Isolated Rabbit Corpus Cavernosum Research Articles

6-Nitrodopamine is an endogenous mediator of the rabbit corpus cavernosum relaxation.

6-Nitrodopamine (6-ND) is a novel endogenous catecholamine that has a potent relaxant action on vascular smooth muscle in vitro. To evaluate the basal release of 6-ND and noradrenaline from rabbit-isolated corpus cavernosum (RbCC) and its relaxing action on this tissue. Rabbit corpus cavernosa were dissected and suspended in a 5-mL organ bath containing oxygenated Krebs-Henseleit's solution. 6-ND and noradrenaline release was quantified by liquid chromatography coupled to tandem mass spectrometry. The relaxant activity of 6-ND was assessed in RbCC strips pre-contracted with endothelin-1 (10nM). Rabbit corpus cavernosum presented basal release of both 6-ND (2.9±0.8ng/mL, n=12) and noradrenaline (1.7±1.3ng/mL, n=12). The 6-ND release was reduced by pre-treatment with Nω -nitro-l-arginine methyl ester (l-NAME) (100 µM), whereas that of noradrenaline was unaffected. Tetrodotoxin (TTX, 1 µM) abolished the noradrenaline release but had no effect on 6-ND release, indicating a non-neurogenic origin for 6-ND. 6-ND and the selective dopamine D2 -agonist L-741,626 caused concentration-dependent RbCC relaxations (pEC50 of 11±0.15 and 11.15±0.28, respectively). Pre-treatment with either l-NAME or the soluble guanylate cyclase inhibitor 1H-[1,2,4] oxadiazolo[4,3-a]quinoxalin-1-on (ODQ) (100 µM) caused a rightward shift of the concentration-response curve to 6-ND, without affecting the L-741,626 responses. In TTX (100nM)-pre-treated preparations, neither l-NAME nor ODQ shifted the 6-ND concentration-response curve. Dopamine, noradrenaline, and adrenaline caused concentration-dependent RbCC contractions. Pre-incubation with 6-ND concentration-dependently inhibited the dopamine-induced contractions, without affecting those induced by either noradrenaline or adrenaline. 6-Nitrodopamine is the most potent endogenous relaxant agent in RbCC ever described and represents a novel mechanism by which NO causes corpus cavernosum smooth muscle relaxation. The finding that 6-ND acts as a truly selective dopamine D2 -receptor antagonist indicates that the balance of dopamine and 6-ND release/synthesis may be the main mechanism that modulates corpus cavernosum smooth muscle tonus in vivo.

Effects of epoxygenases on the nonadrenergic noncholinergic relaxant responses induced by electrical field stimulation in rabbit corpus cavernosum.

We aimed to investigate the effects of epoxygenases on electrical field stimulation (EFS)-mediated nitric oxide (NO)-dependent and NO-independent nonadrenergic noncholinergic (NANC) relaxations in isolated rabbit corpus cavernosum. The tissues of 20 male adult albino rabbits (2.5-3kg) were suspended in organ baths containing aerated Krebs solution, and isometric contractions were recorded. EFS-mediated NANC relaxations were obtained on phenylephrin (3×10-5 M)-contracted tissues in the presence of guanethidine (10-6 M) and atropine (10-6 M). Miconazole (10-9 -10-4 M), 17-octadecynoic acid (ODYA) (10-10 -10-5 M), 14,15-epoxyeicosatrienoic acid (EET) (10-11 -10-8 M), 11,12-EET (10-12 -3×10-8 M) and 20-hydroxyeicosatetraenoic acid (HETE) (10-11 -3×10-8 M) were added cumulatively (n=5-7 for each set of experiments). For NO-independent relaxations, Nω -nitro-l-arginine methyl ester (l-NAME) (10-4 M) was added before a group of experiments. Depending on the concentration, miconazole, 17-ODYA, 14,15-EET, 11,12-EET, and 20-HETE significantly enhanced both NO-dependent and NO-independent EFS-mediated relaxations (p<0.05). Epoxygenases showed similar effect on NO-dependent and NO-independent relaxant responses except 20-HETE which caused significantly more enhanced relaxation on NO-dependent responses (p<0.05). No drug caused a significant relaxation response on tissues contracted with phenylephrine. Epoxygenases contribute to EFS-mediated NO-dependent and NO-independent NANC relaxations by presynaptic mechanisms, offering a new treatment alternative for erectile dysfunction which needs to be explored in further in vivo, molecular and clinical studies.

Effects of eupatilin on the contractility of corpus cavernosal smooth muscle through nitric oxide-independent pathways.

Eupatilin (5,7-dihydroxy-3,4,6-trimethoxyflavone) is one of the main compounds present in Artemisia species. Eupatilin has both antioxidative and anti-inflammatory properties and a relaxation effect on vascular contraction regardless of endothelial function. We evaluated the relaxant effects of eupatilin on the corpus cavernosum (CC) of rabbits and the underlying mechanisms of its activity in human corpus cavernosum smooth muscle (CCSM) cells. Isolated rabbit CC strips were mounted in an organ bath system. A conventional whole-cell patch clamp technique was used to measure activation of calcium-sensitive K+ -channel currents in human CCSM cells. The relaxation effect of eupatilin was evaluated by cumulative addition (10-5 m ~ 3×10-4 m) to CC strips precontracted with 10-5 m phenylephrine. Western blotting analysis was performed to measure myosin phosphatase targeting subunit 1 (MYPT1) and protein kinase C-potentiated inhibitory protein for heterotrimeric myosin light chain phosphatase of 17-kDa (CPI-17) expression and to evaluate the effect of eupatilin on the RhoA/Rho-kinase pathway. Eupatilin effectively relaxed the phenylephrine-induced tone in the rabbit CC strips in a concentration-dependent manner with an estimated EC50 value of 1.2±1.6×10-4 m (n=8, p<0.05). Iberiotoxin and tetraethylammonium significantly reduced the relaxation effect (n=8, p<0.001 and p=0.003, respectively). Removal of the endothelium or the presence of L-NAME or indomethacin did not affect the relaxation effect of eupatilin. In CCSM cells, the extracellular application of eupatilin 10-4 m significantly increased the outward currents, and the eupatilin-stimulated currents were significantly attenuated by treatment with 10-7 m iberiotoxin (n=13, p<0.05). Eupatilin reduced the phosphorylation level of MYPT1 at Thr853 of MLCP and CPI-17 at Thr38. Eupatilin-induced relaxation of the CCSM cells via NO-independent pathways. The relaxation effects of eupatilin on CCSM cells were partially due to activation of BKCa channels and inhibition of RhoA/Rho-kinase.

Neuronal Voltage Gated Potassium Channels May Modulate Nitric Oxide Synthesis in Corpus Cavernosum.

Potassium channels (K+Ch) in corpus cavernosum play an important role in the regulation of erection. Nitric oxide (NO) acts through opening of K+Ch leading to hyperpolarization and relaxation.Aim : This study aims to update knowledge about the role of voltage-gated K+Ch (KV) channels in erectile machinery and investigate their role in the control of NO action &/or synthesis in the corpus cavernosum.Methods : Tension studies using isolated rabbit corpus cavernosum (CC) strips and rat anococcygeus muscle were conducted. Results are expressed as mean ± SEM.Results : Electric field stimulation (EFS, 2–16 Hz) evoked frequency-dependent relaxations of the PE (phenylephrine)-precontracted CC strips. At 2 Hz, EFS-induced relaxation amounted to 73.17 ± 2.55% in presence 4-AP (10−3 M) compared to 41.98 ± 1.45% as control. None of the other selective K+Ch blockers tested inhibited EFS-induced relaxation. 4-AP (10−3M) significantly attenuated ACh-induced relaxation of rabbit CC where dose-response curve was clearly shifted upward, and attenuated SNP- induced relaxation, for example, to 49.28 ± 4.52% compared to 65.53 ± 3.01% as control at 10−6 M SNP. The potentiatory effect of 4-AP on EFS was abolished or reversed in presence of NG-nitro-L-arginine (L-NNA, non-selective nitric oxide synthase inhibitor, 10−5M, and 2 × 10−4M). Same results were observed in rat anococcygeus muscle which is a part of the erectile machinery in rats.Conclusion : This study provides evidence for the presence of prejunctional voltage-gated K+Ch in CC, the blockade of which may increase the neuronal synthesis of NO.

Relaxation Effects of Eucomiae Cortex in Isolated Rabbit Corpus Cavernosum Smooth Muscle

This study was aimed to investigate the relaxation effects of Eucomiae Cortex (EC) extract in isolated rabbit corpus cavernosum smooth muscle and its mechanism. To evaluate the relaxation of EC extract in rabbit corpus cavernosum, EC extract was treated in corporal strips which were precontracted with phenylephrine(PE). To study its mechanism, Nω-nitro-L-arginine (L-NNA) was pretreated after infuse of EC extract and compared with non-treated. In calcium chloride (CaSUP2+/SUP) -free krebs solution, EC extract and CaSUP2+/SUP 1 mM were infused by turns after CaSUP2+/SUP 1 mM was treated into corporal strips contracted by PE. Cell ability, nitric oxide (NO) and epithelial nitric oxide synthase (eNOS) on human umbilical vein endothelial cell (HUVEC) were measured by MTT assay, Griess reagent system and histochemical, immunohistochemical methods. EC extract showed a significant relaxation effects on the corporal strips, this effects were inhibited by pretreatment of L-NNA. EC extract inhibited the increase of contraction by CaSUP2+/SUP influx in CaSUP2+/SUP-free krebs solution, and eNOS positive reaction in corpus cavernosum, NO production in HUVEC increased by treatment of EC extract. These result suggest that the relaxation effects of EC extract in isolated corpus cavernosum smooth muscle are involved in increase of eNOS and NO production, blocking of extracellular CaSUP2+/SUP influx.

腎氣丸의 남성 생식기능 및 성기능 개선효과 : 정자생성 촉진, 고환독성 완화 및 음경해면체 평활근의 이완

Objectives : This study aimed to investigate the effects of Shingi-whan(SG) on the male reproductive and sexual function, so we measured the spermatogenesis and the testicular toxicity in mice and the vasorelaxation in isolated rabbit corpus cavernosum smooth muscle. Methods : To evaluate effect on the spermatogenesis in mice, we prepared two groups, control group and SG group that was orally administered SG(1,000mg/kg) for 20 days, and compared. To analyze testicular toxicity in mice, we also prepared two groups, doxo group that was injected with doxorubicin (3mg/kg) on three times and doxo + SG group that was injected with doxorubicin and SG for 20 days, and compared. To investigate sexual function of SG in mice, we prepared three groups, normal group and aging elicited group consisting of 18-month-old mice, SG treated aging group that was orally administered SG for 60 days, and compared using histochemical staining on mice corpus cavernous tissues. In order to define the relaxation effects of SG, rabbit corpus cavernous tissues were prepared in <TEX>$2{\times}2{\times}6mm$</TEX> sized strip. Then the dose-dependent relaxation responses of SG at 0.01-3.0 mg/ml in contracted strips induced by phenylephrine were measured. Results : The sperm density in dutus epididymis and the diameter of seminiferous tubules of SG group was significantly increased when compared to control group. The testicular weight and the diameter and height of epithelial layer of seminiferous tubules of doxo + SG group was significantly increased when compared to doxo group. The cavernous strips were significantly relaxed by SG extract In SG treated aging group, ratio of smooth muscles to collagen fibers and red blood cell count in venous sinus was increased as compared to aging elicited group. Conclusions : Our findings have shown that SG extract have effect on spermatogenesis and mitigating effect on doxo-induced testicular toxicity. Further, it also have the vasorelaxant effect on rabbit corpus cavernosum.

Effects of Shingi-whan on the Male Reproductive and Sexual Function : Enhancing Spermatogenesis, Reducing Testicular Toxicity, and Relaxing Smooth Muscle of Corpus Cavernosum

Objectives : This study aimed to investigate the effects of Shingi-whan(SG) on the male reproductive and sexual function, so we measured the spermatogenesis and the testicular toxicity in mice and the vasorelaxation in isolated rabbit corpus cavernosum smooth muscle. Methods : To evaluate effect on the spermatogenesis in mice, we prepared two groups, control group and SG group that was orally administered SG(1,000mg/kg) for 20 days, and compared. To analyze testicular toxicity in mice, we also prepared two groups, doxo group that was injected with doxorubicin (3mg/kg) on three times and doxo +SG group that was injected with doxorubicin and SG for 20 days, and compared. To investigate sexual function of SG in mice, we prepared three groups, normal group and aging elicited group consisting of 18-month-old mice, SG treated aging group that was orally administered SG for 60 days, and compared using histochemical staining on mice corpus cavernous tissues. In order to define the relaxation effects of SG, rabbit corpus cavernous tissues were prepared in 2 × 2 × 6 mm sized strip. Then the dose-dependent relaxation responses of SG at 0.01-3.0 mg/ml in contracted strips induced by phenylephrine were measured. Results : The sperm density in dutus epididymis and the diameter of seminiferous tubules of SG group was significantly increased when compared to control group. The testicular weight and the diameter and height of epithelial layer of seminiferous tubules of doxo + SG group was significantly increased when compared to doxo group. The cavernous strips were significantly relaxed by SG extract In SG treated aging group, ratio of smooth muscles to collagen fibers and red blood cell count in venous sinus was increased as compared to aging elicited group. Conclusions : Our findings have shown that SG extract have effect on spermatogenesis and mitigating effect on doxo-induced testicular toxicity. Further, it also have the vasorelaxant effect on rabbit corpus cavernosum.

8-(3-chloro-4-methoxybenzyl)-8H-pyrido[2,3-d]pyrimidin-7-one derivatives as potent and selective phosphodiesterase 5 inhibitors.

A novel series of highly selective phosphodiesterase 5 (PDE5) inhibitors was found. 8H-Pyrido[2,3-d]pyrimidin-7-one derivatives bearing an (S)-2-(hydroxymethyl)pyrrolidin-1-yl group at the 2-position and a 3-chloro-4-methoxybenzyl group at the 8-position exhibited potent PDE5 inhibitory activities and high PDE5 selectivity over PDE6. Among the synthesized compounds, the 5-methyl analogue (5b) showed the most potent relaxant effect on isolated rabbit corpus cavernosum with an EC30 value of 0.85 nM.

Design and synthesis of novel 5-(3,4,5-trimethoxybenzoyl)-4-aminopyrimidine derivatives as potent and selective phosphodiesterase 5 inhibitors: Scaffold hopping using a pseudo-ring by intramolecular hydrogen bond formation

5-(3,4,5-Trimethoxybenzoyl)-4-amimopyrimidine derivatives were found as a novel chemical class of potent and highly selective phosphodiesterase 5 inhibitors. A pseudo-ring formed by an intramolecular hydrogen bond constrained the conformation of 3-chloro-4-methoxybenzylamino and 3,4,5-trimethoxybenzoyl substituents and led to the discovery of T-6932 (19a) with a potent PDE5 inhibitory activity (IC50=0.13nM) and a high selectivity over PDE6 (IC50 ratio: PDE6/PDE5=2400). Further modification at the 2-position of T-6932 resulted in the finding of 26, which exhibited potent relaxant effects on isolated rabbit corpus cavernosum (EC30=11nM) with a high PDE5 selectivity over PDE6 (IC50 ratio: PDE6/PDE5=2800).

Relaxation Effects of Epimedium Koreanum Nakai in Isolated Rabbit Corpus Cavernosum Smooth Muscle

This study aimed to investigate the relaxation effects and its underlying mechanisms of Epimedium koreanum Nakai(EK) in phenylephrine(PE) treated isolated rabbit corpus cavernosum smooth muscle. The dose-dependent relaxation responses of phenylephrine(PE, 1×10<SUP>-6</SUP> M)-precontracted strips to EK at 0.01-3.0 ㎎/㎖ were measured and also observed after endothelial denudation using organ bath. To analyze the underlying mechanisms of EK-induced relaxation, Nω-nitro-L-arginine(L-NNA), methylene blue(MB), tetraethylammonium chloride(TEA), indomethacin(IM) were pretreated before EK extract infused into precontracted strips induced by PE. To investigate cytotoxic activity and nitric oxide(NO) concentration of EK extract on EA.hy926 cells, mitochondrial dehydrogenase activity(MTT) assay and nitric oxide detection kit were used. The cavernous strips were significantly relaxed by EK extract at 0.3 ㎎/㎖, 1.0 ㎎/㎖, 3.0 ㎎/㎖ and the relaxation responses of PE-precontracted strips denuded endothelium also inhibited in comparison with intact endothelium. The pretreatment of L-NNA, MB, TEA reduced EK extract-induced endothelium-dependent relaxation, but the pretreatment of IM didn"t affect EK extract-induced endothelium-dependent relaxation. When EK extract was applicated on EA.hy926 cells, the NO concentration was increased. Our findings have shown that EK extract exerts a relaxing effect on corpus cavernosum in part by suppressing influx of extracellular Ca<SUP>2+</SUP> through activating the NO-cGMP system.

Relaxation Effects of Rubus coreanus in Isolated Rabbit Corpus Cavernosum Smooth Muscle

This study aimed to investigate the relaxation effects and its underlying mechanisms of Rubus coreanus(RC) extract in contracted rabbit corpus cavernous tissues by phenylephrine(PE) 1 μM. In order to define the relaxation effects of RC, rabbit corpus cavernous tissues were prepared in 2×2×6 ㎜ sized strip. The dose-dependent relaxation responses of RC at 0.01-3.0 ㎎/㎖ in contracted strips induced by PE were measured and also observed after endothelial denudation. To analyze the underlying mechanisms of RC-induced relaxation, indomethacin(IM), tetraethylammonium chloride(TEA), Nω-nitro-L-arginine (L-NNA), methylene blue(MB) were treated before RC extract infused into precontracted strips induced by PE. To study the effect of RC extract on influx of extracellular Ca<SUP>2+</SUP> in corpus cavernous strips, calcium chloride(Ca) 1 mM infused into precontracted strips induced by PE after pretreatment of RC extract in Ca<SUP>2+</SUP>-free krebs-ringer solution. To investigate cytotoxic activity and nitric oxide(NO) concentration of RC extract on human umbilical vein endothelial cell(HUVEC), cell viability on HUVEC was measured by MTT assay, and NO concentration was measured by Griess reagent system. The cavernous strips were significantly relaxed by RC extract at 1.0 ㎎/㎖, 3.0 ㎎/㎖ and the relaxation responses to RC were inhibited significantly by endothelial disruption. The pretreatment of IM, TEA didn"t affect RC extract-induced endothelium -dependent relaxation, but the pretreatment of L-NNA, MB reduced RC extract-induced endothelium-dependent relaxation. When Ca<SUP>2+</SUP> was supplied the cavernous strips which were precontracted by PE in a Ca<SUP>2+</SUP>-free krebs-ringer solution, contraction of strips was increased, but pretreatment of RC inhibited contractile response to Ca<SUP>2+</SUP>. When RC extract was applicated on HUVEC, NO concentration was increased. Our findings show that RC extract exerts a relaxing effect on corpus cavernosum in part by suppressing influx of extracellular Ca<SUP>2+</SUP> through activating the NO-cGMP system.

Endothelium-Independent Relaxant Effect of Rubus Coreanus Extracts in Corpus Cavernosum Smooth Muscle

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. To elucidate the direct effects of unripe R. coreanus extract (RCE) on CC smooth muscle cells. 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. The effects of unripe RCE on ion channels and the intracellular Ca(2+) concentration ([Ca(2+)]i ) of CC. 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. RCE acts through endothelium-independent and endothelium-dependent pathways to relax CC. RCE may inhibit VDCCs and Ca(2+) release from sarcoplasmic reticulum.

The relaxant effect of methanolic extract of yohimbe bark (&amp;lt;i&amp;gt;Pausinystalia yohimbe&amp;lt;/i&amp;gt;) on rabbit corpus cavernosum smooth muscles

Background: Development and maintenance of penile erection requires the relaxation of corpus cavernosum smooth muscle (CCSM) cells and it is essentially mediated by nitric oxide (NO). Pausinystalia yohimbe bark is traditionally used for the treatment of erectile dysfunction (ED). Objectives and Methods: This study aimed to evaluate the effects of the methanolic extract of P. yohimbe bark (10 mg/ml) on isolated rabbit corpus cavernosum smooth muscle (CCSM). Results: Methanolic extract of P. yohimbe bark blocked contractions in CCSM induced by adrenergic agonists, through facilitating NO release from the endothelial cells. Conclusion: The obtained results revealed that, methanolic extract of P. yohimbe bark caused relaxation of CCSM that validates its traditional aphrodisiac property, thus it could be used to initiate and maintain erection in cases of erectile dysfunction.

Activation of muscarinic receptors inhibits neurogenic nitric oxide in the corpus cavernosum

The functional role of cholinergic transmission in erection is still far from being fully elucidated. This work aims to further elucidate the modulatory role of neostigmine on NO in the corpus cavernosum and to highlight whether cholinergic transmission in the penis modulates sildenafil action. The isolated rabbit corpus cavernosum and measurement of intracavernosal pressure in the anesthetized rat model were used. Neostigmine (0.02mg/kg) reduced increase of intracavernosal pressure/mean arterial pressure (ICP/MAP) next to cavernous nerve stimulation. Higher doses (0.06 and 0.4mg/kg) potentiated ICP/MAP rise and atropine (1.5 and 10mg/kg) did the opposite. In vitro, neostigmine (10−5 and 10−4M) potentiated neurogenic relaxations and this effect was significantly inhibited by hexamethonium (10−4M) or Nω-propyl-l-arginine (3×10−5M) and partially but significantly reduced in the presence of atropine. Lower dose neostigmine (10−7M), inhibited electrically induced relaxation over the range of 1–4Hz, atropine (10−6M) almost abolished this inhibitory effect as well as NG-nitro-l-arginine (10−5M). It was also significantly reduced by selective nNOS inhibitor Nω-propyl-l-arginine (3×10−5M). Nicotine (10−4M) significantly potentiated electrically induced relaxations amounting to 84.625±8.06% at 1Hz and potentiated the effect of sildenafil synergistically. Hexamthonium did the opposite. The potentiatory effect of sildenafil on neurogenic erection was significantly reduced by low dose neostigmine both in vitro and in vivo. This study provides evidence that muscarinic receptors may modulate NO synthesis in nitrergic nerves by inhibiting nNOS and high level of cholinergic stimulation may activate nicotinic receptors to promote erection probably by potentiating NO synthesis in nitrergic nerves.

Interaction between nitric oxide and dopaminergic transmission in the peripheral control of penile erection

As the peripheral role of dopamine in the mediation of penile erection was recently identified, its potential role in the modulation of NO action and whether D1 or D2 receptors are involved in this potential modulation was investigated. The isolated rabbit corpus cavernosum and measurement of intracavernosal pressure in the anesthetized rat model were used. The selective D1 antagonist SCH23390 but not the D2 antagonist sulpiride reduced the inhibitory effect of N(G)-nitro-L-arginine (L-NNA) and the potentiatory effect of sildenafil on erectile responses. L-NNA did not change the inhibitory effect of SCH23390 or the potentiatory effect of apomorphine but enhanced the effect of high-dose fenoldopam on intracavernosal pressure. Similarly, fenoldopam produced 47.30 ± 6.89% potentiation of relaxation of corpus cavernosum in absence of L-NNA and 80 ± 9.34% potentiation in its presence at 3 Hz. The effect of sildenafil was greatly reduced by pretreatment with SCH23390 or sulpiride and completely abolished by their combination. This study supports the role played by D1 receptors peripherally in the control of penile erection. Absence of NO may potentiate the effect of D1 receptor on erection. Activation of D1 receptors may be involved in the synthesis of NO in the corpus cavernosum or may activate the role of NO in erection.

Enhancement effects of nicotine on neurogenic relaxation responses in the corpus cavernosum in rabbits: The role of nicotinic acetylcholine receptor subtypes

Nicotine acts as an agonist of nicotinic acetylcholine receptors, which belong to a superfamily of neurotransmitter-gated ion channels. We previously demonstrated that nicotine increases the electrical field stimulation (EFS)-evoked nitrergic relaxation responses via activation of nicotinic acetylcholine receptors. The aim of the present study is to investigate the subtypes of nicotinic acetylcholine receptors in rabbit corpus cavernosum. EFS-evoked relaxation responses were recorded from corpus cavernosum strips obtained from rabbits with an isometric force displacement transducers. Effects of nicotine on EFS-evoked relaxations were examined in pre-contracted tissues. Then the effect of nicotine on the EFS-evoked relaxations was examined in the presence of hexamethonium, dihydro-β-erythroidine, mecamylamine or α-bungarotoxin. In our study, nicotine (3 × 10 − 5 , 10 − 4 ) transiently increased nitrergic relaxations induced by EFS in the rabbit isolated corpus cavernosum. While hexamethonium and mecamylamine near totally inhibited or abolished the neurorelaxation response to nicotine (3 × 10 − 5 ) on EFS, dihydro-β-erythroidine and α-bungarotoxin partially inhibited these responses. These findings demonstrated that the alpha3–beta4, alpha4–beta2 and alpha7 subunits of nicotinic acetylcholine receptors play role on the nicotine-induced augmentation in EFS-evoked relaxation responses in rabbit corpus cavernosum.

Effect of hydrogen sulphide‐donating sildenafil (ACS6) on erectile function and oxidative stress in rabbit isolated corpus cavernosum and in hypertensive rats

OBJECTIVE To study the effect of the H(2)S-donating derivative of sildenafil (ACS6) compared to sildenafil citrate and sodium hydrosulphide (NaHS) on relaxation, superoxide formation and NADPH oxidase and type 5 phosphodiesterase (PDE5) expression in isolated rabbit cavernosal tissue and smooth muscle cells (CSMCs), and in vivo on indices of oxidative stress induced with buthionine sulphoximine (BSO). MATERIALS AND METHODS Relaxation was studied in an organ bath in response to carbachol and after incubation with interleukin-1beta for 12 h. CSMCs were incubated with tumour-necrosis factor-alpha or the thromboxane A(2) (TXA(2)) analogue, U46619, with or with no sildenafil citrate, ACS6 or NaHS for 16 h. Superoxide formation and the expression of p47(phox) (an active subunit of the NADPH oxidase complex) and PDE5 protein was then assessed using Western blotting. Rats were also treated with BSO (with or with no sildenafil citrate or ACS6) for 7 days; cavernosal cGMP, cAMP, glutathionine and plasma TXA(2) and 8-isoprostane F(2alpha) was measured by enzyme-linked immunosorbent assay. RESULTS ACS6 and sildenafil citrate relaxed cavernosal smooth muscle equipotently; NaHS alone had little effect at up to 100 microm. The formation of superoxide and expression of p47(phox) and PDE5 was reduced by ACS6, sildenafil citrate and NaHS (order of potency: ACS6 > sildenafil citrate > NaHS). The effects of ACS6 were blocked by inhibitors of protein kinase A (PKA) and PKG. In rats treated with BSO, both ASC6 and sildenafil citrate reduced the increased plasma levels of TXA(2) and 8-isoprostane F(2alpha) but increased cGMP, cAMP and glutathionine levels in corpus cavernosum. CONCLUSIONS By virtue of a dual action on PKA and PKG activation, ACS6 not only promotes erection, acutely, but might also have a long-term beneficial effect through inhibition of oxidative stress and downregulation of PDE5.

Spontaneous Ca2+ Waves in Rabbit Corpus Cavernosum: Modulation by Nitric Oxide and cGMP

Detumescent tone and subsequent relaxation by nitric oxide (NO) are essential processes that determine the erectile state of the penis. Despite this, the mechanisms involved are incompletely understood. It is often assumed that the tone is associated with a sustained high cytosolic Ca(2+) level in the corpus cavernosum smooth muscle cells, however, an alternative possibility is that oscillatory Ca(2+) signals regulate tone, and erection occurs as a result of inhibition of Ca(2+) oscillations by NO. The aim of this study is to determine if smooth muscle cells displayed spontaneous Ca(2+) oscillations and, if so, whether these were regulated by NO. Male New Zealand white rabbits were euthanized and smooth muscle cells were isolated by enzymatic dispersal for confocal imaging of intracellular Ca(2+) (using fluo-4AM) and patch clamp recording of spontaneous membrane currents. Thin tissue slices were also loaded with fluo-4AM for live imaging of Ca(2+). Cytosolic Ca(2+) was measured in isolated smooth muscle cells and tissue slices. Results. Isolated rabbit corpus cavernosum smooth muscle cells developed spontaneous Ca(2+) waves that spread at a mean velocity of 65 microm/s. Dual voltage clamp/confocal recordings revealed that each of the Ca(2+) waves was associated with an inward current typical of the Ca(2+)-activated Cl(-) currents developed by these cells. The waves depended on an intact sarcoplasmic reticulum Ca(2+) store, as they were blocked by cyclopiazonic acid (Calbiochem, San Diego, CA, USA) and agents that interfere with ryanodine receptors and IP(3)-mediated Ca(2+) release. The waves were also inhibited by an NO donor (diethylamine NO; Tocris Bioscience, Bristol, Avon, UK), 3-(5-hydroxymethyl-2-furyl)-1-benzyl indazole (YC-1) (Alexis Biochemicals, Bingham, Notts, UK), 8-bromo-cyclic guanosine mono-phosphate (Tocris), and sildenafil (Viagra, Pfizer, Sandwich, Kent, UK). Regular Ca(2+) oscillations were also observed in whole tissue slices where they were clearly seen to precede contraction. This activity was also markedly inhibited by sildenafil, suggesting that it was under NO regulation. These results provide a new basis for understanding detumescent tone in the corpus cavernosum and its inhibition by NO.

Role of Chloride Channels in the Regulation of Corpus Cavernosum Tone: A Potential Therapeutic Target for Erectile Dysfunction

Recent electrophysiological recordings have identified the existence of outward, excitatory chloride (Cl(-)) currents in rat, rabbit, and human corpus cavernosum (CC) muscle cells. The aim of this study was to investigate the physiological role of Cl(-) currents in the maintenance of cavernosal muscle tone in isolated rabbit CC tissues. CC strips (1 x 1 x 5 mm) were suspended in tissue bath chambers for isometric tension experiments. Spontaneous cavernosal tone and contractions elicited by field stimulation or administration of established smooth muscle constrictors were examined in the presence of chloride channel (ClC) blockers, niflumic acid (NFA), and anthracene-9-carboxylic acid (A9C). Both spontaneous myogenic activity and contractile responses to field stimulation, norepinephrine, histamine, and endothelin-1 were reduced by ClC blockers. Results. In CC strips exhibiting intrinsic myogenic tone, NFA (30 and 100 microM) and A9C (1 mM) caused a relaxation of the tone. In addition, spontaneous contractile activity in CC was abolished in the presence of either ClC blocker. In CC strips precontracted with norepinephrine, histamine, and endothelin-1, both ClC blockers significantly reversed the tone. The ability of NFA and A9C to reverse norepinephrine-induced tone was unaffected by N(omega)-nitro-L-arginine, 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one, and cis-N-[2-phenylcyclopentyl]-azacyclotridec-1-en-2-amine. In addition, neither indomethacin nor tetraethylammonium affected the relaxant response of NFA. NFA and A9C inhibited the neurogenic and norepinephrine-induced contractions in a concentration-dependent manner. While NFA exerted persistent inhibition on neurogenic contraction, inhibition of neurogenic contractions by A9C was readily reversible. On K(+)-depolarized CC, NFA induced a concentration-related relaxation, whereas A9C was inert, suggesting an additional mechanism of NFA on voltage-gated calcium channels. These results underline the importance of Cl(-) currents as a mechanism in the maintenance of cavernosal tone produced by adrenergic and various endogenous constrictors. Thus, the modulation of Cl(-) current, as an attractive and effective approach to regulate penile erection, and specific ClC blockers, as potential erectogenic agents, merits further research.

Effect of hypothyroidism on the purinergic responses of corpus cavernosal smooth muscle in rabbits

Several studies have reported evidence of hormonal abnormalities in 25-35% of impotent men. Hypothyroidism has been reported to occur in 6% of impotent men. In the present study, we examined purinergic relaxation responses in hypothyroidism in an experimental rabbit model and compared them with controls to evaluate the possible involvement of the purinergic pathway. The study comprised 20 male New Zealand white rabbits. The rabbits were divided into two equal groups. We tested the effects of ATP, alpha beta ATP, and adenosine precontracted with phenylephrine on the isolated corpus cavernosum preparations from control and hypothyroid rabbits. We also evaluated the effects of ATP, alpha beta ATP, and adenosine on the cGMP levels in the isolated corpus cavernosum preparations from control and hypothyroid rabbits. T3, T4, and testosterone levels were significantly lower in hypothyroid rabbits. ATP, alpha beta ATP, carbachol, and electrical field stimulation (EFS)-induced frequency-dependent relaxation responses in the isolated rabbit corpus cavernosum strips precontracted with phenylephrine reduced significantly (P<0.05). Adenosine-induced relaxation responses did not change significantly in hypothyroid rabbits. Reduction of relaxation response in hypothyroid rabbits corpus cavernosum can depend on a decreased release of nitric oxide (NO) from nitrergic nerves and endothelium.