Modulation of Ca2+ sensitivity regulates contractility of rabbit corpus cavernosum smooth muscle

PURPOSE: We examined the role of the modulation of Ca2+ sensitivity for regulating the contractility of corpus cavernosum smooth muscle. MATERIALS AND METHODS: We applied simultaneous measurements of intracellular Ca2+ concentration and tension in fura-PE3 loaded intact strips and receptor coupled permeabilization by alpha-toxin. RESULTS: In intact fura-PE3 loaded strips the tension induced by 10 microM. phenylephrine was significantly greater than that produced by depolarization with 118 mM. K+, although the extent of intracellular Ca2+ concentration elevations was similar. During sustained contraction induced by 10 microM. phenylephrine the application of 10 microM. Y-27632 (a Rho kinase inhibitor) induced relaxation with a slight decrease in intracellular Ca2+ concentration, while the application of 3 microM. GF109203X (a protein kinase C inhibitor) induced relaxation without changing intracellular Ca2+ concentration. In alpha-toxin permeabilized strips 10 microM. phenylephrine induced a larger increase in force at a constant intracellular Ca2+ concentration and produced a leftward shift in the intracellular Ca2+ concentration-tension relationship, a response that was partially inhibited by pretreatment with Y-27632 or GF109203X. CONCLUSIONS: These results indicate that in rabbit corpus cavernosum smooth muscle phenylephrine induces contraction not only by increasing intracellular Ca2+ concentration, but also by increasing Ca2+ sensitivity of the contractile apparatus in a Rho kinase and protein kinase C dependent manner. Antagonism of Ca2+ sensitization pathways in the corpus cavernosum smooth muscle represents an alternate target for the treatment of erectile dysfunction.     

Hypercholesterolemia impairs endothelium-dependent relaxation of rabbit corpus cavernosum smooth muscle

The majority of cases of impotence are associated with vascular risk factors such as diabetes, hypercholesterolemia, hypertension and smoking. These factors induce impairment of endothelium-dependent relaxation of blood vessels in man and in experimental animals. In this study the effects of hypercholesterolemia on the reactivity of rabbit corpus cavernosum smooth muscle strips to endothelium-dependent and endothelium-independent agents were investigated. New Zealand White rabbits (n = 14) were randomly divided into control and treatment groups. The control group (n = 7) received a regular diet while the treatment group (n = 7) was fed a diet of 0.5% cholesterol and 4% peanut oil for 10 weeks. Animals were then sacrificed and the corporal tissue studied in organ chambers for isometric tension measurement. Tissue was contracted with phenylephrine and concentration-dependent relaxation to acetylcholine, in the presence and absence of indomethacin, and to nitroprusside were examined. Blood level of cholesterol in the cholesterol-fed group was significantly higher compared to the control group. Contractions to phenylephrine were similar in both groups. Hypercholesterolemia, however, inhibited relaxation to acetylcholine but did not alter relaxation to nitroprusside, a cyclic guanosine monophosphate (cGMP)-dependent, direct smooth muscle dilator. Indomethacin enhanced the relaxations to acetylcholine in both control and cholesterol-fed groups but did not correct the difference in the relaxation to acetylcholine between both groups. It is concluded that hypercholesterolemia impairs endothelium-mediated relaxation of rabbit corpus cavernosum smooth muscle. The mechanism for the endothelial dysfunction does not appear to involve alteration in cyclooxygenase products of arachidonate or the cGMP-dependent relaxation of corporal smooth muscle. Impairment of endothelium-dependent relaxation of corporal smooth muscle may contribute to the pathophysiology of impotence associated with hypercholesterolemia in man.     

兔阴茎海绵体平滑肌细胞的快速分离及鉴定

目的探讨兔阴茎海绵体平滑肌细胞快速分离方法,为应用膜片钳技术研究阴茎勃起机制提供实验材料.方法采用木瓜蛋白酶和胶原酶两步酶解消化法,快速分离出新西兰大白兔阴茎海绵体平滑肌细胞并应用免疫组化鉴定.结果分离的细胞成活率较高,贴壁呈长梭形,胞膜光滑完整,胞浆均匀,可用于膜片钳记录.免疫组化鉴定为兔阴茎海绵体平滑肌细胞.结论酶解消化快速分离兔阴茎海綿体平滑肌细胞为全细胞膜片钳技术研究阴茎勃起功能障碍的电生理机制提供了较好的实验材料.     

Relaxation effects of adrenomedullin in isolated rabbit corpus cavernosum smooth muscle

OBJECTIVES: To clarify the pharmacological effects of adrenomedullin, a potent vasodilator and hypotensive peptide isolated from human phaeochromocytoma cells, on corpus cavernosal smooth muscle in vitro, as the intracavernosal injection of adrenomedullin induces penile erection in the anaesthetized cat. MATERIALS AND METHODS: The effects of adrenomedullin were investigated in isolated muscle strips from New Zealand rabbit corpus cavernosum smooth muscle pre-contracted with phenylephrine alone, in the presence of indomethacin (cyclooxygenase inhibitor), Nomega-nitro l-arginine methyl ester (L-NAME, a nitric oxide synthase inhibitor), and K+-channel blockers. RESULTS: Adrenomedullin caused relaxation of isolated pre-contracted rabbit corpus cavernosum strips in a concentration-dependent manner. The response of corpus cavernosum was unaffected L-NAME, indomethacin and K+-channel blockers. CONCLUSION: The relaxation exerted by adrenomedullin in rabbit corporal tissue may arise from the effect of the drug on its specific receptors and/or calcitonin gene-related peptide-1 receptors. The relaxant effect of adrenomedullin might lead to novel clinical applications for erectile dysfunction.     

Nitric oxide mediates relaxation in rabbit and human corpus cavernosum smooth muscle

Summary We investigated in vitro the relaxant effect of exogenous acetylcholine (ACh) and electric-field stimulation (EFS) on rabbit and human corpus cavernosum smooth muscle strips (CC) precontracted with phenylephrine. The effects of EFS and ACh were monitored alone, after muscarinic receptor blockade and after inhibition of nitric oxide (NO) formation with l-N-nitroarginine (l-NOARG). In rabbit und human CC, both atropine and l-NOARG abolished the relaxant effects of ACh. The relaxant effects of EFS, however, were only slightly reduced by atropine to 97.5±17.5% in human CC and to 89.0±6.1% in rabbit CC. l-NOARG further reduced the EFS effects to 0.8±1.7% in human CC and to 16.2±8.7% in rabbit CC. In strips obtained from impotent patients with diabetes mellitus, the relaxant effects appeared to be significantly less than in strips from nondiabetic impotent men. Tetrodotoxin blocked the relaxant EFS effects in human and rabbit strips completely. The data indicate the important role of NO in cholinergically induced relaxation of cavernous smooth muscle in rabbits and humans. Our findings support the idea of NO as the nonadrenergic noncholinergic neurotransmitter in penile erection in both species. Rabbit erectile tissue might serve as an in vitro animal model for further investigation.     

阴茎海绵体平滑肌细胞培养进展

阐述阴茎海绵体平滑肌细胞（CCSM）培养对目前研究勃起功能障碍的重要性。比较CCSM与其他平滑肌细胞培养的差异,同时比较两种不同的CCSM培养方法结果的异同,为研究勃起功能障碍培养CCSM方法的选择提供参考。     

Diabetes mellitus impairs neurogenic and endothelium-dependent relaxation of rabbit corpus cavernosum smooth muscle.

The effect of alloxan-induced diabetes on the reactivity of corporeal nerves, endothelium and smooth muscle was studied in the New Zealand white rabbit. Fifteen rabbits were randomly divided into treated (n = 6) and control (n = 9) groups. The treated group was maintained for 6 weeks. Two control groups were studied. One control group (n = 3) was maintained for 6 weeks as littermate controls for diabetic group. The second control group (n = 6) was not maintained but was weight matched with the 6 week diabetic group. The reactivity of corpus cavernosum tissue from the diabetic animals and the control animals was studied in organ chambers. When tissue contraction was produced with phenylephrine for the study of relaxation to various stimuli, the tension induced was similar in the diabetic and the control groups. Relaxation of corpus cavernosum tissue to electrical stimulation of autonomic nerves as well as relaxation to the endothelium-dependent vasodilator acetylcholine were comparably unaffected in the weight matched and littermate control groups while significantly inhibited in the diabetic group. Treatment of the corporeal tissue with the cyclooxgenase inhibitor indomethacin enhanced the relaxation to electrical stimulation and to acetylcholine in the control and in the diabetic groups but did not improve the significant difference in relaxation between the two groups. Relaxation of corporeal tissue to endothelium-independent vasodilators, papaverine and nitroprusside was similar in the control groups and the diabetic groups. It is concluded that diabetes impairs neurogenic and endothelium-mediated relaxation of rabbit corpus cavernosum smooth muscle. These findings are comparable to those described in corpus cavernosum tissue from diabetic men, showing the validity of this experimental animal model. The mechanism for the nerve or endothelial dysfunction does not appear to involve alteration in cyclooxygenase products of arachidonate or the ability of the corporeal smooth muscle to relax via a cGMP-dependent mechanism. Since nitric oxide has been shown to act as the nonadrenergic noncholinergic neurotransmitter as well as endothelium-derived relaxing factor (EDRF) of the trabecular smooth muscle, it is possible that impairment of neurogenic and endothelium-dependent relaxation due to diabetes is mediated by alteration in the synthesis or availability of nitric oxide in corporeal tissue.     

Effect of TCDD on corpus cavernosum histology and smooth muscle physiology

A polyhalogenated aromatic hydrocarbon, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), is one of the most potent toxic environmental pollutants. Decreases in spermatogenesis and the ability to conceive and carry a pregnancy to term are the most sensitive signs of reproductive toxicity by TCDD in the mammal, but no report of its effect on the erectile function exists. We performed this study to investigate the effect of TCDD on the erectile function. New Zealand white rabbits were treated intraperitoneally with 1 microg/kg of TCDD. At 4 (Gr I) and 8 (Gr II) weeks after the administration of TCDD, cavernosal tissues were harvested for strip study in the organ bath and testes were prepared for histologic examination. Compared to the maximal amplitude of 17.1+/-4.12 mN in normal control (Gr III), the contractions to cumulative concentrations of NE (10(-8)-10(-4) M) were significantly decreased to 6.57+/-1.34 and 5.45+/-1.01 mN in Groups I and II, respectively. Compared to 51.12+/-7.38% in Gr III, relaxation to cumulative concentration (10(-8)-10(-4) M) of acetylcholine was significantly decreased to 17.25+/-2.17% (Gr I) and 9.73+/-2.17% (Gr II) at a concentration of 10(-4) M, respectively. Compared to 75.12+/-13.18% in Gr III, relaxation to cumulative concentration (10(-8)-10(-4) M) of SNP was significantly decreased to 31.49+/-7.89% (Gr I) and 18.54+/-6.12% (Gr II) at a concentration of 10(-4) M, respectively. Histologically, intracavernosal fibrosis, abnormal subtunical deposition of fat and decreased sinusoidal space with consequent increase of trabecular smooth muscle contents were identified in TCDD-treated groups. In TCDD-treated animals, seminiferous tubules showed a decrease of germ cells with vacuolar degeneration and apoptotic cells. Spermatids were hardly seen. These results suggest that TCDD inhibits spermatogenesis and has a potential harmful effect on erectile function via changes of corpus cavernosum histology and smooth muscle physiology.     

人阴茎海绵体平滑肌细胞的原代培养及鉴定

目的　为研究人阴茎勃起功能提供方便的实验材料。　方法　取新鲜尸体阴茎海绵体平滑肌细胞 ,用含 2 0 %人AB型血清的DMEM液作体外原代培养 ,并以免疫组化法鉴定。　结果　经 10～ 14d培养后 ,6个培养瓶内均铺满梭状细胞 ,免疫组化法鉴定确认为人阴茎海绵体平滑肌细胞。　结论　人阴茎海绵体平滑肌细胞可在体外条件下生长传代 ,并可能成系 ,为阴茎勃起功能的研究提供方便的材料     

低氧对SD大鼠阴茎海绵体平滑肌纤维化的影响

目的:探讨低氧对SD大鼠阴茎海绵体平滑肌纤维化的影响。方法:体外培养阴茎海绵体平滑肌细胞,免疫组化鉴定细胞;常规氧浓度(21%O2浓度)分别培养12、24、48、72h作为对照,低氧(1%O2浓度)干预12、24、48、72h,RT-PCR分别测定各组TGF-β1、Ⅰ型胶原、Ⅲ型胶原的相对表达量。结果:体外培养的阴茎海绵体平滑肌细胞生长良好,抗平滑肌α-肌动蛋白单克隆抗体免疫组化染色阳性;RT-PCR结果提示TGF-β1、Ⅰ型胶原、Ⅲ型胶原的相对表达量在48h内与低氧时间成正相关,时间进一步延长不能增加其相对表达量。结论:在低氧环境下,SD大鼠阴茎海绵体平滑肌细胞的TGF-β1、Ⅰ型胶原、Ⅲ型胶原的相对表达量随时间的延长逐渐增加,48h达到最大值。低氧可导致SD大鼠阴茎海绵体平滑肌纤维化。     

Expression of myosin isoforms in the smooth muscle of human corpus cavernosum

The molecular interaction between smooth muscle (SM) myosin and actin in the corpus cavernosum (CC) determines the erectile state of the penis. A key mechanism regulating this interaction and subsequent development and maintenance of force is alternative splicing of SM myosin heavy chain (MHC) and 17 kDa essential SM myosin light chain (MLC) pre-mRNAs. Our aim was to examine the relative SM myosin isoform composition in human CC. Tissue samples were obtained from 18 patients with erectile dysfunction (ED), Peyronie's disease, or both. One specimen was obtained during a transgender operation. Patients then were stratified according to presence of diabetes mellitus, hypertension, ED, or Peyronie's disease, as well as failure of phosphodiesterase-5 (PDE5) inhibitors and history of previous pelvic or penile surgeries, radiation, or both. Our results revealed that all human CC samples expressed only the SM-A isoform. There was a predominance of SM2 isoform mRNA relative to SM1 across all samples, with a mean of 63.8%, which correlated with protein analysis by gel electrophoresis. A statistically significant difference was found between patients who had undergone previous pelvic surgery, radiation, or both and those who did not. The ratio of LC(17b) to LC(17a) was approximately 1:1 for all patients, with a mean of 48.9% LC(17b). Statistical difference was seen in the relative ratio of LC(17b) to LC(17a) among the group who failed conservative therapy with PDE5 inhibitors compared with all others. In conclusion, we determined the SM myosin isoform composition of human CC and present for the first time differences in relative myosin isoform expression among patients with several risk factors contributing to their cause of ED. Our data reflect the fact that alternative splicing events in the MHC and 17 kDa MLC pre-mRNA may be a possible molecular mechanism involved in the altered contractility of the CCSM in patients with ED.     

人阴茎海绵体平滑肌细胞三维培养模型的建立

目的 观察人阴茎海绵体平滑肌细胞(hCCMCs)在三维培养系统中的生物学特性.方法 体外分离hCCMCs,并用差速贴壁法进行纯化.免疫组化的方法检测α-平滑肌肌动蛋白(α-SMA)的表达,进行hCCMCs的鉴定.在单层贴壁培养的基础上,将hCCMCs在胶原凝胶中培养,形成三维培养系统,并对hCCMCs的形态结构、增殖情况进行研究.结果 原代培养的hCCMCs细胞形态不均一,经过差速贴壁法纯化后,细胞形态均一,免疫组化显示大多数细胞α-SMA阳性表达.三维培养系统中,hCCMCs在不同层面上呈三维生长.hCCMCs在三维培养系统中培养10d后,细胞数量无明显增殖(P>O.05),明显低于贴壁培养条件下细胞增殖速率(P<0.01).结论 hCCMCs三维培养系统可以观察hCCMCs在三维条件下的生长、增殖状况,研究hCCMCs与微环境的相互作用关系,有望为相关研究找到一种更为简单、可控性更强的体内实验替代方法.     

红景天苷对低氧环境下大鼠阴茎海绵体平滑肌细胞α-肌动蛋白和骨桥蛋白表达的影响

目的:探讨红景天苷对低氧SD大鼠阴茎海绵体平滑肌细胞(CCSMC)α-肌动蛋白、骨桥蛋白(OPN)表达的影响。方法:体外培养SD大鼠CCSMC,免疫组化法鉴定细胞;设正常对照组(21%O2浓度)、低氧组(1%O2浓度)、低氧+红景天苷1 mg/L组、低氧+红景天苷3 mg/L组、低氧+红景天苷5 mg/L组、低氧+前列腺素E1(PGE1)0.4μg/L组,分别培养48 h;RT-PCR法分别测定各组α-肌动蛋白、OPN的相对表达量。结果:体外培养的CCSMC生长良好,抗平滑肌α-肌动蛋白单克隆抗体免疫组化染色阳性;与正常对照组比较,低氧组细胞的α-肌动蛋白表达量下降、OPN表达量升高(P<0.01);与低氧组比较,红景天苷5 mg/L组α-肌动蛋白表达量升高、OPN表达量降低(P<0.01),与PGE1作用相当(P﹥0.05)。结论:低氧可引起SD大鼠CCSMC收缩型标志物α-肌动蛋白表达降低,合成型标志物OPN表达升高,推测低氧可能引起CCSMC由收缩型向合成型转化。红景天苷能抑制低氧引起的CCSMCα-肌动蛋白表达降低、OPN表达升高,浓度为5 mg/L时与PGE1作用几乎相当。     

The effects of isolated lipoproteins and triglyceride, combined oxidized low density lipoprotein (LDL) plus triglyceride, and combined oxidized LDL plus high density lipoprotein on the contractile and relaxation response of rabbit cavernous smooth muscle

The aim of this study was to investigate the effects of isolated lipoproteins and triglyceride (TG), and the effects of combined oxidized low density lipoprotein (LDL) plus TG and the combined oxidized LDL plus high density lipoprotein (HDL) on the contractility and relaxation response of rabbit cavernous smooth muscle. Cavernous muscle strips from New Zealand White rabbits were studied in organ chambers for isometric tension measurement. The strips were exposed to HDL, LDL, oxidized LDL, TG, combined oxidized LDL plus TG and combined oxidized LDL plus HDL for 30 min. Both HDL and LDL did not affect contraction and relaxation responses of the cavernous muscles. The oxidized LDL did not affect norepinephrine (NE)-induced contractility of the strips, but significantly ( p < 0.05) decreased the relaxation response to endothelium-dependent agonist, acetylcholine (Ach). Non-specific NO synthase inhibitor (L-NAME) completely inhibited the relaxation response to Ach, and L-arginine partially improved the diminished relaxation. TG did not significantly change the relaxation responses to Ach, but decreased the contractility of cavernous muscle to NE. Neither the combined oxidized LDL plus TG nor oxidized LDL plus HDL had significant synergistic or detoxication effects on the contractility and relaxation responses. In conclusion, oxidized LDL may have acute toxic effects on the endothelium-dependent, NO-mediated relaxation, but not on the contractility, of rabbit cavernous smooth muscle. TG may decrease contractility of the cavernous muscle. There may be neither synergistic nor detoxication effects on the contractility and relaxation response when TG or HDL is added to the oxidized LDL.     

Endothelium-derived nitric oxide and cyclooxygenase products modulate corpus cavernosum smooth muscle tone.

Relaxation of penile corpus cavernosum smooth muscle is controlled by nerve and endothelium derived substances. In this study, endothelium-dependent relaxation of corporal smooth muscle was characterized and the role of arachidonic acid products of cyclooxygenase in endothelium-dependent relaxation was examined. Endothelium removal from rabbit corpora was performed by infusion with 3-[(3-cholamidopropyl)-dimethylammonio]-1-propane sulfonate and was confirmed by transmission electron microscopy. Strips of human and rabbit corporal tissues were studied in the organ chambers for isometric tension measurement. The accumulation of cyclic guanosine monophosphate (cGMP) and the release of eicosanoids from corporal tissue was measured by radioimmunoassay and correlated to smooth muscle relaxation. Our study showed that relaxation of corpus cavernosum tissue to acetylcholine, bradykinin and substance P was endothelium-dependent; potentiated by indomethacin; and inhibited by NG-monomethyl-L-arginine, methylene blue or LY83583. Relaxation to papaverine and sodium nitroprusside was endothelium-independent, and unaffected by NG-monomethyl-L-arginine. Relaxation to vasoactive intestinal polypeptide was partially endothelium-dependent; potentiated by indomethacin; attenuated by NG-monomethyl-L-arginine or methylene blue. The tissue level of cGMP was enhanced by acetylcholine and nitric oxide. Methylene blue inhibited both basal and drug-stimulated levels of cGMP. The release of eicosanoids was enhanced by acetylcholine and blocked by indomethacin. In conclusion, nitric oxide or a closely related substance accounts for the activity of endothelium-derived relaxing factor in the corporal tissue. Inhibition of the release of eicosanoids potentiates the relaxing effect of nitric oxide. Nitric oxide increases tissue cGMP which appears to modulate corporal smooth muscle relaxation.