Upregulation of Ca 2+-Activated Cl - Currents and Tmem16a Expression in Pulmonary Artery of Chronic Hypoxic Rats

Abstract

Calcium-activated chloride Channel (CaCC) has been implicated to play important regulatory roles in vascular functions. Recent studies suggest that CaCC is encoded by TMEM16A. Here we sought to determine the expression of TMEM16A and CaCC currents (ICl.Ca) in pulmonary arterial smooth muscle cells (PASMCs), and their alterations in chronic hypoxic pulmonary hypertension (CHPH). TMEM16A mRNA was quantified using real-time RT-PCR in different arteries of normoxic rat. The expression was most abundant in pulmonary arteries (PA) with the order of large PA>aorta>small PA>cerebral arteries=tail arteries=mesenteric arteries=renal arteries, suggesting an important role of TMEM16A in pulmonary vasculature. The expression of TMEM16B, another closely related putative CaCC, was minimal. ICl.Ca and [Ca2+]i were recorded simultaneously using amphotericin-B perforated patch-clamp technique in Fluo-3/AM-loaded PASMCs. Under K+ free conditions, Ca2+ release activated by caffeine (5-10 mM) evoked robust outward currents at 0 mV (19.9±2.4 pA/pF) and inward current at −80 mV (−21.7±3.1 pA/pF). The reversal potential was −40.3±2.5 mV, similar to the predicted ECl. The current was inhibited by niflumic acid (100 μM), and the reversal potential shifted to 0 mV under symmetrical [Cl-], as consistent with ICl.Ca. Chronic exposure of rat to hypoxia (10% O2, 4 weeks) caused significant upregulation of TMEM16A mRNA, and a five-fold increase in protein levels in rat PA. Upregulation of TMEM16A protein was associated with comparable increase in whole-cell ICl.Ca density, recorded in PASMCs with [Ca2+]i clamped at 750 nM (normoxia: 24.4±3.3 pA/pF; hypoxia: 124.2±24.1 pA/pF at +120 mV). Hence, our results show that TMEM16A and functional CaCC are highly expressed in rat PA. Their upregulation by hypoxia may contribute to the enhanced pulmonary vascular reactivity in CHPH.