Small and Intermediate Calcium-Activated Potassium Channel Openers Improve Rat Endothelial and Erectile Function

Abstract

Modulation of endothelial calcium-activated potassium (K_Ca) channels has been proposed as an approach to restore endothelial function. The present study investigated whether novel openers of K_Ca channels with small (K_Ca2.x) and intermediate (K_Ca3.1) conductance, NS309 and NS4591, improve endothelium-dependent relaxation and erectile function. Rat corpus cavernosum (CC) strips were mounted for isometric tension recording and processed for immunoblotting. Mean arterial pressure (MAP), intracavernosal pressure (ICP), and electrocardiographic (ECG) measurements were conducted in anesthetized rats. Immunoblotting revealed the presence of K_Ca2.3 and large K_Ca conductance (K_Ca1.1) channels in the corpus cavernosum. NS309 and NS4591 increased current in CC endothelial cells in whole cell patch clamp experiments. Relaxation induced by NS309 (<1 μM) was inhibited by endothelial cell removal and high extracellular potassium. An inhibitor of nitric oxide (NO) synthase, and blockers of K_Ca2.x and K_Ca1.1 channels, apamin and iberiotoxin also inhibited NS309 relaxation. Incubation with NS309 (0.5 μM) markedly enhanced acetylcholine relaxation. Basal erectile function (ICP/MAP) increased during administration of NS309. Increases in ICP/MAP after cavernous nerve stimulation with NS309 were unchanged, whereas NS4591 significantly improved erectile function. Administration of NS309 and NS4591 caused small changes in the electrocardiogram, but neither arrhythmic events nor prolongation of the QTc interval were observed. The present study suggests that openers of K_Ca2.x and K_Ca3.1 channels improve endothelial and erectile function. The effects of NS309 and NS4591 on heart rate and ECG are small, but will require additional safety studies before evaluating whether activation of K_Ca2.3 channels has a potential for treatment of erectile dysfunction.

Keywords: KCa2.3; NS309; NS4591; calcium activated potassium channels; corpus cavernosum; erectile function.

Figure 1

Immunoblotting of calcium-activated potassium channels in rat corpus cavernosum and the heart. (A) Immunoblotting of the K_Ca1.1 alpha subunit (n = 11), K_Ca2.3 (n = 14), and K_Ca3.1 (n = 12) channels in corpus cavernosum. (B) Immunoblotting of the K_Ca1.1 alpha subunit (n = 6), K_Ca2.3 (n = 5), and K_Ca3.1 (n = 3) channels in the heart. The results were expressed as a ratio to pan actin and show expression of mainly K_Ca1.1 and K_Ca2.3 immunoreaction in the corpus cavernosum. Data are expressed as means ± S.E.M. *P ≤ 0.05, one-way ANOVA followed by Student's t-test.

Figure 2

K_Ca2.x channels are functionally expressed in endothelial cells from corpus cavernosum. Whole-cell voltage clamp recordings from endothelial cells derived from primary culture of corpus cavernosum (A–D). (A) Current evoked from voltage steps from −140 to +140 in control cells (left) or in presence of NS309 (1 μM; right). (B) Current-Voltage (I-V) relation in basal conditions (filled circles) and in response to 1 μM NS309 (filled squares). Results are means ± SEM. n = 4. (C) Current evoked from voltage steps from −140 to +140 in control cells (left) or in presence of NS4591 (1 μM; right). (D) I-V relation for control cells (filled circles) and in response to 1 μM NS459 (filled triangles). Results are mean ± SEM, n = 4. *P ≤ 0.05.

Figure 3

Involvement of the endothelium and nitric oxide (NO) in NS309 relaxation of rat corpus cavernosum. (A) Average concentration-response curves for NS309 (n = 10) compared with NS4591 (n = 11) in preparations with endothelium, (B) NS309 in preparations with endothelium (+E, n = 5) and without endothelium (−E, n = 5), (C) NS309 relaxation curves in the absence (n = 11) and the presence of nitro-L-arginine (L-NOARG 10⁻⁴ M, n = 7), and (D) NS309 relaxation curves in the absence (n = 6) and the presence of indomethacin (10⁻⁵ M, n = 6). Data are expressed as means ± S.E.M. *P ≤ 0.05, two-way ANOVA compared to preparations without endothelium or control preparations with endothelium.

Figure 4

Involvement of nitric oxide (NO) in acetycholine (ACh) relaxation of rat corpus cavernosum. Concentration-response curves for (A,C) ACh (n = 7,10) and (B,D) sodium nitroprusside (SNP) (n = 7) in the absence and the presence of (A,B) an inhibitor of NO synthase, L-nitro-arginine (L-NOARG, 100 μM) (n = 10,7) or (C,D) cyclooxygenase, indomethacin (10 μM) (n = 7,7). Data are expressed as means ± S.E.M. *P ≤ 0.05, curves were significantly different, two-way ANOVA followed by a Bonferroni's post-test.

Figure 5

Role of potassium blockaged, apamin and iberiotoxin-sensitive channels in NS309 relaxations of rat corpus cavernosum. Average concentration-response curves for NS309 (A) in absence (n = 5) and the presence of KPSS 119 mM plus noradrenaline (n = 5), (B) in absence (n = 5) and the presence of a K_Ca2 blocker apamin (0.5 μM, n = 5), (C) in the absence (n = 4) and the presence of a blocker of K_Ca3.1 channels, TRAM-34 (1 μM, n = 4), (D) in the absence (n = 5) and the presence of a blocker of K_Ca1.1, iberiotoxin (IbTx, 0.1 μM, n = 5). Data are expressed as means ± S.E.M. *P ≤ 0.05, two-way ANOVA compared to control preparations with endothelium.

Figure 6

Involvement of apamin- and iberiotoxin-sensitive channels in acetylcholine (ACh) relaxation of rat corpus cavernosum (CC). Concentration-response curves for ACh in the absence and the presence of (A) control (n = 13) vs. apamin (0.5 μM) (n = 6), (B) control (n = 8) vs. iberiotoxin (0.1 μM) (n = 9), (C) control (n = 8) vs. charybdotoxin (0.1 μM) (n = 10), and (D) control (n = 8) vs. TRAM-34 (1 μM) (n = 9). Data are expressed as means ± S.E.M. *P ≤ 0.05, curves were significantly different, two-way ANOVA followed by a Bonferroni post-test.

Figure 7

An opener of K_Ca2.x and K_Ca3.1 channels, NS309 selectively enhances acetylcholine relaxation in rat corpus cavernosum. Concentration-response curves for (A) acetylcholine (ACh) in the absence (n = 16) and the presence of NS309 (0.5 μM, n = 6), and (B) sodium nitroprusside (SNP) in the absence (n = 5) and in the presence of NS309 (n = 5) in noradrenaline (1 μM)-contracted corpus cavernosum strips. Data are expressed as means ± S.E.M. *P ≤ 0.05, curves were significantly different, two-way ANOVA followed by a Bonferroni's post-test.

Figure 8

Original recordings showing the effect of NS309 and NS4591 on mean arterial pressure (MAP) intracavernous pressure (ICP) in rat penis. The upper traces show mean arterial pressure (MAP) and the lower traces the changes in intracavernous pressure (ICP) induced by submaximal stimulation of the cavernous nerve before and 3 min after (A) NS309 and (B) NS4591 in the rat.

Figure 9

Average effect of NS309 and NS4591 on mean arterial pressure (MAP) and erectile function measured as peak intracavernosal pressure (PICP) over MAP in rats. A maximal and submaximal response to electrical stimulation was obtained before infusion of either vehicles, (A) dimethylsulpoxide (DMSO, 1 mgKg⁻¹, n = 7), (B) polyethylene glycol (PEG, 1 mgKg⁻¹, n = 7), (C) NS309 (1 mgKg⁻¹, n = 5), or (D) of NS4591 (1 mgkg⁻¹, n = 6), and the submaximal stimulations were performed 3, 13, 23, and 33 min after drug infusion. Finally, a maximal stimulation of the cavernous nerve was performed. The results show a facilitating effect on submaximal stimulation 3 min after NS4591 infusion. The direct effects of the drugs on MAP and intracavernosal pressure are depicted in Supplementary Figure S7. Data are expressed as means ± S.E.M. *P ≤ 0.05 compared with submaximal control response in the same animals using one-way ANOVA followed by Dunnett's test.

Figure 10

Original traces showing the effect of NS309 and NS4591 on the electrocardiography of the rat heart. (A,B) Representative ECG obtained as a control value for NS309 and NS4591. (C) Representative ECG obtained during NS309 administration. (D) Representative ECG obtained after NS309 administration. (E) Representative ECG obtained during NS4591 administration. (F) Representative ECG obtained after NS4591 administration.