靶向人PDE5A3基因的shRNA对人阴茎海绵体平滑肌细胞cGMP的影响

目的:观察腺病毒载体介导特异性短发夹RNA(shRNA)对人阴茎海绵体平滑肌细胞环磷酸鸟苷(cGMP)的影响,为应用RNA干扰(RNAi)技术治疗阴茎勃起功能障碍(ED)提供实验依据。方法:成功构建携带3条针对人PDE5A3基因位点特异性shRNA的重组腺病毒(rAd5-shRNA-PDE5A3),并设立阴性对照病毒组和空白对照组,分别转染人阴茎海绵体平滑肌细胞。以放射免疫法分别检测转染重组腺病毒24、48、72h后细胞内cGMP浓度变化,观察rAd5-shRNA-PDE5A3对海绵体平滑肌细胞内cGMP的影响。结果:实验组rAd5-shRNA-PDE5A3转染人阴茎海绵体平滑肌细胞后胞内cGMP水平显著高于阴性对照组和空白对照组,在转染后72h最为显著。结论:3条特异性针对人PDE5A3mRNA靶位点的shRNA可有效地增加海绵体平滑肌细胞内cGMP的水平,增强对PDE5基因的阻抑效果。     

靶向人PDESA3基因的shRNA对人阴茎海绵体平滑肌细胞cGMP的影响

目的：观察腺病毒载体介导特异性短发夹RNA（shRNA）对人阴茎海绵体平滑肌细胞环磷酸鸟苷（cGMP）的影响,为应用RNA干扰（RNAi）技术治疗阴茎勃起功能障碍（ED）提供实验依据。方法：成功构建携带3条针对人PDESA3基因位点特异性shRNA的重组腺病毒（rAd5-shRNA-PDE5A3）,并设立阴性对照病毒组和空白对照组,分别转染人阴茎海绵体平滑肌细胞。以放射免疫法分别检测转染重组腺病毒24、48、72h后细胞内cGMP浓度变化,观察rAd5-shRNA-PDE5A3对海绵体平滑肌细胞内cGMP的影响。结果：实验组rA（15-shRNA-PDE5A3转染人阴茎海绵体平滑肌细胞后胞内cGMP水平显著高于阴性对照组和空白对照组,在转染后72h最为显著。结论：3条特异性针对人PDE5A3mRNA靶位点的shRNA可有效地增加海绵体平滑肌细胞内cGMP的水平,增强对PDE5基因的阻抑效果。     

PDE5基因siRNA对人阴茎海绵体平滑肌细胞cGMP的影响

目的:观察PDE5基因小干扰RNA(siRNA)对人阴茎海绵体平滑肌细胞环磷酸鸟苷(cGMP)的影响,为阴茎勃起功能障碍(ED)的基因治疗提供实验依据。方法:使用美国Amb ion公司提供的设计软件设计并合成人PDE5基因的siRNA序列,合成3对PDE5 siRNA和1对阴性对照siRNA,转染人阴茎海绵体平滑肌细胞,同时设定空白转染组为对照组。以酶联免疫法分别检测转染后不同时间(24、48、72、96 h)点海绵体平滑肌细胞内cGMP浓度变化,观察PDE5 siRNA对海绵体平滑肌细胞内cGMP的影响。结果:siRNA1、siRNA2和siRNA3转染后人阴茎海绵体平滑肌细胞cGMP水平显著高于阴性对照siRNA组和空白对照组(P<0.05),在转染后72 h最为显著,siRNA1、siRNA2、siRNA3、阴性对照siRNA和空白对照组cGMP测定值分别为:5.89±0.19、3.52±0.16、2.88±0.08、0.72±0.12、0.60±0.16 pmol/m l,siRNA1组明显高于siRNA2、siRNA3组(P<0.05)。结论:体外化学合成的PDE5 siRNA能有效地增加海绵体平滑肌细胞内cGMP的水平,不同序列siRNA增加cGMP水平的能力不同,为ED的基因治疗提供了新思路。     

短发夹RNA对大鼠阴茎海绵体平滑肌细胞磷酸二酯酶5型基因表达的抑制作用

目的 观察短发夹RNA(shRNA)对大鼠阴茎海绵体平滑肌细胞磷酸二酯酶5型(PDE5)基因表达的抑制作用,探讨运用RNA干扰(RNAi)技术治疗勃起功能障碍(ED)的可行性.方法 构建靶向大鼠PDE5基因的shRNA重组腺病毒rAd-rPDE5-shRNA,将其转染大鼠阴茎海绵体平滑肌细胞48 h后,通过荧光标签进行显微计数确定转染效率,并以逆转录-聚合酶链反应(RT-PCR)及Western blot检测PDE5基因的表达水平.结果 rAd-rPDE5-shRNA构建成功,转染大鼠阴茎海绵体平滑肌细胞效率达95%以上,并使PDE5基因表达在mRNA水平抑制(80.78±2.30)%,在蛋白水平抑制(67.39±3.33)%.结论 以腺病毒为载体表达的shRNA能稳定、有效地抑制大鼠阴茎海绵体平滑肌细胞PDE5基因的表达.     

腺病毒介导的激活大鼠阴茎海绵体平滑肌细胞中NO／cGMP通路的实验研究

目的：探讨靶向大鼠iNOS基因的shRNA重组腺病毒载体对大鼠阴茎海绵体平滑肌细胞iN0s基因的激活作用,为阴茎勃起功能障碍（ED）的基因治疗提供实验依据。方法：将前期构建的重组腺病毒AdS—iN—OSrshRNA-EGFP（AdU6／shiNOS）和对照病毒AdU6／shControl,分别转染大鼠阴茎海绵体平滑肌细胞,分别在不同病毒MOI（25,50,75）值下72小时后采样检测。采用realtimeRT-PCR半定量检测AdU6／shiNOS对细胞iNOS基因mRNA表达影响;Western—blot法检测海绵体平滑肌细胞iNOS蛋白表达变化。然后培养基中加L—Arg（10mmol／L）,用酶联免疫法检测病毒转染72小时后海绵体平滑肌细胞内cGMP的浓度变化,记录AdU6／shiNOS对平滑肌细胞内cGMP的影响。结果：AdU6／shiNOS转染大鼠阴茎海绵体平滑肌细胞72小时后,和空白对照组、阴性对照组相比iN0s基因在mRNA和蛋白表达水平均显著上调（P〈O．05）,呈剂量依赖性,MOI一75时RNAa效果最好。而且转染72小时后,实验组原代平滑肌细胞内cGMP水平显著高于对照组及空白组（Pd0．05）。结论：利用腺病毒介导的RNAa技术,提高海绵体平滑肌细胞iN0s基因表达获得成功,可以增加阴茎海绵体平滑肌细胞cGMP水平,激活了NO／cGMP通路,这为勃起功能障碍的基因治疗研究开辟了新的方向。     

小干扰RNA抑制人阴茎海绵体平滑肌细胞间隙连接蛋白connexin43的表达

目的:利用小干扰RNA(siRNA)抑制人阴茎海绵体平滑肌细胞间隙连接蛋白connexin43(Cx43)的表达和检测细胞间间隙连接通讯功能,探讨该技术在阴茎海绵体平滑肌细胞间隙连接和阴茎勃起功能研究中的应用。方法:利用Ambion公司设计软件,构建靶向人Cx43基因的siRNA重组质粒,转染人阴茎海绵体平滑肌细胞48h后,逆转录-聚合酶链反应(RT-PCR)和Western印迹检测Cx43基因和蛋白的相对表达水平、划痕标记荧光染料传输技术检测细胞间间隙通讯功能,并分别与siRNA阴性对照、空白对照组比较。结果:酶切和测序证实siRNA真核表达载体构建成功。siRNA重组质粒转染细胞后的Cx43 mRNA和蛋白相对表达水平分别为(0.45±0.08)%、(0.56±0.06)%,与siRNA阴性对照组(0.72±0.04)%、(0.80±0.08)%和空白对照组(0.74±0.09)%、(0.77±0.11)%相比,差异均有显著性(P(0.05);转染后的细胞间间隙连接通讯功能也显著降低。结论:siRNA能有效抑制人阴茎海绵体平滑肌细胞Cx43的表达和阻断间隙连接介导的间隙连接通讯功能。     

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.     

PDE_5基因特异性siRNA改善糖尿病性勃起功能障碍大鼠勃起功能的研究

目的 探讨人鼠PDEs基因siRNA对糖尿病性勃起功能障碍(DM ED)大鼠PDE,基因表达的抑制作用及其对DMED大鼠勃起功能的影响.方法 构建可同时表达2条针对大鼠PDEs基因的shRNA重组腺病毒;50只雄性SD大鼠随机取10只作为正常对照.其余建立DM ED人鼠模型,随机分为实验组、阴性对照组和空白对照组,分别将重组腺病毒rAd-rPDEs-shRNA、腺病毒rAd-mock及生理盐水注射于阴茎海绵体.注射后第7天,电刺激盆神经测定各组大鼠阴茎海绵体内压(ICP)及平均周围动脉压(MAP),然后取海绵体组织通过RT-PCR和Western blot分别检测PDE5基因mRNA及蛋白的表达.结果 ICP/MAP实验组和正常对照组显著高于阴性对照组和窄白对照组(P＜0.05),实验组和正常对照组间无显著性差异(P＞0.05);RT-PCR和Western blot 分析,实验组大鼠PDE5基因mRNA和蛋白表达均显著低于阴性对照组和空白对照组(P＜0.05).结论 PDE5基因shRNA重组腺病毒转染可以改善DMED大鼠的勃起功能,为DMED治疗方法的探索提供了新的思路.     

反义寡脱氧核苷酸对人阴茎海绵体平滑肌细胞PDE5的抑制作用

目的　观察反义寡脱氧核苷酸对人阴茎海绵体平滑肌细胞 5型磷酸二酯酶 (PDE5 )的抑制作用 ,为阴茎勃起功能障碍的基因治疗提供理论和实验依据。　方法　将PDE5基因反义寡脱氧核苷酸与DOTAP转染人阴茎海绵体平滑肌原代细胞 ,Western印迹法检测转染后不同时间 (1～ 4 8h)海绵体平滑肌细胞内PDE5的浓度变化 ,观察反义寡脱氧核苷酸对平滑肌细胞内PDE5的作用。　结果　转染后 1～ 6h ,反义组人阴茎海绵体平滑肌原代细胞内PDE5表达量较对照组显著降低 (P=0 .0 0 0～ 0 .0 14 ) ;转染后 12～ 4 8h ,三组细胞内PDE5的表达比较 ,差别无显著性意义 (P =0 .189～0 .96 2 )。　结论　PDE5基因反义寡脱氧核苷酸对人阴茎海绵体平滑肌细胞PDE5有抑制作用。     

腺病毒载体介导shRNA抑制大鼠阴茎海绵体平滑肌细胞PDE5基因表达的研究

磷酸二酯酶5型(PDE5)作为基因治疗勃起功能障碍(ED)的分子靶点目前已逐渐成为研究热点.我们构建了以大鼠PDE5(rPDE5)为靶基因的重组腺病毒表达载体,将重组腺病毒转染大鼠阴茎海绵体平滑肌细胞后,通过细胞内转录产生的短发夹RNA(shRNA)来诱导针对rPDE5基因的RNA干扰(RNAi),并分别从mRNA和蛋白水平检测其对rPDE基因表达的抑制情况.现报告如下.     

Etomidate attenuates phenylephrine-induced contraction in isolated rat aorta

PURPOSE: A previous study has shown that etomidate inhibits the angiotensin II-induced calcium influx in rat aortic smooth muscle cells. The goals of our current in vitro study were to investigate the effect of etomidate on phenylephrine-induced contraction in rat aorta, and to elucidate the associated signalling pathway. METHODS: Endothelium-denuded aortic rings were suspended for isometric tension recording. Concentration-response curves for phenylephrine (10(-9) to 10(-6) M), 5-hydroxytryptamine (10(-7) to 10(-4) M) and potassium chloride (10 to 60 mM) were generated in the presence and absence of etomidate (5 x 10(-6), 3 x 10(-5), 5 x 10(-5) M). For the rings pretreated with verapamil (10(-5) M), the phenylephrine concentration-response curves were generated in the presence and absence of etomidate (5 x 10(-5) M). In the rings exposed to calcium-free isotonic depolarizing solution, the contractile response induced by the addition of calcium was assessed in the presence and absence of etomidate (5 x 10(-5) M). RESULTS: Etomidate (5 x 10(-5) M) produced a significant rightward shift in the concentration-response curves for phenylephrine, 5-hydroxytryptamine and potassium chloride. Etomidate (5 x 10(-5) M) did not alter phenylephrine-induced contraction in the rings pretreated with verapamil. Etomidate (5 x 10(-5) M) significantly attenuated the contractile response induced by the addition of calcium in the calcium-free isotonic depolarizing solution. CONCLUSION: The results suggest that etomidate, which exceeds the clinically relevant concentration, attenuates the phenylephrine-induced contraction by having an inhibitory effect on the calcium influx by blocking the L-type calcium channels in the rat aortic vascular smooth muscle.     

Cooling-induced bladder contraction: studies on isolated detrusor muscle preparations in the rat

OBJECTIVES: Detrusor muscle contraction and uninhibited micturition after intravesical instillation of ice water is interpreted as a sign of upper motor neuron lesions. The basic mechanism of cooling-induced contraction (CIC) at the level of smooth muscle, however, has not been satisfactorily explained. We therefore designed model experiments with cooling of rat detrusor muscle. METHODS: We recorded isometric tension from strips of rat urinary detrusor muscle in organ baths during stepwise cooling. CIC was tested before and after addition of various standard agents interfering with known neurogenic (autonomic blockers, tetrodotoxin, capsaicin) and myogenic mechanisms of contraction (calcium channel blockers). RESULTS: Stepwise cooling (37 degrees to 5 degrees C) of detrusor muscle induced reproducible graded contractions, inversely proportional to temperature. CIC was not dependent on a neural mechanism (not blocked by tetrodotoxin or capsaicin) or the release of neurotransmitters but was linked to translocation of calcium. It was reduced by calcium channel blockers and Ca(2+)-free solution. Blockage of the Ca(2+)-adenosine triphosphatase pump, which inhibits the extrusion of calcium, also plays a significant role in the process and enhances CIC. CONCLUSIONS: Cooling of detrusor muscle preparations induces a graded myogenic contraction inversely proportional to the temperature. The mechanism is not dependent on local nervous control but is related to calcium translocation.     

Effects of voluntary wheel running on satellite cells in the rat plantaris muscle

This study investigated the effects of voluntary wheel running on satellite cells in the rat plantaris muscle. Seventeen 5-week-old male Wistar rats were assigned to a control (n = 5) or training (n = 12) group. Each rat in the training group ran voluntarily in a running-wheel cage for 8 weeks. After the training period, the animals were anesthetized, and the plantaris muscles were removed, weighed, and analyzed immunohistochemically and biochemically. Although there were no significant differences in muscle weight or fiber area between the groups, the numbers of satellite cells and myonuclei per muscle fiber, percentage of satellite cells, and citrate synthase activity were significantly higher in the training group compared with the control group (p < 0.05). The percentage of satellite cells was also positively correlated with distance run in the training group (r = 0.61, p < 0.05). Voluntary running can induce an increase in the number of satellite cells without changing the mean fiber area in the rat plantaris muscle; this increase in satellite cell content is a function of distance run.

Key points

• There is no study about the effect of voluntary running on satellite cells in the rat plantaris muscle.
• Voluntary running training causes an increase of citrate synthase activity in the rat plantaris muscle but does not affect muscle weight and mean fiber area in the rat plantaris muscle.
• Voluntary running can induce an increase in the number of satellite cells without hypertrophy of the rat plantaris muscle.

Key words: Endurance training, muscle damage, hypertrophy, myonuclear, Pax7.     

雌二醇诱导的慢性非细菌性前列腺炎大鼠前列腺平滑肌细胞电压依赖钙离子通道的定量表达

目的:建立慢性非细菌性前列腺炎大鼠模型,探讨炎症组与正常对照组大鼠前列腺平滑肌细胞电压依赖钙离子通道定量表达的差异性。方法:采用雌二醇诱导大鼠前列腺炎,体外培养纯化前列腺平滑肌细胞(PSMCs),用Trizol提取总RNA,经逆转录,SYBR Green I实时定量PCR测定各亚型钙离子通道α1亚基的mRNA相对表达水平,比较两组间的差异。结果:炎症组和对照组均见L、T、P/Q型钙通道表达,炎症组L型钙通道CaV1.2相对对照组表达量增多,分别为0.048±0.024和0.031±0.015,差异具有显著性(t=2.846,P=0.007),T、P/Q型两组间无统计学差异。结论:慢性前列腺炎PSMCs细胞膜L型钙通道CaV1.2数目上调,钙内流增多,在发病机制中可能具有一定作用。     

Alfentanil attenuates phenylephrine-induced contraction in rat aorta

BACKGROUND AND OBJECTIVES: Alfentanil was reported to relax the rat aorta by direct action on the vascular smooth muscle. The aims of this in vitro study were to examine the effect of alfentanil on phenylephrine-induced contractions in the rat aorta and to determine the cellular mechanism associated with this process. METHODS: Endothelium-denuded aortic rings were suspended in order to record isometric tension. In the rings with or without 10(-6) mol naloxone or 10(-5) mol verapamil, the concentration-response curves for phenylephrine and potassium chloride were generated in the presence or absence of alfentanil (10(-6), 5 x 10(-5), 10(-4) mol). In the rings exposed to a calcium-free isotonic depolarizing solution, the contractile response induced by the addition of calcium was assessed in the presence or absence of alfentanil (5 x 10(-5), 10(-4) mol). RESULTS: Alfentanil (5 x 10(-5), 10(-4) mol) attenuated (P < 0.05) the phenylephrine-induced contraction in the ring with or without 10(-6) mol naloxone but had no effect on the phenylephrine-induced contraction in the rings pretreated with verapamil. Alfentanil (5 x 10(-5), 10(-4) mol) produced a significant rightward shift (P < 0.01) in the potassium chloride dose-response curve, and attenuated the contractile response (P < 0.001) induced by calcium in the calcium-free isotonic depolarizing solution in a dose-dependent manner. CONCLUSIONS: A supraclinical dose of alfentanil attenuates the phenylephrine-induced contraction via an inhibitory effect on calcium influx by blocking the l-type calcium channels in the rat aortic vascular smooth muscle.     

异丙酚舒张血管平滑肌的作用机制

目的:检测异丙酚(Propofol)影响大鼠主动脉平滑肌细胞(ASMC)内游离钙离子浓度([Ca~(2 )]i)作用与影响三磷酸肌醇(inositol 1,4,5-triphosphate,IP_3)合成作用,探讨异丙酚舒张血管平滑肌的作用机制。方法:应用荧光分光光度法,通过荧光剂Fura-2/AM负荷,检测原代培养的ASMC内[Ca~(2 )]i的水平;应用[~3H]肌醇三磷酸测试系统,通过同位素竞争蛋白结合实验,检测ASMC合成IP_3水平。结果:(1)异丙酚与ASMC共同培养72小时后,检测到ASMC的[Ca~(2 )]i基线水平较对照组轻度降低,异丙酚可剂量依赖性抑制去甲肾上腺素(NE)、5-羟色胺(5-HT)引起的[Ca~(2 )]i升高作用;(2)当细胞外液无钙时或钙通道阻滞剂维拉帕米(Virapamil)存在时,异丙酚抑制[Ca~(2 )]i升高作用被部分减弱,但不被取消;(3)异丙酚可剂量依赖性地抑制NE、5-HT的促进IP_3合成作用。结论:异丙酚舒张血管平滑肌作用与抑制IP_3介导的细胞内钙释放作用(IICR)机制密切相关。     

银杏内酯B对缺氧环境下大鼠血管平滑肌细胞线粒体钙离子浓度的影响

目的：探讨银杏内酯B对缺氧环境下大鼠血管平滑肌细胞线粒体内钙离子浓度的调节机制。方法：体外培养大鼠血管平滑肌细胞,分为正常对照组（细胞正常培养）、缺氧处理组（细胞在氮气中培养12h）和1、5、10μmol／L银杏内酯B保护组（细胞加入不同浓度的银杏内酯B后在氮气中培养12h）。细胞经荧光探针Rhod-2／AM染色,激光共聚焦扫描显微镜下观察,并计算各组面积积分吸光度值,以此反映线粒体浓度的变化。结果：与正常对照组比较,缺氧造成大鼠血管平滑肌细胞线粒体钙离子浓度显著升高,各银杏内酯B保护组细胞线粒体钙离子浓度明显低于缺氧处理组,且随着银杏内酯B浓度的升高,线粒体钙离子浓度呈下降趋势。结论：银杏内酯B可以维持线粒体内钙离子浓度的稳定,降低缺氧对细胞造成的损伤。     

Undifferentiated mesenchymal stem cells seeded on a vascular prosthesis contribute to the restoration of a physiologic vascular wall

BACKGROUND: We evaluated the possibility of restoring a physiologic vascular wall using undifferentiated mesenchymal stem cells (MSCs) seeded on a polyurethane vascular prosthesis. METHODS: Undifferentiated MSCs were seeded on a vascular prosthesis and implanted into Wistar male rats (weight, 350 g) to investigate differentiation into smooth muscle cells and to determine graft endothelialization in vivo. RESULTS: Seeded or nonseeded grafts were surgically implanted. Undifferentiated MSCs were first labelled for green fluorescent protein. After 2 weeks in vivo, MSC that were initially self-expanded on the graft in a monolayer were organized in a multicellular layer mimicking media of aortic adjacent wall. They coexpressed green fluorescent protein and smooth muscle proteins that were not present before the in vivo engraftment, indicating that in vivo conditions induced smooth muscle protein maturation. Undifferentiated MSC showed an electrophysiologic profile quite different than mature smooth muscle cells. In both in vitro- and in vivo-differentiated MSCs, adenosine triphosphate, an IP(3)-dependent agonist, induced an increase in calcium similar to that which occurred in mature smooth muscle cells. However, MSCs failed to respond to caffeine, a ryanodine receptor activator, indicating the absence of mature calcium signaling, and finally, contraction was absent. Endothelialization attested by immunohistology and scanning electron microscopy was greater in MSC-seeded grafts that prevent thrombosis. CONCLUSION: Only partial smooth muscle cell differentiation of MSCs resulted when seeded on vascular grafts, but MSCs spontaneously restore a media-like thick wall. Mesenchymal stem cells have a positive impact on in vivo endothelialization in rats that supports their potential for use in vascular surgery. CLINICAL RELEVANCE: Thrombosis of vascular prostheses is a major complication of surgery. We showed on rat aorta that mesenchymal stem cells seeded on polyurethane patch restore endothelium. It also induced incomplete smooth muscle differentiation. In the future, stem cell could prevent thrombosis of vascular prostheses.     

Direct inhibition of rat detrusor muscle contraction by erythromycin

PURPOSE: Detrusor instability is a common problem in the elderly, which is usually treated with anti-cholinergic medication. This study investigates the effect of erythromycin on rat detrusor muscle contractile response to characterise its potential as an alternative inhibitor of bladder muscle contraction. MATERIALS AND METHODS: Strips of rat detrusor muscle were suspended in a perfusion organ bath. The contractile response to direct muscle stimulation, electrical field stimulation (EFS, 0.5-60 Hz), carbachol (10(-5) M), and potassium (10-80 x 10(-3) M) were determined before and after the addition of erythromycin (10(-4)-10(-3) M). The contractile response to carbachol (10(-5) M) in the presence of nifedipine (10(-8) or 10(-6) M) or in calcium-free Kreb's solution was also determined in the absence and presence of erythromycin. RESULTS: Erythromycin 5 x 10(-4) M inhibited the maximum contractile response to EFS, carbachol, and potassium by 38% (P < 0.01), 62% (P < 0.001), and 17% (P < 0.05), respectively, but did not significantly reduce the response to direct muscle stimulation. The atropine-resistant component of EFS-evoked contraction was inhibited by 19.5% (P < 0.01) in the presence of erythromycin. In calcium-free Krebs solution, the maximum contractile response to carbachol was reduced by 42% of control (P < 0.0001) and nifedipine 10(-8) M had no additional effect. When erythromycin 5 x 10(-4) M was added together with nifedipine 10(-8) M, the response to carbachol was inhibited by a further 25% (P < 0.005). CONCLUSIONS: Erythromycin inhibits rat detrusor muscle contraction through the inhibition of calcium influx and the modulation of intracellular calcium movement.     

Effects of isoproterenol on spontaneous excitations in detrusor smooth muscle cells of the guinea pig.

PURPOSE: Because beta-adrenoceptor agonists would be a useful tool for the pharmacological treatment of unstable bladder, we investigated the cellular mechanisms underlying beta-adrenoceptor mediated inhibition on spontaneous excitation in detrusor smooth muscle. MATERIALS AND METHODS: Detrusor smooth muscle bundles were isolated from guinea pig bladders. Changes in membrane potential were recorded using an intracellular recording technique. In preparations loaded with the calcium indicator fura-PE3 changes in the concentration of intracellular calcium ions were measured simultaneously with membrane potential. Effects of isoproterenol on spontaneous changes in the membrane potential and intracellular Ca(2+) were examined RESULTS: Detrusor smooth muscle cells exhibited spontaneous action potentials that were associated with transient increases in intracellular Ca(2+) (calcium transients). Isoproterenol, which hyperpolarized the membrane, prevented action potentials and calcium transients. This induced inhibition of calcium transients was not affected by cyclopiazonic acid. Isoproterenol induced hyperpolarization was inhibited by inhibitors of protein kinase A, N-[2-((p-bromocinnamyl)amino)ethyl]-5-isoquinolinesulfonamide, hydrochloride and Rp-adenosine-3',5'-cyclic phosphorothioate. Hyperpolarization was blocked by a solution containing 30 mM. potassium but not by a range of potassium channel blockers. Ouabain and a solution of 0.5 mM. potassium also inhibited hyperpolarization. CONCLUSIONS: Our results suggest that isoproterenol prevented spontaneous action potential discharges and associated calcium transients through the activation of protein kinase A. The isoproterenol induced inhibition of intracellular Ca(2+) largely depends on the prevention of spontaneous action potentials since the contribution of the intracellular calcium store was small. Isoproterenol hyperpolarizes the membrane, probably by stimulating sodium pump activity.