Abstract
ObjectiveRemodeling of the pulmonary vasculature, involving increased proliferation and migration of pulmonary arterial smooth muscle cells (PASMCs), is a key component of the pathology underlying pulmonary hypertension (PH). Endothelin‐1 (ET‐1) is known to be an important contributor to both pulmonary vasoconstriction and remodeling in PH. We aimed to determine whether ET‐1 modulates PASMC function via a pathway involving Rho kinase (ROCK) and the Na+/H+ exchanger (NHE).MethodsPASMCs were isolated from distal pulmonary arteries of male C57/B6 mice as well as male Wistar rats and expanded in culture. To measure NHE activity, cells were incubated with pH‐sensitive fluorescent dye and the Na+‐dependent recovery of intracellular pH (pHi) following acidification by ammonium pulse challenge was measured via fluorescence microscopy. NHE1 mRNA expression was measured via qRT‐PCR and protein expression was measured via immunoblot. Proliferation was assessed via ELISA for BrdU incorporation over 24 hr. Migration was measured over 24 hr using Transwell filters with 8 μm pores. Cells were treated with 10−8 M ET‐1 versus vehicle for 24–48 hr. NHE was pharmacologically inhibited with 10 μM ethyl‐isopropyl amiloride. ROCK was inhibited with 10 μM Y‐27632.ResultsET‐1 treatment, compared to vehicle, resulted in increased resting pHi and NHE activity in PASMCs. ET‐1 did not result in increased NHE1 mRNA or protein expression. ROCK inhibition prevented ET‐1‐induced NHE activation. PASMC proliferation and migration were both increased by ET‐1 treatment, compared to vehicle. ROCK inhibition attenuated PASMC proliferation and migration in the presence of ET‐1. Similarly, NHE inhibition also attenuated ET‐1‐induced PASMC proliferation and migration.ConclusionsET‐1 results in increased PASMC proliferation and migration via a pathway involving ROCK and NHE activation. This suggests a novel pathway which may contribute to vascular remodeling in PH.Support or Funding InformationNIH K08HL133475 & R01HL073859This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Highlights
Three isoforms of endothelin (ET-1, ET-2, ET-3) have been identified, of which ET-1 is the most widely distributed (Rubin 2012)
We demonstrated that prolonged exposure to ET-1 increased resting pHi and Na+/H+ exchange (NHE) activity in rat pulmonary arterial smooth muscle cell (PASMC)
We showed that ET-1 enhanced PASMC proliferation and migration in vitro
Summary
Three isoforms of endothelin (ET-1, ET-2, ET-3) have been identified, of which ET-1 is the most widely distributed (Rubin 2012). Secreted from, vascular endothelium, ET-1 is one of the most potent and abundant endothelial-derived constricting factors identified to date. In the lung, both the synthesis and release of ET-1 are upregulated under a variety of conditions. Physiological Reports published by Wiley Periodicals, Inc. on behalf of The Physiological Society and the American Physiological Society.
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