Epithelial Sodium Channel in Human Arteries – an Emerging Player in Blood Pressure Regulation

Abstract

The epithelial sodium channel (ENaC) is a trimer composed of α, β and γ subunits. A fourth ENaC subunit δ has been reported in humans and is known to form a functional channel. There is growing evidence for the function of ENaC in the vasculature contributing to vascular tone and blood pressure regulation. Studies in mouse and rat models identified the association of vascular ENaC with endothelial dysfunction and hypertension. This indicates that vascular ENaC could be a potential drug target for antihypertensive medications. However, the absence of the δ subunit in these animal models makes it difficult to translate these findings into clinical settings. Therefore, this study aimed to characterise the expression and function of ENaC subunits in freshly isolated human arteries and ENaC’s association with hypertension in a group of cardiovascular patients. Human internal mammary arteries (IMA) and aortic punches from patients undergoing coronary artery bypass graft (CABG) surgery were collected through the HeartOtago network. The patients were divided into three groups: normotensive, uncontrolled hypertensive, and controlled hypertensive based on their hypertensive history and blood pressure measurements. The expression of ENaC subunits was analysed by RT-qPCR and western blot analysis, whereas, the localisation of ENaC subunits was detected with immunohistochemistry analysis. Primary endothelial cells isolated and cultured from IMA (MAECs) were used for the functional analysis of ENaC channels using whole-cell and single-channel patch-clamp electrophysiology. Finally, human umbilical vein endothelial cells (HUVECs) and MAECs were treated with common β-blockers (Metoprolol and Carvedilol) and the angiotensin-converting enzyme (ACE) inhibitor (Cilazapril) to reveal if common antihypertensive medications affect ENaC expression. The expression analysis detected, for the first time, both mRNA and protein expression of α-, β-, γ- and δ-ENaC subunits in human arteries. Further, the immunohistochemistry analysis localised the expression of all four ENaC subunits in cells of both endothelial and vascular smooth muscle layers of IMA. In primary MAECs, whole-cell patch-clamp electrophysiology revealed the presence of amiloride-sensitive current. Single-channel patch-clamp experiments identified channels with two different conductances ~6pS and ~12.5pS, which are similar to the reported conductances of αβγ- and δβγ-ENaC channels respectively. Interestingly, in IMA, the δ-ENaC subunit was significantly elevated in the uncontrolled hypertensive group compared to controlled hypertensive groups at both mRNA and protein levels. However, in aortic punches, the protein levels of β- and γ-ENaC subunits were significantly downregulated in controlled hypertensive patients compared to uncontrolled hypertensive and normotensive patients. Finally, the antihypertensive drugs differentially downregulated the mRNA and protein expression levels of α- and δ-ENaC subunits in HUVECs. However, these drugs did not change the protein expression levels of α and δ subunits in MAECs. The findings of this study suggest that functional ENaC channels (αβγ and δβγ) are expressed in human arteries and there is evidence that ENaCs are associated with hypertension. Furthermore, the δ-ENaC subunit in human arteries could play a major role in vascular pathology. This implicates that vascular ENaCs are potential drug targets for hypertension. However, the tissue/cell and subunit-specific regulation of ENaC subunits provide more challenges to find a potential drug that specifically targets vascular ENaCs.