Role of Connexin 43 increased expression in pulmonary arterial hyperreactivity induced by the nerve growth factor NGF

Abstract

We have previously shown that the nerve growth factor (NGF) plays a critical role in pathophysiology of pulmonary hypertension (PH) by promoting pulmonary arterial (PA) inflammation, remodelling and hyperreactivity. To characterize mechanisms activated by NGF to induce PA hyperreactivity, focusing in particular on Connexin 43 (Cx43), a gap junction protein essential for vascular reactivity. Cx43 expression was evaluated by Western blotting after NGF treatment (100 ng/ml, 24 h) ex vivo on rat pulmonary arteries or in vitro in primary human PA smooth muscle cells (hPASMC). Cx43 sub-cellular localization in hPASMC was evaluated by cell surface biotinylation and mitochondrial isolation. Contractions of control rat pulmonary arteries were induced ex vivo by phenylephrine (PHE, 10-10-10-4 M) in absence or presence of NGF (100 ng/ml, 24 h). The role of Cx43 in NGF-induced rat PA reactivity was evaluated ex vivo by use of 43Gap26 (Cx43 blocking peptide, 300 μM), or in vivo by use of an anti-Cx43 siRNA (10nmol IV). NGF increases Cx43 total expression in rat pulmonary arteries and in hPASMC. This effect is totally abolished after treatment with K252a (TrkA kinase inhibitor), wortmannin (PI3 K inhibitor) or PD98059 (ERK pathway inhibitor). NGF-induced PA hyperreactivity to PHE is inhibited ex vivo by treatment with 43Gap26 or in vivo after administration of Cx43 siRNA. Sub-cellular localization experiments in hPASMC show, as expected, Cx43 increased expression by NGF at the plasma membrane, but surprisingly also in mitochondria. These results show that NGF increases Cx43 PA expression through activation of its TrkA receptor and a PI3 K/ERK-dependent signalling pathway. Cx43 increased expression at hPASMC plasma membrane seems to participate in NGF-induced PA hyperreactivity. The role of Cx43 increased mitochondrial expression by NGF in hPASMC needs to be further elucidated.