Increased TMEM16A-Mediated Ca2+-Activated Cl- Currents in Portal Vein Smooth Muscle Cells of Caveolin 1-Deficient Mice.

Ca2+-activated Cl- (ClCa) channels regulate membrane excitability and myogenic tone in vascular smooth muscles. TMEM16A-coding proteins are mainly responsible for functional ClCa channels in vascular smooth muscles, including portal vein smooth muscles (PVSMs). Caveolae are cholesterol-rich and Ω-shaped invaginations on the plasma membrane that structurally contributes to effective signal transduction. Caveolin 1 (Cav1) accumulates in caveolae to form functional complexes among receptors, ion channels, and kinases. The present study examined the functional roles of Cav1 in the expression and activity of ClCa channels in the portal vein smooth muscle cells (PVSMCs) of wild-type (WT) and Cav1-knockout (KO) mice. Contractile experiments revealed that the amplitude of spontaneous PVSM contractions was larger in Cav1-KO mice than WT mice. Under whole-cell patch-clamp configurations, ClCa currents were markedly inhibited by 1 µM Ani9 (a selective TMEM16A ClCa channel blocker) in WT and Cav1-KO PVSMCs. However, Ani9-sensitive ClCa currents were significantly larger in Cav1-KO PVSMCs than in WT PVSMCs. Expression analyses showed that TMEM16A expression levels were higher in Cav1-KO PVSMs than in WT PVSMs. Therefore, the caveolar structure formed by Cav1 negatively regulates the expression and activity of TMEM16A-mediated ClCa channels in vascular smooth muscle cells.

Ca2+ Signaling and Proliferation via Ca2+-Sensing Receptors in Human Hepatic Stellate LX-2 Cells.

Hepatic stellate cells (HSCs) play a significant role in the development of chronic liver diseases. Hepatic damage activates HSCs and results in hepatic fibrosis. The functions of activated HSCs require an increase in the cytosolic Ca2+ concentration ([Ca2+]cyt). However, the regulatory mechanisms underlying Ca2+ signaling in activated HSCs remain largely unknown. In the present study, functional analyses of Ca2+-sensing receptors (CaSRs) were performed using activated human HSCs, LX-2. Expression analyses revealed that CaSR proteins were expressed in α-smooth muscle actin-positive LX-2 cells. Extracellular Ca2+ restoration (from 0 to 2.2 mM) increased [Ca2+]cyt in these cells. The extracellular Ca2+-induced increase in [Ca2+]cyt was reduced by the CaSR antagonists, NPS2143 and Calhex 231. Furthermore, the growth of LX-2 cells was blocked by NPS2143 and Calhex 231 in concentration-dependent manners (IC50 = 6.0 and 9.5 µM, respectively). LX-2 cell proliferation was also attenuated by NPS2143 and Calhex 231. In conclusion, CaSRs are functionally expressed in activated HSCs and regulate Ca2+ signaling and cell proliferation. The present results provide insights into the molecular mechanisms underlying hepatic fibrosis and will contribute to the development of potential therapeutic targets.

Downregulation of Ca2+-Activated Cl- Channel TMEM16A Mediated by Angiotensin II in Cirrhotic Portal Hypertensive Mice.

Portal hypertension is defined as an increased pressure in the portal venous system and occurs as a major complication in chronic liver diseases. The pathological mechanism underlying the pathogenesis and development of portal hypertension has been extensively investigated. Vascular tone of portal vein smooth muscles (PVSMs) is regulated by the activities of several ion channels, including Ca2+-activated Cl- (ClCa) channels. TMEM16A is mainly responsible for ClCa channel conductance in vascular smooth muscle cells, including portal vein smooth muscle cells (PVSMCs). In the present study, the functional roles of TMEM16A channels were examined using two experimental portal hypertensive models, bile duct ligation (BDL) mice with cirrhotic portal hypertension and partial portal vein ligation (PPVL) mice with non-cirrhotic portal hypertension. Expression analyses revealed that the expression of TMEM16A was downregulated in BDL-PVSMs, but not in PPVL-PVSMs. Whole-cell ClCa currents were smaller in BDL-PVSMCs than in sham- and PPVL-PVSMCs. The amplitude of spontaneous contractions was smaller and the frequency was higher in BDL-PVSMs than in sham- and PPVL-PVSMs. Spontaneous contractions sensitive to a specific inhibitor of TMEM16A channels, T16Ainh-A01, were reduced in BDL-PVSMs. Furthermore, in normal PVSMs, the downregulation of TMEM16A expression was mimicked by the exposure to angiotensin II, but not to bilirubin. This study suggests that the activity of ClCa channels is attenuated by the downregulation of TMEM16A expression in PVSMCs associated with cirrhotic portal hypertension, which is partly mediated by increased angiotensin II in cirrhosis.

Involvement of TREK1 channels in the proliferation of human hepatic stellate LX-2 cells.

Activation of hepatic stellate cells (HSCs) causes hepatic fibrosis and results in chronic liver diseases. Although activated HSC functions are facilitated by an increase in the cytosolic Ca2+ concentration ([Ca2+]cyt), the pathophysiological roles of ion channels are largely unknown. In the present study, functional analyses of the two-pore domain K+ (K2P) channels, which regulate the resting membrane potential and [Ca2+]cyt, were performed using the human HSC line, LX-2. Expression analyses revealed that TREK1 (also known as KCNK2 and K2P2.1) channels are expressed in LX-2 cells. Whole-cell K+ currents were activated by 10 µM arachidonic acid and the activation was abolished by 100 µM tetrapentylammonium, which are pharmacological characteristics of TREK1 channels. The siRNA knockdown of TREK1 channels caused membrane depolarization and reduced [Ca2+]cyt. In addition, TREK1 knockdown downregulated the gene expression of collage type I and platelet-derived growth factor. Furthermore, TREK1 knockdown inhibited the proliferation of LX-2 cells. In conclusion, the activity of TREK1 channels determines the resting membrane potential and [Ca2+]cyt, which play a role in extracellular matrix production and cell proliferation in HSCs. This study may help elucidate the molecular mechanism underlying hepatic fibrosis in HSCs and provide a potential therapeutic target for hepatic fibrosis.