*3.2. Ca2+-Activated Chloride Channels (CaCCs) in Vascular Endothelial Cells*

In addition to their expression in vascular smooth muscle cells, CaCCs have been reported to be present in some vascular endothelial cells [81–84]. Although the physiological role of endothelial CaCCs is still not well understood, the endothelial CaCCs may contribute to the regulation of the resting membrane potential of the endothelial cells. Indeed, in mouse brain capillary endothelial cells, pharmacological blockade or knockdown of TMEM16A with siRNA induced membrane hyperpolarization, suggesting that the activation of endothelial CaCCs acts to depolarize the membrane potential of the endothelial cells [83]. Further support for this notion comes from the study by Yamamoto et al. [85]. They found that, in the isolated endothelium of guinea pig mesenteric arteries, ACh increased the intracellular concentration of Ca2+, which subsequently activated endothelial small conductance Ca2+-activated K+ channels (SKCas), intermediate conductance KCa (IKCa) and CaCC simultaneously, and the endothelium-dependent hyperpolarization (EDH) through the activation of both SKCa and IKCa was counteracted by the opposing membrane depolarization evoked by the activation of CaCCs [85].

With respect to the alteration of endothelial CaCCs in hypertension, we have previously shown a functional upregulation of endothelial CaCCs in mesenteric resistance arteries of SHRs [86]. In that study, after blockade of EDH with KCa channel inhibitors, iontophoresed acetylcholine (ACh) evoked a rapid and substantial membrane depolarization in mesenteric resistance arteries of SHRs, but only negligible slow depolarization was detected in those of WKY rats [86,87] (Figure 1).

As the estimated reversal potential of the ACh-evoked depolarization in that study was −18 mV [86], which agrees closely with that reported for Cl<sup>−</sup> ions [23,88], and the ACh-evoked depolarization was abolished by endothelium denudation, or reduced either by replacement of external Cl− ions with impermeant anions or by treatment with the CaCC inhibitors niflumic acid or flufenamic acid, the ACh-evoked depolarization appears to be, at least in part, generated through the activation of endothelial CaCCs in the mesenteric resistance arteries of SHRs [86]. Moreover, the inhibition of the ACh-evoked depolarization by CaCC inhibitors improved the impaired ACh-induced EDH in mesenteric arteries of SHRs, suggesting that an increased activity of endothelial CaCCs may be responsible for the impairment of EDH (Figure 2) (Table 2).

**Figure 1.** Acetylcholine (ACh)-evoked depolarization in mesenteric arteries of spontaneously hypertensive rats (SHRs). (**a**) A hidden depolarization emerged after blockade of endothelium-dependent hyperpolarization (EDH) with apamin (0.5 μmol/L, a small-conductance Ca2+-sensitive K+ channel (KCa) inhibitor) plus TRAM34 (100 nmol/L, an intermediate-conductance KCa inhibitor) in mesenteric arteries of SHRs. All recordings were from the same cell. (**b**) ACh-evoked depolarization in the presence of apamin (0.5 μmol/L) plus charybdotoxin (60 nmol/L, a large and intermediate-conductance KCa inhibitor) was larger in amplitude and faster in time course in SHRs than in Wistar Kyoto (WKY) rats. Each paired recording was from the same preparation. Indomethacin (10 μmol/L) and Nω-nitro-L-arginine methyl ester (100 μmol/L) were present throughout. Arrows, application of ACh. Modified from Goto et al. [87].

**Figure 2.** Effects of niflumic acid on acetylcholine-induced, endothelium-dependent hyperpolarization (EDH) in mesenteric arteries of spontaneously hypertensive rats (SHRs) and Wistar Kyoto (WKY) rats. Niflumic acid (50 μmol/L) improved EDH in SHRs but not in WKY rats. Each paired recording was from the same preparation. Indomethacin (10 μmol/L) and Nω-nitro-L-arginine methyl ester (100 μmol/L) were present throughout.


**Table 2.** Alterations in vascular endothelial Ca2+-activated Cl<sup>−</sup> channels during hypertension.

CaCCs, Ca2+-activated Cl−channels; SHRs, spontaneously hypertensive rats; EDH, endothelium-dependent hyperpolarization; Ang II, angiotensin II.

As endothelial cells and adjacent smooth muscle cells are electrically coupled via myoendothelial gap junctions in rat mesenteric arteries [89–91], the impaired EDH leads to attenuated EDH-mediated relaxation and hence to endothelial dysfunction in SHRs [86]. Although some studies have reported that the inhibition of volume-activated Cl− channels potentiates K+-induced, EDH-mediated relaxation in rat mesenteric arteries [92,93], the involvement of the volume-activated Cl− channels in the ACh-evoked depolarization in mesenteric arteries of SHRs is not likely because the volume-regulated Cl− channel inhibitor NPPB had no effect on the ACh-evoked depolarization in this vascular bed [86].

A negative causal link between the activity of endothelial CaCCs, specifically TMEM16A, and endothelial function has also been reported in other studies [82,84]. Thus, in Ang IIinduced hypertensive mice, in which the expression of vascular endothelial TMEM16A is increased, the endothelial-specific TMEM16A knockout ameliorated endothelial function and lowered the systolic blood pressure, whereas the endothelial-specific TMEM16A overexpression deteriorated endothelial function and further elevated the systolic blood pressure [82], and these interactions appear to be related to the facilitating effects of TMEM16A on reactive oxygen species generation via Nox2-containing NADPH oxidase [82] (Table 2).

Another study showed that overexpression of TMEM16A in human pulmonary endothelial cells led to a decrease in ACh-induced NO production [84]. Taken together, these findings suggest that upregulation of endothelial CaCC TMEM16A may contribute to the impaired endothelial function, and if so, that it likely does so via a reduction in the activity of EDH and/or NO; finally, the results suggest that such a reduction in EDH and/or NO activity may be at least partly responsible for the elevated blood pressure in hypertension (Figure 3).
