Apelin Regulation of K-Cl Cotransport in Vascular Smooth Muscle Cells as a Potential Target for Cardiovascular Disease

Background/Aims: Apelin and its signaling through the G-protein coupled receptor (APJ, gene symbol APLNR) regulate cardiovascular function via two mechanisms: 1) By promoting nitric oxide (NO)-mediated vasodilation, impaired by oxidized low-density lipoproteins (oxLDL); and 2) By inducing cell proliferation via the phosphatidylinositol-3-kinase (PI3K)/protein kinase B/Akt pathway (PI3K/Akt) and mitogen activated protein kinase (MAPK) pathways. The potassium chloride cotransporter (KCC1,3,4; SLC12A4,6,7) controls cell volume, and regulates cardiovascular function through proliferation, migration, and blood pressure control. Importantly, KCC regulatory mechanisms and apelin/APJ signaling pathways overlap placing KCC as a potential target for apelin/APJ to elicit its cardioprotective effects. Therefore, apelin’s action on KCC activity was examined in contractile and synthetic rat aortic vascular smooth muscle cells (VSMCs). Methods: KCC activity was measured by atomic absorption spectrophotometry in chloride (Cl-) and Cl--free medium with sulfamate (Sf-) as Cl- replacement, and with rubidium (Rb+) as a potassium (K+) congener. The calculated difference between Rb+ transport in the presence of chloride (Cl-) and sulfamate (Sf-) is the Cl--dependent Rb+ influx (i.e., K-Cl cotransport activity). Apelin-13 (1 µM) was added either during flux (acute effect) and/or in the growth media (chronic effect) based on the experimental goals. KCC activity was characterized with respect to the VSMC phenotypes, in the presence or absence of apelin and corresponding inhibitors of the signaling pathways, oxLDL, and as a function of various physiological factors described below. Results: The APJ receptor was expressed in both contractile and synthetic VSMC phenotypes, the former also possessing the soluble guanylyl cyclase-coupled protein kinase G (PKG) receptor, critical for NO-mediated signaling. In general, KCC activity was higher in synthetic vs. contractile VSMCs, consistent with enhanced migration and proliferation in the former. In addition, apelin-mediated activation of KCC was modulated by extracellular sodium [Na+]o, osmolality, length of apelin treatment (acute or chronic) and VSMC phenotype (contractile vs synthetic). Based on selective inhibitors, apelin activated KCC through the (NO)/soluble guanylate cyclase (sGC)/protein kinase G (PKG) (NO/sGC/PKG)-, PI3K/Akt- and MAPK-dependent pathway(s). Furthermore, apelin rescued the inhibition of KCC by oxLDL. Altogether, results suggest apelin/APJ as an important modulator of KCC activity to sustain cell volume regulation and cardiovascular function. More recently, the apelinergic system has been proposed as a novel target for the treatment of Corona virus disease 2019 (COVID-19) and, given the significant overlap between the regulatory mechanisms of this system and KCC, and their role in cardiovascular disease (CVD), this study opens new avenues to identify potential targets for diverse implementation strategies. Conclusion: A better understanding of apelin effects on KCC will help design a novel therapeutic approach to treat atherosclerosis-linked cardiovascular diseases, including COVID-19-associated mortality.