H2O2 is the transferrable factor mediating flow-induced dilation in human coronary arterioles

Abstract

Rationale: Endothelial derived hydrogen peroxide (H(2)O(2)) is a necessary component of the pathway regulating flow-mediated dilation (FMD) in human coronary arterioles (HCAs). However, H(2)O(2) has never been shown to be the endothelium-dependent transferrable hyperpolarization factor (EDHF) in response to shear stress.

Objective: We examined the hypothesis that H(2)O(2) serves as the EDHF in HCAs to shear stress.

Methods and results: Two HCAs were cannulated in series (a donor intact vessel upstream and endothelium-denuded detector vessel downstream). Diameter changes to flow were examined in the absence and presence of polyethylene glycol catalase (PEG-CAT). The open state probability of large conductance Ca(2+)-activated K(+) (BK(Ca)) channels in smooth muscle cells downstream from the perfusate from an endothelium-intact arteriole was examined by patch clamping. In some experiments, a cyanogen bromide-activated resin column bound with CAT was used to remove H(2)O(2) from the donor vessel. When flow proceeds from donor to detector, both vessels dilate (donor:68±7%; detector: 45±11%). With flow in the opposite direction, only the donor vessel dilates. PEG-CAT contacting only the detector vessel blocked FMD in that vessel (6±4%) but not in donor vessel (61±13%). Paxilline inhibited dilation of endothelium-denuded HCAs to H(2)O(2). Effluent from donor vessels elicited K(+) channel opening in an iberiotoxin- or PEG-CAT-sensitive fashion in cell-attached patches but had little effect on channel opening on inside-out patches. Vasodilation of detector vessels was diminished when exposed to effluent from CAT-column.

Conclusions: Flow induced endothelial production of H(2)O(2), which acts as the transferrable EDHF activating BK(Ca) channels on the smooth muscle cells.

Figure 1

Two HCAs were connected in series with donor (with endothelium, +E) upstream and detector vessel (without endothelium, -E) downstream. A. When flow proceeds from donor to detector, both donor and detector vessel dilated. B. With flow was reversed, only the donor dilated, but not detector vessels.

Figure 2

Effect of PEG-CAT (500 U/ml) on FMD in donor vessels (A) and detector vessels (B). When PEG-CAT was placed in detector vessel chamber (cross bar), FMD was inhibited in detector vessels, but was reserved in donor vessels. When PEG-CAT was placed in the perfusate traversing both vessels (gray bar), FMD was eliminated in both donor and detector vessels.

Figure 3

Effect of paxilline on H₂O₂ induce dilation in human coronary arterioles. H₂O₂ elicited dose-dependent vasodilation, which was significantly inhibited by 100 nM paxilline.

Figure 4

Effect of effluent from donor vessel on K⁺ channel activity in cell-attached patches of coronary smooth muscle cells. As indicated in sample traces (A) and summarized data (B), effluent from donor vessel greatly enhanced open state probability in cell-attached patches of smooth muscle cells. The enhancement was blocked by 100 nmol/L iberiotoxin.

Figure 5

Effect of PEG-CAT on K⁺ channel opening induced by effluent from donor vessel in cell-attached patches of coronary smooth muscle cells. The increased opening of K⁺ channels by effluent was abolished in the presence of PEG-CAT as illustrated in sample traces (A) and summarized data (B).

Figure 6

Effect of effluent from donor vessel on K⁺ activity in inside-out patches of coronary smooth muscle cells. K⁺ channel activity was significantly reduced in response to vessel effluent after elimination of intracellular components. A. Sample traces. B. Data summarized from 6 experiments.

Figure 7

A. A significant reduction in H₂O₂ concentration was observed in effluent from CAT-column compared to effluent from CF-column. B. Sample images comparing fluorescence intensity of DCFH representing the production of H₂O₂. DCFH fluorescence intensities were increased in HCA exposed to effluent from donor vessel. Fluorescence intensities were greatly reduced in HCA incubated with effluent passing through the CAT-column. C. Summary of fluorescence intensities (normalized to control) in 4 arteries of each group. Donor effluent-induced increases in DCFH fluorescence were reduced in vessels exposed to effluent from CAT-column.

Figure 8

Comparison of dilator response of detector vessels to effluent from donor vessel, CF-column or CAT-column. Detector vessel dilated to effluent from donor vessels or CF-column in as volume of donor effluent increase. The dilator response was significantly reduced in detector vessels incubated in effluent from CAT-column.