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(Arteriosclerosis, Thrombosis, and Vascular Biology. 2007;27:2496.)
© 2007 American Heart Association, Inc.

Editorials

Epoxyeicosatrienoic Acids, TRP Channels, and Intracellular Ca²⁺ in the Vasculature

An Endothelium-Derived Endothelium-Hyperpolarizing Factor?

Brandon T. Larsen; David X. Zhang; David D. Gutterman

From the Departments of Pharmacology and Toxicology (B.T.L., D.D.G.), Medicine (D.X.Z., D.D.G.), and the Cardiovascular Center (B.T.L., D.X.Z., D.D.G.), Medical College of Wisconsin, and Veterans Administration Medical Center (D.D.G.), Milwaukee, Wisc.

Correspondence to David D. Gutterman, MD, Northwestern Mutual Professor of Medicine, Senior Associate Dean for Research, Medical College of Wisconsin, 8701 Watertown Plank Road, Milwaukee, WI 53226. E-mail dgutt{at}mcw.edu

Epoxyeicosatrienoic acids (EETs) are cytochrome P450-derived metabolites of arachidonic acid that function as endothelium-derived hyperpolarizing factors (EDHFs) in many species, including humans. Strictly speaking, an EDHF is a substance derived from endothelial cells that stimulates hyperpolarization of the underlying vascular smooth muscle cells (VSMCs) to elicit vasorelaxation. Although a physiological role of EETs in vasomotor¹ and nonvasomotor² regulation of the vasculature is now increasingly recognized, the underlying signaling mechanisms and cell types involved remain incompletely understood.

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EETs are potent vasodilators that activate large-conductance Ca²⁺-activated potassium (BK_Ca) channels to induce membrane hyperpolarization.^3,4 In VSMCs, this hyperpolarization decreases Ca²⁺ influx through voltage-sensitive Ca²⁺ channels, ultimately leading to a global decrease in intracellular Ca²⁺ ([Ca²⁺]_i) with subsequent vasorelaxation (Figure). Electrophysiological studies indicate that EET-mediated activation of BK_Ca may involve an indirect membrane-delimited mechanism that involves ADP-ribosylation of G_s^4,5; however, specific EET receptors, if any, have not yet been isolated or cloned. Recent evidence suggests that nonselective cation "transient receptor potential" (TRP) channels may function as EET receptors per se, particularly the vanilloid type 4 (TRPV4) channel, which is directly activated by EETs.^6,7 Importantly, EET-induced Ca²⁺ influx through TRPV4 channels indirectly augments BK_Ca channel activity by stimulating localized ryanodine receptor-dependent Ca²⁺ release events (Ca²⁺ sparks) from the sarcoplasmic reticulum that then activate the BK_Ca channel⁷ without a concomitant increase in global cytosolic [Ca²⁺]_i (Figure).

View larger version (23K): [in this window] [in a new window]   Figure. Proposed signaling pathways of EET-mediated vasodilation. On activation by bradykinin, endothelial cells release Ca2+ from intracellular stores to promote formation of the vasodilator compounds NO or EETs. As outlined in the present study by Fleming et al,16 EETs act in an autocrine manner (bold arrows) to promote endothelial hyperpolarization by inducing PKA-mediated translocation of TRPC6 channels to the endothelial cell surface, a key step in TRPC6 activation and subsequent Ca2+ entry. Localized TRPC6-mediated increases in endothelial Ca2+ may activate K+ channel–mediated K+ efflux and hyperpolarization, which may promote alternative mechanisms of vasodilation. In this way, EETs may function as an EDEHF. Alternatively, EETs may function as a traditional EDHF by directly hyperpolarizing VSMCs. EETs induce a localized TRPV4 channel–mediated Ca2+ influx that activates additional localized Ca2+ release events (Ca2+ sparks) from intracellular stores, an important mechanism of activation of BKCa channels on VSMCs. K+ efflux through BKCa channels leads to membrane hyperpolarization and decreased Ca2+ influx via VDCCs. The subsequent drop in global [Ca2+]i initiates relaxation. AA indicates arachidonic acid; B2R, bradykinin type 2 receptor; B/I/SKCa, large/intermediate/small conductance Ca2+-activated K+ channel; cGMP, cyclic guanosine monophosphate; CYP, cytochrome P450; EET, epoxyeicosatrienoic acid; ER, endoplasmic reticulum; IP3, inositol triphosphate; NO, nitric oxide; NOS, nitric oxide synthase; PKA, protein kinase A; PLA2, phospholipase A2; RyR, ryanodine receptor; SR, sarcoplasmic reticulum; TRP, transient receptor potential channel; VDCC, voltage-dependent Ca2+ channel.

Although many studies indicate that BK_Ca channels on VSMCs are the primary effectors of EETs, this is not always the case, as EETs also activate BK_Ca channels on endothelial cells.⁸ Interestingly, EETs directly hyperpolarize endothelial cells and have been linked to endothelial [Ca²⁺]_i regulation,⁹ possibly by lowering membrane potential and increasing the driving force for Ca²⁺ entry. However, many endothelial cell types do not express BK_Ca channels,^10–12 suggesting that EETs may also modulate endothelial [Ca²⁺]_i by another unknown mechanism. Interestingly, a large number of TRP channel subtypes are expressed on endothelial cells, including TRPV4.¹³ Endothelial TRPV4 is mechanosensitive and may play a role in flow-induced vasodilation.¹⁴ In addition, endothelial TRPV4 is, like its VSMC counterpart, activated by EETs.¹⁵ However, the mechanism underlying EET-mediated TRPV4 activation in endothelial cells is not fully understood. In addition, it is not known whether other TRP channel subtypes also function as receptors or effectors of EETs.

As reported in the current issue of Arteriosclerosis, Thrombosis, and Vascular Biology, I. Fleming et al¹⁶ provide convincing new evidence that supports an autocrine role of EETs in endothelial cells as modulators of endothelial [Ca²⁺]_i. In addition, the authors propose a novel mechanism whereby EETs regulate endothelial [Ca²⁺]_i by inducing the translocation of TRPC6 channel proteins to caveolin-1-rich areas of the endothelial cell membrane (Figure). This observation is important, as it suggests that EETs may activate more than 1 TRP channel subtype. Unlike TRPV4 that is directly activated by EETs,^6,7 TRPC6 may function not as an EET receptor, but as an EET effector. Although the specific mechanism linking EETs to the stimulation of TRPC6 translocation remains unclear, EET-induced Ca²⁺ influx and TRPC6 translocation are cAMP-dependent, suggesting that EET-induced protein kinase A (PKA) activity may promote channel phosphorylation on residues that are critical for membrane targeting. Thus, EETs may indirectly modulate endothelial [Ca²⁺]_i by promoting membrane recruitment of TRPC6 channels; however, it is not known whether EETs increase TRPC6 channel current in endothelial cells or whether TRPC6 translocation influences vasomotor tone. In addition, the relative importance of TRPV4 versus TRPC6 in endothelial [Ca²⁺]_i regulation remains unexplored.

Athough significant evidence now supports a role for EETs as EDHFs,^1,17 the identity of the specific mediator of the EDHF phenomenon remains controversial. A central focus of the argument¹⁷ against EETs as EDHFs is the fact that EETs activate BK_Ca channels, whereas the EDHF phenomenon is sensitive to the combination of charybdotoxin and apamin, which blocks not only BK_Ca channels, but also intermediate- and small-conductance Ca²⁺-activated K⁺ (IK_Ca and SK_Ca) channels and voltage-dependent K⁺ (K_v) channels. Although bioassay studies indicate that endothelium-derived EETs diffuse to VSMCs to elicit hyperpolarization and vasodilation,¹⁸ it has been proposed that EETs may primarily function in an autocrine manner as an intracellular amplifier of endothelial cell hyperpolarization to promote the release of other distinct mediators of the EDHF phenomenon.¹⁹

The present studies provide compelling evidence for an autocrine role of EETs in endothelial cells and suggest that promotion of TRP channel translocation to the cell membrane may underlie some of the vascular effects of EETs. Localized TRPC6-mediated increases in endothelial Ca²⁺ may activate IK_Ca- and SK_Ca-mediated K⁺ efflux and hyperpolarization (Figure). In this way, EETs may function as an endothelium-derived endothelium-hyperpolarizing factor, or "EDEHF". Given that endothelial membrane hyperpolarization increases the driving force for Ca²⁺ entry, EETs may play a critical role in modulating endothelial [Ca²⁺]_i to promote alternative mechanisms of vasodilation. Interestingly, it was recently reported that IK_Ca- and SK_Ca-mediated hyperpolarization is a critical early event in endothelial NO production by regulating Ca²⁺ influx.²⁰ Although it is possible that EETs indirectly induce IK_Ca and SK_Ca activity by promoting TRP-mediated Ca²⁺ influx, this has not yet been demonstrated, and the functional significance of EET-mediated endothelial [Ca²⁺]_i regulation remains unknown.

There are potential clinical ramifications of the study by Fleming et al. Importantly, dysregulation of TRPC channel family members has been identified as a potential contributing factor in a multitude of cardiovascular diseases.²¹ Interestingly, TRPC6 knockout mice display significantly elevated blood pressure and agonist-induced vascular contractility, suggesting that TRPC6 plays an important role in the regulation of blood pressure and vascular resistance.²² In addition, TRPC6 expression is downregulated by high glucose; thus, modulation of TRPC6 may play a role in the cardiovascular complications of diabetes.²³ The present study expands our understanding of the roles of TRPC6 in endothelial biology to include a role as an EET effector, and suggests that a loss of EET signaling may partially explain the detrimental effects of TRPC6 dysregulation. Furthermore, this study suggests that targeted pharmacological intervention to enhance TRPC6 or EET function may be useful for improving endothelial function.

In conclusion, the present report by Fleming et al¹⁶ indicates that EETs play an important role in endothelial [Ca²⁺]_i regulation and identifies TRPC6 as a potential novel effector of EETs in the vascular system. EETs may therefore function not only as traditional EDHFs, but may also function as EDEHFs in the vasculature. This study broadens our understanding of the complex signaling mechanisms underlying the vascular effects of EETs. Future studies are indicated to determine the physiological significance of EET-mediated [Ca²⁺]_i regulation and TRPC6 translocation in the endothelial cell.

	Acknowledgments

 
This work was supported by a Veterans Administration Merit Review Award (to D.D.G.), a fellowship award from the American Heart Association (to B.T.L.), and by the National Institutes of Health (HL68769, HL080704; to D.D.G.).

Disclosures

None.

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Epoxyeicosatrienoic Acids Regulate Trp Channel–Dependent Ca²⁺ Signaling and Hyperpolarization in Endothelial Cells

Ingrid Fleming, Alexandra Rueben, Rüdiger Popp, Beate Fisslthaler, Susanne Schrodt, Anna Sander, Judith Haendeler, John R. Falck, Christophe Morisseau, Bruce D. Hammock, and Rudi Busse
Arterioscler. Thromb. Vasc. Biol. 2007 27: 2612-2618. [Abstract] [Full Text] [PDF]

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