Activation of endothelial BK Ca channels causes pulmonary vasodilation Alexander Vang a , Jeffrey Mazer a,b , Brian Casserly a,b , Gaurav Choudhary a,b, ⁎ a Vascular Research Laboratory, Providence VA Medical Center, Providence, RI, USA b Department of Medicine, Warren Alpert Medical School of Brown University, Providence, RI, USA abstract article info Article history: Received 21 December 2009 Received in revised form 28 April 2010 Accepted 6 May 2010 Keywords: Large-conductance calcium activated potassium channel Endothelial cells Lung Vasodilation Background: Large-conductance Ca 2+ -activated K + (BK Ca ) channels cause hyperpolarization and can regulate vascular tone. In this study, we evaluated the effect of endothelial BK Ca activation on pulmonary vascular tone. Methods: The presence of BK Ca channels in lung microvascular endothelial cells (LMVEC) and rat lung tissue was confirmed by RT-PCR, immunoblotting and immunohistochemistry. Isolated pulmonary artery (PA) rings and isolated ventilated-perfused rat lungs were used to assay the effects of BK Ca channel activation on endothelium-dependent vasodilation. Results: Immunoblotting and RT-PCR revealed the presence of BK Ca channel α- and β 4 -subunits in LMVEC. Immunohistochemical staining showed BK Ca channel α-subunit expression in vascular endothelium in rat lungs. In arterial ring studies, BK Ca channel activation by NS1619 enhanced endothelium-dependent vasodilation that was attenuated by tetraethylammonium and iberiotoxin. In addition, activation of BK Ca channels by C-type natriuretic peptide caused endothelial-dependent vasodilation that was blocked by iberiotoxin, L-NAME, and lanthanum. Furthermore, BK Ca activation by NS1619 caused a dose-dependent reduction in PA pressures that was attenuated by L-NAME. In vitro, BK Ca channel activation in LMVEC caused hyperpolarization and increased NO production. Conclusions: Pulmonary endothelium expresses BK Ca channels. Activation of endothelial BK Ca channels causes hyperpolarization and NO mediated endothelium-dependent vasodilation in micro- and macrovasculature in the lung. Published by Elsevier Inc. 1. Introduction The vascular endothelium is composed of a single layer of cells situated between circulating blood and vessel wall. This cell monolayer is responsible for regulating many different physiological functions, such as angiogenesis, metabolism, synthesis and degrada- tion of the extracellular matrix, and vascular tone and permeability (Aird, 2007). Endothelial membrane potential (E m ) plays an im- portant role in the regulation of many of these functions especially vascular tone (Nilius and Droogmans, 2001; Nilius et al., 1997). Endothelial cell hyperpolarization can increase the gradient for ex- tracellular Ca 2+ entry via non-specific cation channels or trp channels (Adams and Hill, 2004). Increased [Ca 2+ ] i stimulates the release of nitric oxide (NO), prostacyclin, and endothelium-derived hyperpolar- izing factors (EDHF) that act on vascular smooth muscle cells resulting in vasodilation (Himmel et al., 1993). In addition, the presence of myo-endothelial gap-junctions facilitates the hyperpolarization of vascular smooth muscle cells causing vasorelaxation (Chaytor et al., 1998; Larson and Sheridan, 1985; Ungvari et al., 2002; Yamamoto et al., 1999). Large-conductance calcium activated potassium (BK Ca ) channels are comprised of a pore-forming α-subunit that may be associated with a regulatory β-subunit (Ghatta et al., 2006). Each channel exists as a tetramer, composed of 4 α-subunits either alone or in association with β-subunit pair (Garcia-Calvo et al., 1994; Knaus et al., 1994). The α-subunit in BK Ca channels opens in response to depolarization and Ca 2+ binding, causing efflux of K + ions and hyperpolarization (Eichhorn and Dobrev, 2007). The β-subunits play important regulatory roles in modulating the activity of the channel (Brenner et al., 2000b; Cox, 2005; Ungvari et al., 2002). There have been several reports on the presence and role of vascular smooth muscle BK Ca channels in the regulation of pulmonary vascular tone (Barman et al, 2004; Bonnet et al., 2003a,b; Dubuis et al., 2005); however, there is no evidence yet supporting the role of endothelial BK Ca channels affecting pulmonary vascular tone. BK Ca channels have been observed in other endothelial cells (Faehling et al., 2001; Frieden et al., 2002; Kawasaki et al., 2004; Kim et al., 2006; Kuhlmann et al., 2005; Papassotiriou et al., 2000; Wang et al., 2005; Wrzosek et al., 2009) and we recently demonstrated that C-type natriuretic peptide (CNP) activated BK Ca channels in Vascular Pharmacology 53 (2010) 122–129 Abbreviations: BK Ca , Large-conductance Ca 2+ -activated K + channel; LMVEC, Lung microvascular endothelial cells; PA, Pulmonary artery; NO, Nitric oxide; EDHF, Endothelium-derived hyperpolarizing factors; CNP, C-type natriuretic peptide; IbTx, Iberiotoxin; TEA, Tetraethylammonium. ⁎ Corresponding author. Providence VA Medical Center, 830 Chalkstone Avenue, Providence, RI 02908, USA. Tel.: +1 401 273 7100x2029; fax: +1 401 457 3305. E-mail address: gaurav_choudhary@brown.edu (G. Choudhary). 1537-1891/$ – see front matter. Published by Elsevier Inc. doi:10.1016/j.vph.2010.05.001 Contents lists available at ScienceDirect Vascular Pharmacology journal homepage: www.elsevier.com/locate/vph