Hexane fraction of Prunus japonica thunb. Seed extract enhances boar sperm motility via CatSper ion channel.

Introduction: Mammalian sperm motility is facilitated by flagellar beating, which depends on active ion movement through ion channels and their regulation. Prunus japonica Thunb., also known as oriental bush cherry, is a widely used traditional medicinal plant. However, its significance in improving fertility and sperm quality has not been fully elucidated yet. One of our previous reports revealed that P. japonica seed extract (PJE) can improve human sperm motility through intracellular pH modulation. Aim of the study: The present study was designed to investigate the effects of PJE on boar spermatozoa and potential underlying mechanisms. Materials and methods: Sperm motility changes were examined using a computer-assisted sperm analysis (CASA) system under both capacitated and non-capacitated conditions. Intracellular calcium concentration was measured using either confocal microscopy or a fluorescent microplate reader with Fluo-4AM calcium fluorescent dye. Sperm capacitation-related proteins were analyzed using western blotting. Results: A significant increase in rapid motility, velocity, and linear displacement of sperm was observed in PJE-treated capacitated boar sperm, whereas the effect was insignificant in the non-capacitated counterparts. Intracellular calcium levels were significantly elevated upon PJE treatment (20-100 µg/L) in a concentration-dependent manner. The increase in intracellular calcium levels was inhibited when the sperm were treated with a CatSper (cation channel of sperm) channel inhibitor, 10 µM Mibefradil, indicating the involvement of the ion channel in the PJE modulatory mechanism. In addition, western blotting revealed an increased level of protein phosphorylation (p-tyrosine and p-PKA), which is a hallmark of sperm capacitation. Conclusions: PJE treatment resulted in a combination of increased motility, intracellular calcium concentration, and capacitation, thereby indicating its potential to ameliorate sperm motility parameters and induce capacitation of boar spermatozoa as a result of intracellular calcium elevation via the CatSper channel. Our observations further elaborate ion channel-related underlying mechanisms and show putative implications of the seed extract of traditionally used P. japonica Thunb. in ameliorating sperm quality.

In vitro assessment of Prunus japonica seed extract on human spermatozoa hypermotility and intracellular alkalization.

Prunus japonica var. nakaii is used in traditional Korean medicine to treat various conditions; however, it has not been investigated for treating male infertility. In this study, we investigated the in vitro effects of the ethanolic extract of P. japonica seeds on human sperm motility and identified its mechanism of action. Eleven male volunteers were selected, and the effects of the extract on human spermatozoa were assessed through a computer-assisted semen analysis. The P. japonica seed extract increased the percentage of total and progressive motility of spermatozoa. To understand the mechanism of action, we monitored intracellular alkalization using flow cytometry and obtained electrophysiological recordings of human voltage-gated proton channels hHv1 that were overexpressed in HEK-293 cells. The extract shifted the activation curves in a concentration-dependent manner. Two major constituents of the extract, linoleic acid and oleic acid, exhibited proton channel activity. Our in vitro experiments suggested that P. japonica seed extract could be potentially used to rescue sperm motility in idiopathic infertility patients via pharmacological modulation of the proton channels during capacitation. Therefore, our results indicate the therapeutic potential of P. japonica seed extract for treating male infertility.

Perspectives on Potential Fatty Acid Modulations of Motility Associated Human Sperm Ion Channels.

Human spermatozoan ion channels are specifically distributed in the spermatozoan membrane, contribute to sperm motility, and are associated with male reproductive abnormalities. Calcium, potassium, protons, sodium, and chloride are the main ions that are regulated across this membrane, and their intracellular concentrations are crucial for sperm motility. Fatty acids (FAs) affect sperm quality parameters, reproductive pathologies, male fertility, and regulate ion channel functions in other cells. However, to date the literature is insufficient to draw any conclusions regarding the effects of FAs on human spermatozoan ion channels. Here, we aimed to discern the possible effects of FAs on spermatozoan ion channels and direct guidance for future research. After investigating the effects of FAs on characteristics related to human spermatozoan motility, reproductive pathologies, and the modulation of similar ion channels in other cells by FAs, we extrapolated polyunsaturated FAs (PUFAs) to have the highest potency in modulating sperm ion channels to increase sperm motility. Of the PUFAs, the ω-3 unsaturated fatty acids have the greatest effect. We speculate that saturated and monounsaturated FAs will have little to no effect on sperm ion channel activity, though the possible effects could be opposite to those of the PUFAs, considering the differences between FA structure and behavior.

The agonistic action of URO-K10 on Kv7.4 and 7.5 channels is attenuated by co-expression of KCNE4 ancillary subunit.

KCNQ family constitutes slowly-activating potassium channels among voltage-gated potassium channel superfamily. Recent studies suggested that KCNQ4 and 5 channels are abundantly expressed in smooth muscle cells, especially in lower urinary tract including corpus cavernosum and that both channels can exert membrane stabilizing effect in the tissues. In this article, we examined the electrophysiological characteristics of overexpressed KCNQ4, 5 channels in HEK293 cells with recently developed KCNQ-specific agonist. With submicromolar EC50, the drug not only increased the open probability of KCNQ4 channel but also increased slope conductance of the channel. The overall effect of the drug in whole-cell configuration was to increase maximal whole-cell conductance, to prolongate the activation process, and left-shift of the activation curve. The agonistic action of the drug, however, was highly attenuated by the co-expression of one of the ß ancillary subunits of KCNQ family, KCNE4. Strong in vitro interactions between KCNQ4, 5 and KCNE4 were found through Foster Resonance Energy Transfer and co-immunoprecipitation. Although the expression levels of both KCNQ4 and KCNE4 are high in mesenteric arterial smooth muscle cells, we found that 1 µM of the agonist was sufficient to almost completely relax phenylephrine-induced contraction of the muscle strip. Significant expression of KCNQ4 and KCNE4 in corpus cavernosum together with high tonic contractility of the tissue grants highly promising relaxational effect of the KCNQspecific agonist in the tissue.

Characterization and functional roles of KCNQ-encoded voltage-gated potassium (Kv7) channels in human corpus cavernosum smooth muscle.

The group of KCNQ-encoded voltage-gated potassium (Kv7) channels includes five family members (Kv7.1-7.5). We examined the molecular expression and functional roles of Kv7 channels in corporal smooth muscle (CSM). Isolated rabbit CSM strips were mounted in an organ bath system to characterize Kv7 channels during CSM relaxation. Intracellular Ca2+ levels were measured in the CSM using the Ca2+ dye Fluo-4 AM. The expression of the KCNQ1-5 (the encoding genes for Kv7.1-7.5) and KCNE1-5 subtypes was determined by quantitative real-time PCR. Electrophysiological recordings and an in situ proximity ligation assay (PLA) were also performed. ML213 (a Kv7.2/7.4/7.5 activator) exhibited the most potent relaxation effect. XE911 (a Kv7.1-7.5 blocker) significantly inhibited the relaxation caused by ML213. Removal of the endothelium from the CSM did not affect the relaxation effect of ML213. H-89 (a protein kinase A inhibitor) and ESI-09 (an exchange protein directly activated by cAMP inhibitor) significantly inhibited ML213-induced relaxation (H-89: 31.3%; ESI-09: 52.7%). XE991 significantly increased basal [Ca2+]i in hCSM cells. KCNQ4 (the Kv7.4-encoding gene) and KCNE4 in CSM were the most abundantly expressed subtypes in humans and rats, respectively. KCNQ4 and KCNE4 expression was significantly decreased in diabetes mellitus rats. ML213 significantly increased the outward current amplitude. XE991 inhibited the ML213-induced outward currents. ML213 hyperpolarized the hCSM cell membrane potential. Subsequent addition of XE991 completely reversed the ML213-induced hyperpolarizing effects. A combination of Kv7.4 and Kv7.5 antibodies generated a strong PLA signal. We found that the Kv7.4 channel is a potential target for ED treatment.

Onion peel extract and its constituent, quercetin inhibits human Slo3 in a pH and calcium dependent manner.

Sperm function and male fertility are closely related to pH dependent K+ current (KSper) in human sperm, which is most likely composed of Slo3 and its auxiliary subunit leucine-rich repeat-containing protein 52 (LRRC52). Onion peel extract (OPE) and its major active ingredient quercetin are widely used as fertility enhancers; however, the effect of OPE and quercetin on Slo3 has not been elucidated. The purpose of this study is to investigate the effect of quercetin on human Slo3 channels. Human Slo3 and LRRC52 were co-transfected into HEK293 cells and pharmacological properties were studied with the whole cell patch clamp technique. We successfully expressed and measured pH sensitive and calcium insensitive Slo3 currents in HEK293 cells. We found that OPE and its key ingredient quercetin inhibit Slo3 currents. Inhibition by quercetin is dose dependent and this degree of inhibition decreases with elevating internal alkalization and internal free calcium concentrations. Functional moieties in the quercetin polyphenolic ring govern the degree of inhibition of Slo3 by quercetin, and the composition of such functional moieties are sensitive to the pH of the medium. These results suggest that quercetin inhibits Slo3 in a pH and calcium dependent manner. Therefore, we surmise that quercetin induced depolarization in spermatozoa may enhance the voltage gated proton channel (Hv1), and activate non-selective cation channels of sperm (CatSper) dependent calcium influx to trigger sperm capacitation and acrosome reaction.

Persistent Erectile Dysfunction after Discontinuation of 5-Alpha Reductase Inhibitor Therapy in Rats Depending on the Duration of Treatment.

PURPOSE: The current study is aimed to assess whether a longer duration of 5α-reductase inhibitor (5α-RI) exposure was associated with higher rate of permanent erectile dysfunction (ED) in a rat model. MATERIALS AND METHODS: Male Sprague-Dawley rats (n=76) were assigned to five groups: (i) normal control group; (ii) dutasteride (0.5 mg/rat/d) for 4-weeks group; (iii) dutasteride for 4-weeks plus 2-weeks of resting group; (iv) dutasteride for 8-weeks group; and (v) dutasteride for 8-weeks plus 2-weeks of resting group. In vivo erectile responses to electrical stimulation, and changes of fibrotic factors and smooth muscle/collagen contents in the corpus cavernosum were evaluated in each group. RESULTS: Dutasteride administration for 4 and 8 weeks significantly decreased erectile parameters compared with the control group. Reduced erectile responses were recovered during 2 weeks of drug-free time in the 4-week treatment group, but were not in the 8-week group. Protein levels of fibrosis-related factors transforming growth factor (TGF)-ß1, TGF-ß2, and p-Smad/Smad (Smad 2/3) in the corpus cavernosum showed no significant change after 4 weeks of dutasteride oral administration, but were enhanced after 8 weeks. Dutasteride markedly decreased smooth muscle content and increased collagen after 4 and 8 weeks of use, but no nuclear size changes; however, neither group showed significant improvement in the smooth muscle to collagen ratio after the rest period. CONCLUSIONS: Our study showed that recovery from ED depended on the duration of medication, and administration of dutasteride for more than 8-weeks in rats could result in irreversible ED even after discontinuation of medication.

Effect of BKCa Channel Opener LDD175 on Erectile Function in an In Vivo Diabetic Rat Model.

INTRODUCTION: The development of novel therapeutic options is imperative in patients with erectile dysfunction, especially those non-responsive to phosphodiesterase type 5 inhibitors. LDD175, a potent BKCa channel opener, has a relaxation effect on the in vitro cavernosal smooth muscle strip. AIM: To investigate the effect of LDD175 on erectile function using in vivo animal disease model. METHODS: Male Sprague-Dawley rats were assigned to a normal control group and seven diabetic groups: diabetic control, sildenafil (1 and 5 mg/kg), LDD175 (5 and 10 mg/kg), LDD175 5 mg/kg plus sildenafil 1 mg/kg, and LDD175 10 mg/kg plus tetraethylammonium. MAIN OUTCOME MEASURES: Intracavernosal pressure (ICP), ratio of ICP to mean arterial pressure (MAP), and the area under curve of ICP/MAP of eight groups were compared using in vivo pelvic nerve stimulation. RESULTS: The ICP, ICP/MAP ratio, and area under curve of the ICP/MAP ratio of the normal control rats increased with an increase in electrical field stimulation voltage. All parameters in the diabetic control group were significantly lower than those in the normal control rats, with an electrical field stimulation ranging from 1 to 5 V (P < .05). LDD175 improved the erectile response in diabetic rats in a dose-dependent manner. The combination of sildenafil (1 mg/kg) and LDD175 (5 mg/kg) showed a significant additive effect (P < .05) on the improvement of erectile function compared with sildenafil (1 mg/kg) alone. The enhancement of erectile function by LDD175 was completely blocked by tetraethylammonium. CONCLUSION: The results showed that the BKCa channel opener LDD175 improved erectile function in an in vivo diabetic rat model. Furthermore, combination therapy of LDD175 and sildenafil had an additive effect on the improvement of erectile function in diabetic rats. LDD175 could be a new candidate for the treatment of erectile dysfunction.

Gene expression profile comparison in the penile tissue of diabetes and cavernous nerve injury-induced erectile dysfunction rat model.

PURPOSE: To investigate the effects of cavernous nerve injury (CNI) on gene expression profiles in the cavernosal tissue of a CNI-induced erectile dysfunction (ED) model and to provide a basis for future investigations to discover potential target genes for ED treatment. MATERIALS AND METHODS: Young adult rats were divided randomly into 2 groups: sham operation and bilateral CN resection. At 12 weeks after CNI we measured erectile responses and performed microarray experiments and gene set enrichment analysis to reveal gene signatures that were enriched in the CNI-induced ED model. Alterations in gene signatures were compared with those in the diabetes-induced ED model. The diabetic-induced ED data is taken from GSE2457. RESULTS: The mean ratio of intracavernosal pressure/blood pressure for the CNI group (0.54±0.4 cmH2O) was significantly lower than that in the sham operation group (0.73±0.8 cmH2O, p<0.05). Supervised and unsupervised clustering analysis showed that the diabetes- and CNI-induced ED cavernous tissues had different gene expression profiles from normal cavernous tissues. We identified 46 genes that were upregulated and 77 genes that were downregulated in both the CNI- and diabetes-induced ED models. CONCLUSIONS: Our genome-wide and computational studies provide the groundwork for understanding complex mechanisms and molecular signature changes in ED.

Urinary Bladder-Relaxant Effect of Kurarinone Depending on Potentiation of Large-Conductance Ca2+-Activated K+ Channels.

The large-conductance calcium-activated potassium channel (BKCa channel) plays critical roles in smooth muscle relaxation. In urinary bladder smooth muscle, BKCa channel activity underlies the maintenance of the resting membrane potential and repolarization of the spontaneous action potential triggering the phasic contraction. To identify novel BKCa channel activators, we screened a library of natural compounds using a cell-based fluorescence assay and a hyperactive mutant BKCa channel (Lee et al., 2013). From 794 natural compounds, kurarinone, a flavanone from Sophora flavescens, strongly potentiated BKCa channels. When treated from the extracellular side, this compound progressively shifted the conductance-voltage relationship of BKCa channels to more negative voltages and increased the maximum conductance in a dose-dependent manner. Whereas kurarinone strongly potentiated the homomeric BKCa channel composed of only the α subunit, its effects were much smaller on heteromeric channels coassembled with auxiliary ß subunits. Although the activation kinetics was not altered significantly, the deactivation of BKCa channels was dramatically slowed by kurarinone treatment. At the single-channel level, kurarinone increased the open probability of the BKCa channel without affecting its single-channel conductance. Kurarinone potently relaxed acetylcholine-induced contraction of rat bladder smooth muscle and thus decreased the micturition frequency of rats with overactive bladder symptoms. These results indicate that kurarinone can directly potentiate BKCa channels and demonstrate the therapeutic potentials of kurarinone and its derivatives for developing antioveractive bladder medications and supplements.

New Methodology for Investigating Ejaculation Dysfunction: Measuring Intraluminal Seminal Vesicle Pressure in Rats with a Telemetric Device.

INTRODUCTION: Ejaculation dysfunction is one of the most common male sexual disorders. Despite its prevalence and adverse impact on patients, little attention has been given to investigating ejaculation dysfunction. AIM: We introduce a new method for evaluating ejaculation dysfunction in rats with a telemetric device. METHODS: A pressure transducer was surgically implanted in the seminal vesicles of 7-week-old male Sprague-Dawley rats. One week later, the rats were subcutaneously administered tamsulosin 3 µg/kg, and intra-seminal vesicle pressure (ISVP) was recorded in freely moving rats after an injection of apomorphine (80 µg/kg). Same rats repeated experiment with tamsulosin 10 µg/kg, silodosin 1 mg/kg, and normal saline with 3-day intervals. MAIN OUTCOME MEASURE: Sexual events were visually identified and recorded. Ejaculation was confirmed by visualization of a copulatory plug in the tip of the penis. We compared the maximal ISVP and area under the curve (AUC) of the ISVP. RESULTS: Adequate ISVP data were easily recorded and available in 66.6% rats (10/15) over a 6-week telemetric recording period (12 recordings). The mean number of ejaculations during an inspection time of 30 minutes was 1.5 ± 0.1. The maximal ISVP values in rats receiving 3 µg/kg (30.0 ± 5.2 mm Hg) and 10 µg/kg tamsulosin (15.1 ± 1.6 mm Hg) and 1 mg/kg silodosin (12.9 ± 2.2 mm Hg) were significantly lower than that in control rats (61.4 ± 13.4 mm Hg, P < 0.05). The AUC values in rats receiving 3 µg/kg (72.7 ± 18.9 mm Hg × s) and 10 µg/kg tamsulosin (23.5 ± 6.1 mm Hg) and 1 mg/kg silodosin (23.9 ± 8.0 mm Hg) were also lower than that of control rats (162.6 ± 34.3 mm Hg, P < 0.05). CONCLUSIONS: Telemetric ISVP assessment is reliable and feasible for investigating apomorphine-induced ejaculation in rats. Tamsulosin (3 µg/kg and 10 µg/kg) and silodosin 1 mg/kg decreased the ISVP during ejaculation.

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.

Endothelium-independent relaxant effect of Rubus coreanus extracts in corpus cavernosum smooth muscle.

INTRODUCTION: Rubus coreanus is a perennial shrub native to the southern part of the Korean peninsula. Although it is known that R. coreanus has a dose-dependent relaxation effect on rabbit corpus cavernosum (CC), the exact mechanism of action by which R. coreanus work is not fully known. AIMS: To elucidate the direct effects of unripe R. coreanus extract (RCE) on CC smooth muscle cells. METHODS: Dried unripe R. coreanus fruits were pulverized and extracted with 95% ethanol. Isolated rabbit CC strips were mounted in an organ-bath system, and the effects of RCE were evaluated. To estimate [Ca(2+)]i , we used a Fura-2 fluorescent technique. MAIN OUTCOME MEASURES: The effects of unripe RCE on ion channels and the intracellular Ca(2+) concentration ([Ca(2+)]i ) of CC. RESULTS: RCE effectively relaxed phenylephrine (PE)-induced tone in rabbit CC, and removal of the endothelium did not completely abolish the relaxation effect of RCE. Tetraethylammonium (1 mM) did not inhibit RCE-induced relaxation in strips precontracted by PE in the organ bath. However, CaCl2 -induced constriction of CC strips, bathed in Ca(2+)-free buffer and primed with PE, was abolished by RCE. In addition, RCE decreased basal [Ca(2+)]i in corporal smooth muscle cells. The increases of [Ca(2+)]i evoked by 60 mM K(+)-containing solution in A7r5 cells were suppressed by RCE, and RCE relaxed KCl-induced tone in endothelium-free CC, which indicated that RCE blocked the voltage-dependent Ca(2+) channels (VDCCs). RCE decreased basal [Ca(2+)]i and the [Arg8]-vasopressin-induced [Ca(2+)]i increases in A7r5 cells, and RCE inhibited the contraction of endothelium-free CC induced by PE in Ca(2+)-free solution, which suggested that RCE might act as a modulator of corporal smooth muscle cell tone by inhibiting Ca(2+) release from sarcoplasmic reticulum. CONCLUSION: RCE acts through endothelium-independent and endothelium-dependent pathways to relax CC. RCE may inhibit VDCCs and Ca(2+) release from sarcoplasmic reticulum.

Molecular and functional characterization of ORAI and STIM in human corporeal smooth muscle cells and effects of the transfer of their dominant-negative mutant genes into diabetic rats.

PURPOSE: We investigated the molecular identity and functional activity of STIM1 and ORAI in human cavernous smooth muscle. We also determined whether transferring dominant negative mutants of the STIM1 or ORAI gene would correct diabetes related erectile dysfunction in a rat model. MATERIALS AND METHODS: Reverse transcriptase-polymerase chain reaction was done to identify ORAI and STIM in human cavernous smooth muscle. For the in vivo study intracavernous pressure, blood pressure and their ratio were assessed after cavernous nerve stimulation to diabetic rats transfected with pcDNA encoding the ORAI1(DN) or the STIM1(DN) gene. RESULTS: ORAI (1, 2 and 3) and STIM (1 and 2) were identified in human cavernous smooth muscle cells. After [Ca(2+)] depletion by thapsigargin and cyclopiazonic acid we recorded store operated Ca(2+) entry in human cavernous smooth muscle cells. Entry was decreased by the store operated Ca(2+) channel blockers La(3+) and SKF96365. Mean ± SE intracavernous pressure/blood pressure in rats with ORAI1(DN) or STIM1(DN) gene transfer was 78.8% ± 2.2% and 77.1% ± 1.2% in 11 and 10, respectively. This result was significantly higher than that in 10 diabetic controls (51.0% ± 3.7%) and similar to that in 9 normal controls (85.8% ± 2.6%). Using reverse transcriptase-polymerase chain reaction we confirmed transgene expression in rat cavernous tissue. CONCLUSIONS: Transfer of ORAI(DN) or STIM1(DN) genes restored erectile function in diabetic rats. It might be applicable to develop new therapy for erectile dysfunction.

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

UNLABELLED: What's known on the subject? And what does the study add? Schisandra chinensis extract (SCE) has been known to have relaxative effects on penile smooth muscle. A recent study showed that SCE could enhance slidenafil citrate-induced relaxation of penile corpus cavernosum. The current study investigated the mechanism of action of SCE and its constituents on corporal smooth muscle cells. And this study shows that SCE induced relaxation of CSM primarily through an endothelium independent pathway and the relaxation effects of SCE on corporal smooth muscle are, in part, due to the activation of K(+) channels and inhibition of TRPC6 channels, resulting in decreased [Ca(2+)]. OBJECTIVE: • To evaluate the relaxant effects of Schisandra chinensis extract (SCE) on corporal tissue in the penis and to investigate the mechanism of action of SCE and its constituents on corporal smooth muscle (CSM) cells. MATERIALS AND METHODS: • The fruit of SC was collected and extracted with ethanol. Six SC lignans (schisandrol A, schisandrol B, schisandrin A, schisandrin B, gomisin N, and schisandrin C) were isolated and purified, and the chemical structures were confirmed by (1)H-nuclear magnetic resonance (NMR) and (13)C-NMR data. • Isolated rabbit CSM strips were mounted in an organ-bath system, and the effects of SCE were evaluated. • To estimate the intracellular Ca(2+) level ([Ca(2+)](i)), we used a Fura-2 fluorescent technique, and a conventional whole-cell patch-clamp technique was used to measure the calcium-sensitive K(+) channels (K(Ca)), inward rectifier K(+) channels (K(IR)), and canonical transient receptor potential cation channel 6 (TRPC6) currents. RESULTS: • SCE induced concentration-dependent relaxation in contracted CSM tissue, and the removal of the endothelium did not significantly affect their relaxation potencies. • In CSM cells, extracellular application of SCE significantly increased whole-cell K(Ca) currents (117.4%) and K(IR) currents (110.0%). These effects were completely abolished by charybdotoxin or BaCl(2). • In contrast, carbachol-induced TRPC6 channel activity was significantly inhibited (87.3%) by SCE in green fluorescent protein-TRPC6 pcDNA transfected HEK 293 cells. [Ca(2+)](i) measurements showed that SCE effectively reduced basal [Ca(2+)](i) in both cell lines (CSM cells and A7r5 cells) and the [Arg8]-vasopressin (AVP)-induced [Ca(2+)](i) increase in A7r5 cells. • Among the six SC lignans, schisandrin A and schisandrin B most effectively attenuated the AVP-induced [Ca(2+)](i) increase. CONCLUSIONS: • SCE induced relaxation of CSM that occurred primarily via an endothelium-independent pathway. • The relaxation effects of SCE on CSM were, in part, due to the activation of K(+) channels and inhibition of TRPC6 channels, resulting in decreased [Ca(2+)](i).

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.

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.

Gene transfer of TRPC6 (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 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.