Testosterone rapidly increases Ca 2+ -activated K + currents causing hyperpolarization in human coronary artery endothelial cells Katesirin Ruamyod a , Wattana B. Watanapa a, *, Chairat Shayakul b a Department of Physiology Faculty of Medicine Siriraj Hospital, Mahidol University, Bangkok, 10700, Thailand b Department of Medicine, Faculty of Medicine Siriraj Hospital, Mahidol University, Bangkok, 10700, Thailand A R T I C L E I N F O Article history: Received 21 November 2016 Received in revised form 14 February 2017 Accepted 17 February 2017 Available online xxx Keywords: Androgen Endothelial ion channel Hyperpolarization Surface membrane receptor A B S T R A C T Testosterone has endothelium-dependent vasodilatory effects on the coronary artery, with some reports suggesting endothelial ion channel involvement. This study employed the whole-cell patch clamp technique to investigate the effect of testosterone on ion channels in human coronary artery endothelial cells (HCAECs) and the mechanisms involved. We found that 0.03–3 mM testosterone significantly induced a rapid, concentration-dependent increase in total HCAEC current (EC 50 , 71.96  1.66 nM; maximum increase, 59.13  8.37%; mean  SEM). The testosterone-enhanced currents consisted of small- and large-conductance Ca 2+ -activated K + currents (SK Ca and BK Ca currents), but not Cl  and nonselective cation currents. Either a non-permeant testosterone conjugate or the non-aromatizable androgen dihydrotestosterone (DHT) could increase HCAEC currents as well. The androgen receptor antagonist flutamide prevented this testosterone, testosterone conjugate, and DHT effect, while the estrogen receptor antagonist fulvestrant did not. Incubating HCAECs with pertussis toxin or protein kinase A inhibitor H-89 largely inhibited the testosterone effect, while pre-incubation with phospholipase C inhibitor U-73122, prostacyclin inhibitor indomethacin, nitric oxide synthase inhibitor L-NAME or cytochrome P450 inhibitor MS-PPOH, did not. Finally, testosterone application induced HCAEC hyperpolarization within minutes; this effect was prevented by SK Ca and BK Ca current inhibitors apamin and iberiotoxin. This is the first electrophysiological demonstration of androgen-induced K Ca current increase, leading to hyperpolarization, in any endothelial cell, and the first report of SK Ca as a testosterone target. Our data show that testosterone rapidly increased whole-cell HCAEC SK Ca and BK Ca currents via a surface androgen receptor, G i/o protein, and protein kinase A. This mechanism may explain rapid testosterone-induced coronary vasodilation seen in vivo. © 2017 Elsevier Ltd. All rights reserved. 1. Introduction The vascular system is a target for androgen actions. Studies have demonstrated that testosterone decreases blood pressure [1], and enhances coronary perfusion [2]. Testosterone deficiency, found in men with hypogonadism and 30% of 40–79-year-old men, is associated with hypertension and increased cardiovascular risks [3]. Interestingly, acute intravenous testosterone application (30 min) improved exercise-induced myocardial ischemia in men with coronary artery disease [4,5]. Moreover, a study in coronary artery disease patients found that intracoronary admin- istration of testosterone at a physiological dose caused coronary artery vasodilation within 3 min [2]. In experimental animals, acute androgen exposure elicited a hypotensive effect in both Abbreviations: ANOVA, analysis of variance; BK Ca , large conductance Ca 2+ -activated K + channel; DHT, dihydrotestosterone; DIDS, 4,4 0 -diisothiocyanatostilbene-2,2 0 - disulfonic acid; DMSO, dimethyl sulfoxide; EET, epoxyeicosatrienoic acid; EGTA, ethylene glycol-bis(b-aminoethyl ether)-N,N,N',N'-tetraacetic acid; eNOS, endothelial nitric oxide synthase or NOS3; FBS, fetal bovine serum; G i/o protein, G i (inhibitory G) or G o protein; H-89, N-[2-(p-bromocinnamylamino)ethyl]-5-isoquinolinesulfonamide; HCAEC, human coronary artery endothelial cell; HEPES, 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid; IK Ca , intermediate-conductance Ca 2+ -activated K + channel; K ATP , ATP- sensitive K + channel; K Ca , Ca 2+ -activated K + channel; K ir , inward rectifier K + channel; L-NAME, N v -nitro-L-arginine methylester; MS-PPOH, N-(methylsulfonyl)-2-(2- propynyloxy)-benzenehexanamide; NO, nitric oxide; NOS, nitric oxide synthase; NSC, nonselective cation channel; PKA, protein kinase A; PLC, phospholipase C; PTX, pertussis toxin; SEM, standard error of the mean; SK Ca , small-conductance Ca 2+ -activated K + channel; TEA, tetraethylammonium; testosterone-BSA, testosterone conjugated with bovine serum albumin; TRP, transient receptor potential channel; U-73122, 1-[6-[((17b)-3-methoxyestra-1,3,5[10]-trien-17-yl)amino]hexyl]-1H-pyrrole-2,5-dione. * Corresponding author at: Department of Physiology, Faculty of Medicine Siriraj Hospital, Mahidol University, 2 Wanglang Road, Bangkoknoi, Bangkok, 10700, Thailand. E-mail addresses: katesirin.ruy@mahidol.ac.th (K. Ruamyod), wattana.wat@mahidol.ac.th (W.B. Watanapa), chairat.sha@mahidol.ac.th (C. Shayakul). http://dx.doi.org/10.1016/j.jsbmb.2017.02.014 0960-0760/© 2017 Elsevier Ltd. All rights reserved. Journal of Steroid Biochemistry & Molecular Biology xxx (2016) xxx–xxx G Model SBMB 4894 No. of Pages 9 Please cite this article in press as: K. Ruamyod, et al., Testosterone rapidly increases Ca 2+ -activated K + currents causing hyperpolarization in human coronary artery endothelial cells, J. Steroid Biochem. Mol. Biol. (2017), http://dx.doi.org/10.1016/j.jsbmb.2017.02.014 Contents lists available at ScienceDirect Journal of Steroid Biochemistry & Molecular Biology journal homepage: www.else vie r.com/locate /jsbmb