*3.1. Ca2+-Activated Chloride Channels (CaCCs) in Vascular Smooth Muscle Cells*

It is generally accepted that essential hypertension is characterized by an increased peripheral resistance [49,50]. The increased peripheral resistance in hypertension is determined by an integral and complex interplay between various pathogenic factors, including increased sympathetic nervous activity, enhanced calcium ion mobilization in vascular smooth muscle cells, increased calcium sensitivity of vascular smooth muscle cells and reduced production of endothelium-derived relaxing factors, to name a few [50,51]. Among these factors, alterations in the function of vascular ion channels during hypertension contribute to the increased peripheral resistance by shifting the membrane potential to depolarized levels [22,50,52].

While many studies have demonstrated downregulation of the expression and/or function of vascular potassium (K+) channels in hypertension [50,51,53,54], emerging evidence reveals an upregulation of expression and/or function of CaCCs in vascular smooth muscle cells of spontaneous hypertensive rats (SHRs), a genetic model of human essential hypertension. Although a previous study suggested an increased activity of CaCCs in vascular smooth muscle cells of SHRs [55], the molecular identity of the CaCCs observed in that study was unclear at the time. A subsequent study by Wang et al. for the first time revealed that TMEM16A is the molecular counterpart for the increased activity of CaCCs in vascular smooth muscle cells of SHRs, and that TMEM16A protein expression is significantly upregulated in the aorta, the carotid arteries, the hindlimb arteries and the mesenteric arteries of SHRs compared to those of normotensive Wistar Kyoto (WKY) rats [56] (Table 1). Consistent with the seminal findings of Wang and colleagues [56], the increased TMEM16A expression levels and the resultant potentiation of vasoconstrictions have also been reported in smooth muscle cells of the coronary arteries [57] and the renal arterioles [47] of SHRs (Table 1).

**Table 1.** Alterations in vascular smooth muscle Ca2+-activated Cl<sup>−</sup> channels during hypertension.


CaCCs, Ca2+-activated Cl<sup>−</sup> channels; SHRs, spontaneously hypertensive rats; 2K2C, 2-kidney, 2-clip.

Importantly, the increased expression and function of TMEM16A appear to be associated with blood pressure elevation in SHRs: the in vivo knockdown of TMEM16A by small interfering RNA (siRNA) transfection prevented blood pressure rise, and the in vivo inhibition of TMEM16A activity by T16Ainh-A01, a TMEM16A inhibitor, reduced blood pressure in SHRs [56] (Table 1). Similarly, a recent study in SHRs showed that in vitro treatment of mesenteric resistance arteries with TMinh-23, a small molecule inhibitor of vascular smooth muscle TMEM16A, blocked vascular smooth muscle constriction in response to vasoconstrictor stimuli, and in vivo treatment with TMinh-23 reduced blood pressure in SHRs with minimal blood pressure change in normotensive rats and mice [58] (Table 1). Although the greater blood pressure lowering effect of TMinh-23 in SHRs appears to be due to an increased sensitivity of TMEM16A to TMinh-23 [58], the mechanisms underlying the increased sensitivity of TMEM16A are unclear and warrant further investigations. Together, these findings implicate vascular smooth muscle CaCC TMEM16A as a possible contributor in the pathogenesis of hypertension in SHRs.

In rat basilar arteries of 2-kidney, 2-clip (2K2C) renal hypertensive rats, exogenously applied angiotensin II (Ang II) induced vasoconstriction that was sensitive to T16Ainh-A01, and Ang II evoked TMEM16A-mediated CaCC currents in rat basilar smooth muscle cells [59]. These findings suggest that CaCC TMEM16A modulates the vasocontractility of basilar arteries of 2K2C renal hypertensive rats; however, in sharp contrast with SHRs, the activity of CaCCs was decreased gradually during the development of hypertension, and the CaCCs' current density was negatively correlated with blood pressure levels, in basilar arteries of 2K2C renal hypertensive rats [60] (Table 1). Moreover, the TMEM16A protein expression in the smooth muscle layer of the basilar artery decreased during the development of hypertension in 2K2C renal hypertensive rats [59,60] (Table 1).

It is not clear why the activity and the expression of CaCC TMEM16A changed in the opposite direction between SHRs and 2K2C renal hypertensive rats, but the difference might be explained by the different levels of activity of the renin–angiotensin system (RAS) in the vasculature: while the plasma and tissue RASs are suppressed in SHRs [61], the RAS components—particularly the vascular Ang II concentration—are increased in 2K2C renal hypertensive rats [62]. As Ang II decreased TMEM16A expression in some vascular smooth muscle cells, including those from rat basilar arteries [59,60,63], an increase in vascular Ang II concentration in the basilar arteries of 2K2C renal hypertensive rats might downregulate TMEM16A expression and hence reduce the CaCCs' current in this model.

It has been reported that the perivascular sympathetic nerves exert an abnormal trophic influence on the vascular smooth muscle membrane properties of SHRs [64], and a recent report showed that the expression and contractile function of the CaCC TMEM16A in rat arteries were reduced due to the trophic influence of sympathetic nerves during postnatal maturation [65]. Therefore, we speculate that the expression and function of CaCC TMEM16A might also be decreased along with the longer duration of hypertension in SHRs because of the persistent abnormal trophic influence of the sympathetic nerves. This hypothesis might be supported by the observation that the contribution of CaCCs to norepinephrine-induced vasoconstriction in the femoral arteries was decreased in 12 month-old SHRs compared to that of 6-month-old SHRs [66].

TMEM16A may modulate vascular contractility in cooperation with other ion channels in certain vascular beds. Thus, in rat mesenteric and tail arteries, TMEM16A modulates vascular contractility, at least in part, by positively regulating the expression and function of vascular L-type Ca2+ channels [67,68]. In another study in rat cerebral arterial smooth muscle cells, transient receptor potential canonical 6 channel (TRPC6) and TMEM16A were found to be spatially localized, and TRPC6 activation led to a local elevation of Ca2+, which in turn activated nearby TMEM16A, leading to vasoconstriction [69]. As the function and expression of both L-type Ca2+ channels [70,71] and TRPC6 [72] have been reported to be upregulated in hypertensive rats, it is intriguing to speculate that these mutual interactions of TMEM16A with other vascular ion channels function cooperatively to augment vasoconstriction and hence increase blood pressure in hypertension.

It has been reported that phosphatidylinositol 4,5-bisphosphate (PIP2), a phospholipid of the plasma membrane, regulates ion channel activity in various cell types [73], and several studies reported that PIP2 acts as a positive modifier of TMEM16A [74–76]. By contrast, the TMEM16A-mediated CaCC current was not augmented, but rather inhibited by PIP2 in rat pulmonary artery smooth muscle cells [77]. The reason for the discrepancy is not clear. Nevertheless, a previous report suggests that a significant difference exists between WKY and SHR aortas regarding the PIP2 hydrolysis response following stimulation with norepinephrine [78], indicating the need for further research to understand the possible regulation of TMEM16A by PIP2 in blood vessels in hypertension.

Recent evidence suggests that inositol 1,4,5-trisphosphate receptors (IP3Rs) are spatially colocalized with TMEM16A proteins in nociceptive sensory neurons [79]. If the same holds true in vascular smooth muscle cells, IP3-induced Ca2+ release from intracellular Ca2+ stores would activate nearby TMEM16A, and alterations in this signaling pathway might contribute to the TMEM16A-mediated vasoconstriction in SHRs. Indeed, it has been reported that IP3R channels are upregulated in vascular smooth muscle in hypertension, resulting in enhanced IP3-induced Ca2+ release and increased vasoconstriction [80].

To sum up, while there is a growing body of evidence that CaCC TMEM16A contributes to the increased vascular contractility and elevated blood pressure in SHRs, it is currently unclear whether the upregulation of TMEM16A is specific to SHRs or is present in other hypertensive animal models, and further studies will be needed to clarify the molecular mechanisms that regulate TMEM16A activity during hypertension.
