Testosterone-induced relaxation of coronary arteries: activation of BKCa channels via the cGMP-dependent protein kinase

Abstract

Androgens are reported to have both beneficial and detrimental effects on human cardiovascular health. The aim of this study was to characterize nongenomic signaling mechanisms in coronary artery smooth muscle (CASM) and define the ionic basis of testosterone (TES) action. TES-induced relaxation of endothelium-denuded porcine coronary arteries was nearly abolished by 20 nM iberiotoxin, a highly specific inhibitor of large-conductance, calcium-activated potassium (BK(Ca)) channels. Molecular patch-clamp studies confirmed that nanomolar concentrations of TES stimulated BK(Ca) channel activity by ∼100-fold and that inhibition of nitric oxide synthase (NOS) activity by N(G)-monomethyl-L-arginine nearly abolished this effect. Inhibition of nitric oxide (NO) synthesis or guanylyl cyclase activity also attenuated TES-induced coronary artery relaxation but did not alter relaxation due to 8-bromo-cGMP. Furthermore, we detected TES-stimulated NO production in porcine coronary arteries and in human CASM cells via stimulation of the type 1 neuronal NOS isoform. Inhibition of the cGMP-dependent protein kinase (PKG) attenuated TES-stimulated BK(Ca) channel activity, and direct assay determined that TES increased activity of PKG in a concentration-dependent fashion. Last, the stimulatory effect of TES on BK(Ca) channel activity was mimicked by addition of purified PKG to the cytoplasmic surface of a cell-free membrane patch from CASM myocytes (∼100-fold increase). These findings indicate that TES-induced relaxation of endothelium-denuded coronary arteries is mediated, at least in part, by enhanced NO production, leading to cGMP synthesis and PKG activation, which, in turn, opens BK(Ca) channels. These findings provide a molecular mechanism that could help explain why androgens have been reported to relax coronary arteries and relieve angina pectoris.

Fig. 1.

Testosterone (TES)-induced coronary artery relaxation requires nitric oxide (NO)-stimulated large-conductance, calcium-activated potassium (BK_Ca) channel activity. A: representative traces of TES (25 μM)-induced maximal relaxation of endothelium-denuded coronary arteries precontracted with 10 μM prostaglandin F_2α (PGF_2α), in the presence or absence of iberiotoxin (20 nM; 30 min). The period of drug exposure is indicated by the line above the tracing or by arrow. B: typical BK_Ca channel recordings from the same cell-attached patch on a porcine coronary artery smooth muscle (CASM) cell (+40 mV) before (con) and 20 min after exposure to 100 nM TES and then after cumulative addition of 20 μM N^G-monomethyl-l-arginine (l-NMMA) and 500 μM 8-bromo (Br)-cGMP. Channel openings are upward deflections from the baseline (closed) state, indicated by dashed line (c). The opening level amplitude of a single BK_Ca channel is indicated by the dashed line at open level 1 (O1), whereas simultaneous opening of 2 channels is indicated by the dashed line at open level 2 (O2).

Fig. 2.

TES increases NO production in porcine and human CASM. A: average nitrite accumulation in porcine CASM before (con) and 30 min after 10 μM TES and in the presence of 20 μM l-NMMA or l-NMMA and 2 mM l-arginine (l-arg). Bars represent mean values ± SE (n = 4). P < 0.05 compared with control (*) and compared with TES alone (#). B: 4-amino-5-methylamino- 2′,7′-difluorescein (DAF-2) fluorescence recorded from human CASM myocytes before (con) and 20 min after 100 nM TES. The time course plot is a moving average of fluorescence intensity after addition of TES. C: DAF-2 fluorescence recorded from human CASM myocytes in the presence of 10 μM N^ω-propyl-l-arginine (l-NPA) and then 20 min after subsequent addition of 100 nM TES. The time course plot is a moving average of fluorescence intensity in the presence of l-NPA and TES normalized to control levels (i.e., 1.0). Inset: immunoblot detection of nitric oxide synthase isoforms expressed in human CASM (representative of n = 4 preparations). eNOS, endothelial nitric oxide synthase; nNOS, neuronal nitric oxide synthase.

Fig. 3.

A: the stimulatory effect of TES on BK_Ca channel activity is not easily reversed. Representative recordings (+40 mV) before (con) and 10 min after exposure to 100 nM TES and then 20 min after washout (w/o) of TES. Channel activity was only minimally reversed even 20–60 min after TES removal (n = 3 cell-attached patches). B: TES-stimulated BK_Ca channel activity is inhibited by wortmannin. Summary analysis of channel open probability (NP_o) before (con) and 10 min after exposure to 100 nM TES. In the presence of 50 nM wortmannin (Wt), TES had no significant effect on channel activity (n = 6; *P < 0.05 compared with control). Each bar represents the mean channel NP_o ± SE.

Fig. 4.

Summary of inhibitor action on TES-induced coronary artery relaxation. Relaxation response to 25 μM TES was measured in the presence of 20 μM l-NMMA (n = 6), 20 μM LY-83583 (LY, n = 6), 10 μM 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ, n = 6), or 100 μM SQ-22536 (SQ, n = 4). Relaxation to 500 μM 8-Br-cGMP was used as a positive control. Each bar represents the mean relaxation response ± SE (n = 4–6). *P < 0.05 compared with TES alone.

Fig. 5.

The cGMP-dependent protein kinase (PKG) is involved in TES-stimulated BK_Ca channel activity. A: summary of average BK_Ca channel activity (NP_o) from cell-attached patch recordings (porcine CASM myocyte, +40 mV) before (con) and 20 min after exposure to 100 nM TES and then after cumulative addition of 300 nM KT-5823. Each bar represents the mean channel NP_o ± SE (n = 3). P < 0.05 compared with control (*) and compared with TES alone (#). B: average activity of either PKG or cAMP-dependent protein kinase (PKA) measured in porcine CASM before (con) and 30 min after treatment with 1 or 10 μM TES (n = 6). Bars represent mean values ± SE. *P < 0.05 compared with control.

Fig. 6.

Purified PKG opens BK_Ca channels. Typical recordings from the same cell-free (inside-out) patch (+40 mV) before (control) and 5 min after exposure to purified PKG (400 U/ml; activated with 50 μM cGMP). Channel openings are upward deflections from baseline (closed) state, indicated by dashed line.

Fig. 7.

Summary model of nongenomic steroid [TES and 17β-estradiol (E2)] signal transduction in CASM. ER, estrogen receptor; AR, putative androgen receptor; PI3, phosphoinositide 3-kinase; Akt, protein kinase B; sGC, soluble guanylyl cyclase; BK, BK_Ca channel. Broken line indicates that TES may also stimulate production of NO from endothelial nitric oxide synthase (44).