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

Abstract

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.

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

“…We previously reported that spontaneous contractions were reduced by 30 µM T16A inh -A01 (a specific TMEM16A Cl Ca channel blocker). 13) In Cav1-KO PVSMs, their amplitude was increased (0.345 ± 0.071 mN, n = 4, p = 0.038; Figs. 1A, B), whereas their frequency was not altered (25.9 ± 3.1 min −1 , n = 4, p = 0.357; Figs.…”

“…Enhanced Spontaneous PVSM Contractions in Cav1-KO Mice PVSMs exhibit spontaneous and periodic contractions without any stimulations and spontaneous contractions are sensitive to Cl Ca blockers. 13) To demonstrate the direct involvement of Cav1 in spontaneous PVSM contractions, contractile responses were recorded in the PVSMs of WT and Cav1-KO mice. In WT PVSMs, the mean amplitude of spontaneous contractions was 0.163 ± 0.030 mN (n = 5; Figs.…”

“…Contractile Measurements The portal vein was dissected from WT mice (C57BL/6, male/female, 8 to 16 weeks old; Japan SLC, Hamamatsu, Japan) and Cav1-KO mice (C57BL/6 background, male/female, age-matched; Jackson Laboratory, Bar Harbor, ME, U.S.A.) in aerated Krebs' solution (112 mM NaCl, 4.7 mM KCl, 2.2 mM CaCl 2 , 1.2 mM MgCl 2 , 25 mM NaHCO 3 , 1.2 mM KH 2 PO 4 , 14 mM glucose, and pH 7.4 by gassing with a mixture of 95% O 2 /5% CO 2 ) and the endothelium was stripped out by water flow. 13) Portal vein smooth muscles (PVSMs; length of 5 mm) were set in a tissue chamber filled with aerated Krebs' solution at 37 °C and then stretched to 1 mN of tension. Contractile responses were measured using an isometric transducer (UM-203; Iwashiya Kishimoto Medical Instruments, Kyoto, Japan) and the data acquisition system (PowerLab 2/26; ADInstruments, Bella Vista, Australia).…”

“…Cell Isolation PVSMs were incubated in physiological saline solution (PSS; 125 mM NaCl, 5.4 mM KCl, 0.05 mM CaCl 2 , 15.4 mM NaHCO 3 , 0.33 mM Na 2 HPO 4 , 0.34 mM KH 2 PO 4 , 10 mM glucose, and 11 mM N-(2-hydroxyethyl) piperazine-N′-2-ethanesulfonic acid (HEPES)) containing 0.3% protease (type XIV; Sigma-Aldrich, St. Louis, MO, U.S.A.) at 37 °C for 5 min. 13) Tissues were then incubated in PSS containing 0.6% collagenase (type IA; Sigma-Aldrich) at 37 °C for 5 min. After the incubation, single PVSMCs were obtained by mechanical dispersion in PSS.…”

“…Quantitative Real-Time PCR mRNA expression levels in PVSMs were examined using the real-time PCR system (ABI PRISM 7000; Applied Biosystems, Foster City, CA, U.S.A.). 13) Specific primers were designed: Tmem16a (NM_178642, 2583-2715), Tmem16b (NM_153589, 2779-2899), Tmem16c (NM_001128103, 2716-2827), Tmem16d (NM_001277188, 3121-3233), Tmem16e (NM_177694, 2492-2592), Tmem16f (NM_001253813, 2592-2697), Tmem16g (NM_207031, 2338-2442), Tmem16h (NM_001164679, 2429-2536), Tmem16j (NM_178381, 2096-2199), Tmem16k (NM_133979, 1735-1836), and Gapdh (glyceraldehyde-3-phosphate dehydrogenase; NM_001289726, 776-879). Values for each unknown sample relative to the standard curve for specific primers were calculated and yielded transcriptional quantitation of gene products relative to the endogenous standard (Gapdh).…”