Research Of NFA’s Effect For TMEM16A On Pasmcs In Rats With High Pulmonary Blood Flow-Induced Pulmonary Hypertension

Objective To study the mechanism of NFA’s effect for TMEM16A (Transmembrane Protein 16A) in pulmonary artery smooth muscle cells (PASMCs) in rats with high pulmonary blood flow-induced pulmonary hypertension.Methods 40 male or female Sprague-Dawley rats (180-230g) were randomly divided into four groups:(1) Normal group:n=10; (2) sham group: n=10, only exposed the abdominal aorta and inferior vena about 10-20min; (3)Model group:n=10, rats were subjected to an abdominal aorta-inferior vena cava shunt to create animal models of high pulmonary; (4) NFA group:n=10, rats were subjected to the same surgery with Model group, then they were drenched by the inhibitor of calcium-activated chloride channels (CaCCs), niflumic acid (NFA), every day. All the rats were reared under the same conditions for 11 weeks. After that, the mean pulmonary artery pressure (mPAP) of every rat was determined by right heart catheterization. The pulmonary artery angiotasis was determined by the isolated organ perfusion system, and the expression of TMEM16A mRNA and protein were determined by Real-Time RT PCR and Western-Blot in pulmonary artery tissue in rats.Results (1) There was no significantly differ between Normal and Sham about the mPAP (P>0.05). The mPAP of Model and NFA groups were significantly higher than Sham group (all P＜0.001). In contrast to Model, the NFA decreased significantly, but it was still higher than Normal (P<0.001). (2) Under the same concentration of phenylephrine stimulation, the contraction percentage of pulmonary artery rings of Model group were significantly greater than the group of Normal, Sham and NFA (P＜0.01, respectively). In contrast to Normal and Sham group, the contraction percentage of pulmonary artery rings of NFA group were greater (P<0.01, respectively). But there was no statistically significant difference between group of Normal and Sham (P>0.05).With increasing of concentration of phenylephrine, the tension of pulmonary artery ring also increased. (3) Compared with Sham, the mRNA expression level of TMEM16A was increased in groups of Model and NFA (P＜0.001, respectively), but the degree of increase was inhibited in treatment with NFA (P＜0.001).In addition, Real-time RT-PCR showed no statistically significant with either group of Normal or Shan (P>0.05). (4) Western blotting analysis showed that the protein expression level of TMEM16A was significantly higher in Model and NFA group than in Normal and Sham group (P＜0.001), while there was significant difference between the group of Normal and Sham (P>0.05), and compared with Model group, the protein expression level was lower after NFA administration (P＜0.003).Conclusions (1) The up-regulation of TMEM16A’s expression in PASMCs was brought about the rise of pulmonary artery angiotasis in high pulmonary blood flow-induced pulmonary hypertension. It prompts that TMEM16A may probably participate in the process of the pulmonary hypertension induced by high pulmonary blood flow. (2) The pulmonary artery angiotasis of rats in high pulmonary blood flow-induced pulmonary hypertension was reduced by the application of NFA in vivo. Meanwhile, the expression of TMEM16A in PASMCs was also lowered by NFA. Therefore, we propose the idea that the regulatory role of CaCCs for high pulmonary blood flow-induced pulmonary hypertension was mediated by TMEM16A.