhibitor of intermediate-conductance calcium-sensitive K+ channels (IKCa), alone has little effect on the EDHF-mediated relaxation to acetylcholine; when combined, however, apamin and charybdotoxin together abolish the EDHF-mediated relaxation.13 15 46 Our results confirm that the EDHF-mediated relaxation in human subcutaneous resistance arteries is similarly resistant to apamin or charybdotoxin alone but is abolished by the combination of the 2 toxins. Interestingly, substitution of charybdotoxin with iberiotoxin, a selective inhibitor of BKCa, in the presence of apamin had no effect on the relaxation response.47 Interpretation of these findings is difficult. Inhibition of IKCa or BKCa alone clearly does not inhibit the actions of EDHF, suggesting that there is a BKCa/IKCa channel codependency involved in this mechanism of vasorelaxation. Alternatively, there may be an as yet unidentified BKCa/IKCa-like heteromultimeric K+ channel associated with an EDHF-mediated response.48 Charybdotoxin has inhibitory action at BKCa and a number of voltage-sensitive K+ channels.49 The fact that charybdotoxin, as opposed to iberiotoxin, is required in combination with apamin to block the relaxation response insensitive to L-NOARG and indomethacin suggests that Ca2+-sensitive K+ channels and voltage-sensitive K+ channels are involved in this response.
Elevation of the extracellular potassium concentration,
[K+]o, has been
shown to dilate isolated arteries from rat cerebral, coronary,
hepatic, and mesenteric vascular
beds.15 21 The
mechanistic basis of this response involves
K+ efflux from vascular smooth muscle via
the inwardly rectifying potassium channel
(KIR) and the
Na+,K+-ATPase
pump, leading to hyperpolarization and
vasorelaxation. Using the protocols established by Edwards and
colleagues, we mimicked the effects of EDHF by increasing
[K+]o. In our
study, elevation of
[K+]o resulted in a
small vasorelaxation of preconstricted arteries. The maximum relaxation
response occurred at 14 to 16 mmol/L
[K+]o; increasing
[K+]o further
resulted in vasoconstriction. The
[K+]o-dependent
vasodilation observed was sensitive to the combination of
Ba2+, an inhibitor of
KIR, plus ouabain, an inhibitor of
the Na+,K+-ATPase
pump, but not Ba2+ or ouabain alone. In
fact, elevation of
[K+]o in the
presence of Ba2+ plus ouabain resulted in a
concentration-dependent vasoconstriction at all increments of
[K+]o and in all
arteries studied. This observation confirms
K+ as having vasodilator properties and
confirms the role of the KIR and the
Na+,K+-ATPase
pump in K+-dependent vasodilation in these
vessels. Ba2+ plus ouabain, however, had no
effect on the EDHF-mediated relaxation to acetylcholine. This
observation shows that EDHF is insensitive to inhibition of the
KIR and the
Na+,K+-ATPase
pump.
These results demonstrate that the EDHF component of the acetylcholine-mediated endothelium-dependent relaxation is mediated via mechanisms different from those through which K+ mediates vasorelaxation. If K+ were an EDHF in human subcutaneous resistance arteries, this response should have been sensitive to the combination of Ba2+ plus ouabain.
Phospholipase A2–dependent metabolism of membrane-bound phospholipids is a primary source of arachidonic acid and contributes to EDHF-mediated vasorelaxation in rabbit mesenteric arteries.50 In turn, the metabolism of arachidonic acid by P450 enzymes into vasoactive substances is a commonly identified phenomenon in vascular physiology.51 52 53 54 55 In the present study, luminal incubation with OOPC, a specific inhibitor of phospholipase A2, had a profound effect on endothelium-dependent relaxation to EDHF. Likewise, luminal incubation with the P450 enzyme inhibitor ketoconazole resulted in a concentration-dependent inhibition. The OOPC and ketoconazole results provide strong evidence that the endothelium-dependent relaxation to EDHF is a product of phospholipase A2/arachidonic acid/P450 enzyme metabolism in human subcutaneous resistance arteries.
We are confident that these results reflect specifically endothelium-dependent mechanisms, because the vasorelaxation response to the endothelium-independent vasodilator sodium nitroprusside was unaffected by the highest concentrations of OOPC and ketoconazole. Moreover, the endothelium-independent mechanisms of hyperpolarization were unaffected by this treatment, because the KATP channel opener pinacidil and the Ca2+-sensitive K+ channel opener EBIO produced potent vasorelaxation unaffected by either OOPC or ketoconazole.
In this study, we were unable to measure smooth muscle membrane potential and therefore have no direct evidence that the acetylcholine-mediated relaxation insensitive to L-NOARG and indomethacin is definitely the result of hyperpolarization. Raising [K+]o to 25 mmol/L, however, completely blocked the L-NOARG/indomethacin-insensitive response to acetylcholine. Previous studies have shown that raising [K+]o has little effect on NO and PGI2-mediated vasorelaxation, but rather it specifically antagonizes the actions of EDHF by counterbalancing smooth muscle cell membrane potential.
Experimental evidence suggests that the commonly used concentrations of NO synthase inhibitors may not be completely effective in blocking all NO production.56 57 This is important, because NO has been shown to have a hyperpolarizing effect via cGMP in vascular smooth muscle and the response insensitive to L-NOARG may be as a consequence of residual NO production. L-NOARG (100 µmol/L) in this tissue, however, abolished the acetylcholine-dependent liberation of cGMP. Therefore, we are confident that there was no residual NO and that the relaxation responses observed were independent of NO. Moreover, NO has been shown to hyperpolarize vascular tissue via cGMP- and cAMP-dependent mechanisms sensitive to iberiotoxin and glibenclamide, respectively. Both iberiotoxin and glibenclamide had no effect on the relaxation response insensitive to L-NOARG and indomethacin. Therefore, we are confident that the acetylcholine-mediated relaxation insensitive to L-NOARG and indomethacin is truly an NO/prostanoid-independent phenomenon and is likely to be mediated by an EDHF.
This study shows that endothelium-dependent relaxation to EDHF is mediated via mechanisms different from those mediating K+ vasorelaxation; therefore, it is unlikely that K+ is an EDHF in human subcutaneous resistance arteries. Moreover, this mechanism is sensitive to phospholipase A2 and P450 enzyme inhibition, thus indicating that EDHF in human subcutaneous resistance arteries is likely to be a P450 enzyme–dependent product of arachidonic acid metabolism. Also, vasodilation depends largely on K+ channels, suggesting that selective use of appropriate potassium channel drugs may provide a useful therapeutic approach for the treatment of vascular pathologies.
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Acknowledgments |
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This study was supported by the Barnwood House Trust. Dr Coats was supported by a Glasgow Caledonian University (UK) Research Studentship. The authors thank Dr David Bunton for his assistance with cGMP radioimmunoassay.
Received July 13, 2000; revision received October 4, 2000; accepted October 16, 2000.
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