TRPV4 and the regulation of vascular tone

Abstract

Recent studies have introduced the importance of Transient Receptor Potential Vanilloid Subtype 4 (TRPV4) channels in the regulation of vascular tone. TRPV4 channels are expressed in both endothelium and vascular smooth muscle cells and can be activated by numerous stimuli including mechanical (e.g. shear stress, cell swelling, and heat) and chemical (e.g. epoxyeicosatrienoic acids (EETs), endocanabinoids, 4α-phorbol esters). In the brain, TRPV4 channels are primarily localized to astrocytic endfeet processes which wrap around blood vessels. Thus, TRPV4 channels are strategically localized to sense hemodynamic changes and contribute to the regulation of vascular tone. TRPV4 channel activation leads to smooth muscle cell hyperpolarization and vasodilation. Here we review recent findings on the cellular mechanisms underlying TRPV4-mediated vasodilation, TRPV4 channel interaction with other proteins including Transient Receptor Potential Channel 1 (TRPC1), small conductance (K_Ca2.3) and large conductance (K_Ca1.1) calcium-activated, potassium-selective channels and the importance of caveolin-rich domains for these interactions to take place.

Introduction

Transient receptor potential (TRP) channels are non-selective cation channels expressed in almost all cells and permeable to Ca²⁺and Na⁺ions. The TRP channel superfamily is divided according to DNA and protein sequence homology ¹; while this superfamily encompasses a large number of channels, the purpose of this review is to highlight recent findings on the role of TRPV4 channels in the regulation of vascular tone. TRPV4 channels are members of the vanilloid receptor subfamily and expressed in various tissues including lung, spleen, heart, endothelium, cochlear, liver, testes, fat and brain 2-5. TRPV4 channel currents carry Ca²⁺and Mg²⁺with permeability ratios of 6-10 P_Ca/P_Naand 2-3 P_Mg/P_Na, respectively ^6-9 . The single channel conductance for TRPV4 channels is in the range of 90-100 pS (outward currents) and 50-60 pS (inward currents) ^{7, 9, 10}.

At the structural level, the TRPV4 channel has six transmembrane spanning segments (TM1-6) with the pore region located between TM5 and TM6 ¹⁰. The protein has 871 amino acids with intracellular C- and N-termini ¹⁰. Four TRPV4 subunits are needed to assemble a functional channel ¹⁰with part of its volume (~ 30%) in the plasma membrane and the rest (~70%) exposed intracellularly or extracellularly, allowing interactions with associated proteins ¹¹. These include inositol trisphosphate (IP₃) receptors ¹², actin filaments ^{13, 14}, microtubule-associated protein (MAP) 7 ¹⁵, aquaporin (AQP) 5 ¹⁶and AQP4 ¹⁷, human osteosarcoma (OS) 9 ¹⁸, transient receptor potential polycystic (TRPP) 2 ¹⁹, caveolin 1 ²⁰, Cystic fibrosis transmembrane conductance regulator (CFTR) 21 and large conductance Ca²⁺activated K⁺channels (BK or K 1.1) ^{22, 23} Ca .

Functionally, TRPV4 channels stand out due to the broad range of stimuli that lead to their activation, including physical (cell swelling ⁵, heat ^{9, 24}, mechanical ²⁵) and chemical stimuli (endocannabinoids, arachidonic acid (AA), and 4-α-phorbol esters ^{26, 9}). Table 1 and 2 provide a pharmacological overview of TRPV4 agonists and antagonists. Activation by swelling and endocanabinoids involves cytochrome P450 epoxygenase-dependent AA metabolism to epoxyeicosatrienoic acids (EETs) ^{26, 27}, potent endogenous agonists for TRPV4 channels ^{2, 5, 22, 28-30}. EETs cause vascular smooth muscle cell hyperpolarization, leading to vascular relaxation. The effect of EETs on smooth muscle cell hyperpolarization persists when the production of nitric oxide (NO) and prostacyclin are inhibited ³¹. Thus, EETs are commonly referred to as one of the endothelium-derived hyperpolarizing factors (EDHFs). The role of EETs is especially important in some key vascular beds, including the coronary circulation ³¹. EETs may directly bind to TRPV4 to exert their action. A putative arachidonate recognition site, where EETs could bind, is located at N-terminal cytoplasmic domain of TRPV4 ⁴. However, there are some controversies regarding EET regioisomer selectivity for TRPV4. 11,12-EET and 14,15-EET are two predominant endogenous EET isoforms ³². However, one report showed that 5,6-EET and 8,9-EET but not 11,12-EET and 14,15-EET activate TRPV4 in TRPV4-overexpressing HEK293 cells ²⁷. In contrast, several other studies demonstrated that 11,12-EET and 14,15-EET are able to activate TRPV4 in native smooth muscles ^{26, 30}.

Table 1. Data summary for TRPV4 agonists ^(a).

Agonist	Potency (IC50, μM)	Species	Cross-reactivity
4α-PDD	0.16-0.92 0.16-0.93 4.4	human mouse rat	---
5,6-EET, 8,9-EET,11,12- EET	0.15 for 5,6-EET	human mouse Rat	activates G protein coupled receptor
Bisandrographolide	0.79-0.95	mouse	--
RN-1747	0.77 4.0 4.1	human mouse rat	activates TRPV1 at 100 μM, inhibits TRPM8 (IC50=4μM)
GSK-1016790A	0.003-0.005 0.0185 0.010	human mouse rat	activates TRPV1 (EC50=5nM)

^(a)modified from ^89,31

Table 2. Data summary for TRPV4 antagonists ^(a).

Antagonist	Potency (IC50, μM)	Species	Cross-reactivity
RN-1734	2.3 5.9 3.2	human mouse rat	---
RN-9893	<0.12 <0.06 <0.12	human mouse rat	---
HC-067047	0.048 0.017 0.133	human mouse rat	inhibits TRPM8, HERG at submicromolar
Ruthenium Red	<0.086-1 <0.21-1 <0.2-0.33	human mouse rat	inhibits all TRPVs, TRPM6, TRPM8, TRPA1, RyR1-3
Capsazepine	18.6 13.5	human rat	Inhibits TRPV1, TRPM8
Citral	32	mouse	Inhibits TRPA1, activates TRPV1, TRPV3, TRPM8

^(a)modified from ^{89, 90}

TRPV4 is unequivocally important for the regulation of vascular tone. However, the underlying molecular mechanisms remain unclear. Here, we describe recent advances on the role of TRPV4 channels in the peripheral circulation as well as the cerebral circulation, where TRPV4 channel expression is prominent in astrocytes.

TRPV4 is expressed in vascular smooth muscle cells

Immunostaining, Western blot and reverse transcription-polymerase chain reaction (RTPCR) showed the expression of TRPV4 in smooth muscle cells of rat cerebral arteries ³³, smooth muscle of human and rat lung extraalveolar vessels ²⁶, endothelium-denuded rat intralobar pulmonary arteries ^{34, 35}, rat mesenteric artery smooth muscle cells ³⁶, and rat and mouse aortic smooth muscle cells ³⁷.

Dependent on the vascular bed and animal species, EET may act on TRPV4 channels expressed either in vascular smooth muscle cells, endothelial cells or both ³¹. In rat cerebral arteries, the endothelium-derived EETs diffuse to nearby smooth muscle cells, activating TRPV4 in smooth muscle cells ²². Resultant Ca²⁺entry stimulates Ca²⁺release from ryanodine receptors, causing an increased frequency of Ca²⁺sparks ²². The Ca²⁺sparks in turn activate K_Ca1.1 to hyperpolarize vascular smooth muscle cells, leading to vascular relaxation ²². A 11,12 EET- and 4α-PDD-activated TRPV4-like current was recorded in smooth muscle cells of mouse small mesenteric arteries. The current was absent in myocytes from TRPV4 knockout mice. EETs, via their action on smooth muscle TRPV4, were also found to induce smooth muscle hyperpolarization and vascular relaxation in mouse mesenteric arteries, the effect of which was absent in TRPV4 knockout mice ³⁰. Endothelial disruption only caused a moderate reduction in 11,12-EET-induced smooth muscle hyperpolarization and vascular relaxation (by ~50%) in these arteries ³⁰. Thus, the authors reasoned that the remaining 50% was endothelium-independent and could be attributed to direct EET action on smooth muscle TRPV4. In agreement, the authors also found that inhibiting K_Ca1.1 in smooth muscle cells could reduce the EET-induced responses by ~50%, further supporting the notion that the endothelium-independent component was ~50%. These data suggest a link between EET, smooth muscle TRPV4 and K_Ca1.1 in mouse mesenteric arteries. Because EETs can be produced by endothelial cells in response to physiological stimuli such as bradykinin, acetylcholine, pulsatile stretch and shear stress, the functional coupling of smooth muscle TRPV4 with K_Ca1.1 may play a major role in vascular tone control under different physiological conditions.

Up to the present, the function of smooth muscle TRPV4 has only been reported in rat cerebral arteries and mouse mesenteric arteries ^{22, 30}. However, it is likely that a similar mechanism exists in other vascular beds. It is well documented that, in a great variety of artery types, EETs stimulate the activity of K_Ca1.1 channels in smooth muscle cells causing smooth muscle hyperpolarization and vascular relaxation ³¹. This mechanism has been documented in mouse skeletal arteries, human internal mammary arteries and coronary arteries from many species ^{31, 32}. However, EETs do not directly act on K_Ca1.1 channels. Thus, the well-characterized EET-TRPV4-K_Ca1.1 axis may provide an attractive mechanistic explanation for smooth muscle relaxation in these arteries.

Recent studies found that TRPV4 may heteromerize with TRPC1 or TRPP2 to form heteromeric channels in vascular endothelial cells and renal cortical collecting duct cells ^{38, 39}. TRPC1 is ubiquitously expressed in many cell types including vascular smooth muscle cells from many arteries ³⁷. TRPP2 expression has also been identified in some artery types ³⁷. In the future, it will be important to determine whether heteromeric TRPV4-C1 and/or TRPV4-P2 exist in vascular smooth muscle cells and whether EETs act on homomeric or heteromeric TRPV4 to initiate hyperpolarizing responses in vascular smooth muscle cells. Interestingly, studies have shown that TRPC1 and K_Ca1.1 can form a physical complex in vascular smooth muscle cells ⁴⁰and that the complex plays an important role in smooth muscle hyperpolarization and the control of vascular tone ⁴⁰. Based on this evidence, it is reasonable to propose the existence of a TRPV4-TRPC1-K_Ca1.1 complex in vascular smooth muscle cells. EETs may act on this complex to induce smooth muscle hyperpolarization and vascular relaxation.

TRPV4 is expressed in endothelial cells

In the endothelium, TRPV4 was first identified in mouse aorta by Bernd Nilius’s group, ⁷and since then, it has been shown to be ubiquitously expressed in endothelial cells of both large conductance vessels and small resistance vessels. Indeed, RT-PCR, western blot analysis and intracellular calcium measurements demonstrate that TRPV4 is functionally expressed in mouse aortic endothelial cells ²⁹. Köhler’s group investigated the expression and function of TRPV4 in rat carotid artery and arteria gracilis endothelial cells by using in situ patch-clamp techniques, single-cell RT-PCR and pressure myography, ⁴¹whereas Alvarez and coworkers studied TRPV4 in rat pulmonary artery and microvascular endothelium ²⁶. More recently, TRPV4 localization was examined by Willette et al.⁴²in a variety of rat tissues, and a generalized pattern of immunoreactive TRPV4 staining was identified in the endothelium and epithelium ⁴².

From a functional perspective, as shown by Zhang et al. ⁴³, acetylcholine-induced nitric oxide (NO) production was significantly reduced in vascular endothelial cells and EDHF-mediated relaxation was also attenuated in small mesenteric arteries of TRPV4 knockout mice. These results are in agreement with previous data from Köhler et al.⁴⁴, showing that in large vessels, like carotid arteries, the inhibition of nitric oxide synthase almost completely abolished 4α-PDD induced vasodilation whereas in small vessels selective inhibition of calcium activated potassium channels (SKCa/K_Ca2.3 and IKCa/K_Ca3.1) inhibited the TRPV4-induced vasodilation. Very recently, Sonkusare et al. ⁴⁵ demonstrated that even a small number of active TRPV4 channels were able to mediate local calcium signals that activated IK and SK channels and induced maximal dilation of resistance arteries, thereby contributing to the regulation of vascular function ⁴⁵. Thus intracellular calcium increases mediated by TRPV4 channels trigger both NO-and/or EDHF-dependent vasodilatation, an effect that appears to be dependent on the vascular bed. Interestingly, in several cell types including endothelial and smooth muscle cells, calcium handling proteins are located in caveolae. K_Ca2.3 ⁴⁶and K 1.1 ⁴⁷ Ca channels have been shown to reside in caveolin-rich lipid domains. Direct measurement of calcium waves in endothelial cells have suggested that caveolae could be the sites that initiate calcium entry and calcium dependent signal transduction ⁴⁸. Recent data demonstrate that, similar to TRPC1 ⁴⁹, TRPV4 may interact physically with the structural caveolar protein caveolin-1 and that the interaction is functionally important for 4α-PDD-evoked calcium increase ²⁰. The fact that TRPV4 may heteromerize with TRPC1 ³⁹, as well as the work of Graziani and coworkers showing that caveolar integrity is essential for AA recruitment and EDHF signaling in porcine arteries ⁵⁰, provides additional evidence in favor of a potential involvement of caveolar microdomains in TRP-mediated calcium signaling and subsequent vasodilation.

TRPV4 expression in astrocytes

In the brain, TRPV4 mRNA is expressed in both neuronal and non-neuronal cell types including astrocytes and microglia ^51-54, endothelial cells and vascular smooth muscle cells ³³. Importantly, and relevant to the control of vascular tone, Marrelli et al ³³showed TRPV4 channel expression in endothelial cells of middle cerebral arteries and demonstrated its regulation by PLA₂activation. Similar to the potential polarized expression of TRPV4 channels in the abluminal face of the endothelium 33, Benfenati et al. ⁵⁵reported that expression of TRPV4 channels is localized mostly to astrocytic membranes at the interface between brain parenchyma and extracerebral liquid spaces and on astrocytic endfeet abutting pial and parenchymal blood vessels ⁵⁵. The unique arrangement of TRPV4 channels on perivascular astrocyte processes was also reported by Butenko et al. ⁵⁶ in the rat hippocampal CA1 region. Given that cell-cell communication by astrocytes is primarily mediated through dynamic intracellular Ca²⁺changes, information regarding the activity of TRPV4 channels in astrocytes is important to further our understanding of the physiological function of these cells in the CNS.

Moreover, the important observation that astrocytic endfeet processes possess parallel vasoactive mechanisms to those described in vascular cells, particularly endothelial cells, supports the notion that cerebral vascular smooth muscle cells are modulated from their luminal and abluminal sides by both endothelial cell and astrocyte signaling pathways, respectively. As with endothelial cells, astrocytes modulate vascular tone through K⁺signaling ⁵⁷and via the release of AA metabolites such as EETs ^58-60, 20-HETEs ⁶¹ and prostacyclin ^{62, 63}. The resemblance of these vasoactive pathways to those described in vascular cells, in addition to the expression of TRPV4 channels in astrocytic endfeet processes at the gliovascular interface, points to the possibility that astrocytic TRPV4 channels are also involved in the regulation of vascular tone. In response to neuronal activity, glutamate released at the synapse activates metabotropic glutamate receptors (mGluR) in astrocytes leading to an increase in intracellular Ca²⁺which in turn activates PLA₂⁶⁴. The resulting production of AA and its metabolism follows similar pathways to those described in endothelial cells (e.g. conversion to metabolites such EETs). As described above, EETs are endogenous activators of TRPV4 channels. Blanco et al. ⁶⁵ showed that 11, 12 EET increased Ca²⁺oscillations in cortical astrocytes. The study, however, did not evaluate whether these Ca²⁺responses in astrocytes were indeed mediated via TRPV4 channel activation. mGluR-induced increases in Ca²⁺also has been shown to increase the single channel open probability of BK channels expressed in astrocytic endfeet processes ^{65, 57}. Depending on the K⁺concentration released, efflux of K⁺from astrocytes results in vasodilation ^{57, 66} or vasoconstriction ⁶⁶.

To date, TRPV4 channel function in astrocytes is associated with osmosensation, thus playing an important role in the maintenance of brain volume ⁶⁷17 as achieved through the activity of AQP4 channels expressed in endfeet processes ^{17, 68, 69}. It has been demonstrated that AQP4 channels colocalize with TRPV4 channels in astrocytic endfeet, providing evidence for their co-participation in regulatory volume decrease ¹⁷.

Although a role for astrocytic TRPV4 channels has yet to be demonstrated in the control of vascular tone, given their association with K⁺channels (also preferentially expressed in astrocytic endfeet processes ^{65, 70}), it is tempting to speculate that the activation of TRPV4 channels in astrocytes contributes to K⁺channel signaling and neurovascular coupling. Along these lines, Higashimori et al.⁷¹showed that the synthetic EET analog 11-nonyloxy-undec-8(Z)-enoic acid or the mGluR agonist,t-ACPD significantly increased K_Ca1.1 channel currents in perivascular astrocytes. EETs-induced outward currents were also associated with an increase in the frequency of Ca²⁺oscillations in astrocytes, supporting the idea that EETs-induced intracellular Ca²⁺changes contribute to K⁺signaling in astrocytes ⁷¹and likely the control of vascular tone ⁵⁷.

In addition to EETs, glutamate-mediated activation of mGluR in astrocytes results in the release a number of vasoactive signals (i.e. NO, ATP, adenosine), which could also contribute to astrocyte TRPV4 channel regulation. Among them, NO is of particular interest. TRPV4 channel activation is linked to NO production ^{43, 72}, which in turn can lead to sustained increases in astrocyte Ca²⁺levels ⁷³. A recent study showed that TRVP4 channel activation resulted in endothelial Ca²⁺increase and NO-mediated vasodilation ⁴¹. In addition, NO has been shown to induce sustained increases in astrocytic Ca²⁺in cultured astrocytes ⁷³. Based on these studies and the fact that NO can readily cross the blood brain barrier and alter astrocyte ⁷³and neuronal activity ⁷⁴, another unresolved role for TRPV4 channels in the control of vascular tone may be linked to NO signaling. Thus, given their distinctive molecular and biophysical properties, their strategic expression within the neurovascular unit and their ability to respond to a variety of vascular- and glial-derived signals in the brain, TRPV4 channels expressed in astrocytes may be regarded as ideal candidates to sense and/or transduce hemodynamic information into a glial response (changes in intracellular calcium). Comparable to our current knowledge on TRPV4-channel induced activation in the endothelium and vascular smooth muscle cells, additional work is needed to determine whether astrocytic TRPV4 channels contribute to the regulation of vascular tone via similar mechanisms.

TRPV4 expression in perivascular nerves

TRPV4 channels have shown to be expressed in sensory nerves and to co-localize with calcitonin gene-related peptide (CGRP) as well as substance P ^{75, 76}. Gao and Wang showed that the depressor effect of the TRPV4 channel, 4αPDD was attenuated following degeneration of capsaicin-sensitive sensory nerves or in the presence of CGRP_8-37(an antagonist of CGRP). Moreover, they showed that intravenous administration of 4αPDD increased plasma CGRP; the hypotensive effect of TRPV4 channel activation was, at least in part, mediated by the activation of Ca²⁺-activated K⁺channels ³⁶. Using a model of baroreflex impairment, McHugh et al. showed a role for TRPV4 channels as osmosensors in the portal region and their potential participation in the afferent input of the pressor response ⁷⁷. The authors suggested that spinal afferents may relay information from the hepatic/portal environment to dorsal root ganglion neurons which express TRPV4 channels ^{78, 79}resulting blood pressure regulation through sympathetic output⁷⁷.

TRPV4 in disease

Although TRPV4 channels appear to have an important role in the regulation of vascular tone, TRPV4^−/− mice do not show altered blood pressure at rest ³⁰. Earley et al., suggested the participation of TRPV4 channels in a negative feedback mechanism which opposes hypertension in the presence of a hypertensive challenge ^{30, 43, 80}. Using the synthetic TRPV4 activator, GSK1016790A, Willette et al provided evidence that circulatory collapse induced by exogenous TRPV4 activation is mediated by a NO-independent failure of the endothelial-epithelial permeability barrier in the lung and other tissues ⁴². Impaired pressure and stretch sensing has been reported in C-fibers of the dorsal root ganglia ¹⁵and retinal ganglion cells ⁸¹, respectively, in TRPV4^−/− mice. Several reports suggest that TRPV4 channels are likely involved in hypoxia-induced pathogenesis. Following cerebral hypoxia/ischemia, TRPV4 channel expression is increased in hippocampal astrocytes resulting in augmented astrocytic Ca²⁺oscillatory frequency and possibly astroglial reactivity in the brain ⁵⁶. In chronic hypoxic pulmonary hypertension, Yang and coworkers identified TRPV4 channels as an obligatory calcium entry pathway that is upregulated ³⁵. In mouse mesenteric arteries, TRPV4 activity is favored by hypoxic insult that is associated with an increased Ca²⁺response in endothelial cells upon agonist stimulation, contributing to a potentiated EDH-mediated dilation.

As flow-activated channels in vascular endothelial cells, TRPV4 are good candidates for shear stress activation and, consequently, have been investigated in different models of arteriogenesis. In rats, after femoral artery ligation, TRPV4 participates in collateral remodeling and growth ⁸². The same group also provided evidence that pharmacological TRPV4 activation enhanced cerebral arteriogenesis⁸³. TRPV4 channel activation has been associated with pulmonary hypertension ⁸⁴, bone disorders ⁸⁵, neurodegenerative skeletal muscle dysplasias ⁸⁶ and hyponatremia ^{87, 88}to name a few.

Summary and Perspectives

In summary, current studies suggest that activity of TRPV4 channels in endothelial and vascular smooth muscle cells contribute to the regulation of vascular tone. Importantly, TRPV4-induced Ca²⁺increases in endothelial and vascular smooth muscle cells contribute to vasodilation. The broad range of stimuli activating TRPV4 channels, along with their strategic location in the endothelium, favors flow and shear-stress mediated release of EDHF and vasodilation. Moreover, recent studies have shed light on the interaction between TRPV4 channels and SK_Caand IK_Ca, suggesting a key cellular mechanism by which TRPV4-mediated Ca²⁺increases in endothelial cells induce vasodilatory responses ⁴⁵. The structural arrangement of TRPV4 channels allows for interaction with a number of proteins including K⁺channels and other members of the TRP channel family (e.g TRPC1). Particular interest has also been placed on caveolin, as several calcium handling proteins from endothelial and smooth muscle cells reside in caveolae (e.g. K_Ca2.3 ⁴⁶and K_Ca1.1 ⁴⁷channels); the interaction between TRPV4 and caveolin-1 appear to be an important component of 4α-PDD-evoked calcium increase ²⁰.

Studies have demonstrated the importance of TRPV4 channels expressed in vascular smooth muscle cells as mediators of vasodilation via EET. TRPV4-induced Ca²⁺increases lead to Ca²⁺sparks and subsequent activation of K_Ca1.1 channels, causing, in turn, smooth muscle hyperpolarization and vasodilation ²². In addition, TRPV4 channel interaction with other proteins such as the TRPV4-TRPC1-K_Ca1.1 complex in vascular smooth muscle cells may prove to be yet another mechanism for smooth muscle hyperpolarization and vascular relaxation.

Clearly, the wide expression of TRPV4 channels in various tissues along with the broad range of stimuli which can activate them, give rise to a multiplicity of mechanisms and pathologies associated with TRPV4 channel dysregulation. TRPV4 channels, thus, may represent a novel pharmacotherapeutic target in a wide range of diseases.

Figure 1. Contribution of TRPV4 channels to the regulation of vascular tone.

As shown, heteromeric TRPV4-TRPC1 channels expressed in endothelial cells can be activated by shear stress, agonists (4αPDD, GSK1016790A) and epoxyeicosatrienoic acids (EETs) resulting in an increase in intracellular Ca²⁺and the release of various vasoactive substances such as EETs, nitric oxide (NO) and prostaglandin (PGI₂) leading to vasodilation. In addition, TRPV4 channels in caveolae interact with small conductance potassium channels (SK) contributing to the release of K⁺from endothelial cells. In smooth muscle cells, K⁺-induced hyperpolarization is mediated through the activation of the Na/K pump as well as inwardly rectifying potassium channels (Kir). TRPV4-TRPC1 channels in smooth muscle cells are activated by EETs which trigger Ca²⁺sparks from ryanodine receptors and the subsequent activation of large conductance calcium-activated potassium-selective channels (BK) resulting in smooth muscle cell hyperpolarization and vasodilation. In cerebral parenchymal arterioles, the abluminal side of the vessel is surrounded by astrocytic endfeet processes which also modulate vascular tone. Glutamate-mediated rise in intracellular Ca²⁺leads to vasodilation through activation of KCa1.1 channels, K⁺release and activation of Kir channels in smooth muscle cells. The rise in intracellular Ca²⁺stimulates phospholipase A₂(PLA₂) and mobilizes arachidonic acid (AA) which then is metabolized to form EETs (among other signals); EETs released at the gliovascular interface activates TRPV4 channels in astrocytic endfeet processes further contributing to the rise in intracellular Ca²⁺and K⁺channel signaling. In perivascular nerves, TRPV4 channel activation has been associated with the release of calcitonin gene-related peptide (CGRP) activation of G-protein coupled receptors (GPCR) in smooth muscle cells and vasodilation.