Endothelium-derived hyperpolarizing factor determines resting and stimulated forearm vasodilator tone in health and in disease

Abstract

Background: We assessed the contribution of endothelium-derived hyperpolarizing factors to resting and agonist-stimulated vasodilator tone in health and disease. Tetraethylammonium chloride (TEA) was used to inhibit K(+)(Ca) channel activation and fluconazole was used to inhibit cytochrome P450 2C9-mediated epoxyeicosatrienoic acid synthesis. We hypothesized that endothelium-derived hyperpolarizing factors contribute to resting vascular tone by K(+)(Ca) channel activation and epoxyeicosatrienoic acid release and that endothelium-derived hyperpolarizing factors compensate for reduced nitric oxide bioavailability at rest and with endothelium-dependent vasodilators.

Methods and results: In 103 healthy subjects and 71 nonhypertensive subjects with multiple risk factors, we measured resting forearm blood flow (FBF) using venous occlusion plethysmography before and after intra-arterial infusions of N(G)-monomethyl-l-arginine (L-NMMA), TEA, fluconazole, and their combination. The effects of these antagonists on resting FBF and on bradykinin- and acetylcholine-mediated vasodilation were studied. Resting FBF decreased with TEA and L-NMMA in all subjects (P<0.001); however, the vasoconstrictor response to L-NMMA was greater (P=0.04) and to TEA was lower (P=0.04) in healthy subjects compared with those with risk factors. Fluconazole decreased resting FBF in all subjects, and the addition of TEA further reduced FBF after fluconazole, suggesting that cytochrome P450 metabolites and other hyperpolarizing factor(s) activate K(+)(Ca) channels. Both L-NMMA and TEA attenuated bradykinin-mediated vasodilation in healthy and hypercholesterolemic subjects (P<0.001). In contrast, acetylcholine-mediated vasodilation remained unchanged with TEA in healthy subjects but was significantly attenuated in hypercholesterolemia (P<0.04).

Conclusions: First, by activating TEA-inhibitable K(+)(Ca) channels, endothelium-derived hyperpolarizing factors, together with nitric oxide, contribute to resting microvascular dilator tone. The contribution of K(+)(Ca) channel activation compared with nitric oxide is greater in those with multiple risk factors compared with healthy subjects. Second, activation of K(+)(Ca) channels is only partly through epoxyeicosatrienoic acid release, indicating the presence of other hyperpolarizing mechanisms. Third, bradykinin, but not acetylcholine, stimulates K(+)(Ca) channel-mediated vasodilation in healthy subjects, whereas in hypercholesterolemia, K(+)(Ca) channel-mediated vasodilation compensates for the reduced nitric oxide activity. Thus, enhanced endothelium-derived hyperpolarizing factor activity in conditions of nitric oxide deficiency contributes to maintenance of resting and agonist-stimulated vasodilation. Clinical Trial Registration- URL: http://www.clinicaltrials.gov. Unique identifier: NCT00166166.

Figure 1. Study Design for Protocol 4

Aspirin (975mg) was administered 1hour prior to commencement of the study. FBF was measured with bradykinin (100, 200 and 400 ng/min) or acetylcholine (7.5, 15 and 30 μg/min), followed by sodium nitroprusside (1.6, 3.2 μg/min). Measurements were repeated after L-NMMA (A), K⁺_Ca channel blockade with TEA (B), and after combined blockade with L-NMMA and TEA.

Figure 2. Contribution of NO and K⁺_Ca channel activation to resting FBF and vascular resistance

Change in resting FBF and vascular resistance in healthy subjects and those with risk factors; Upper panels depict responses to TEA alone and combined TEA and L-NMMA infusions. Lower panels show responses with L-NMMA alone followed by L-NMMA and TEA infusions. Data presented as mean ± SEM.

Figure 2. Contribution of NO and K⁺_Ca channel activation to resting FBF and vascular resistance

Change in resting FBF and vascular resistance in healthy subjects and those with risk factors; Upper panels depict responses to TEA alone and combined TEA and L-NMMA infusions. Lower panels show responses with L-NMMA alone followed by L-NMMA and TEA infusions. Data presented as mean ± SEM.

Figure 3. Relationship between contribution of NO and K⁺_Ca channel activation to resting vasodilator tone

Correlation between the percent change in FBF with L-NMMA and TEA (n=131).

Figures 4. Contribution of cytochrome P450 metabolites and K⁺_Ca channel activation to resting FBF and vascular resistance

Change in (a) resting FBF and (b) vascular resistance with fluconazole alone and combined infusions of fluconazole and TEA. Mean ± SEM.

Figure 5. Contribution of NO and K⁺_Ca channel activation to bradykinin-mediated vasodilation

FBF and vascular resistance in response to bradykinin in healthy and hypercholesterolemic subjects. Left panels demonstrate effect of L-NMMA, and combined blockade with L-NMMA and TEA, in 17 healthy and 16 hypercholesterolemic subjects. Right panels depict effects of TEA and combined infusions of TEA and L-NMMA, in 12 healthy and 10 hypercholesterolemic subjects. Mean ± SEM

Figure 5. Contribution of NO and K⁺_Ca channel activation to bradykinin-mediated vasodilation

FBF and vascular resistance in response to bradykinin in healthy and hypercholesterolemic subjects. Left panels demonstrate effect of L-NMMA, and combined blockade with L-NMMA and TEA, in 17 healthy and 16 hypercholesterolemic subjects. Right panels depict effects of TEA and combined infusions of TEA and L-NMMA, in 12 healthy and 10 hypercholesterolemic subjects. Mean ± SEM

Figure 6. Contribution of NO and K⁺_Ca channel activation to acetylcholine-stimulated vasodilation in healthy and hypercholesterolemic forearm vasculature

FBF and vascular resistance in response to acetylcholine in healthy and hypercholesterolemic subjects. Left panels demonstrate effect of L-NMMA, and combined blockade with L-NMMA and TEA, in 10 healthy and 8 hypercholesterolemic subjects. Right panels depict effects of TEA and combined infusions of TEA and L-NMMA, in 15 healthy and 10 hypercholesterolemic subjects. Mean ± SEM

Figure 6. Contribution of NO and K⁺_Ca channel activation to acetylcholine-stimulated vasodilation in healthy and hypercholesterolemic forearm vasculature

FBF and vascular resistance in response to acetylcholine in healthy and hypercholesterolemic subjects. Left panels demonstrate effect of L-NMMA, and combined blockade with L-NMMA and TEA, in 10 healthy and 8 hypercholesterolemic subjects. Right panels depict effects of TEA and combined infusions of TEA and L-NMMA, in 15 healthy and 10 hypercholesterolemic subjects. Mean ± SEM

Figure 7. Contribution of NO and K⁺_Ca channel activation to nitroprusside-stimulated vasodilation

FBF in response to sodium nitroprusside alone and after combined blockade with L-NMMA and TEA in healthy (n=42) and hypercholesterolemic (n=38) subjects. Mean ± SEM.