Effects of verapamil on excitation-contraction coupling in single crab muscle fibers

Summary The effects of verapamil and its optical isomers on the electrical and mechanical characteristics of single muscle fibers ofCallinectes danae were studied. Verapamil (10–20 g/ml) blocked the procaine-and TEA-induced spikes; the blockade was preceded by reduction in the rate of rise of the up-stroke and increase in the duration of the action potentials. Inhibition of Ba-spikes required higher concentrations of verapamil (>50 g/ml). These concentrations reduced the amplitude of the normally occurring graded electrogenic membrane responses and reduced the rate of development of the current-induced tensions. With lower concentrations (10–30 g/ml) verapamil enhanced the negative afterpotentials and the peak amplitude of the local contractions elicited by depolarizing current pulses, while the graded membrane responses were not markedly modified. Verapamil (1–100 g/ml) did not affect the resting membrane potential but increased the effective membrane resistance. Determination of the cable characteristics by DC pulses indicated that verapamil (1–10 g/ml) shortens the membrane length constant, increases the specific resistivity of the sarcoplasm and, in most cases, increases the membrane time constant. Verapamil (10 g/ml) induced tension in these crab fibers. The contractions were potentiated in Na-deficient media, by increase in [Ca]₀, and by membrane depolarization; Ca-free salines depressed, and procaine abolished these contractions. The results suggest that verapamil affects both Ca and K conductances and interferes with the Ca-sequestering mechanisms of these fibers. The (–)-isomer of verapamil was more effective than the (+)-isomer with respect to tension development, prolongation and subsequent blockade of procainespikes and enhancement of current-induced after-potentials and contractions.This work was supported in part with grants from CNP_q (TC-16.897) and from CEPG-UFRJ     

Prolonged hypoxia modulates platelet activating factor receptor-mediated responses by fetal ovine pulmonary vascular smooth muscle cells

Hypoxia augments PAF receptor (PAFr) binding and PAFr protein expression in venous SMC (SMC-PV). We compared effect of acute and prolonged hypoxia (pO₂ < 40 torr) on PAFr-mediated responses in arterial SMC (SMC-PA) and SMC-PV. Cells were studied for 30 min (acute) or for 48 h (prolonged) hypoxia and compared to normoxic (pO₂ ~ 100 torr) conditions. PAF binding was quantified in fmol/10⁶ cells (mean ± SEM). PAF binding in normoxia were SMC-PA, 5.2 ± 0.2 and in SMC-PV, 19.3 ± 1.1; values in acute hypoxia were SMC-PA, 7.7 ± 0.4 and in SMC-PV, 27.8 ± 1.7. Prolonged hypoxia produced 6-fold increase in binding in SMC-PA, but only 2-fold increase in SMC-PV, but binding in SMC-PV was still higher. Acute hypoxia augmented inositol phosphate release by 50% and 40% in SMC-PA and SMC-PV, respectively. During normoxia, PAFr mRNA expression by both cell types was similar, but expression in hypoxia by SMC-PA was greater. In SMC-PA, hypoxia and PAF augmented intracellular calcium flux. Re-exposure of cells to 30 min normoxia after 48 h hypoxia decreased binding by 45–60%, suggesting immediate down-regulation of hypoxia-induced PAFr-mediated effects. We speculate that re-oxygenation immediately reverses hypoxia effect probably due to oxygen tension-dependent reversibility of PAFr activation and suggest that exposure of the neonate to prolonged state of hypoxia will vilify oxygen exchange capacity of the neonatal lungs.     

Induction of endothelin-1 expression by oxidative stress in vascular smooth muscle cells

Atherosclerosis is based on endothelial dysfunction leading to impaired vasomotor function. This is partially due to nitric oxide (NO) depletion caused by oxidative stress. Since the vasoconstrictor endothelin-1 (ET-1) might also be involved in endothelial dysfunction, we investigated whether oxidative stress regulates ET-1 expression in vascular smooth muscle cells (VSMC). Human aortic VSMC were treated with H₂O₂ (200 μM) for up to 8 h. mRNA expression of preproendothelin (prepro-ET) was analyzed by RT-PCR. ET-1 protein and the marker for oxidative stress, 8-isoprostane, were determined by ELISA. Activity of cytosolic phospholipase A2 (cPLA₂) as an indicator of ET-1 autocrine activity was measured photometrically. Stimulation of VSMC with H₂O₂ resulted in increased expression of prepro-ET mRNA after 1 h with a maximum after 6 h (fourfold), similar to treatment with angiotensin II. ET-1 protein was significantly increased by H₂O₂ treatment with a maximum after 8 h (P<.05). This effect was inhibited by the antioxidants resveratrol (100 μM) and quercetin (50 μM). In quiesced VSMC, incubation with H₂O₂-conditioned medium resulted in increased cPLA₂ activity compared to the controls (P<.05). This activity was partially inhibited by the ET_A-receptor antagonist, PD 142893 (10 μM), indicating functional ET-1 in the conditioned medium. The presence of oxidative stress in H₂O₂-treated VSMC was associated by significantly increased formation of 8-isoprostane (P<.05). The data indicate for the first time that oxidative stress increases ET-1 generation and autocrine ET-1 activity in VSMC, a mechanism that might contribute to endothelial dysfunction in atherosclerosis.     

失血性休克大鼠血管平滑肌收缩功能变化研究

目的：研究失血性休克后大鼠胸主动脉血管平滑肌收缩功能变化并初步探讨其可能机制。方法：采用生物张力换能器及生理记录仪等技术体外测定失血性休克大鼠在休克后2 h、4 h VSM环对去甲肾上腺素（NE）、苯肾上腺素（PE）、咖啡因（caffeine）及氯化钾（KCl）等的收缩反应张力。结果：VSM环在休克后2 h、4 h对NE的最大反应张力分别为对照组的76.17%和66.50%；休克2 h后对高浓度（大于20 mmol/L）K⁺ 和20 mmol/L caffeine的反应张力明显下降；休克后4 h对 3×10^-6 mol/L PE的反应张力亦显著下降。结论：失血性休克后大鼠VSM收缩功能下降，反应性降低，其初步机制可能部分与休克后VSM细胞胞外钙内流及胞内钙释放功能下降等有关。     

Insulin enhances angiotensin II induced DNA synthesis in vascular smooth muscle cells of the rat

Summary Hypertension has a high prevalence among subjects with decreased insulin sensitivity and/or hyperinsulinemia. Furthermore, angiotensin II plays a pivotal role in the regulation of vascular tone and is known to induce hypertrophy and/or hyperplasia in vascular smooth muscle cells. In the present study, the effect of insulin on angiotensin II induced smooth muscle cell growth (Wistar-Kyoto rat) was investigated. Cell growth was assessed by the measurement of [³H]thymidine incorporation into cell DNA. Insulin in a concentration range of 1.7 × 10^–10–1.7 × 10^–6 M lacked any effect on cell DNA synthesis. However, insulin enhanced the angiotensin 11 induced DNA synthesis in a concentration-dependent manner. This effect was similar in cells with a weak and in cells with a marked response in DNA synthesis to stimulation with 100 nM angiotensin 11. In conclusion, insulin is able to enhance angiotensin 11 induced DNA synthesis and may therefore function as a growth cofactor in vascular smooth muscle cells.Abbreviations AngII angiotensin II - EGF epidermal growth factor - bFGF basic fibroblast growth factor - PDGF-BB platelet-derived growth factor-BB - VSMC vascular smooth muscle cells Dedicated to Prof. Dr. N. Zöllner on the occasion of his 70th birthday     

A simple method for differentiating vascular smooth muscle cells and fibroblasts in tissue culture

Summary The morphologic differentiation of vascular smooth muscle cells and fibroblasts in tissue culture is difficult if not impossible. By direct immunofluorescence, it is possible to distinguish between vascular smooth muscle cells and fibroblasts after 6 to 10 days in tissue culture. Microfilaments appear from the 6th to the 10th day. After an incubation period of 30 minutes with antibody against smooth muscle actomyosin at room temperature, microfilaments are demonstrable in smooth muscle cells. In contrast, fibroblasts, if incubated for the same period, show strong nuclear fluorescence and a primary fluorescence of the cytoplasm, but filaments are not visible. If fibroblasts are incubated with antiactomyosin for one hour at 37 °C, however, microfilaments are easily detectable.With this method it is possible to differentiate in a simple manner vascular smooth muscle cells from fibroblasts in a heterologous tissue culture.These studies were supported by the Deutsche Forschungsgemeinschaft, SFB 90,Cardiovasculäres System.     

Modulation of calcium movements by nitroprusside in isolated vascular smooth muscle cells

Using the patch-clamp technique, we have characterised the inward current from enzymatically dispersed rabbit pulmonary arterial cells, and investigated the effects of the vasodilator, nitroprusside (NP), on these and other membrane currents. With Cs⁺-filled pipettes, inward currents were recorded during brief depolarizing voltage steps in both physiological Ca²⁺ and 10 mM Ba²⁺. The threshold for current activation was positive to -40 mV and the current peaked at 0 mV for Ca²⁺ and +10mV for Ba²⁺. During the first few minutes of recording, inward currents increased or ran-up. This could not be attributed to blockade of outward current or the inclusion of adenosine triphosphate (ATP) in the patch pipette. Experiments revealed that all the inward current was carried through a single type of voltage-activated Ca²⁺ channel, namely the high-threshold, dihydropyridine-sensitive channel. It was unaffected by tetrodotoxin but was abolished at all potentials by low concentrations of Cd²⁺ (100 M) or nifedipine (1–2M). NP (1 M) suppressed peak inward Ba²⁺ current at +10 mV by approximately 45%. Higher concentrations (50 M) did not produce further blockade of the current. This decrease was associated with increased inactivation of the current, and both effects required the presence of ATP in the patch pipette. In physiological Ca²⁺, using K⁺-filled pipettes, NP was found to induce spontaneous bursts of outward currents, which are probably activated by the release of Ca²⁺ from Ca²⁺-overloaded stores. These results are consistent with NP lowering cytosolic Ca²⁺, and hence causing vasodilation, by inhibiting Ca²⁺ influx through voltage-gated Ca²⁺ channels and by promoting Ca²⁺ uptake into the sarcoplasmic reticulum.     

Canstatin RNA转染对兔血管平滑肌细胞体外增殖的抑制作用

目的:探讨canstatin对体外培养的兔血管平滑肌细胞(VSMC)增殖的影响。方法:将阳离子脂质体介导的canstatinRNA转染VSMC,应用细胞计数,[³H]-TdR掺入率测定观察canstatin基因对VSMC增殖的作用。结果:阳离子脂质体介导的canstatinRNA可有效地转染VSMC并能显著地抑制VSMC的增殖及其DNA的合成率。结论:Canstatin基因可抑制体外培养的VSMC的增殖.     

牛磺酸减轻β-甘油磷酸诱导的大鼠血管平滑肌细胞钙化氧化应激在反流物所致食管粘膜损伤中的作用

目的：观察牛磺酸对钙化的形成及逆转作用的影响，探讨牛磺酸在血管钙化发生中的作用。方法：利用β-甘油磷酸制备钙化血管平滑肌细胞（VSMCs），测定细胞钙含量、碱性磷酸酶活性、[⁴⁵Ca]沉积及[³H]-胸腺嘧啶。结果：与对照组相比，钙化细胞的钙含量、ALP活性和[⁴⁵Ca]沉积均明显升高（P<0.05），而牛磺酸与β-甘油磷酸同时孵育呈剂量依赖性地抑制钙化发生。在牛磺酸逆转实验中，钙化细胞的钙含量、ALP活性和[⁴⁵Ca]沉积均较换液前明显降低（P<0.01）；且牛磺酸呈剂量依赖性地逆转已钙化细胞。与对照组相比，钙化细胞的细胞数量和[³H]-TdR掺入量增加（P<0.01），牛磺酸早期干预组显著抑制钙化细胞的增殖，但牛磺酸对已钙化的细胞,增殖抑制效应不明显。结论：牛磺酸可抑制细胞钙化形成且能逆转已形成钙化。     

Inhibition of angiotensin II and platelet-derived growth factor-induced vascular smooth muscle cell proliferation by calcium entry blockers

Summary Structural changes within the blood vessel wall such as hyperplasia and hypertrophy of vascular smooth muscle cells are important factors in the pathogenesis of hypertension. Humoral growth factors such as angiotensin II (AII) and platelet-derived growth factor BB (PDGF-BB) may participate in the remodelling of the blood vessel wall. Whether and by which mechanisms antihypertensive treatment is capable of influencing the structural blood vessel alterations to date remains unclear. In the present study, the effect of nifedipine and diltiazem on AII- and PDGF-BB-induced vascular smooth muscle cell proliferation was examined. Nifedipine and diltiazem at a concentration of 10 M did not affect baseline DNA synthesis in isolated vascular smooth muscle cells in culture. AII (final concentration 100 nM) and PDGF-BB (50 ng/ml) stimulated DNA synthesis by approximately 9.0- and 4.6-fold, respectively. Both AII- and PDGF-BB-induced DNA synthesis was significantly blunted by diltiazem and nifedipine in a concentration of 10 M, while no significant influence was seen with concentrations from 10 nM up to 1 M. In contrast, no significant influence of these drugs could be observed on fetal calf serum 5%-induced DNA synthesis. The findings indicate that calcium antagonists possess antimitogenic potential and that they may thus contribute to the regression of structural changes of the blood vessels associated with hypertension.Abbreviations PDGF-BB platelet-derived growth factor BB - AII angiotensin II - FCS fetal calf serum - VSMC vascular smooth muscle cells - ACE angiotensin I converting enzyme This work was supported by HEXAL-PHARMA, Holzkirchen, Federal Republic of Germany     

血管紧张素II及其受体在血管平滑肌细胞迁移中的作用

目的:探讨血管紧张素II(AngII)及其受体(ATRs)在局部血管损伤后血管平滑肌细胞(VSMC)迁移中的作用及其机制。方法:以体外培养VSMC为基础,采用细胞化学和改良Boyden'schamber的方法,观察AngⅡ干预VSMC后AngII受体的表达、VSMC迁移能力的变化、肌动蛋白纤维丝的动态组装变化,并探讨AT₁R拮抗剂、AT₂R拮抗剂对上述观测指标的影响。结果:AngII10^-7mol/L可以刺激VSMC发生迁移,该作用是通过影响VSMC内应力纤维动态组装而实现的;AngII干预VSMC后可使AT₁R表达上调,随着作用时间延长AT₁R表达水平下降。AT₁R拮抗剂可下调AT₁R表达。AngII通过AT₁R的介导发挥其影响VSMC迁移能力的生物学效应。AT₂R对此无明显影响。结论:AngII通过AT₁R介导来调节VSMC内肌动蛋白微丝的动态组装,进而改变VSMC的迁移能力,从而发挥其介导VSMC迁移的生物学效应。     

Length-tension relationships in resting and activated vascular smooth muscle fibres

Summary The length-tension relationships of resting or activated (by 130 mM of potassium) helical strips of the pig coronary artery were studied. The distensibility of the preparations was expressed by approximated exponential function. In non-activated strips, an average lengthening of 9.64% was necessary for doubling the resting tension. The maximum of active tension (isometric contractions) occurred at high degrees of muscle stretch, whereas the maximum of shortening (isotonic free-loaded contractions) was found at lower values of resting tension. If the theory of the sliding mechanism is a correct assumption for the contraction process of vascular smooth muscle, the maximum of active tension observed at a muscle stretch of about 8000 dynes/mm² is obviously caused by an optimal overlapping of actin and myosin filaments. At this high degree of muscle stretch, however, a great number of cross-linkages is necessary to overcome the passive tension and only few cross-linkages are available for shortening. Therefore, in isotonic contractions the amount of shortening is diminished and the time to peak of contraction is augmented with elevated resting tension exceeding 1000 dynes/mm².     

钙依赖中性蛋白酶和半胱天冬酶-3在辛伐他汀诱导大鼠血管平滑肌细胞凋亡中的作用

目的:观察辛伐他汀(simvastatin)诱导大鼠血管平滑肌细胞(VSMC)凋亡及可能参与其中的凋亡信号通路的研究, 探讨他汀类药物可能的非调脂抗动脉粥样硬化作用机制。方法:体外培养VSMC, 荧光分光光度仪检测钙依赖中性蛋白酶(calpain)活性;Western印迹杂交法检测半胱天冬酶-3(caspase-3)的激活;基因组DNA凝胶电泳、流式细胞仪PI/AnnexinV双重染色等鉴定细胞凋亡。结果:30μmol/Lsimvastatin刺激8h后, calpain活性显著高于对照组(P<0.05, n=4), 12h后达对照的3倍以上(P<0.01);caspase-3的20kD活性亚基在30μmol/Lsimvastatin刺激12h后直至48h均可检测到, 而对照组及simvastatin刺激2h均未见caspase-3的20kD条带;calpain特异性抑制剂PD150606可显著抑制simvastatin诱导的VSMC凋亡, 100μmol/LPD150606与30μmol/Lsimvastatin共同刺激细胞24h后, 流式细胞仪检测凋亡率为9.5%±1.9%, 显著低于simvastatin单独刺激的24.2%±1.7%(P＜0.01, n=4)。DNA梯度现象亦被抑制。同时PD150606也能有效抑制simvastatin诱导的caspase-3激活。结论:Simvastatin诱导大鼠VSMC凋亡可能是通过calpain并进而激活caspase-3来达到的。     

Calcium channel currents in isolated smooth muscle cells from the basilar artery of the guinea pig

Ca²⁺ channel currents were studied in smooth muscle cells from the basilar artery of the guinea pig using the whole-cell patch-clamp technique, 10 mM Ba²⁺ as the charge carrier and strong buffering of intracellular Ca²⁺ with EGTA (pCa_i=8). Cell capacitance was 18.8 ± 6.6 pF (n= 96) and maximum current density at +10 to +20 mV (holding potential < –55 mV), measured early in dialysis, was –14.8 ± 4.9 pA/pF (n= 83). Currents reversed at approximately +95 mV and, at more positive potentials, outward Cs⁺ currents were recorded that were blocked by either external Cd²⁺ or Ca²⁺. One component of current was identified that had properties consistent with L-type channels. On the basis of measurements of tail currents, its threshold for activation was –15 mV, its voltage dependence of activation was steep and it was half-activated at +8.5 mV. It inactivated very slowly at +15 mV (2787 ± 511 ms) and it deactivated rapidly (251 ± 55 s) at –55 mV. It was quickly lost during dialysis and was largely blocked by 1 nM nifedipine (1-s pulses, holding potential = –55 mV). A second component, termed B-type current, was identified that had properties inconsistent with those of T-type channels. On the basis of tail currents, its threshold for activation was –30 mV, its voltage dependence of activation was less steep and it was half-activated at +33.7 mV. It was half-inactivated at –32.1 mV, it inactivated with a time constant of 162 ± 13 ms at +15 mV and it deactivated relatively slowly (3.8 ± 0.8 ms) at –55 mV. It was less sensitive than L-type current to dialysis and to block by nifedipine.     

A comparison of two different indicators: quin 2 and aequorin in isolated single cells and intact strips of ferret portal vein

A comparison between the fluorescent indicator quin 2 and the bioluminescent indicator aequorin was performed in the same smooth muscle cell type. Aequorin was loaded into intact strips and quin was loaded into enzymatically isolated single cells from ferret portal vein. Both indicators gave qualitatively the same calcium profiles when the tissue was challenged with agonists. Quin loading caused a dramatic shift to the right in dose response curves to potassium and phenylephrine. The ED₅₀ values for quin loaded cells were significantly different from those for control cells for both agonists. Intracellular calcium levels at rest were not significantly different with quin and aequorin. Cells stimulated with potassium gave significantly different intracellular calcium values with the two indicators suggesting a change in the stimulated steady state level due to the introduction of an additional calcium buffer (quin2) into the cell.     

血管紧张素转换酶抑制剂抑制血管平滑肌细胞增殖的机制

目的: 探讨血管紧张素转换酶抑制剂(ACEI)抑制动脉平滑肌细胞增殖及向内膜迁徙的机理。方法: 球囊导管损伤Wistar大鼠颈总动脉, 实验组于术前2 d开始给与ACEI(temocapril-HCl 10 mg·kg^-1·d^-1), 术后2 d、3 d、5 d分批处死。用抗人PDGF-A、-B及其受体, 抗人MMP-1、MMP-9等抗体以ABC法行免疫染色。动脉组织行放射自显影乳剂原位酶谱分析。电镜下观察细胞质内小器官及细胞周围弹性、胶原纤维的密度。结果: 给ACEI后, PDGF及其受体、MMP-1、-9蛋白阳性细胞率及明胶酶活性显著降低, 并抑制了中膜平滑肌细胞的表型转换。结论: ACEI可能通过继发地抑制PDGFs、MMPs蛋白表达, 阻碍细胞表型转换, 从而阻止中膜平滑肌细胞增殖及向内膜迁徙。     

碱性成纤维细胞生长因子对人脑动静脉畸形血管平滑肌细胞增殖和表达骨桥蛋白的影响

目的：研究碱性成纤维细胞生长因子（bFGF）对体外培养的人脑动静脉畸形（CAVM）血管平滑肌细胞（VSMC）增殖和表达骨桥蛋白的影响。方法： 应用胶原酶消化法培养人CAVM的VSMC，MTT法检测VSMC的增殖，免疫印迹法检测VSMC骨桥蛋白的表达。结果：bFGF作用于VSMC后，细胞增殖活性明显增加。实验组骨桥蛋白的表达显著高于对照组 (P<0.05)。结论： 体外条件下，bFGF可以刺激CAVM的VSMC增殖，并能促进其表达骨桥蛋白。