Mee Ree Chae

Effect of Testosterone on Potassium Channel Opening in Human Corporal Smooth Muscle Cells

INTRODUCTION In humans, the role of testosterone in sexual functions, including sexual desire, nocturnal penile erections, and ejaculatory volume, has been relatively well established. However, the effects of testosterone on intrapenile structure in humans remains controversial. AIM We assessed the direct effects of testosterone on potassium channels in human corporal smooth muscle cells, in an effort to understand the mechanisms inherent to the testosterone-induced relaxation of corporal smooth muscle cells at the cellular and molecular levels. Methods. We conducted electrophysiologic studies using cultured human corporal smooth muscle cells. We evaluated the effects of testosterone on potassium channels-BK(Ca) and K(ATP) channels-by determining the whole-cell currents and single-channel activities. For the electrophysiologic recordings, whole-cell and cell-attached configuration patch-clamp techniques were utilized. MAIN OUTCOME MEASURES Changes in whole-cell currents and channel activities of BK(Ca) and K(ATP) channels by testosterone. Results. Testosterone (200 nM) significantly increased the single-channel activity of calcium-activated potassium (BK(Ca)) channels and whole-cell K(+) currents by 443.4 +/- 83.4% (at +60 mV; N = 11, P < 0.05), and this effect was abolished by tetraethylammonium (TEA) (1 mM), a BK(Ca) channel blocker. The whole-cell inward K(+) currents of the K(ATP) channels were also increased by 226.5 +/- 49.3% (at -100 mV; N = 7, P < 0.05). In the presence of a combination of vardenafil (10 nM) and testosterone (200 nM), the BK(Ca) channel was activated to a significantly higher degree than was induced by testosterone alone. CONCLUSIONS The results of patch-clamp studies provided direct molecular evidence that testosterone stimulates the activity of BK(Ca) channels and K(ATP) channels. An understanding of the signaling mechanisms that couple testosterone receptor activation to potassium channel stimulation will provide us with an insight into the cellular processes underlying the vasorelaxant effects of testosterone.

Lysophosphatidylcholine, a component of atherogenic lipoproteins, induces the change of calcium mobilization via TRPC ion channels in cultured human corporal smooth muscle cells.

Hypercholesterolemia is a major risk factor for erectile dysfunction. To understand the mechanism(s) of hypercholesterolemia-induced erectile dysfunction, we studied the effect of lysophosphatidylcholine (LPC) on the membrane conductance of corporal smooth muscle cells. We used cultured human corporal smooth muscle cells. The intracelluar Ca2+ concentration ([Ca2+]i) and the influx of divalent cation was monitored by the ratio of fura-2 fluorescence (F340/380) and by the Mn2+-induced quenching rate of fura-2, respectively. The LPC-induced membrane current was characterized by the whole-cell patch-clamp technique and the molecular identity of suspected channels was probed by RT-PCR. LPC (20 μM) induced a statistically significant increase in F340/380 to 119.9±3.9% of initial control (n=6) in corporal smooth muscle cells. The addition of 20 μM LPC accelerated the quenching rate of F360 by 59.5±11.8% (n=5). LPC activated nonselective cationic current (ILPC), similar to the known effects of phenylephrine in corporal myocytes. The size of ILPC at −60 mV was −55.3±6.3 pA (n=8). The transcript of transient receptor potential channel 6 (TRPC6) was detected in human corporal myocytes. We also found one splicing variant of TRPC6, TRPC6α. In conclusion, the present study suggests that the LPC, a major component of oxidized low-density lipoprotiens, increases calcium in corporal smooth muscle cells probably through activation of a TRPC6 channel and the increased [Ca2+]i by LPC via TRP channels is one of mechanisms for hypercholesterolemia-induced erectile dysfunction.

Effects of Schisandra chinensis extract on the contractility of corpus cavernosal smooth muscle (CSM) and Ca2+ homeostasis in CSM cells.

Whats known on the subject? and What does the study add?

Gene Transfer of TRPC6DN (Dominant Negative) Restores Erectile Function in Diabetic Rats

INTRODUCTION Transient receptor potential (TRP) channels play an important role in modulating intracellular Ca(2+) ([Ca(2+)](i)) levels. AIM We examined the hypothesis that overexpression of TRPC6(DN) (dominant negative) may contribute to decreased [Ca(2+)](i) levels in corporal smooth muscle (CSM). We also investigated whether gene transfer of TRPC6(DN) could restore erectile function in diabetic rats. METHODS For the in vitro study, the K(Ca), K(ATP), and TRPC6(DN) channel genes were transferred using cDNA, into cultured human CSM cells and human embryonic kidney cells. For the in vivo study, young adult rats were divided into three groups: normal controls; diabetic controls transfected with vector only; and a diabetic group transfected with pcDNA of the TRPC6(DN) gene. MAIN OUTCOME MEASURES After gene transfer, the effects of reducing [Ca(2+)](i) levels were assessed by Fura-2-based imaging analysis. The intracavernosal pressure (ICP) response to cavernosal nerve stimulation was assessed after intracorporal injection of TRPC6(DN) pcDNA. The transgene expression of the TRPC6(DN) was examined by reverse transcription polymerase chain reaction (RT-PCR) in rats transfected with TRPC6(DN) pcDNA. RESULTS Gene transfer of ion channels effectively reduced [Ca(2+)](i). Among these channels, transfer of the TRPC6(DN) gene resulted in the greatest reduction of [Ca(2+)](i) in human CSM. The mean (+/-standard error of the mean) ratio of ICP to mean arterial pressure (BP) in the gene-transfer rats was 79.4 +/- 2.4% (N = 8). This was significantly higher than that in control rats (55.6 +/- 3.7% [N = 8]), and similar to that in the young control rats (83 +/- 2.2% [N = 12]). The RT-PCR showed expression of TRPC6(DN) genes in the transfected rats. CONCLUSION Gene transfer of TRPC6(DN) not only reduced [Ca(2+)](i) in human CSM but also restored erectile function in diabetic rats. These results suggest that pcDNA transfer of TRPC6(DN) may represent a promising new form of therapy for the treatment of male erectile dysfunction in the future.

Effect of the Novel BKCa Channel Opener LDD175 on the Modulation of Corporal Smooth Muscle Tone

INTRODUCTION The BKCa channel has been reported to play an important role in erectile function. Recently, novel BKCa channel activator, LDD175, was introduced. AIM This study aims to investigate whether LDD175 relaxes corporal smooth muscle (CSM) via BKCa channel activation. METHODS After isolation of CSM strip from a male rabbit model, contraction studies using organ bath was performed. Isolating human tissue and cell cultures, electrophysiological studies were done via whole-cell patch-clamp recording. MAIN OUTCOME MEASURES Vasodilatory effects of LDD175 were evaluated by cumulative addition ranging from 10(-7) to 10(-4) M in corpus cavernosal strips after precontraction with 10(-5) M phenylephrine via organ bath system. Using cultured human CSM cells, patch-clamp recording was performed. Erectile function was measured by in vivo rat cavernous nerve stimulation. RESULTS LDD175 caused an endothelium-independent relaxation of corporal tissues, and this effect was abolished by pretreatment with iberiotoxin. The relaxation effect of 10(-4) M LDD175 was greater than that of 10(-6) M udenafil (54.0 ± 3.1% vs. 34.5 ± 3.9%, P < 0.05); 10(-5) M LDD175 with 10(-6) M udenafil caused a greater relaxation effect on strips than 10(-5) M LDD175 or 10(-6) M udenafil alone (50.7%, 34.1%, vs. 20.7%, respectively, P < 0.001). In patch-clamp recordings, LDD175 increased K(+) currents in a dose-dependent manner, and washout of LDD175 or the addition of iberiotoxin fully reversed the increase. Intravenous LDD175 improved erectile function measured by area under the curve (AUC) of the intracavernosal pressure (ICP)/arterial blood pressure (ABP) ratio (1,612.1 ± 135.6 vs. 1,093.7 ± 123.1, P < 0.05). There was no difference between 10 mg/kg LDD175 and 1 mg/kg udenafil regarding maximal ICP, maximal ICP/ABP ratio, and the AUC of the ICP/ABP ratio (P > 0.05). CONCLUSIONS LDD175 leads to an endothelium-independent relaxation of erectile tissue, primarily through the opening of BKCa channels. The results suggest that LDD175 might be a new candidate treatment for erectile dysfunction.

Effects of Schisandra chinensis extract on the relaxation of isolated human prostate tissue and smooth muscle cell

ETHNOPHARMACOLOGICAL RELEVANCE Schisandra chinensis has been commonly used as a traditional herbal medicine to treat various diseases including body weakness, dysentery, impotence, enuresis and frequent urination in many countries including Korea, China and Russia. Benign prostate hyperplasia is a common disease for the elderly men and it induces lower urinary tract symptoms which hinder general activity and quality of life. We evaluated the therapeutic potential of Schisandra chinensis extract (SCE) in benign prostate hyperplasia using human prostate tissue. MATERIALS AND METHODS Schisandra chinensis fruit was collected and extracted with ethanol. Human prostate tissues were obtained from 14 prostate cancer patients. Macroscopically normal tissue was excised from the transition zone and the periurethral regions. Isolated prostate tissue strips were mounted in an organ-bath system, and the relaxation effect of SCE was evaluated by cumulative addition to prostate strips pre-contracted with 10(-5)M norepinephrine. The effect of tamsulosin was compared, and the additive effect was evaluated. Electrophysiological studies using cultured human prostate smooth muscle cells (HPrSMC) were conducted. RESULTS Cumulative dosing of SCE induced concentration-dependent relaxation in contracted prostate tissue (n=18, P<0.05). Simultaneous dosing of SCE and tamsulosin showed an additive relaxation effect. The relaxation effect of SCE was abolished by inhibition of K+ channels by pre-treatment with tetraethylammonium. In HPrSMC, extracellular application of 100 μg/mL SCE significantly increased outward currents, and this effect was significantly attenuated by treatment with 100 nM Iberiotoxin. CONCLUSIONS SCE showed a dose dependent relaxation effect on human prostate tissue as well as an additive effect with tamsulosin. The relaxation effects of SCE on HPrSMC were, in part, due to the activation of K+ channels.

Effects of Ginkgo biloba extracts with mirodenafil on the relaxation of corpus cavernosal smooth muscle and the potassium channel activity of corporal smooth muscle cells

In this study, we investigated the effects of a combination of Ginkgo biloba extracts (GBE) and phosphodiesterase type 5 (PDE-5) inhibitors on the muscular tone of the corpus cavernosum and potassium channel activity of corporal smooth muscle cells. Strips of corpus cavernosum from male New Zealand white rabbits were mounted in organ baths for isometric tension studies. After contraction with 1×10⁻⁵ mol l⁻¹ norepinephrine, GBE (0.01-1 mg ml⁻¹) and mirodenafil (0.01-100 nmol l⁻¹) were added together into the organ bath. In electrophysiological studies, whole-cell currents were recorded by the conventional patch-clamp technique in cultured smooth muscle cells of the human corpus cavernosum. The corpus cavernosum was relaxed in response to GBE in a dose-dependent manner (from 0.64%±8.35% at 0.01 mg ml⁻¹ to 52.28% ± 11.42% at 1 mg ml⁻¹). After pre-treatment with 0.03 mg ml⁻¹ of GBE, the relaxant effects of mirodenafil were increased at all concentrations. After tetraethylammonium (TEA) (1 mmol l⁻¹) administration, the increased effects were inhibited (P<0.01). Extracellular administration of GBE increased the whole-cell K(+) outward currents in a dose-dependent fashion. The increase of the outward current was inhibited by 1 mmol l⁻¹ TEA. These results suggest that GBE could increase the relaxant potency of mirodenafil even at a minimally effective dose. The K(+) flow through potassium channels might be one of the mechanisms involved in this synergistic relaxation.

Increased expression of TRPC4 channels associated with erectile dysfunction in diabetes

In recent reports, an association between altered TRPC channel function and the development of various diabetic complications has drawn the attention of many investigators. The aim of this study was to investigate the expression of TRPC4 channels of corpus smooth muscle (CSM) cells in diabetes, and to evaluate the association between erectile dysfunction (ED) and altered TRPC4 channel function. The expression of TRPC4 in the penile tissue of human, normal and diabetic rat was investigated using RT‐PCR, western blotting and immunohistochemistry (IHC). In vivo gene transfer of dominant negative (DN) TRPC4 into the CSM of rat was conducted. In vivo pelvic nerve stimulation was performed to measure erectile function. Expression of TRPC1, TRPC3, TRPC4 and TRPC6 in human and rat CSM tissues was confirmed by RT‐PCR, western blot and IHC. In the diabetic rat, the expression levels of mRNA and protein of the TRPC4, and TRPC6 were significantly increased compared to control rats (p < 0.05). The change in TRPC4 expression in the diabetic rats was higher than those of the other TRPC subunits (p < 0.05). The IHC showed that only TRPC4 expression had a higher intensity in the diabetes compared to normal rats (p < 0.05). Gene transfection with TRPC4DN into the diabetic rats restored erectile function to levels similar to that of normal controls. Gene expression of TRPC4DN in CSM tissue was confirmed by RT‐PCR 2 weeks after transfection. This study demonstrated that TRPC4 channel expression increased in the penile CSM cells of diabetic rats. The down‐regulation of TRPC4 with DN form restored erectile function in the diabetic rats. The alteration of TRPC4 channel is one of pathophysiology of ED and could be a target for drug development for ED.

Effects of ethanol on the tonicity of corporal tissue and the intracellular Ca2+ concentration of human corporal smooth muscle cells

Heavy alcohol consumption is associated with an increased risk of erectile dysfunction (ED); however, the acute effects of ethanol (EtOH) on penile tissue are not fully understood. We sought to investigate the effects of EtOH on corporal tissue tonicity, as well as the intracellular Ca(2+) concentration ([Ca(2+)](i)) and potassium channel activity of corporal smooth muscle. Strips of corpus cavernosum (CC) from rabbits were mounted in organ baths for isometric tension studies. Electrical field stimulation (EFS) was applied to strips precontracted with 10 μmol L(-1) phenylephrine as a control. EtOH was then added to the organ bath and incubated before EFS. The [Ca(2+)](i) levels were monitored by the ratio of fura-2 fluorescence intensities using the fura-2 loading method. Single-channel and whole-cell currents were recorded by the conventional patch-clamp technique in short-term cultured smooth muscle cells from human CC tissue. The corpus cavernosal relaxant response of EFS was decreased in proportion to the concentration of EtOH. EtOH induced a sustained increase in [Ca(2+)](i) in a dose-dependent manner, Extracellular application of EtOH significantly increased whole-cell K(+) currents in a concentration-dependent manner (P < 0.05). EtOH also increased the open probability in cell-attached patches; however, in inside-out patches, the application of EtOH to the intracellular aspect of the patches induced slight inhibition of Ca(2+)-activated potassium channel (KCa) activity. EtOH caused a dose-dependent increase in cavernosal tension by alterations to [Ca(2+)](i). Although EtOH did not affect KCa channels directly, it increased the channel activity by increasing [Ca(2+)](i). The increased corpus cavernosal tone caused by EtOH might be one of the mechanisms of ED after heavy drinking.

Comparisons of apomorphine‐induced erection and spontaneous erection in rats by telemetric assessment of intracavernosal pressure

Although there are several methods for assessing erectile function in rats, the standard methods for telemetric monitoring have not been established. Theoretically assessment of spontaneous erection (SE) seems to be a physiologic method but it needs long measuring time and additional efforts. Apomorphine‐induced erection (AIE) is one available and simple method; however, the correlation with SE has not been assessed. We compared erection profiles of AIE and SE in normal and two disease rat models using telemetric assessment of intracavernosal pressure (ICP). Seven‐week‐old male Sprague–Dawley rats were assigned to normal control, diabetes mellitus (DM) and hypercholesterolemia (HC) group. After 19 weeks a telemetric pressure sensor (C40; Data Sciences) was surgically implanted in the corpus cavernosum. One week later, ICP was recorded in freely moving rats after intraperitoneal apomorphine (100 μg/kg) injection (AIE) or during SE. Sexual events were visually identified and recorded. Only the pressure increases that occurred during sexual behavior were analyzed. We compared the erectile profiles such as duration, maximal ICP and the area under the curve (AUC, area under time × ICP curves). Two‐way anova revealed no significant effect of the measuring methods on the mean AUC (F1,43 = 2.756, p‐value = 0.104), but a significant effect of different disease models on mean AUC (two‐way anova: F2,43 = 12.929, p‐value < 0.001) was observed. The mean AUC of normal control rats was significantly higher than that of DM and HC rats (Bonferroni post hoc test: p < 0.001 and p = 0.001, respectively). ICP measurements using a telemetric device showed no significant difference in AUC between AIE and SE. AIE is easy and requires less time than SE measurements. Therefore, AIE could be a useful method to evaluate ICP in rats.