2-NBDG ic50 br Transparency document br Introduction Hyperch
Introduction Hypercholesterolemia, high plasma Cho, is very common in elderly human beings and acts as a risk factor for cardiovascular diseases. Both hypercholesterolemia and aging are often associated with hypertension. However, it remains unclear how they increase the incidence of hypertension. ENaC plays an important role in maintaining Na+ homeostasis and controls systemic blood pressure. Gain-of-function mutations of ENaC cause hypertension, as seen in Liddle Syndrome [, , ]. The important role of ENaC in regulating blood pressure has encouraged investigators to determine whether ENaC can be regulated by Cho and account for hypercholesterolemia-induced hypertension. The initial studies show that exogenous Cho and its sequestering agent, methyl‑β‑cyclodextrin (MβCD), are unable to alter ENaC activity [4,5]. However, Cho in the outer leaflet of the apical membrane of renal epithelial 2-NBDG ic50 is tightly packed with sphingolipids [6,7]. Due to this fact, Cho in the outer leaflet is difficult to be extracted by MβCD or high-density lipoprotein [7,8]. Indeed, we have shown that a relatively high concentration of MβCD is required for MβCD to efficiently extract Cho out of the apical membrane and regulate ENaC . However, it still remains unclear whether the Cho in the outer leaflet is more important for ENaC activity than the Cho in the inner leaflet, because extraction of the outer leaflet Cho may facilitate Cho efflux, resulting in a reduction of the inner leaflet Cho. It is also unknown whether Cho directly interacts with ENaC or indirectly stimulates ENaC by interacting with other membrane components. Our recent studies suggest that Cho can regulate the distribution of PIP2 in the apical membrane to alter the activity of ROMK channels . The apical membrane of epithelial nephron cells contains protrusions called microvilli to increase the surface area for its transporting function. Scanning ion conductance and atomic force microscopy studies suggest that microvilli exist in the distal nephron A6 cells [11,12] and that ENaC may be mainly located in the microvilli . Our previous studies have shown that inhibition of Cho efflux from the inner leaflet of A6 cell apical membrane with ABCA1 inhibitors increases intracellular Cho and ENaC activity . Therefore, Cho may regulate ENaC activity via its inner leaflet localization. It is well known that anionic phospholipids in the inner leaflet of the plasma membrane, especially PIP2, stimulate ENaC [, , ]. We have shown that the stimulation appears to be caused by a direct interaction between anionic phospholipids and all three ENaC subunits [14,17]. Cho is localized both in the outer leaflet and in the inner leaflet of the plasma membrane. In the outer leaflet, Cho interacts with sphingolipids via hydrogen bonds to form membrane microdomains, also referred as lipid rafts [, , ]. Atomic force microscopy shows that the rafts in artificial membranes exist in a Cho-dependent manner [20,21]. In the inner leaflet, however, it has been suggested that Cho may also interact with PIP2 to form PIP2 microdomains or to localize PIP2 in lipid rafts [, , ]. We have shown that PIP2 is predominantly localized in microvilli where lipid rafts are located . These studies together indicate that Cho may stimulate ENaC by interacting with PIP2. Cho levels in the inner leaflet are precisely controlled by the Cho transporters in the plasma membrane. As a potent Cho transporter, ABCA1 can transport Cho from the inner leaflet to the outer leaflet of the cell membrane [25,26]. A previous report has shown that CsA acts as a potent inhibitor of ABCA1 activity and inhibits ABCA1-mediated Cho efflux in mouse macrophage cell . Our following studies have shown that pharmacological blockade of ABCA1 with CsA elevates intracellular Cho and stimulates ENaC in the distal nephron cells , suggesting that increased sodium reabsorption via elevated ENaC activity may account for CsA-induced hypertension. However, it remains unknown whether inhibition of Cho synthesis with statins can attenuate CsA-induced hypertension. In addition, several lines of evidence suggest that impairment of Cho efflux due to reduced ABCA1 function also participates in the pathogenesis of age-related macular degeneration, cardiovascular diseases, and Alzheimer's disease [, , ]. Therefore, elevated ENaC activity due to reduced ABCA1 function may account for the age-related hypertension. In the present study, we show that intracellular Cho stimulates ENaC by promoting the interaction between ENaC and its activator PIP2 and that this mechanism mediates CsA-induced hypertension. We also show that intracellular Cho is elevated in aging mice due to reduced ABCA1 expression. Therefore, the stimulation of ENaC by intracellular Cho may also account for age-related hypertension.