**1. Introduction**

Hypertension is the most prevalent and important risk factor for cardiovascular disease around the world [1], and cardiovascular complications associated with hypertension accounted for 8.5 million deaths worldwide in 2015 [2]. Nevertheless, global control (<140/90 mmHg) rates among subjects with hypertension in 2019 were only 23% for women and 18% for men [3], and thus more effective treatment strategies for hypertension control are urgently needed.

Lifestyle modifications are recommended for the treatment and prevention of hypertension and hypertension-associated cardiovascular diseases for all subjects, including subjects with high normal blood pressure and patients who are taking antihypertensive agents [4]. In particular, the restriction of dietary sodium chloride (NaCl) has been one of the major focus points among lifestyle modifications for the treatment and prevention of hypertension [5,6]. Indeed, numerous animal and human studies have established a causal relationship between dietary NaCl intake and hypertension as well as hypertensionassociated cardiovascular diseases [7–9].

While it is generally assumed that sodium ions (Na+) but not chloride ions (Cl<sup>−</sup>) play a critical role in NaCl-induced hypertension [10,11], the copresence of Na<sup>+</sup> and Cl<sup>−</sup> has been reported to be requisite for the development or progression of hypertension in some animal models of hypertension, including desoxycorticosterone-induced hypertensive rats [12],

**Citation:** Goto, K.; Kitazono, T. Chloride Ions, Vascular Function and Hypertension. *Biomedicines* **2022**, *10*, 2316. https://doi.org/10.3390/ biomedicines10092316

Academic Editors: Josef Zicha and Ivana Vanˇeˇcková

Received: 17 August 2022 Accepted: 15 September 2022 Published: 18 September 2022

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**Copyright:** © 2022 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

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Dahl salt-sensitive hypertensive rats [13,14] and stroke-prone spontaneously hypertensive rats [15]. Likewise, several studies have suggested the importance of Cl− in NaCl-induced hypertension in humans [16–18]. These animal and human studies suggest that Na<sup>+</sup> alone may not be sufficient, and that Cl− may be indispensable or may act cooperatively with Na+ to give rise to NaCl-induced hypertension. A detailed description of the role of Cl<sup>−</sup> in NaCl-induced hypertension in animals and humans can be found in an excellent review by McCallum et al. [19].

The precise mechanisms by which Cl− contributes to the blood pressure rise in the above studies are yet to be determined, but the ability of Cl− to modify vascular contractility may play a role. In vascular smooth muscle cells, the intracellular concentration of Cl− is accumulated by anion exchangers and the anion–proton cotransporter system [20,21]. As the resting membrane potential of smooth muscle in vivo (e.g., −38 mV in the rat caudal artery [22]) is more negative than the reversal potential for Cl<sup>−</sup> (e.g., −18 mV in the guinea pig vas deferens [23]), the opening of Cl− channels leads to an efflux of Cl− and depolarizes the membrane potential, which would then increase the open probability of L-type Ca2+ channels to trigger smooth muscle constriction [20,24].

Thus, in situations with increased intracellular Cl− concentration or increased Cl− channel activity in vascular smooth muscle cells, the driving force for the efflux of Cl− is expected to increase, which in turn could facilitate membrane depolarization and vasoconstriction, and emerging evidence suggests that this scenario is indeed the case in some animal models of hypertension. In this review, we will discuss the possible involvement of Cl− in the pathogenesis of hypertension. Particular emphasis is given to the roles of Ca2+-activated Cl<sup>−</sup> channel transmembrane membrane 16A (TMEM16A; also known as Ano1) and Na+–K+–2Cl<sup>−</sup> cotransporter 1 (NKCC1) in the increased vascular contractility during hypertension.

#### **2. Role of Chloride Ions in Regulation of Vascular Tone and Blood Pressure**

The vascular tone in vivo is regulated by perivascular nerves, including sympathetic, parasympathetic and non-adrenergic non-cholinergic nerves, and the corelease of norepinephrine and ATP from the sympathetic nerve terminals causes vascular smooth muscle membrane depolarization and subsequent constriction [25–29]. Although multiple ionic mechanisms would underpin the nerve-mediated vascular smooth muscle depolarization, several previous studies have suggested that nerve-mediated and exogenously applied norepinephrine-evoked smooth muscle depolarization could be at least partly due to the generation of Ca2+-activated Cl<sup>−</sup> currents triggered by the Ca2+ release from the intracellular Ca2+ stores [24,30–32].

In addition to perivascular nerve-mediated regulation, myogenic response-mediated vascular smooth muscle depolarization and constriction in response to intravascular pressure change also contribute to the regulation of vascular tone [33]: in rat cerebral arteries, intravascular pressure-induced depolarization and constriction have been shown to be inhibited by two distinct Cl− channel blockers, indanyloxyacetic acid (IAA-94) and 4,4 diisothiocyanatostilbene-2,2 -disulphonic acid (DIDS), suggesting that the efflux of Cl− ions through Cl− channels could contribute to the myogenic response-mediated vasoconstriction [34]. Indeed, in support of this observation, efflux of Cl− ions was associated with the myogenic constriction in the rat cerebral vascular bed [35]. Nevertheless, because subsequent studies performed in the rat cerebral arteries revealed that IAA-94 depresses L-type calcium current [36], and both IAA-94 and DIDS depress non-selective cationic current [37], the validity of the contribution of Cl− currents to the myogenic response was called into question.

As such, despite a significant amount of physiological and pharmacological evidence showing that vascular Cl− channels play a crucial role in regulating vascular tone, the absence of specific inhibitors and the lack of the molecular identities of the channels make it difficult to reach indisputable conclusions. Among other things, there has been a debate regarding the molecular identity of CaCCs ever since the initial report by Byrne and Large in 1987 [38]. Indeed, several proteins have been proposed as the molecular counterpart of CaCCs, and these include CLCA, CLC-3, TWEENTY and bestrophins [39]. However, three independent groups revealed in 2008 that the TMEM16A protein is a molecular counterpart for CaCCs [40–42].

Since these 2008 reports, many studies have confirmed that TMEM16A generates functional CaCC currents in a number of vascular smooth muscle cells and thereby regulates agonist-induced vasoconstriction [21,43–45]. Moreover, it has been revealed that TMEM16A also contributes to intravascular pressure-induced myogenic depolarization and vasoconstriction in the cerebral arteries and renal arterioles of rats [46,47]. Thus, it appears likely that the TMEM16A in vascular smooth muscle cells plays a critical role in regulating vascular tone and blood pressure. Support for this notion comes from the fact that conditional knockout mice of TMEM16A in vascular smooth muscle cells shows a complete deficiency of CaCC currents, decreased responsiveness to vasoconstrictor stimuli and reduced systemic blood pressure [48].
