Chloride channels play an important role in regulating smooth muscle contraction and proliferation, and contribute to the enhanced vasoconstriction in pulmonary hypertension (PH). The intracellular Cl- concentrations ([Cl-]i) determine the driving force for Cl- conductance, thereby the functional consequence of channel activation. We have previously found that the [Cl-]i was increased in pulmonary artery (PA) smooth muscle cells of rats with chronic hypoxic PH (CHPH), which may lead to an increased depolarizing Cl- efflux during channel activation. Yet how the [Cl-]i is regulated in PA is unknown. Here we examined the expression profile in rat PAs of SLC12 family cation-coupled Cl- cotransporters (CCCs) and SLC4 family of Cl/HCO3 anion exchangers (AEs) that have been shown to regulate the intracellular Cl- homeostasis in other tissues. CHPH was induced by exposing rats to 10% O2 for 3 weeks. Conventional and real-time PCR were used to detect and quantify the mRNA expression in endothelium-denuded intralobal PAs. Normoxic PAs expressed all 9 CCCs and both Na+- dependent (SLC4A8) and -independent AEs (SLC4A1-3). SCL12A2 encoding for Na-K-2Cl cotransporter 1 (NKCC1) showed 45- and 12-fold higher expression than NKCC2-encoding SCL12A1 and Na-Cl cotransporter-encoding SLC12A3, respectively. Among the 4 genes that encode for K-Cl cotransporters (KCC) 1-4, the expression of SLC12A4, 6 and 7 was about 100-1000 fold higher than that of SCL12A5. Control PAs also showed higher SLC4A2 and 3 expression than SLC4A1 and 8. In hypoxic PAs, the expression of SCL12A2 encoding for Cl--accumulating NKCC1 was increased (copy number normalized to that of cyclophilin A served as an internal control: 1.23x10-2±.001, n=6 vs. 6.19x10-3±.0002, n=6, p<0.001) while that of SLC12A7 encoding for Cl--extruding KCC4 was reduced (4.92x10-3±.0005, n=6 vs. 1.14x10-2±.002, n=6, p<0.01), consistent with our previous functional findings. This study provides the first characterization of Cl- transporter expression profile and the first evidence of CHPH-associated transcriptional alterations in Cl- transporters in pulmonary arterial smooth muscle.
Altered expression of PPAR-γ and TRPC in neonatal rats with persistent pulmonary hypertension
