Contribution of endothelium-derived hyperpolarizing factor to exercise-induced vasodilation in health and hypercholesterolemia

Abstract

The role of endothelium-derived hyperpolarizing factor (EDHF) in either the healthy circulation or in those with hypercholesterolemia is unknown. In healthy and hypercholesterolemic subjects, we measured forearm blood flow (FBF) using strain-gauge plethysmography at rest, during graded handgrip exercise, and after sodium nitroprusside infusion. Measurements were repeated after l-NMMA, tetraethylammonium (TEA), and combined infusions. At rest, l-NMMA infusion reduced FBF in healthy but not hypercholesterolemic subjects. At peak exercise, vasodilation was lower in hypercholesterolemic compared to healthy subjects (274% vs 438% increase in FBF, p=0.017). TEA infusion reduced exercise-induced vasodilation in both healthy and hypercholesterolemic subjects (27%, p<0.0001 and −20%, p<0.0001, respectively). The addition of l-NMMA to TEA further reduced FBF in healthy (–14%, p=0.012) but not in hypercholesterolemic subjects, indicating a reduced nitric oxide and greater EDHF-mediated contribution to exercise-induced vasodilation in hypercholesterolemia. In conclusion, exercise-induced vasodilation is impaired and predominantly mediated by EDHF in hypercholesterolemic subjects. Clinical Trial Registration Identifier: NCT00166166

Keywords

endothelial function endothelium-derived hyperpolarizing factor exercise hypercholesterolemia nitric oxide vasodilation

Introduction

The endothelium plays a dynamic role in regulating vasomotor tone and blood flow in response to physiologic and chemical signals by releasing paracrine vasoactive factors that include nitric oxide (NO), prostacyclin, and endo-thelium-derived hyperpolarizing factors (EDHF). Even after inhibition of NO and cyclooxygenase, endothelium-dependent vasodilation persists, revealing the existence of a substantial NO- and prostaglandin-independent component that has been attributed to EDHF release.^1–4 The hallmark of EDHF-mediated responses is their abolition by the combination of apamin (a specific inhibitor of K⁺_Ca channels of small conductance vessels) plus charybdotoxin (a non-selective inhibitor of large-conductance and intermediate-conductance channels, as well as some voltage-dependent K⁺ channels), as noted in pre-clinical studies.^5,6 Although potential EDHFs may differ by species and vascular bed,^6–10 they are thought to promote vasodilation via a common pathway of activation of calcium-dependent potassium (K⁺_Ca) channels.^11,12 Recently, we demonstrated using tetraethylammonium chloride (TEA) – a specific K⁺_Ca channel antagonist that EDHF contributes to both resting vasodilator tone and to bradykinin-stimulated vasodilation.^13,14

Microvascular forearm vasodilation during exercise is a multi-factorial phenomenon that includes contribution from local muscle metabolites such as lactate, adenine nucleotides, acidosis, decreased tissue oxygen tension, increased carbon dioxide tension, and osmolality.¹⁵ The magnitude of the contribution of NO and prostacyclin to exercise-induced vasodilation appears to be modest; inhibition of NO and prostacyclin causes an 11% and 13% reduction in exercise-induced vasodilation, respectively.^2,16–22 The contribution of EDHF to exercise-induced forearm microvascular vasodilation in humans remains unknown, with a variable contribution observed in previous studies in leg muscles.^16,17 Importantly, the impact of cardiovascular risk factors that are known to impair NO activity on the contribution of EDHF to vasodilation during exercise remains un-known.^23–27 We investigated the contribution of NO and EDHF to exercise-induced forearm vasodilation in healthy humans and in those with hypercholesterolemia. We hypothesized that endothelium-dependent hyperpolarization contributes to exercise-mediated forearm microvascular vasodilation via activation of K⁺_Ca channels, and that the contribution of NO and EDHF varies between the healthy and hypercholesterolemic vasculature.

Methods

Subjects

Twenty-six healthy individuals and 19 subjects with hypercholesterolemia aged between 21 and 60 years (47% male) were enrolled into two separate protocols (Figure 1). All subjects were non-smokers and free from hypertension, cardiovascular disease, systemic disorders, and any oral medications. Pregnant females were excluded. Hyper-cholesterolemia was defined as a low-density lipoprotein (LDL)-cholesterol level of >140 mg/dL. The study was approved by the Emory University Institutional Review Board and all subjects provided informed consent.

Figure 1.

Study design. Aspirin (975 mg) was administered 1 hour prior to commencement of the study for cyclooxygenase inhibition. Forearm blood flow measurements were performed after handgrip exercise (15%, 30% and 45% of maximum grip strength) and after endothelium-independent vasodilation with intra-arterial sodium nitroprusside (SNP; 1.6, 3.2 µg/min). Measurements were repeated after l-NMMA (16 µmol/min) or TEA (1 µmol/min) and after combined blockade with l-NMMA and TEA.

Measurement of forearm blood flow at rest and during exercise

Measurements were performed after an overnight fast in a quiet temperature-controlled (22°C to 24°C) room. Subjects refrained from exercise, alcohol and caffeine for at least 24 hours previously. After insertion of an intra-brachial arterial cannula for arterial pressure monitoring and delivery of drug infusions, subjects received oral aspirin (975 mg) to inhibit prostacyclin synthesis at least 1 hour prior to the study.²⁸ Simultaneous forearm blood flow (FBF) measurements were obtained in both arms using a dual-channel venous occlusion strain-gauge plethysmograph (model EC6; DE Hokanson, Bellevue, WA, USA) as described previously.¹⁹ Flow measurements were recorded for approximately 7 seconds, every 15 seconds up to eight times, and a mean FBF value in mL·min⁻¹·100 mL⁻¹ was computed. Forearm vascular resistance (FVR) was calculated as the mean arterial pressure ÷ FBF and expressed as mmHg per mL·min⁻¹·100 mL⁻¹.

The instrumented forearm was exercised with intermittent handgrip by squeezing an inflated pneumatic bag, as previously described.^2,29 Exercise was performed at 15%, 30%, and 45% of maximum voluntary grip strength, which was determined in each subject. Each contraction lasted for 5 seconds followed by relaxation for 10 seconds. The contraction–relaxation sequence was repeated for a total of 5 minutes at each workload and FBF measurements were made in the final 2 minutes at each workload. Preliminary studies and previous studies in our laboratory demonstrated that FBF remained constant after 2 minutes of exercise.²

Contribution of K⁺_Ca channel activation and NO to exercise-induced vasodilator tone

Protocol 1

All agents were administered intra-arterially after resting measurements were made during saline infusion (2.5 mL·min⁻¹). To investigate the effect of inhibition of K⁺_Ca channels without prior inhibition of NO on exercise-induced vasodilation, we studied 10 healthy and nine hypercholesterolemic subjects. FBF was measured during infusion of saline (1 mL·min⁻¹), after exercise, and after intra-arterial infusions of the endothelium-independent vasodilator sodium nitroprusside at 1.6 and 3.2 mg/min for 8 minutes each (Figure 1). After repeating resting flow measurements, FBF was measured during an 8-minute infusion of intra-arterial tetraethylammonium chloride (TEA; Clinalfa, Switzerland) at 1 mg/min. While continuing the infusion of TEA, exercise was repeated as before. After a 30-minute rest period, FBF measurements were repeated during an 8-minute infusion of l-NMMA (Clinalfa) at 16 µmol/min to inhibit NO synthesis. While continuing l-NMMA and TEA infusions, exercise and infusions of sodium nitroprusside were repeated as before. The l-NMMA dose has been previously shown to be effective in attenuating resting and agonist-stimulated FBF.² When given at 0.25, 0.5 and 1 mg/min, TEA is known to selectively inhibit K⁺_Ca channels, but it reduced FBF at rest and after bradykinin only at the highest rate of infusion in previous experiments. Also, at a 1 mg/min infusion rate, the intra-arterial concentration of <0.6 µmol/min is known to specifically inhibit K⁺_Ca channels.^11,30–32

Arterial blood pressure and FBF measurements were repeated in the last 2 minutes of each intervention (Figure 1). This protocol enabled measurements of the contribution of NO and combined NO and EDHF blockade on exercise-induced vasodilation.

Protocol 2

To investigate the effect of inhibition of K⁺_Ca channels after NO inhibition on exercise-induced vasodilation, protocol 1 was repeated in separate experiments in 16 healthy and 10 hypercholesterolemic subjects. During saline infusions, FBF was measured at rest, after graded handgrip exercise, and after infusions of sodium nitroprusside. Measurements were repeated after an 8-minute infusion of l-NMMA at 16 µmol/min followed by exercise. This was followed by the addition of TEA to the l-NMMA. FBF was repeated at rest, after graded exercise, and after sodium nitroprusside infusions (Figure 1). Arterial blood pressure and FBF were measured in the last 2 minutes of each intervention.

Statistical analysis

FBF and FVR were log-transformed into a normal distribution. The effects of l-NMMA and TEA on resting FBF and FVR with single and combined blockade within each group (healthy and hypercholesterolemic) were then compared using linear mixed effects modeling for repeated measures, with the blocker (TEA or l-NMMA or combined blockade) as a fixed effect. Similarly, to assess the impact of blockade on exercise-induced vasodilation, FBF and FVR were compared between healthy and hypercholesterolemic subjects using linear mixed effects modeling for repeated measures, with the group (healthy or hypercholesterolemic), blocker (l-NMMA, TEA or combined blockage) and exercise intensity as fixed effects. All comparisons were adjusted for age, sex, race, body mass index (BMI), mean arterial pressure and baseline FBF or FVR. The Bonferroni correction was used to adjust for multiple comparisons. Data in the text are presented as mean ± SD and as mean ± SEM in the figures. A value of p<0.05 was considered statistically significant.

Results

Baseline characteristics

Hypercholesterolemic subjects had higher total and LDL-cholesterol and triglyceride levels. They were also older, had a higher diastolic blood pressure and a greater BMI compared to healthy subjects (Table 1).

Table 1.

Characteristics of study subjects.

	Healthy (n=26)	Hypercholesterolemic (n=19)
Age, years	33±10	47±8^a
Male, n (%)	10 (38.5)	11 (57.9)
Systolic BP, mmHg	116±12	117±13
Diastolic BP, mmHg	67±11	74±9^a
Height, cm	168±9	172±11
Weight, kg	71±13	86±20^a
Body mass index, kg/m²	25±5	29±5^a
Total cholesterol, mg/dL	172±25	246±18^a
LDL-cholesterol, mg/dL	99±23	160±14^a
HDL-cholesterol, mg/dL	58±11	57±15
Triglycerides, mg/dL	73±39	144±63^a
Glucose, mg/dL	84±8	86±13

Data are mean ± SD.

LDL, low-density lipoprotein; HDL, high-density lipoprotein.

Indicates a statistically significant (p<0.05; t-test) difference between groups.

Effects of l-NMMA and TEA on resting vasodilator tone

There were no significant differences in baseline FBF and FVR between healthy and hypercholesterolemic subjects. FBF decreased and FVR increased significantly with l-NMMA in healthy individuals (p<0.001 for both) but not in hypercholesterolemic subjects (p=0.3 for FBF; p=0.4 for FVR) (Figure 2). In contrast, TEA did not change the FBF (p=0.2) or FVR (p=0.2) significantly in healthy individuals, but decreased FBF (p=0.018) and increased FVR (p=0.037) in hypercholesterolemic subjects compared to baseline (Figure 2). Combined blockade of NO and K⁺_Ca with l-NMMA and TEA led to additional vasoconstriction in both groups independent of the order of administration of the antagonists, with a lower FBF and higher FVR (p<0.001 for both) (Figure 2). As previously described, these findings indicated that, in comparison to healthy individuals, the contribution of NO to resting vasodilator tone is lower in hypercholesterolemic subjects and is partially compensated by an increased contribution of EDHF.¹³

Figure 2.

Contribution of NO and K⁺_Ca channel activation to resting vascular tone in healthy and hypercholesterolemic subjects. Resting forearm blood flow (FBF) (A and C) and forearm vascular resistance (FVR) (B and D) in response to l-NMMA, TEA, and combined l-NMMA and TEA infusions in 26 healthy (A and B) and 19 hypercholesterolemic subjects (C and D). Data are shown as mean ± 95% confidence intervals. *p-value <0.05 for comparison with baseline.

Exercise-induced vasodilation in healthy and hypercholesterolemic subjects

Handgrip exercise produced increasing vasodilation that was similar at the lower two levels of exercise (15% and 30% of maximal handgrip) in both groups, but was lower at peak exercise (45% of maximal handgrip) in hypercholesterolemic compared to healthy subjects (change in FBF: +274% vs +438%, p=0.017; FVR: –69% vs –78%, p=0.004, respectively) (Figure 3). Throughout the experiments, the handgrip exercise did not cause significant change in mean arterial blood pressure, heart rate, or blood flow in the contralateral control arm.

Figure 3.

Forearm vasodilator response to exercise-induced vasodilation in healthy and hypercholesterolemic subjects. (A) Forearm blood flow (FBF) and (B) forearm vascular resistance (FVR) responses to handgrip exercise in 26 healthy and 19 hyperlipidemic subjects. Data shown as mean ± SEM. *p<0.05, **p<0.005.

Effect of TEA alone and in combination with l-NMMA on forearm vascular tone during exercise

Healthy subjects (n=10)

Exercise-induced vasodilation was attenuated by TEA in the absence of l-NMMA; mean FBF was 27.3% (p<0.0001) lower and mean FVR increased 41.6% (p<0.0001), indicating the important contribution of K⁺_Ca channel activation (i.e. EDHF activity) to exercise-mediated microvascular dilation (Figure 4A, 4C). Exercise-induced vasodilation was further attenuated after addition of l-NMMA to TEA, with an additional 13.6% (p=0.012) reduction in mean FBF and 20.6% (p=0.0004) increase in mean FVR compared with TEA infusion alone (Figure 4A, 4C).

Figure 4.

Contribution of K⁺_Ca channel activation, in the presence and absence of nitric oxide, to exercise-induced forearm vasodilation in healthy and hypercholesterolemic subjects. Forearm blood flow (FBF) and forearm vascular resistance (FVR) changes in response to increasing handgrip exercise before and after TEA (1 µmol/min) and combined administration of TEA and l-NMMA (16 µmol/min) in (A) + (C) 10 healthy and (B) + (D) nine hypercholesterolemic subjects. Data shown as mean ± SEM. ⁺p<0.01, **p<0.001.

Hypercholesterolemic subjects (n=9)

Infusion of TEA alone attenuated exercise-induced vasodilation in hypercholesterolemic subjects; mean FBF was reduced by 20.3% (p<0.0001) and mean FVR was increased by 24.9% (p<0.0001) (Figure 4B, 4D). However, co-infusion of l-NMMA with TEA produced no further reduction in exercise-stimulated vasodilation (p=0.5), indicating a decreased contribution of NO in subjects with hypercholesterolemia in whom K⁺_Ca channel activity was preserved.

Effect of l-NMMA alone and in combination with TEA on forearm vasodilation during exercise

Healthy subjects (n=16)

l-NMMA attenuated exercise-induced vasodilation with a 19.3% (p<0.0001) lower mean FBF, and a 36.5% (p=0.0007) higher mean FVR (Figure 5A, 5C). Exercise-induced vasodilation was further attenuated after the addition of TEA to l-NMMA, with an additional 8.5% (p=0.002) reduction in mean FBF and 11.7% (p=0.0001) increase in mean FVR compared with l-NMMA infusion alone (Figure 5A, 5C).

Figure 5.

Contribution of nitric oxide, in the presence and absence of K⁺_Ca channel activation, to exercise-induced forearm vasodilation in healthy and hypercholesterolemic subjects. Forearm blood flow (FBF) and forearm vascular resistance (FVR) changes in response to handgrip exercise before and after l-NMMA (16 µmol/min) and after combined administration of l-NMMA and TEA (1 µmol/min) in (A) + (C) 16 healthy and (B) + (D) 10 hypercholesterolemic subjects. Data shown as mean ± SEM. ⁺p<0.05, *p<0.01, **p<0.001.

Hypercholesterolemic subjects (n=10)

l-NMMA attenuated exercise-induced vasodilation with a 17.6% (p=0.0001) lower mean FBF and a 35.9% (p=0.0006) higher mean FVR (Figure 5B, 5D). Exercise-induced vasodilation was further attenuated after the addition of TEA to l-NMMA, with an additional 18.3% (p=0.005) reduction in mean FBF and a 17.5% (p=0.02) increase in mean FVR compared with l-NMMA infusion alone (Figure 5B, 5D). The reductions in exercise-induced vasodilation with NO blockade and with the addition of TEA were similar in magnitude to that observed in healthy subjects.

Vasodilator responses to sodium nitroprusside

Infusion of sodium nitroprusside produced a stepwise vasodilator response that was similar (FBF, p=0.37; FVR, p=0.70) in healthy and hypercholesterolemic subjects. Combined infusions of l-NMMA and TEA did not alter forearm vasodilation with sodium nitroprusside. Age, sex, systolic blood pressure and BMI did not influence the results of NO or K⁺_Ca channel inhibition.

Discussion

Herein we demonstrate that both NO and K⁺_Ca channel activation (EDHF) contribute equally and in concert to exercise-induced microvascular vasodilation in the healthy human forearm circulation. Dual inhibition of these endothelium-derived factors caused further inhibition of microcirculatory vasodilation during exercise, demonstrating the independent and additive contribution of NO and EDHF to physiologic vasodilation. In contrast, in hypercholesterolemia, K⁺_Ca channel activation appeared to be the predominant mechanism for exercise-induced vasodilation; after K⁺_Ca channel blockade, inhibition of NO had no further effect on the vasodilator response to exercise in these subjects. Finally, the lack of inhibition of sodium nitroprusside-mediated endothelium-independent vasodilation by l-NMMA and TEA indicates that the blockade observed during exercise with these antagonists was specifically endothelium-dependent.

NO and exercise-induced vasodilation

The mechanisms underlying exercise-induced vasodilation are complex and the understanding of the potential mediators remains incomplete. Skeletal muscle blood flow increases in proportion to the metabolic demand.^33–37 The precise contribution of the various vasoactive compounds to exercise-induced vasorelaxation may also differ by muscle beds studied and involve the interaction of the autonomic nervous system.^37–40 Prostacyclins contribute modestly and transiently to exercise-induced vasodilation²⁰ and, for the purposes of this study, cyclooxygenase was inhibited prior to the study. Previous studies have either shown modest inhibition of exercise hyperemia with NO blockade^2,17,20 or have failed to demonstrate any contribution of NO to exercise,^21,41,42 raising the concept of redundancy, where compensatory release of other vasodilators may explain the lack of effect of NO inhibition.¹⁶ Differences in experimental methods, sample size, and muscle beds examined may also account for the differences.^21,41,42 Herein, we have demonstrated a 19.3% reduction in mean vasodilation in response to exercise in healthy individuals after inhibition of NO, consistent with, or more pronounced than the 11% reduction observed previously.² This difference may be due to the comparatively younger age and consequently greater NO activity in the current healthy cohort. There was a contribution of NO to exercise-induced vasodilation even after K⁺_Ca channel blockade, with a further 13.6% reduction in flow with l-NMMA in healthy subjects. This confirms the importance of NO in physiological vasodilator responses and demonstrates it is additive to the contribution of K⁺_Ca channel activation.

EDHF and exercise-induced vasodilation in healthy subjects

In a previous study of seven healthy subjects, inhibition of cytochrome P450 2C9 with sulphaphenazole did not alter exercise-induced vasodilation in thigh muscles, suggesting lack of contribution of epoxyeicosatrienoic acids to exercise hyperemia.¹⁶ Our results provide the first evidence for the contribution of EDHF, and in particular of K⁺_Ca channel activation, to physiological vasodilation of the forearm microvasculature during exercise. We have demonstrated a 27.3% reduction in exercise-induced vasodilation with TEA, even in the absence of inhibition of NO, and an additional reduction in FBF with TEA after NO blockade. Taken together with the findings of the previous study, it would appear that EDHFs stimulated by exercise that lead to K⁺_Ca channel stimulation may not be cytochrome P450-derived peptides, and potentially involve hydrogen peroxide and other EDHF mechanisms. In contrast to our findings, Mortensen et al. failed to demonstrate an effect of K⁺_Ca channel blockade with TEA when measuring blood flow changes in response to leg exercise.¹⁷ Variations in muscle beds studied and techniques (thermodilution versus plethysmography) may have accounted for the observed differences. The intravascular concentration of TEA (0.5 mmol/L) used was similar in this study, and has previously been demonstrated to specifically inhibit K⁺_Ca channels.^30,43–46

The contribution of both NO and K⁺_Ca channel activation to exercise hyperemia was similar in magnitude in the healthy subjects. This was unexpected based on experimental studies, demonstrating that the contribution of EDHF is only evidenced after NO blockade.^16,46–48 Even after inhibition of NO with l-NMMA, K⁺_Ca channel activation continued to contribute to exercise-induced microvascular dilation. Similarly, after the initial inhibition of K⁺_Ca channels with TEA, further inhibition of exercise-vasodilation with l-NNMA was observed, with an additional 13.6% reduction in mean exercise flow, suggesting an independent and additive effect of dual blockade.

EDHF and exercise-induced vasodilation in hypercholesterolemia

In hypercholesterolemic subjects, the attenuation of exercise-induced vasodilation after NO blockade, when EDHF was not inhibited, was similar to the responses observed in healthy subjects, suggesting that NO contributes to physiologic vasodilation. This is consistent with our previous observations of preserved endothelium-dependent vasodilation with bradykinin in hypercholesterolemia.¹³ However, in contrast to healthy subjects and the effects on bradykinin responses, after EDHF blockade with TEA, no further attenuation of exercise-induced vasodilation was observed with NO blockade in subjects with hypercholesterolemia. This suggests that in vasculature exposed to hypercholesterolemia, K⁺_Ca channel activity may be the predominant mechanism for exercise-induced vasodilation. It also suggests that NO and K⁺_Ca channels may interact such that inhibition of K⁺_Ca channels may inhibit the NO pathway in hypercholesterolemia, an observation that needs further investigation.

We have further demonstrated that hypercholesterolemic individuals have an attenuated forearm vasodilator response to exercise at the highest handgrip effort when compared to healthy individuals. Also, in contrast to the healthy microvasculature, there is a lack of contribution of NO to exercise-induced vasodilation after K⁺_Ca channel blockade, and this NO deficiency may contribute to the lower vasodilator response in the hypercholesterolemic subset. This reduction of NO activity was as a result of decreased NO production or availability and not from vascular smooth muscle dysfunction, as the sodium nitroprusside responses were similar in both groups.

It may be argued that the reduced vasodilation observed during exercise with l-NMMA and TEA is due to the vasoconstrictor effect of these antagonists on resting tone. However, resting vasoconstriction was entirely overcome by the endothelium-independent vasodilator sodium nitroprusside. Thus, if an endothelium-independent stimulus were entirely responsible for the exercise-induced vasodilation, then exercise hyperemia would also be expected to overcome the effect of the inhibitors on resting vascular tone.

Limitations

Our findings are limited to the forearm microcirculation and thus other vascular beds, including conductance arteries, warrant further investigation. Nevertheless, it is known that the contribution of EDHF is less in con-ductance vessels than in the microvessels.^8,12,49 Second, l-NMMA and TEA are competitive inhibitors and may not completely inhibit NO and K⁺_ca channels and thus may underestimate the importance of these mediators in vivo. Third, other endothelium-derived vasoactive mediators to exercise hyperemia may also play a compensatory role after the blockade of NO and K⁺_Ca channels and need to be investigated.³⁷ Fourth, our investigations were conducted on a background of cyclooxygenase inhibition. Although we have previously demonstrated that vasodilator prostanoids contribute to resting vasodilator tone, we found that cyclooxygenase products did not contribute to the endothelial dysfunction of hypertensive or hypercholesterolemic patients.⁵⁰ Fifth, we employed an intermittent isometric handgrip exercise that produced no systemic hemodynamic changes. Other muscle beds and different intensities of exercise that alter shear stress variably will need to be explored in future studies.^51,52 The older age and higher BMI in our hypercholesterolemic subjects may have also contributed to the observed abnormalities, but statistical correction for these factors did not alter our findings. Finally, we measured blood flow with plethysmography immediately after terminating handgrip exercise, and thus report on flow during early recovery from exercise.

Conclusions

We have demonstrated an important contribution of EDHF, via the activation of K⁺_Ca channels, to forearm microvascular vasodilation during exercise in healthy subjects with a greater contribution compared to NO in hypercholesterolemic subjects. Whether enhancing EDHF in conditions with impaired NO activity would be of therapeutic value, and whether agents that improve endothelial dysfunction, such as statins and angiotensin antagonists, also enhance EDHF bioactivity remains to be studied.

Footnotes

Declaration of conflicting interest

The authors declare that there is no conflict of interest.

Funding

National Institutes of Health Research Grant RO1 HL79115, PHS Grant UL1 RR025008 from the Clinical and Translational Science Award Program, and PHS Grant M01 RR00039 from the General Clinical Research Center Program, National Institutes of Health, National Center for Research Resources, British Cardiovascular Society Research Fellowship, and the National Blood Foundation.

References

Quyyumi

Dakak

Andrews

Gilligan

Panza

Cannon

3rd . Contribution of nitric oxide to metabolic coronary vasodilation in the human heart. Circulation 1995; 92: 320–326.

Gilligan

Panza

Kilcoyne

Waclawiw

Casino

Quyyumi

. Contribution of endothelium-derived nitric oxide to exercise-induced vasodilation. Circulation 1994; 90: 2853–2858.

Joannides

Richard

Haefeli

Linder

Luscher

Thuillez

. Role of basal and stimulated release of nitric oxide in the regulation of radial artery caliber in humans. Hypertension 1995; 26: 327–331.

Mullen

Kharbanda

Cross

. Heterogenous nature of flow-mediated dilatation in human conduit arteries in vivo: relevance to endothelial dysfunction in hypercholesterolemia. Circ Res 2001; 88: 145–151.

Scotland

Madhani

Chauhan

. Investigation of vascular responses in endothelial nitric oxide synthase/cyclooxygenase-1 double-knockout mice: key role for endothelium-derived hyperpolarizing factor in the regulation of blood pressure in vivo. Circulation 2005; 111: 796–803.

Feletou

Vanhoutte

. Endothelium-dependent hyperpolarizations: past beliefs and present facts. Ann Med 2007; 39: 495–516.

Shimokawa

Yasutake

Fujii

. The importance of the hyperpolarizing mechanism increases as the vessel size decreases in endothelium-dependent relaxations in rat mesenteric circulation. J Cardiovasc Pharmacol 1996; 28: 703–711.

Urakami-Harasawa

Shimokawa

Nakashima

Egashira

Takeshita

. Importance of endothelium-derived hyperpolarizing factor in human arteries. J Clin Invest 1997; 100: 2793–2799.

Busse

Edwards

Feletou

Fleming

Vanhoutte

Weston

. EDHF: bringing the concepts together. Trends Pharmacol Sci 2002; 23: 374–380.

10.

Coleman

Tare

Parkington

. Endothelial potassium channels, endothelium-dependent hyperpolarization and the regulation of vascular tone in health and disease. Clin Exp Pharmacol Physiol 2004; 31: 641–649.

11.

Inokuchi

Hirooka

Shimokawa

. Role of endothelium-derived hyperpolarizing factor in human forearm circulation. Hypertension 2003; 42: 919–924.

12.

Miura

Wachtel

Liu

. Flow-induced dilation of human coronary arterioles: important role of Ca²⁺-activated K⁺ channels. Circulation 2001; 103: 1992–1998.

13.

Ozkor

Murrow

Rahman

. Endothelium-derived hyperpolarizing factor determines resting and stimulated forearm vasodilator tone in health and in disease. Circulation 2011; 123: 2244–2253.

14.

Ozkor

Rahman

Murrow

. Differences in vascular nitric oxide and endothelium-derived hyperpolarizing factor bioavailability in blacks and whites. Arterioscler Thromb Vasc Biol 2014; 34: 1320–1327.

15.

Laughlin

McAllister

Jasperse

Crader

Williams

Huxley

. Endothelium-medicated control of the coronary circulation. Exercise training-induced vascular adaptations. Sports Med 1996; 22: 228–250.

16.

Hillig

Krustrup

Fleming

Osada

Saltin

Hellsten

. Cytochrome P450 2C9 plays an important role in the regulation of exercise-induced skeletal muscle blood flow and oxygen uptake in humans. J Physiol 2003; 546(Pt 1): 307–314.

17.

Mortensen

Gonzalez-Alonso

Damsgaard

Saltin

Hellsten

. Inhibition of nitric oxide and prostaglandins, but not endothelial-derived hyperpolarizing factors, reduces blood flow and aerobic energy turnover in the exercising human leg. J Physiol 2007; 581(Pt 2): 853–861.

18.

Schrage

Dietz

Joyner

. Effects of combined inhibition of ATP-sensitive potassium channels, nitric oxide, and prostaglandins on hyperemia during moderate exercise. J Appl Physiol 2006; 100: 1506–1512.

19.

Gilligan

Guetta

Panza

Garcia

Quyyumi

Cannon

3rd . Selective loss of microvascular endothelial function in human hypercholesterolemia. Circulation 1994; 90: 35–41.

20.

Schrage

Joyner

Dinenno

. Local inhibition of nitric oxide and prostaglandins independently reduces forearm exercise hyperaemia in humans. J Physiol 2004; 557(Pt 2): 599–611.

21.

Shoemaker

Halliwill

Hughson

Joyner

. Contributions of acetylcholine and nitric oxide to forearm blood flow at exercise onset and recovery. Am J Physiol 1997; 273(5 Pt 2): H2388–2395.

22.

Radegran

Calbet

. Role of adenosine in exercise-induced human skeletal muscle vasodilatation. Acta Physiol Scand 2001; 171: 177–185.

23.

Halcox

Schenke

Zalos

. Prognostic value of coronary vascular endothelial dysfunction. Circulation 2002; 106: 653–658.

24.

Panza

Casino

Kilcoyne

Quyyumi

. Role of endothelium-derived nitric oxide in the abnormal endothelium-dependent vascular relaxation of patients with essential hypertension. Circulation 1993; 87: 1468–1474.

25.

Panza

Garcia

Kilcoyne

Quyyumi

Cannon

3rd . Impaired endothelium-dependent vasodilation in patients with essential hypertension. Evidence that nitric oxide abnormality is not localized to a single signal transduction pathway. Circulation 1995; 91: 1732–1738.

26.

Quyyumi

Dakak

Mulcahy

. Nitric oxide activity in the atherosclerotic human coronary circulation. J Am Coll Cardiol 1997; 29: 308–317.

27.

Taddei

Galetta

Virdis

. Physical activity prevents age-related impairment in nitric oxide availability in elderly athletes. Circulation 2000; 101: 2896–2901.

28.

Heavey

Barrow

Hickling

Ritter

. Aspirin causes short-lived inhibition of bradykinin-stimulated prostacyclin production in man. Nature 1985; 318: 186–188.

29.

Zelis

Longhurst

Capone

Mason

. A comparison of regional blood flow and oxygen utilization during dynamic forearm exercise in normal subjects and patients with congestive heart failure. Circulation 1974; 50: 137–143.

30.

Honing

Smits

Morrison

Rabelink

. Bradykinin-induced vasodilation of human forearm resistance vessels is primarily mediated by endothelium-dependent hyperpolarization. Hypertension 2000; 35: 1314–1318.

31.

Stanfield

. Tetraethylammonium ions and the potassium permeability of excitable cells. Rev Physiol Biochem Pharmacol 1983; 97: 1–67.

32.

Langton

Nelson

Huang

Standen

. Block of calcium-activated potassium channels in mammalian arterial myocytes by tetraethylammonium ions. Am J Physiol 1991; 260(3 Pt 2): H927–934.

33.

Armstrong

Laughlin

. Rat muscle blood flows during high-speed locomotion. J Appl Physiol 1985; 59: 1322–1328.

34.

Laughlin

Armstrong

. Muscular blood flow distribution patterns as a function of running speed in rats. Am J Physiol 1982; 243: H296–306.

35.

Mackie

Terjung

. Blood flow to different skeletal muscle fiber types during contraction. Am J Physiol 1983; 245: H265–275.

36.

Mohrman

Regal

. Relation of blood flow to VO₂, PO₂, and PCO₂ in dog gastrocnemius muscle. Am J Physiol 1988; 255(5 Pt 2): H1004–1010.

37.

Clifford

Hellsten

. Vasodilatory mechanisms in contracting skeletal muscle. J Appl Physiol 2004; 97: 393–403.

38.

Hamann

Buckwalter

Valic

Clifford

. Sympathetic restraint of muscle blood flow at the onset of dynamic exercise. J Appl Physiol 2002; 92: 2452–2456.

39.

Buckwalter

Clifford

. Alpha-adrenergic vasoconstriction in active skeletal muscles during dynamic exercise. Am J Physiol 1999; 277(1 Pt 2): H33–39.

40.

Buckwalter

Mueller

Clifford

. Sympathetic vasoconstriction in active skeletal muscles during dynamic exercise. J Appl Physiol 1997; 83(5): 1575–1580.

41.

Radegran

Saltin

. Nitric oxide in the regulation of vasomotor tone in human skeletal muscle. Am J Physiol 1999; 276(6 Pt 2): H1951–1960.

42.

Bradley

Kingwell

McConell

. Nitric oxide synthase inhibition reduces leg glucose uptake but not blood flow during dynamic exercise in humans. Diabetes 1999; 48: 1815–1821.

43.

Nelson

Quayle

. Physiological roles and properties of potassium channels in arterial smooth muscle. Am J Physiol 1995; 268(4 Pt 1): C799–822.

44.

Honing

Smits

Morrison

Burnett

Jr Rabelink

. C-type natriuretic peptide-induced vasodilation is dependent on hyperpolarization in human forearm resistance vessels. Hypertension 2001; 37: 1179–1183.

45.

Pickkers

Hughes

Russel

Thien

Smits

. In vivo evidence for K_Ca channel opening properties of acetazolamide in the human vasculature. Br J Pharmacol 2001; 132: 443–450.

46.

Bellien

Joannides

Iacob

Arnaud

Thuillez

. Calcium-activated potassium channels and NO regulate human peripheral conduit artery mechanics. Hypertension 2005; 46: 210–216.

47.

Alonso-Galicia

Drummond

Reddy

Falck

Roman

. Inhibition of 20-HETE production contributes to the vascular responses to nitric oxide. Hypertension 1997; 29(1 Pt 2): 320–325.

48.

Udosen

Jiang

Hercule

Oyekan

. Nitric oxide-epoxygenase interactions and arachidonate-induced dilation of rat renal microvessels. Am J Physiol Heart Circ Physiol 2003; 285: H2054–2063.

49.

Miura

Gutterman

. Human coronary arteriolar dilation to arachidonic acid depends on cytochrome P-450 monooxygenase and Ca²⁺-activated K⁺ channels. Circ Res 1998; 83: 501–507.

50.

Campia

Choucair

Bryant

Quyyumi

Cardillo

Panza

. Role of cyclooxygenase products in the regulation of vascular tone and in the endothelial vasodilator function of normal, hypertensive, and hypercholesterolemic humans. Am J Cardiol 2002; 89: 286–290.

51.

Halcox

JPJ

Narayanan

Cramer-Joyce

Mincemoyer

Quyyumi

. Characterization of endothelium-derived hyperpolarizing factor in the human forearm microcirculation. Am J Physiol Heart Circ Physiol 2001; 280: H2470–2477.

52.

Pohl

Holtz

Busse

Bassenge

. Crucial role of endothelium in the vasodilator response to increased flow in vivo. Hypertension 1986; 8: 37–44.

Contribution of endothelium-derived hyperpolarizing factor to exercise-induced vasodilation in health and hypercholesterolemia

Abstract

Keywords

Introduction

Methods

Subjects

Measurement of forearm blood flow at rest and during exercise

Contribution of K+Ca channel activation and NO to exercise-induced vasodilator tone

Protocol 1

Protocol 2

Statistical analysis

Results

Baseline characteristics

Effects of l-NMMA and TEA on resting vasodilator tone

Exercise-induced vasodilation in healthy and hypercholesterolemic subjects

Effect of TEA alone and in combination with l-NMMA on forearm vascular tone during exercise

Healthy subjects (n=10)

Hypercholesterolemic subjects (n=9)

Effect of l-NMMA alone and in combination with TEA on forearm vasodilation during exercise

Healthy subjects (n=16)

Hypercholesterolemic subjects (n=10)

Vasodilator responses to sodium nitroprusside

Discussion

NO and exercise-induced vasodilation

EDHF and exercise-induced vasodilation in healthy subjects

EDHF and exercise-induced vasodilation in hypercholesterolemia

Limitations

Conclusions

Footnotes

Declaration of conflicting interest

Funding

References

Contribution of K⁺_Ca channel activation and NO to exercise-induced vasodilator tone