Abstract

Calcium signaling is key for contraction, differentiation and proliferation of pulmonary artery smooth muscle cells (PASMC). Further, calcium influx through store operated channels (SOC) is particularly important in the vasoconstrictor response to hypoxia. We have previously observed a reduction of pulmonary hypertension and remodeling in newborn lambs exposed to perinatal chronic hypoxia, when treated with 2-aminoethyldiphenylborinate (2-APB), a SOC inhibitor, among other targets (1). Nevertheless, the effect of 2-APB on pulmonary artery reactivity and mitogen expression is poorly characterized in whole animal models. Here, we studied the effect of a 2-APB treatment on the response of pulmonary arteries function, and the expression of transcripts and proteins involved in signaling regulation of contractile and proliferative response to hypoxia. Twelve 3-4 days-old newborn lambs with 66% of gestation at highlands (3600 m) and returned to lowlands 2 days after delivery simulating in part the condition of a newborn whose pulmonary hypertension persists despite oxygen therapy, were treated with a daily single dose of 2-APB (10 mg.kg-1, i.v.) or its vehicle (DMSO:saline 1:10) during 10 days. At the end of the treatment, lung samples were collected for ex vivo and in vitro experiments. We determined the responses of small pulmonary arteries to the vasoconstrictors endothelin-1 (ET-1) and a thromboxane A2 mimetic (U46619), and the vasodilators PKG activator (8Br-cGMP) and fasudil (Rho-kinase inhibitor), by wire myography. Further, we determined the pulmonary expression of TP receptor, PKG1, ROCK 1, 2 and PKG1 by immunoblot, the expression of calcium-regulated mitogens ppET1, PDGF and VEGF-A by RT-PCR, and the proliferation marker Ki67 by immunohistochemistry. All procedures were approved by the Bioethical Committee of the Faculty of Medicine, University of Chile. 2-APB treatment produced: a) a reduction of the maximal contraction to ET-1 and U46619 without modification of TP receptor expression; b) an increase of the maximal relaxation to 8BrcGMP, and the maximal relaxation and sensitivity to fasudil, without effect on PKG-1 expression but with an increase in both ROCK1 and ROCK2 proteins; c) a decrease of VEGF-A without modification of PDGF and ppET1 transcripts; and d) a decrease of Ki67 positive nuclei in the medial layer of pulmonary arteries. These results indicate that the reduction of mPAP and remodeling after 2-APB treatment is the result of a new balance between vasodilator and vasoconstrictor function and the reduction of mitogen expression and proliferation of PASMC, despite an increase of ROCK function, suggesting a potential role of SOC inhibitors for neonatal pulmonary hypertension treatment.