雄激素对兔阴茎海绵体eNOS和PDE5蛋白表达的影响

目的研究雄激素对eNOS和PDE5蛋白在同一动物模型中表达的影响。方法用化学发光法测定新西兰白兔睾酮基线水平和去势后各个时间点的睾酮水平；用Western blot和免疫组化方法分析各组兔阴茎海绵体eNOS和PDE5蛋白的表达。结果假手术对照组（C组）兔血清睾酮水平和基线值没有显著性差异；去势组（Ox组）睾酮水平在2、4、8周时下降为基线值的17％、14％和13％；睾酮补充治疗组（OxT组）睾酮水平在不同剂量睾酮补充之后有所回升。C组eNOS和PDE5蛋白的表达不随时间的推移而变化；Ox组中，eNOS的表达速度、程度和时间推移的关系不明显，但是，PDE5蛋白的表达水平随去势时间的延长而降低；OxT组中eNOS蛋白表达水平没有明显差异，PDE5蛋白的表达水平在补充睾酮前后差异明显。结论在去势兔模型中，eNOS和PDE5蛋白表达不均衡，致使cGMP的生成和降解不能保持动态平衡，提示运用雄激素维持cGMP的动态平衡是ED治疗机理上的新视角。     

应用RNA干扰技术抑制PDE5A3基因在人阴茎海绵体平滑肌细胞表达

目的应用RNA干扰(RNAi)技术抑制人阴茎海绵体平滑肌细胞PDE5A3基因的表达,探讨应用该技术治疗勃起功能障碍(ED)的可行性.方法构建6个靶向人PDE5A3基因的短发夹RNA(shRNA)重组质粒,转染人阴茎海绵体平滑肌细胞48 h后,逆转录-聚合酶链反应(RT-PCR)及Western blot检测PDE5A3基因的表达抑制效果.结果抑制率最高的1、2、4号重组质粒使PDE5A3基因表达在mRNA水平分别抑制(66.26±4.02)%,(54.90±3.06)%,(23.83±3.61)%;在蛋白质水平分别抑制(64.14±3.32)%,(49.21±2.96)%,(29.85±4.91)%.结论RNAi能明显抑制人阴茎海绵体平滑肌细胞PDE5A3基因的表达,且抑制率具有序列相关性,是潜在的ED基因治疗新方法.     

In vivo and in vitro response of corpus cavernosum to phosphodiesterase-5 inhibition in the hypercholesterolaemic rabbit

OBJECTIVE

To investigate the effects of hypercholesterolaemia (HC) on rabbit corpus cavernosa in vivo and in vitro, and evaluate the efficacy of vardenafil and sildenafil in normal and HC rabbits, as the phosphodiesterase‐5 (PDE‐5) inhibitors vardenafil and sildenafil are widely used for treating erectile dysfunction (ED) and most organic causes of ED are associated with vascular risk factors like HC.

MATERIALS AND METHODS

Male New Zealand White rabbits were randomly divided into two groups; 11 HC rabbits were fed a 2% cholesterol diet, and 12 age‐matched control rabbits received a regular diet. After 12–14 weeks, erectile responses to intravenous sodium nitroprusside (SNP) and PDE‐5 inhibitors were evaluated for 2 h in conscious rabbits. Penile length was measured and the area under the curve calculated. Relaxant responses of corpus cavernosal strips to electrical‐field stimulation (EFS) were measured before and after exposure to PDE‐5 inhibitors and the nitric oxide synthase inhibitor N′‐nitro‐ l ‐arginine methyl ester.

RESULTS

HC rabbits had a lower erectile response to SNP than controls; in both control and HC rabbits there was a greater erectile response after simultaneous exposure to SNP and vardenafil, or SNP and sildenafil. However, the responses of the HC rabbits were still significantly less than those of the controls. Corpora from control rabbits responded to EFS with greater relaxations at all frequencies, except 1 Hz. Corpora from both HC and control rabbits had greater responses to EFS after exposure to vardenafil and sildenafil; N′‐nitro‐ l ‐arginine methyl ester diminished the response to EFS.

CONCLUSIONS

There was a significantly lower in vivo and in vitro erectile response in HC rabbits than in controls; erectile function measured in conscious rabbits can be used to assess quantitatively the efficacy of different agents, e.g. sildenafil and vardenafil, in pathological animals. In addition, both agents improve in vitro responses of erectile tissue from HC rabbits to EFS.

     

雄激素调控大鼠阴茎海绵体组织中eNOS表达的分子机制

目的:研究雄激素是否通过蛋白激酶B-3(AKT3)、Ⅰ类磷脂酰肌醇-3-激酶的p110催化亚单位(PIK3CA)、钙调蛋白(CALM)、小窝蛋白1(CAV1)调控大鼠阴茎海绵体组织内皮型一氧化氮合酶(e NOS)的表达,并影响阴茎勃起功能。方法:8周龄健康雄性SD大鼠36只,随机均分为6组:4周对照组(A组)、6周对照组(B组)、4周去势组(C组)、6周去势组(D组)、4周去势+睾酮(T)替代组(E组)、6周去势+T替代组(F组)。C、D、E、F组切除双侧睾丸及附睾,1 d后E、F组隔日1次丙酸睾酮3 mg/kg皮下注射,其余各组等量植物油皮下注射。4周后(A、C、E组)、6周后(B、D、F组)分别检测各组大鼠海绵体内压(ICP_(max))/平均动脉压(MAP)、血清T,通过免疫组化法及Western印迹法分别测定e NOS、P-e NOS、AKT3、PIK3CA、CALM、CAV1在各组大鼠阴茎海绵体中的表达。结果:各组实验大鼠体重、MAP无显著差异。血清T值和ICP_(max)/MAP比值:去势组(C、D组)较对照组(A、B组)及去势+T替代组(E、F组)极显著降低(P均0.01),且去势6周组(D组)较去势4周组(C组)显著降低(P均0.05),去势+T替代组(E、F组)及对照组(A、B组)各组间无显著差异。免疫组化结果显示:e NOS、Pe NOS主要表达在血管内皮细胞胞膜和海绵窦血管腔内;AKT3、PIK3CA、CALM、CAV1主要表达于血管内皮细胞胞质和胞膜,少数表达于平滑肌细胞。e NOS、P-e NOS、AKT3、PIK3CA、CALM、CAV1Western印迹结果显示:去势组(C、D组)与对照组(A、B组)、去势+T替代组(E、F组)相比极显著降低(P均0.01),去势+T替代组(E、F组)与对照组(A、B组)相比无显著差异,且6周去势组(D组)比4周去势组(C组)表达显著降低(P均0.05),6周去势+T替代组(F组)与4周去势+T替代组(E组)相比无显著差异,6周对照组(B组)与4周对照组(A组)相比无显著差异。结论:雄激素可能通过上调AKT3、PIK3CA、CALM、CAV1蛋白的表达,磷酸化e NOS后激活e NOS,促进勃起;然而,确切的机制还应该采用AKT3、PIK3CA、CALM、CAV1等的阻滞剂或者激活剂,以及采用基因转染或者基因敲除等方法,进一步明确分子机制。     

Effects of the recreational use of PDE5 inhibitors on the corpus cavernosum of young,healthy rats

Purpose

PDE5 inhibitors are widely used for the treatment of erectile dysfunction. However, these drugs have recently become popular among men without erectile dysfunction as a means of enhancing sexual performance and improving sexual desire. The aim of this study was to investigate the histopathological and ultrastructural effects of PDE5 inhibitors on the corpus cavernosum in young, healthy male rats.

Methods

Twenty-four 4-month-old male rats were divided into four groups: group 1 was the control group, group 2 rats received sildenafil citrate, group 3 rats received vardenafil hydrochloride, and group 4 rats received tadalafil. All drugs were administered for 4 weeks. Penile tissue was collected for electron microscopy and tissue collagen measurements. Electron microscopic analysis indicated that the number of active fibroblasts and macrophages and the synthesis of new collagen fibers increased in treated rats.

Results

Cavernous tissue collagen levels were significantly higher in the sildenafil-, vardenafil-, and tadalafil-treated groups than in controls (46.16 ± 4.9, 42.06 ± 2.4, 41.07 ± 2.4, and 29.20 ± 3.3, respectively) (p < 0.001).

Conclusions

Young men who use these drugs to enhance performance in the absence of erectile dysfunction may experience irreversible damage to the cavernosal tissue. However, more studies are needed to evaluate the molecular mechanisms by which PDE5 inhibitors affect the corpus cavernosum.     

Effect of avanafil on rat and human corpus cavernosum

We compared the activity of a new phosphodiesterase‐5 inhibitor (PDE5i) avanafil with sildenafil and tadalafil in human and rat corpus cavernosum (CC) tissues. The effect of avanafil with several inhibitors and electrical field stimulation (EFS) was evaluated on CC after pre‐contraction with phenylephrine. With the PDE5i, sildenafil and tadalafil, concentration–response curves were obtained and cyclic guanosine monophosphate (cGMP) levels were measured in tissues. Avanafil induced relaxation with maximum response of 74 ± 5% in human CC. This response was attenuated by NOS inhibitor and soluble guanylate cyclase (sGC) inhibitor. Avanafil potentiated relaxation responses to acetylcholine and EFS in human CC and enhanced SNP‐induced relaxation and showed 3‐fold increase in cGMP levels. When compared with sildenafil, avanafil and tadalafil were effective at lower concentrations in human CC. In addition, Sprague–Dawley rats underwent in vivo intracavernosal pressure (ICP) and mean arterial pressure (MAP) measurements. Avanafil increased ICP/MAP that was enhanced by SNP and cavernous nerve (CN) stimulation in rat CC tissues. Also avanafil showed maximum relaxation response of 83 ± 7% in rat CC with 3‐fold increase in cGMP concentration. Taken together, these results of our in vivo and in vitro studies in human and rat suggest that avanafil promotes the CC relaxation and penile erection via NO‐cGMP pathway.     

Gene expression of the phosphodiesterases 3A and 5A in human corpus cavernosum penis

OBJECTIVE: The following study was performed to evaluate the importance of phosphodiesterases 3A (PDE3A) and 5A (PDE5A) for the regulation of penile smooth muscle tone. Furthermore, indications of side effects of specific inhibitors in certain tissues as well as of a possible relation between the level of PDE3A and PDE5A gene expression and erectile dysfunction should have been deduced from the results obtained. METHODS: Total ribonucleic acid was isolated from different human tissues (urogenital tract, gastrointestinal tract, cardiovascular system and central nervous system) and subjected to RTPCR analysis and Northern blotting using primers and probes specific for PDE3A and PDE5A. RESULTS: As shown by RT-PCR and Northern blotting, PDE3A and PDE5A mRNAs exhibit a distinct distribution throughout the tissues examined but were 2-fold higher in cavernous tissue than in all other tissues investigated. However, there were no significant differences in the levels of gene expression between the two subgroups of patients. CONCLUSIONS: Very high expression levels of PDE3A and PDE5A in human cavernous tissue underscore the physiological importance of these enzymes for the regulation of penile erection, emphasizing their therapeutical and pharmacological relevance. The distribution pattern of the mRNA for the isoenzymes PDE3A and PDE5A may explain the pharmacological effects as well as the side effects of milrinone and sidenafil.     

Kallistatin基因在大鼠阴茎海绵体中的表达研究

目的:探讨人组织激肽释放酶结合蛋白(kallistatin)在阴茎海绵体中的表达情况,为勃起功能调控和ED治疗寻找新的分子靶点。方法:采用qRT-PCR、Western印迹和免疫荧光技术检测kallistatin在大鼠阴茎海绵体组织的表达情况,同时检测主动脉中的表达水平进行对照。结果:qRT-PCR和Western印迹检测结果显示,Kallistatin mRNA和蛋白在大鼠阴茎海绵体组织中均有表达,且其表达水平与主动脉相比均无统计学差异(P>0.05);免疫荧光检测显示,Kallistatin表达于阴茎海绵体内皮细胞和平滑肌细胞,且定位于细胞胞质,与主动脉相比,两者间表达亦无明显差异(P>0.05)。结论:Kallistatin基因高表达于阴茎海绵体且定位于海绵体内皮细胞和平滑肌细胞内,提示kallistatin可能在海绵体内皮细胞和平滑肌细胞的细胞功能中发挥作用,从而参与阴茎勃起功能调控。     

CD34及Sca-1蛋白在不同月龄大鼠阴茎海绵体中的表达

目的 检测CD34和干细胞抗原-1(Sca-1)在不同月龄组SD大鼠阴茎海绵体组织中的表达,对阴茎海绵体干细胞进行初步的观察.方法 随机选取2月龄、5月龄、20月龄不同窝别SD大鼠各10只分别作为幼龄组、壮龄组和老龄组,乙醚麻醉下取阴茎海绵体组织,分别采用免疫组织化学和RT-PCR技术对CD34和Sca-1进行测定.结果 阴茎海绵体组织中存在CD34和Sca-1这两种干细胞抗原标志物的表达.CD34表达以血管及血窦内皮为主,并可见部分散在表达;Sca-1散在表达,主要沿血窦及动脉血管分布;亦可见极少量双阳性染色细胞(CD34/Sca-1),主要表达于海绵体血窦周围.CD34、Sca-1、CD34/Sca-1表达量于组间两两比较均有显著统计学差异(P＜0.01),且均与月龄呈明显负相关(P＜0.01).结论 SD大鼠阴茎海绵体内CD34及Sca-l蛋白表达随月龄增长明显下降,并有双阳性染色细胞的存在.海绵体组织中有内源性干细胞存在的可能.     

5-HT4 receptors in isolated human corpus cavernosum?

The novel serotonin subtype-4 (5-HT4) receptor agonist, SC53116 (SC), produced a limited relaxation of noradrenaline (NA) pre-contracted human corpus cavernosum (CC) smooth muscle in vitro. This effect was not significantly attenuated by the 5-HT4 antagonist SDZ250557 (SDZ). In the presence of (+/-) pindolol (1 microM) and methysergide (1 microM), employed to mask 5-HT1 and beta-adrenergic, and 5-HT2 receptors respectively, SC failed to relax NA pre-contracted CC strips to a greater extent than saline. Functional cAMP dependent relaxation pathways were demonstrated by a significant reduction in NA induced tone by prostaglandin E1 (PGE1) and isopropylnoradrenaline (IPNA), the action of the latter compound was effectively eliminated in the presence of (+/-) pindolol. Relaxation of NA induced tone caused by the nitric oxide donor nitro-glycerine (NTG) was significant and similar in the absence and presence of the 5-HT and beta-adrenergic antagonists. The results of this present study indicate that human corporal smooth muscle does not contain 5-HT4 receptors and that, although compounds like SC act to relax non-vascular smooth muscle via cAMP dependent mechanisms, 5-HT4 receptor agonists may be expected to be of limited utility in triggering cAMP dependent relaxation responses in human CC.     

NCX-911, a novel nitric oxide-releasing PDE5 inhibitor relaxes rabbit corpus cavernosum in the absence of endogenous nitric oxide

Phosphodiesterase type 5 (PDE5) inhibitors have reduced efficacy in treating erectile dysfunction (ED) in conditions where there is a lack of endogenous nitric oxide (NO). Therefore, NO-releasing PDE5 inhibitors have been developed. Here we report the effect of such a compound, NCX-911, on the tone and nitrergic relaxations of rabbit corpus cavernosum in the absence or presence of a NO synthase inhibitor, N(G)-nitro-L-arginine methyl ester (L-NAME; 500 microM). NCX-911 was found to be as potent as sildenafil at inducing relaxation of rabbit cavernosum (EC(50) values 997.8+/-195.7 and 1000.5+/-140.8 nM, respectively). The potency of NCX-911 was not altered, but that of sildenafil decreased five-fold in the presence of L-NAME (EC(50) values 1281.2+/-268.3 and 4959.1+/-882.1, nM respectively, P<0.001 for sildenafil). Both compounds potentiated nitrergic relaxations with similar potencies. These results suggest that NO-releasing PDE5 inhibitors could potentially be more useful than PDE5 inhibitors in the treatment of ED in conditions where there is a lack of endogenous NO.     

尼古丁对大鼠阴茎海绵体内源性CO浓度及NOS活性的影响

目的:观察尼古丁对成年雄性大鼠阴茎海绵体内源性一氧化碳(CO)浓度及一氧化氮合酶(NOS)活性的影响,探讨吸烟对勃起功能损害的可能机制。方法:40只成年雄性Wistar大鼠分为4组,尼古丁注射1个月组、2个月组、3个月组和对照组,尼古丁注射组尼古丁0.5 mg/(kg.d)皮下分别注射1、2、3个月,对照组注射生理盐水。处理后,取阴茎海绵体,用改良双波长分光光度法检测CO浓度,改良Griess法检测NOS活性。结果:对照组CO浓度为(13.66±0.40)μmol/mg prot,NOS活性为(9.72±0.47)U/mg prot。尼古丁注射1个月,CO浓度和NOS活性分别下降为(12.43±0.56)μmol/mg prot和(8.44±0.69)U/mg prot,显著低于对照组(P均<0.01);尼古丁注射2个月,CO浓度和NOS活性分别下降为(11.41±0.52)μmol/mg prot和(7.53±0.24)U/mg prot,显著低于对照组和尼古丁注射1个月组(P<0.01);尼古丁注射3个月,海绵体CO浓度和NOS活性分别下降为(10.52±0.59)μmol/mg prot和(6.64±0.31)U/mg prot,均显著低于对照组和尼古丁注射1个月、2个月组(P均<0.01)。结论:尼古丁可导致成年雄性大鼠阴茎海绵体内源性CO浓度及NOS活性下降,提示内源性CO及NOS参与吸烟引起勃起功能障碍的病理生理过程。     

Responses to serotonin (5HT) in isolated corpus cavernosum penis of rabbit

This study was aimed at determining serotonin (5-hydroxytryptamine: 5HT) receptor subtypes participating in 5HT-induced response in the isolated corpus cavernosum penis (CCP) of rabbits. 5HT contracted the CCP in a concentration-dependent manner. Both WAY100635 (5HT(1A) antagonist) and LY53857 (5HT(2) antagonist) concentration-dependently suppressed the 5HT-induced contraction. The suppression of the 5HT-induced contraction by ketanserin (5HT(2A) antagonist) was weaker than that by LY53857. LY278584 (5HT(3) antagonist) did not affect the 5HT-induced contraction. SDZ205557 (5HT(4) antagonist) showed a tendency to potentiate the 5HT-induced contraction. The above results suggest that 5HT(1A) and 5HT(2) receptor subtypes partially participate in the contractile response to 5HT in rabbit CCP, and the potentiation by SDZ205557 of the 5HT-induced contraction implies the existence of dual contractile and relaxing responses to 5HT via 5HT(1) and 5HT(2), and 5HT(4) receptors, respectively. The relaxing response to 5HT(4) receptor stimulation may be masked by 5HT-induced contraction.     

大鼠阴茎海绵体肌源性干细胞的分离与表型鉴定

目的 探讨阴茎海绵体中肌源性干细胞(MDSCs)的分离与表型鉴定的方法.方法 取2个月龄SD大鼠阴茎海绵体组织,用0.5％Ⅰ型胶原酶、0.1％胰蛋白酶进行酶消化初步分离.应用改良的Preplate差速贴壁法进一步行细胞纯化,细胞培养1h,贴壁细胞为PP1;未贴壁细胞转瓶培养2h,贴壁细胞为PP2;未贴壁细胞再次转瓶培养18h,贴壁细胞为PP3;此后,每次间隔24h转瓶培养依次获得的贴壁细胞为PP4、PP5、PP6,观察细胞形态变化.流式细胞仪检测PP3、PP6细胞中干细胞抗原l (Sca-l)和结蛋白(Desmin)的阳性表达量;Western blot检测PP1 ～ PP6细胞中Sca-1和Desmin的阳性表达;免疫荧光细胞化学法检测Sca-1、Desmin、Sca-1/Desmin在PP6细胞中的表达.结果 PP1～ PP6细胞贴壁能力逐渐减慢,PP6细胞贴壁最慢,2～3d后才开始贴壁成圆形或短梭形.流式细胞仪检测结果显示,PP3细胞中Sca-1阳性表达量为(0.3±0.2)％,与同型对照组比较差异无统计学意义(P＞0.05),Desmin阳性表达量为(15.3±1.5)％,与同型对照组比较差异有统计学意义(P＜0.05);PP6细胞中Sca-1、Desmin阳性表达量分别为(5.7±0.7)％、(41.1±1.6)％,与PP3比较差异均有统计学意义(P＜0.05).Western blot显示PP1 ～ PP5细胞中未检测到Sca-1蛋白明显表达,但在PP6细胞中则检测到Sca-1蛋白的表达,Desmin蛋白在PP1 ～ PP6细胞中表达逐渐增强.免疫荧光细胞化学显示在PP6细胞中有少量细胞表达Sca-1、散在分布、以胞质表达为主;表达Desmin细胞聚集、数量较多、主要表达于胞质;亦发现有极少量双阳性标记细胞(Sca-1/Desmin),共同表达于同一细胞的胞质.结论 在大鼠阴茎海绵体中分离出既有于细胞表型又有肌源性表型的细胞,提示此类细胞为MDSCs.     

The effects of 5HT(1) agonists on erection in rats in vivo and rabbit corpus cavernosum in vitro

We have examined the effects of the selective 5-HT(1a) and 5-HT(1b) agonists 8-hydroxy-2-(di-n-propylamino)tetralin (8-OHDPAT) and 7-trifluoromethyl-4-(4-methyl-1-piperazinyl)-pyrrolo[1,2-a]quinoxaline (CGS12066b), respectively on erection in rats in vivo and rabbit corpus cavernosum in vitro. Apomorphine (0.1 mg/kg) induced 3.1+/-0.4 erections in vehicle-pretreated animals. At the highest doses tested 8-OHDPAT (0.4-0.64 mg/kg) and CGS12066b (1.0-10.0 mg/kg) significantly reduced apomorphine erection to 0.9+/-0.3 erections and 0.5+/-0.2 erections respectively. The nonselective 5-HT agonist metachlorophenylpiperazine (m-CPP; 0.1 mg/kg) elicited characteristic increases in cavernous nerve activity (CNA) and intracavernous pressure responses (ICP) in anesthetized rats. 8-OHDPAT (0.64 mg/kg) and CGS12066b (1.0 mg/kg) failed to elicit CNA or ICP responses. CGS12066b reduced ICP responses resulting from the direct stimulation of the cavernous nerve whereas 8-OHDPAT did not. CGS12066b reduced the CNA and ICP responses to m-CPP administration whereas 8-OHDPAT potentiated m-CPP induced CNA and ICP responses. In isolated rabbit corpus cavernosum (CC) 8-OHDPAT and CGS12066b both failed to alter noradrenergic induced contraction and non-adrenergic non-cholinergic relaxation. Our results indicate that selective 5-HT(1a) and 5-HT(1b) agonists have different effects in different models of erection.