TRPC1 channels are non-selectively permeable to Ca²⁺ (a unitary conductance of ~5pS) [40]. Although TRPC1 channel is known to be expressed in the endothelium, it is debatable that TRPC1 channels show homomeric channel in intact endothelial cells. In contrast, it is definitely elucidated that TRPC1 channels form heteromeric channels with TRPC3 [41], TRPC4 [40], TRPC5 [40], and TPRV4 [42,43] channels. With regard to the role of TRPC1 channels in endothelial intracellular Ca²⁺ signaling, the activation of Ca²⁺-sensing receptor (CaSR) stimulates Ca²⁺ influx through TRPC1 channels and leads to NO production in the endothelium [44]. Furthermore, once CaSR is activated by the elevation of extracellular Ca²⁺ concentration, TRPC1 channels that are co-localized with TRPV4 channels showed vasorelaxation, which was significantly abrogated in the presence of TRPC1 and TRPV4 inhibitors [42]. Therefore, it is suggested that the heteromeric TRPC1/TRPV4 channels stimulated by CaSR activation induce NO-mediated vasorelaxation.

TRPC3 channels are expressed in arteriolar myocytes and contribute to membrane depolarization-induced vasoconstriction (i.e., permeability: Ca²⁺ = Na⁺ > K⁺) [45-48]. Meanwhile, growing evidence has shown that TRPC3 cation channels are expressed in ECs and are responsible for EC hyperpolarization-mediated vasodilation. The vasoreactivity of rat small mesenteric arteries was considerably blunted in response to flow or bradykinin in the treatment of TRPC3 antisense oligonucleotide [49]. Subsequent pharmacological studies with a specific inhibitor of TRPC3 channels (i.e., Pyr3) have consistently demonstrated that the ACh-induced vasodilation of human internal mammary arteries or rat mesenteric arteries is markedly reduced with Pyr3 pretreatment [50,51], implying that EC GPCR activation by shear stress or exogenous agonists is coupled to TRPC3-induced downstream signaling for vasodilation. Senadheera and colleagues used ultra-structural immunohistochemistry to show that TRPC3 channels are spatially restricted at MEPs (i.e., five-fold higher expression compared to non-MEPs) in resistance mesenteric arteries [51]. This investigation also revealed that TRPC3 inhibition with Pyr3 causes a significant reduction in EC hyperpolarization in response to ACh [51]. More recently, studies have shown that EC TRPC3 channels in cerebral arteries are translocated to the plasma membrane following purinergic receptor activation with ATP and are then involved in EDH-mediated vasodilation [52]. Collectively, it is speculated that second messengers (e.g., DAG) produced by GPCR activation stimulate Ca²⁺ influx via TRPC3 channels and in turn lead to EDH-mediated vasodilation in resistance small arteries.

TRPC4 channels preferably allow Ca²⁺ influx in the endothelium through store-operated Ca²⁺ entry and are stimulated by GPCR downstream signaling [32,53]. Freichel and colleagues [54] demonstrated that endothelium-dependent vasodilation occurs through the activation of TRPC4 channels in mouse aorta. The specific molecular mechanism underlying the vasodilation is attributed to endothelial NO synthase (eNOS) activation and NO production followed by the activation of muscarinic acetylcholine receptors [54,55]. Even though subsequent studies failed to show that endothelial store-operated Ca²⁺ entry is mediated by TRPC4 channels [53,56], it is noteworthy to understand that TRPC4 channels could be involved in endothelium-dependent vasodilation in conduit arteries, but not small-sized resistance arteries.

TRPC5 channels show a unitary conductance of 40–60 pS and are more permeable to Ca²⁺ than Na⁺ (P_Ca:P_Na = 1.8–9.5) [57,58]. Although the role of TRPC5 channels in endothelial function has not been clearly identified to date, previous investigations found that TRPC5 channels are distributed in the endothelium from mouse aorta, human coronary, and cerebral arteries [59-61]. Mori and colleagues revealed that Ca²⁺ influx through endothelial TRPC5 channel is regulated by post-translational modifications. Specifically, S-nitrosylation, the covalent attachment of an NO to cysteine groups, enhances the activity of TRPC5 channels and Ca²⁺ influx in cultured ECs [62]. However, the alteration of vasodilatory response to ACh was not monitored in mouse aortic rings originated from TRPC5 knockout models [60]. This suggests that NO-mediated vasodilation in the presence of ACh was not augmented by TRPC5 channels, which is inconsistent with previous studies [62]. Thus, it is necessary in future studies to explore if Ca²⁺ entry via TRPC5 channels elicits vasodilation and via what supporting mechanisms.

TRPM2 channels that have a similar permeability of Ca²⁺ and Na⁺ ions display the unitary conductance of 58–76 pS [63]. The subtype of TRPM2 channels is gated by several activators including ADP-ribose, arachidonic acid, hydrogen peroxide [64-66]. The Ca²⁺ influx of TRPM2 channels is also clarified to induce endothelium-dependent vasodilation. It was found in rat cremaster resistance arteries that exogenous hydrogen peroxide application leads to Ca²⁺ entry and subsequent vasodilation [67]. However, those Ca²⁺ signaling and vascular reactivity were markedly diminished by the treatments of TRPM2 antibody or IK_Ca/SK_Ca channel inhibitors. These results indicate that hydrogen peroxide-mediated Ca²⁺ entry through TRPM2 channels evokes endothelial vasodilation in the manner dependent of EDH [67].

TRPV1 nonselective cation channels are expressed in arterial ECs [68,69] and are favorably permeable to Ca²⁺ compared with Na⁺ (P_Ca:P_Na ≈ 9.6) [32]. Capsaicin, endocannabinoids, and noxious heat activate TRPV1 channels [70,71]. Anandamide-evoked TRPV1 activation increased NO in the endothelium of rat mesenteric arteries [72]. A synthetic cannabinoid compound (VSN16) induced vasorelaxation of the mesenteric arteries, which was markedly attenuated by capsazepine (a TRPV1 antagonist), L-NNA (an NOS inhibitor), charybdotoxin (an IK_Ca/BK_Ca channel inhibitor), or apamin (a SK_Ca blocker) [73]. In gracilis skeletal muscle arterioles, the capsaicin-mediated stimulation of TRPV1 channels produced vasodilation that was substantially attenuated by L-NAME (an NOS inhibitor), which suggests that EC TRPV1-mediated vasodilation occurs in an NO-dependent manner [74]. Interestingly, NO-mediated vasorelaxation/dilation following TRPV1 activation differs from vasodilatory mechanisms of other TRP subfamilies (i.e., TRPV4, TRPA1) that display EDH-dependent vasodilation. In accordance with previous studies showing a relationship between EC TRPV1 activation and NO production, capsaicin treatment increased intracellular EC Ca²⁺ levels and elicited the phosphorylation of eNOS in cultured mouse aortic ECs [75]. Additionally, the chronic administration of capsaicin profoundly increased eNOS phosphorylation at Ser¹¹⁷⁷, NO generation, and protein kinase A (PKA) activation in mouse mesenteric arteries; these changes were absent in TRPV1 knockout mice [75]. As expected, the chronic activation of TRPV1 channels was coupled to the enhancement of EC-dependent vasorelaxation in freshly isolated mesenteric arteries [75]. Surprisingly, this study showed convincing clinical evidence that chronic capsaicin application significantly reduces blood pressure in spontaneously hypertensive rats. This finding implies that TRPV1 channels participate in the regulation of vascular tone and arterial pressure. The following study in bovine aortic ECs found that capsaicin-induced Ca²⁺ entry via TRPV1 channels stimulates Akt-calmodulin-dependent protein kinase II (CaMKII)-eNOS pathway; in turn, phosphorylated eNOS physically interacts with TRPV1 channels to produce NO [76]. However, despite consistent findings, a recent study elucidated that large renal arteries in mice do not respond to a relatively higher concentration of capsaicin (i.e., a 100-fold higher level of EC₅₀ is required for significant vasodilation), whereas mesenteric small arteries showed a marked vasodilation in response to 25 nM of capsaicin [77]. Thus, the magnitude of TRPV1-dependent vasoreactivity is specific to vascular beds.

TRPV3 channels display a large unitary conductance (≈ 150–200 pS) [78]. Dietary monoterpenoid compounds including carvacrol, eugenol, and thymol stimulate TRPV3 channels in the oral/nasal epithelium [79]. With regard to the roles of TRPV3 in EC-dependent vasodilation, Earley and co-workers revealed for the first time that carvacrol that is typically found in the Mediterranean diet, induces vasodilation in rat cerebral arteries [80]. Increased endothelial Ca²⁺ concentrations and TRPV3-like currents have been found in the presence of carvacrol [80]. Moreover, TRPV3-dependent cerebral vasodilation was found to result from VSMC hyperpolarization that was inhibited by TRAM-34, apamin, or BaCl₂ (an inhibitor of inwardly rectifying K⁺ (K_ir) channels), but not by L-NNA or indomethacin. This suggests that TRPV3 activation-induced K⁺ efflux through IK_Ca or SK_Ca leads to the K_ir-mediated hyperpolarization of VSMCs and cerebral vasodilation [80]. Recently, it was demonstrated that EC TRPV3 Ca²⁺ sparklets (i.e., localized Ca²⁺ influx through EC TRPV3 channels) are directly and optically detected with carvacrol in the ECs of rat cerebral parenchymal arterioles [27]. The unitary Ca²⁺ events in ECs were significantly attenuated by a selective TRPV3 antagonist, isopentenyl pyrophosphate (IPP). This study also noted that TRPV3 sparklet-mediated vasodilation is not sensitive to NOS or COX inhibitors. By contrast, the inhibition of IK_Ca or SK_Ca channels markedly reduced the dietary compound-induced vasodilation. Importantly, the authors also suggested that the amplitude of TRPV3 channels (∆F/F₀ = 0.20; peak fluorescence [F] is normalized to baseline fluorescence [F₀]) is greater than that of TRPA1 (∆F/F₀ = 0.13) and TRPV4 (∆F/F₀ = 0.06) channels. TRPV3 Ca²⁺ sparklets were implicated to play a key role in EC Ca²⁺ signaling in cerebral resistance arterioles [27]. However, to the best of our knowledge, there have been few observations on the influence of TRPV3 channels on vasomotor reactivity. Thus, further observations are required for a better understanding of the contribution of EC TRPV3 for the modulation of vascular function.

TRPV4 channels were shown to have a specific permeability to Ca²⁺, Na⁺, and Mg²⁺ (P_Mg:P_Na ≈ 2–3:1; P_Ca:P_Na ≈ 6–10:1) [68,81,82]. TRPV4 channels are stimulated by a variety of endogenous activators or synthetic agonists including 4α-phorbol 12,13-didecanoate (4α-PDD; 200 nM of EC₅₀; [83]), GSK1016790A (18 nM of EC₅₀; [84]), RN-1747 (4 μM of EC₅₀; [85]), or arachidonic acid metabolites (e.g., 5,6-EET; 130 nM of EC₅₀; [86]). When Ca²⁺-permeable TRPV4 channels were pharmacologically activated, increased whole-cell currents or Ca²⁺ influx were observed in isolated ECs [87-90]. Previous investigations demonstrated that in small arteries, EC TRPV4-mediated alterations in Ca²⁺ levels following the activation of muscarinic or purinergic receptors in ECs contribute to marked vasodilation. ACh fails to dilate mesenteric arteries with a genetic deletion of TRPV4 channels [90]. In this study, ACh-mediated Ca²⁺ influx and NO production were also significantly blunted in TRPV4^-/- mice. Similarly, a potent TRPV4 antagonist (RN-1734) pretreatment considerably disrupted the ACh-evoked vasodilation of radial uterine arteries [91].

Sullivan et al. [92] and Sonkusare et al. [15] marked the beginning of a new area of endothelial Ca²⁺ influx through TRPV4 channels, called TRPV4 Ca²⁺ sparklets, by using improved and sophisticated optical imaging techniques including high-speed/resolution confocal or TIRF microscopy. The Sonkusare and Nelson group sought to delineate the properties of elementary TRPV4 Ca²⁺ sparklets spatially identified at MEPs. TRPV4 channels were revealed to exhibit quantal events of Ca²⁺ entry, and the quantal amplitudes were shown to be dependent on the electrochemical gradient. Surprisingly, three to eight TRPV4 sparklet sites per EC was considered firmly sufficient for maximal vasodilation [15,92]. Extensive monitoring also showed that Ca²⁺ signaling through EC TRPV4 channels is explicitly coupled to IK_Ca and SK_Ca activation-mediated hyperpolarization at MEPs and vasodilation [15]. Further, K⁺ efflux via IK_Ca and SK_Ca channels enhanced vasodilation by stimulating K_ir channels in ECs and VSMCs and by amplifying the hyperpolarization of both cells [23,93]. Next, one of the novel characteristics of TRPV4 sparklets is channel cooperative behavior [94]. Ca²⁺-permeable TRPV4 channels are interestingly designed with a four-channel (tetramer) metastructure. The clustered TRPV4 channels locally distributed at MEPs are cooperatively gated, which is determined by protein kinase C-anchoring protein 150 (AKAP150) and intracellular Ca²⁺ [94]. It is conceivable that opening only one single TRPV4 channel may induce a minimal Ca²⁺ influx and activate a small number of IK_Ca and/or SK_Ca channels at the microdomains. However, increased cooperativity of the clustered TRPV4 channels (i.e., quantal events) gives rise to augmented Ca²⁺ amplitudes and enhanced hyperpolarization [94]. Angiotensin II-induced hypertension was shown to impair AKAP150-dependent cooperative activity of TRPV4 channels by disrupting the scaffolding protein expression at MEPs [94]. It is speculated that EC TRPV4 and AKAP150 play a critical role in preventing pathophysiological circumstances in the cardiovascular system.

With respect to the roles of TRPV channels in mechanotransduction, since ECs directly interact with shear stress induced by blood flow velocity and blood viscosity due to their unique anatomical position, considerable attention was given to flow (shear stress)-mediated EC TRPV4 activation and subsequent vasodilation in small resistance arteries. Further, because most previous studies of TRPV4 Ca²⁺ imaging were implemented in the presence of chemical agonist compounds (e.g., 4α-PDD, GSK1016790A), the impact of physiological modulators including shear stress or intravascular pressure on EC TRPV4 activation and downstream signaling for vasodilation requires further exploration. TRPV4 channels activated by shear stress resulted in increased Ca²⁺ entry in the ECs of mesenteric arteries and vasodilation that was significantly suppressed in TRPV4-deficient mice [38]. Pharmacological inhibition to NOS entirely suppressed shear stress (10 dyn cm^-2)-mediated vasodilation in mesenteric arteries [38], which was consistently shown in gracilis muscle arterioles [88]. These studies suggest that the mechanoactivation of TRPV4 channels exerts vasodilation through NO production; however, it is not completely clear as to how the channels are directly or indirectly activated by shear stress or metabolites (e.g., epoxyeicosatrienoic acids [EETs]) produced by shear stress. Conversely, the perfusion of 4α-PDD and 11,12-EET still evoked vasodilation in mesenteric arteries even in the presence of NOS and COX inhibitors [72,95]. It is implied that different physiological or pharmacological forms of activation of TRPV4 channels may result in disparate downstream signaling for vasodilation [88], presumably due to the unique polymodal nature of TRPV4 channels. Further, it was delineated in human coronary arteries that flow-dependent activation of TRPV4 channels generates mitochondrial ROS (i.e., hydrogen peroxide and superoxide) that play an important role in vasodilation in human coronary arteries [96].

As mentioned above, although VSMCs are constantly exposed to intraluminal pressure, biological processes in ECs are mechanically affected by shear stress. Nonetheless, EC TRPV4 channel activity has been demonstrated to be modulated by low intravascular pressure (i.e., below 50 mmHg) in rat cremaster or mesenteric arterioles [97]. Specifically, the frequency of EC Ca²⁺ events via EC TRPV4 channels was further augmented at a low intraluminal pressure (two- to three-fold higher than those at 60–80 mmHg of arterial intraluminal pressure). Both pharmacological (i.e., selective TRPV4 agonists) and physiological (i.e., intraluminal pressure) TRPV4 activations were markedly diminished by RN1734 or HC067047 [97]. Although they revealed that TRPV4, IK_Ca, and SK_Ca channels are clustered at perforations in the IEL, the inhibition of IK_Ca with TRAM-34 (but not the inhibition of SK_Ca with apamin) considerably enhanced the basal myogenic tone in cremaster arterioles. Additionally, the blockade of PLC and the IP₃ receptor with U-73122 and xestospongin fully abrogated the low pressure-mediated increase in Ca²⁺ sparklet events. Thus, Ca²⁺ influx via EC TRPV4 channels in response to low intraluminal pressure stimulates IK_Ca channels through PLC and IP₃R signaling, which in turn leads to EC/VSMC hyperpolarization-dependent vasodilation [97].

Arachidonic acid and its metabolites are potential physiological modulators for the activation of TRPV4 channels in ECs. Arachidonic acid is converted to EETs such as 5,6-EET, 8,9-EET, 11,12-EET, and 14,15-EET through cytochrome P450 (CYP) epoxygenase [86]. It has been well known that EETs have anti-hypertensive/inflammatory effects and contribute to vasodilation as a prevailing EDHF [98-100]. Watanabe and colleagues [86] determined that EETs stimulate TRPV4 channels in aortic ECs and may induce vasodilation. A subsequent study elucidated that exogenous 11,12-EET application in mesenteric arteries elicited vasodilation via hyperpolarization in VSMCs, and this vasomotor activity was substantially abrogated by TRPV4 knockout or the inhibition of Ca²⁺-sensitive K⁺ channels, but not by the blockade of NOS and COX [95]. More recently, pre-constricted human coronary arteries with endothelin-1 exhibited vasodilation in the presence of arachidonic acid, which was inhibited by RN-1734 [101]. In addition, the arachidonic acid-induced increase in Ca²⁺ concentration and vasodilation was profoundly reduced by the combination of TRAM-34 and apamin (inhibitors of IK_Ca and SK_Ca channels, respectively), the removal of extracellular Ca²⁺, or pretreatment with CYP inhibitors, suggesting that CYP-dependent metabolite generation from arachidonic acid increases Ca²⁺ influx (but not Ca²⁺ release from the sarcoplasmic reticulum) through EC TRPV4 channels and subsequently requires IK_Ca and SK_Ca-mediated hyperpolarization for vasodilation in human coronary arteries [101].

As most studies on TRPV4 channels have been performed under ex vivo preparations, many investigators have raised questions regarding the regulatory effect of TRPV4 channels on the cardiovascular system. The administration of GSK1016790A (a selective agonist for TRPV4 channels) interestingly decreased mean arterial pressure [102], which implies that the systemic activation of TRPV4 channels may contribute to the attenuation of peripheral resistance [32]. Consistently, there are studies showing that TRPV4-deficient mice did exhibit a higher mean arterial pressure compared with that of control mice [103]. Moreover, in the presence of the concurrent administration of an NOS inhibitor (L-NAME) and angiotensin II, TRPV4 knockout mice showed a much greater mean arterial pressure [104], indicating that TRPV4 may provide an alternative compensatory pathway in pathophysiological circumstances to prevent cardiovascular diseases (e.g., hypertension) by reducing peripheral resistance or inducing vasodilation [105].

TRPA1 channels are nonselective cation (i.e., Na⁺, Ca²⁺) channels (but specifically permeable to Ca²⁺ compared with Na⁺, P_Ca:P_Na ≈ 7.9) that have a large unitary conductance (≈ 98 pS) [106,107]. As the name of the ion channel implies, TRPA1 channels have a distinguished structure that exhibits approximately 14–19 ankyrin repeat domains in the intracellular N-terminus of the ion channels [108]. The ankyrin repeat domains participate in protein-protein interactions and modulate channel activity in response to agonists or heat [109,110]. It is well established that diverse pungent dietary compounds such as allyl isothiocyanate (AITC) from mustard, allicin from garlic, eugenol from cloves, or cinnamaldehyde from cinnamon [111] activate TRPA1 channels by exerting covalent modifications on cysteine residues of the N-terminus of the channels [112,113]. Along with the dietary activators, oxidative stress and specific metabolites of lipid peroxidation act as physiological regulators for TRPA1 channels. Previous studies determined that endogenous aldehydes, 4-hydroxy-2-nonenal (4-HNE), 4-oxononenal (4-ONE), or 4-hydroxyhexenal (4-HHE) derived from the peroxidation of ω6 polyunsaturated fatty acids in the plasma membrane activate TRPA1 channels [114-118]. Further, hydrogen peroxide (H₂O₂) resulted in the oxidative modification of cysteine residues and subsequently activated TRPA1 channels in neuron cells [114,116]. Therefore, oxidative stress metabolites play a vital role in stimulating TRPA1 channels.

In light of the physiological consequences of TRPA1 activation, it was first noted that AITC application leads to vasorelaxation in rat mesenteric arteries by activating the target ion channels in perivascular nerves [119]. Interestingly, the AITC-mediated relaxant response was markedly attenuated after the pretreatment of the calcitonin gene-related peptide (CGRP) receptor antagonist (i.e., CGRP 8–37). Previous findings demonstrated that the dietary TRPA1 activators caused cultured trigeminal neurons to release CGRP, which was abolished by a TRPA1 channel blocker [120]. Therefore, it is likely that CGRP released from perivascular nerve endings elicits vasodilation. Subsequent studies further showed that cinnamaldehyde-mediated vasodilation in mesenteric arteries is considerably abrogated in TRPA1-deficient mice, whereas endothelial denudation has minor effects on vasodilation [121]. This suggests that pungent dietary activators of TRPA1 channels elicit vasodilation by inducing Ca²⁺ influx-dependent CGRP release from perivascular nerves and subsequently binding the peptide to CGRP receptors in VSMCs. Recently, it was reported that TRPA1 channels regulate the cold-induced vascular response [122]. Specifically, sequential vasoconstriction and vasodilation occur following local cold exposure to prevent cutaneous tissues from suffering from cold injury. Although TRPA1 channel-induced superoxide generation provoked vasoconstriction through α₂-adrenoreceptor activation and myosin light chain phosphorylation immediately after environmental cold exposure, neuropeptide CGRP released from sensory nerves evoked TRPA1 channel-dependent vasodilation for the restoration of reduced blood flow [122].

Meanwhile, TRPA1 channels are identified in ECs and in perivascular or sensory nerves. However, the distribution of EC TRPA1 channels is limited to only the cerebral arteries, not the mesenteric, coronary, or renal arteries [28,37]. The direct influence of EC TRPA1 channels on the vasomotor response following the treatment of an electrophilic TRPA1 activator derived from mustard oil (e.g., AITC) was first assessed in pressurized cerebral arteries [37]. AITC concentration-dependent vasodilation of cerebral arteries was observed, which was accompanied by a significant reduction in the intracellular Ca²⁺ levels of cerebral arterial myocytes. Because VDCCs are a key determinant for Ca²⁺ dynamics in VSMCs, TRPA1 activation was suggested to induce VSMC hyperpolarization and vasodilation. TRPA1 channel-mediated vasodilation was diminished in EC-denuded arteries or after TRPA1 channel blocker (HC-030031) pretreatment [37], indicating that AITC-dependent vasodilation in cerebral arteries is EC-dependent and distinct from CGRP-induced vasorelaxation in mesenteric arteries [119]. Moreover, in intact ECs, TRPA1 channels are detected at MEPs where IK_Ca channels, but not SK_Ca channels, are locally expressed [37]. Pharmacological approaches have substantiated that although TRPA1-induced cerebral vasodilation is not affected by the inhibition of NOS and COX, the application of Ca²⁺-sensitive K⁺ channel blockers or a K_ir channel inhibitor markedly suppresses AITC-induced vasodilation. Thus, EC TRPA1-dependent Ca²⁺ entry (called TRPA1 Ca²⁺ sparklets) elicits a K⁺ efflux via IK_Ca/SK_Ca channels and provokes vasodilation through EC and VSMC hyperpolarization, which is amplified by K_ir channels in cerebral arteriolar myocytes [37]. Earley and co-workers extended their novel hypotheses on TRPA1 channel-induced vasodilation and revealed innovative cerebral dilatory mechanisms. They found that EC TRPA1 channels frequently display a binary channel gating behavior (i.e., two TRPA1 channels open simultaneously) [28]. Their study focused on the ROS-mediated activation of TRPA1 channels in cerebral arteries [28]. NADPH (nicotinamide adenine dinucleotide phosphate) oxidase (NOX) was shown to generate ROS [123,124], and TRPA1 channels in neurons are stimulated by ROS or ROS-derived metabolites [114,116,125]. In cerebral arteries, the administration of exogenous NADPH-induced vasodilation that was profoundly decreased in the presence of HC-030031 or TRAM-34 [28]. Consistent with their prior study [37], the combination of L-NNA and indomethacin did not affect NADPH-induced vasodilation [28]. Collectively, NADPH-NOX downstream signaling was shown to stimulate Ca²⁺ entry via EC TRPA1 channels and subsequently led to IK_Ca channel-dependent vasodilation. To investigate specific signaling pathways, diverse pharmacological interventions including apocynin (an NOX inhibitor), catalase (a hydrogen peroxide-metabolizing enzyme), and deferoxamine (an inhibitor for the hydroxyl radical and lipid peroxidation) were employed in that study [28]. As a result, NADPH-mediated vasodilation was completely abolished in the presence of those inhibitors, suggesting that NOX-derived H₂O₂ causes the production of lipid peroxidation metabolites, which stimulate EC TRPA1 Ca²⁺ sparklets and provoke IK_Ca activation-dependent vasodilation in cerebral arteries [28]. Therefore, since NOX-derived ROS production was reported to be much greater in the cerebral vasculature than in other vascular beds [126], it is strongly noted that ROS and lipid peroxidation products may serve as endogenous modulators of TRPA1 channels and physiological regulators for vasomotor reactivity in the cerebral circulation.