doi: 10.1161/01.RES.0000196741.99904.e4
© 2005 American Heart Association, Inc.
Editorials |
EDHF Redux: EETs, TRPV4, and Ca2+ Sparks
From the Department of Biomedical Sciences, College of Veterinary Medicine, Cornell University, Ithaca, NY.
Correspondence to Michael I. Kotlikoff, Department of Biomedical Sciences, College of Veterinary Medicine, Cornell University, Ithaca, NY 14853. E-mail mik7{at}cornell.edu
See related article, pages 1270–1279
Key Words: K channel • ryanodine receptor • EET • epoxygenase
Over the past two decades the complexity of the molecular processes underlying endothelial-dependent vascular relaxation has gradually become apparent. After the ground breaking discovery of the role of NO as a major endothelium-derived relaxant factor (EDRF), evidence emerged indicating the existence of a hyperpolarizing factor (endothelium-derived hyperpolarizing factor [EDHF]) with actions distinct from the cyclase-dependent relaxations associated with endothelial production of NO and prostacyclin.1–3 The specific molecules and vascular relaxation mechanisms underlying this intensely studied phenomenon have been difficult to establish, however. H2O2, K ions, and epoxygenase products have all been implicated as the EDHF, and evidence has suggested in turn that the mechanism by which EDHF relaxes arteries involves diffusion of the substance to smooth muscle and direct gating effects on calcium-activated K+ (maxiK) channels,4,5 actions mediated through endothelial small and intermediate conductance Ca2+-activated K+ (SK and IK) channels,6–8 and passive ionic effects associated with an extracellular accumulation of K+ ions.9 To further muddy these dark waters, recent evidence has implicated TRPV4 channels in EDHF signaling in several vascular preparations. In this issue of Circulation Research, Earley and colleagues10 identify a new putative mechanism and one that links EDHF to the regulation of Ca2+ release by ryanodine receptors (RYR), thereby forging a mechanistic link between EDHF and the well described local gating of large conductance Ca2+-activated K+ (maxiK) channels by Ca2+ sparks.11
Several lines of evidence in diverse vascular beds have implicated endothelial epoxygenase products in the process of endothelial hyperpolarization.12,13 While multifunctional cytochrome P450 (CYP) epoxygenases constitute a large and diverse family of enzymes primarily directed toward cellular detoxification, the ability of CYP to metabolize arachidonic acid in vitro has been known for almost 25 years.14 This activity appears to reside largely in the CYP 2 group of this large enzyme family, which metabolizes membrane arachidonate generating epoxyeicosatrienoic acids (EETs), particularly 5,6-, 8,9-, and 11,12-EETs. Although the degree to which vascular relaxation coincides with an increase in EET metabolism in arteries under physiological conditions is not well established, the potent vasodilating effects of EETs have provoked intense speculation regarding their role as an EDHF. Two principal mechanisms have been advanced by which EETs might relax arteries: (1) diffusion of EETs to vascular smooth muscle and the direct activation of maxiK channels,15,16 and (2) activation of TRPV4 channels and Ca2+ influx in endothelial cells,17 leading to opening of SK and IK channels and the subsequent hyperpolarization of smooth muscle through direct coupling of endothelium and smooth muscle or subsequent to the accumulation of K+ ions in the extracellular space.1
TRPV4 channels are widely expressed in the vasculature and are gated by numerous stimuli including cell swelling, heat, and extracellular and intracellular ligands.18 Of note, TRPV4 channels are activated by arachidonic acid metabolites in a CYP-dependent manner.17 Moreover, recent work published in Circulation Research demonstrates the activation of Ca2+ influx by 5,6- and 8,9-EET and the absence of this response in endothelial cells from TRPV4 null mice.19 The report by Earley et al in the current issue10 expands and perhaps generalizes the concept that endothelial CYP metabolites activate TRP channels to affect vascular dilation.
Earley and colleagues investigated the effects of 11,12-EET on myocytes isolated from cerebral vessels. They demonstrate the expression of TRPV4 in these cells and activation by 11,12-EET to produce a typical nonselective current. Surprisingly, they show that the hyperpolarizing effects of 11,12-EET occur through the activation of TRPV4 and subsequent augmentation of Ca2+ spark frequency, an effect not predicted by the previously postulated actions of EETs on K+ channel (endothelial or smooth muscle) or TRPV4 channel (endothelial) gating. The increase in Ca2+ sparks increases the frequency of the ubiquitous spontaneous transient outward currents (STOCs), a hallmark of spontaneous electrical activity in smooth muscle cells20 that results from the local coupling of Ca2+ sparks to maxiK channels.11 The Brayden group uses several approaches to demonstrate that maxiK channel activation is indirect, requiring TRPV4 channel gating and RYR-mediated Ca2+ release; maxiK currents are stimulated by TRPV4 channel agonists, but 11,12-EET coupling to STOCs is eliminated by pharmacological inhibition of TRPV4 channels, in the absence of extracellular Ca2+, or by RYR channel antagonism. Moreover, TRPV4 targeting by specific antisense oligonucleotides largely abolishes the 11,12-EET cation current, as well as the augmentation of Ca2+ spark and STOC frequency. Finally, and most critically, antisense inhibition of TRPV4 channel expression eliminates both the hyperpolarization and vasodilation observed when intact pressurized arteries are exposed to 11,12-EET. These last experiments represent an elegant demonstration of the physiological importance of the described pathway.
These data provide strong evidence for a linkage between EDHF and local Ca2+ release events in smooth muscle, and offer some hints of a specialized communication system in specific arterial beds. Although diversity of mechanisms will likely remain a defining feature of endothelial relaxation of smooth muscle, actions of locally-generated epoxygenase products that target specialized Ca2+ release mechanisms within smooth muscle may go some way to explaining how these ubiquitously-expressed multifunctional enzymes target widely-expressed K+ channels to specifically alter vascular tone. Several critical questions remain, however. First, as has been emphasized by past reviews, NO-independent, endothelial relaxation is not blocked by iberiotoxin in many arterial preparations,1 raising questions as to the generality of the current findings. Second, the existence of TRPV4 channels on endothelial cells, and the suggested coupling mechanisms linking increases in Ca2+ in endothelial cells to smooth muscle hyperpolarization, represents a competing mechanism that will require careful physiological dissection. Third, inconsistency remains regarding the action of specific EET molecules on TRPV4 channels, given the finding that 5,6-EET, but not 11,12-EET, activates heterologously expressed channels.17 Finally, the mechanism proposed by Earley et al raises critical questions regarding the mechanism by which the influx of Ca2+ through TRPV4 channels couples to SR release and Ca2+ sparks without causing a global increase in [Ca2+]i within the myocyte. This coupling remains somewhat mysterious, as TRPV4 activation produces robust increases in global [Ca2+]i within endothelial cells and such rises would be expected to produce contraction of vascular smooth muscle. The authors invoke localized Ca2+-induced Ca2+ release (CICR) to support this exquisite compartmentalization, but studies of CICR in smooth muscle to date have indicated that the process is far less compartmentalized than in cardiac muscle, comprising a "loosely coupled" system despite the involvement of many of the same molecular elements.21,22 Nonetheless, continuous spatially-localized Ca2+ release events are a prominent feature of vascular smooth muscle within intact vessels, and there is much that we do not understand regarding the regulation of these events. The work by Earley and colleagues provides substantial motivation to further understand the coupling between endothelial cell epoxygenase activity and Ca2+ sparks in smooth muscle.
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Acknowledgments |
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The author gratefully acknowledges support from National Heart, Lung, and Blood Institute (HL45239) and National Institute of Diabetes and Digestive and Kidney Disorders (DK065992).
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Footnotes |
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The opinions expressed in this editorial are not necessarily those of the editors or of the American Heart Association.
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References |
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