Effects of nitric oxide on large‐conductance Ca2+‐activated K+ currents in human cardiac fibroblasts through PKA and PKG‐related pathways

The human cardiac fibroblast (HCF) is the most abundant cell type in the myocardium, and HCFs play critical roles in maintaining normal cardiac function. However, unlike cardiomyocytes, the electrophysiology of HCFs is not well established. In the cardiovascular system, Ca²⁺‐activated K⁺ (KCa) channels have distinct physiological and pathological functions, and nitric oxide (NO) plays a key role. In this study, we investigated the potential effects of NO on KCa channels in HCFs. We recorded strong oscillating, well‐maintained outward K⁺ currents without marked inactivation throughout the test pulse period and detected outward rectification in the I‐V curve; these are all characteristics that are typical of KCa currents. These currents were blocked with iberiotoxin (IBTX, a BKCa blocker) but not with TRAM‐34 (an IKCa blocker). The amplitudes of the currents were increased with SNAP (an NO donor), and these increases were inhibited with IBTX. The SNAP‐stimulating effect on the BKCa currents was blocked by pretreatment with KT5823 (a protein kinase G [PKG] inhibitor) or 1 H‐[1,‐2, ‐4] oxadiazolo‐[4,‐3‐a] quinoxalin‐1‐one (ODQ; a soluble guanylate cyclase inhibitor). Additionally, 8‐bromo‐cyclic guanosine 3’,5’‐monophosphate (8‐Br‐cGMP) stimulated the BKCa currents, and pretreatment with KT5720 (a protein kinase A [PKA] inhibitor) and SQ22536 (an adenylyl cyclase inhibitor) blocked the NO‐stimulating effect on the BKCa currents. Furthermore, 8‐bromo‐cyclic adenosine 3’,5’‐monophosphate (8‐Br‐cAMP) activated the BKCa currents. These data suggest that BKCa current is the main subtype of the KCa current in HCFs and that NO enhances these currents through the PKG and PKA pathways.     

Thiol reagents and nitric oxide modulate the gating of BKCa channels from the guinea-pig taenia caeci

1. The site of the direct modulation of the gating of BKCa channels by the nitric oxide donor s-nitroso-l-cysteine (NOCys) was examined in excised membrane patches of the guinea-pig taenia caeci by the use of various thiol (sulphydryl)-specific reagents, including N-ethylmaleimide (NEM) and three charged methanethiosulphonate (MTS) reagents, namely positively charged 2-aminoethyl MTS hydrobromide (MTSEA) and [2-(trimethylammonium)ethyl] MTS bromide (MTSET) and negatively charged sodium (2-sulphonatoethyl) MTS (MTSES), which all specifically convert sulphydryls to a disulphide. 2. At 10 micro mol/L, NOCys transiently increased the probability of opening (N.Po) of the BKCa channels (at 0 mV) after a delay of 1-2 min. 3. Disulphide-reducing agents, such as dithiothreitol (10 micro mol/L), increased N.Po in a manner that was reversed by the sulphide-oxidizing agent thimerosal (10 micro mol/L). Both positively charged MTSET (2.5 mmol/L) and negatively charged MTSES (2.5 mmol/L) rapidly increased N.Po. However, only the MTSES-evoked increase in N.Po remained after a prolonged washout period. 4. The specific alkylating agent of cysteine thiols NEM (1 mmol/L) and the positively charged, but membrane permeable, MTSEA (2.5 mmol/L) decreased N.Po (at 0 mV). 5. Pre-exposure of excised membrane patches to NEM or MTSES prevented the excitatory actions of NOCys (10 micro mol/L). 6. We conclude that MTSES and NOCys must modify thiols on cysteine residues within basic regions of the channel protein that would electrostatically exclude MTSEA and MTSET. A consensus sequence of various mammalian alpha-subunits of the BKCa channel reveals two pairs of cysteine residues surrounded by basic amino acids that could be the site of action for NOCys and MTSES.     

一氧化氮对大鼠肝脏缺血再灌注细胞凋亡的影响

目的:探讨一氧化氮在大鼠肝脏缺血—再灌注细胞凋亡中的作用机制。方法:采用动物分组对照实验,研究了L—精氨酸(NO供体)对大鼠肝缺血再灌注细胞凋亡的影响,测量不同状态下动物肝脏组织中NO- 3/NO- 2 含量和细胞凋亡百分率(AI)的变化。结果:肝组织中一氧化氮代谢产物NO- 3/NO- 2 含量明显增加,并且肝组织中细胞凋亡百分率(AI)明显减小。结论:一氧化氮对肝脏缺血再灌注细胞凋亡有抑制作用,本实验为一氧化氮在肝缺血再灌注损伤的临床治疗提供实验基础。     

心肌肽素对豚鼠心室肌细胞L型钙通道的影响

目的研究心肌肽素对豚鼠心室肌细胞L型钙通道的影响,探讨心肌肽素在离子通道水平的药理作用机制。方法用急性酶解分离法获得豚鼠心室肌细胞,标准的全细胞膜片钳技术记录L型钙电流(ICa-L)。结果心肌肽素1、5、10、50、100、500 mg.L-1使豚鼠心室肌细胞ICa-L分别增加(5±4)%、(21±5)%、(30±5)%、(55±8)%、(76±11)%、(80±9)%,半最大效应浓度(EC50)为(18±6)mg.L-1。心肌肽素50 mg.L-1使ICa-L激活时间(TTP)从(6.7±0.9)m s缩短为(5.9±0.7)m s(P<0.01);使ICa-L电流密度-电压曲线下移,但激活电压、峰电压和I-V曲线的形状不变;激活曲线向负电压方向变化,半数激活电压从(-4.3±0.4)mV减少至(-8.6±0.4)mV(P<0.05);不影响稳态失活曲线和稳态失活后恢复曲线。结论心肌肽素浓度依赖性增强豚鼠心室肌细胞ICa-L。     

Nanomolar level of ouabain increases intracellular calcium to produce nitric oxide in rat aortic endothelial cells

Changes in [Ca(2+)](i) across the cell membrane and/or the sarcoplasmic reticulum regulate endothelial nitric oxide (NO) synthase activity. In the present study, we investigated the effect of ouabain, a specific inhibitor of Na(+)/K(+)-ATPase, on NO release and [Ca(2+)](i) movements in cultured rat aortic endothelial cells (RAEC) by monitoring NO production continuously using an NO-specific real-time sensor and by measuring the change in [Ca(2+)](i) using a fluorescence microscopic imaging technique with high-speed wavelength switching. The t((1/2)) (half-time of the decline of [Ca(2+)](i) to basal levels after stimulation with 10 micro mol/L bradykinin) was used as an index of [Ca(2+)](i) extrusion. A very low concentration of ouabain (10 nmol/L) did not increase the peak of NO production, but decreased the decay of NO release and, accordingly, increased integral NO production by the maximal dose-response concentration induced by bradykinin. The same dose of ouabain affected [Ca(2+)](i) movements across the cell membrane and/or sarcoplasmic reticulum induced by bradykinin with a time-course similar to that of NO release. Moreover, the t((1/2)) was significantly increased. Pretreatment of RAEC with Na(+)-free solution, an inhibitor of the Na(+)/Ca(2+) exchanger, and nickel chloride hexahydrate prevented the effects induced by bradykinin and ouabain. These observations using real-time recording indicate that a small amount of ouabain contributes to the bradykinin-stimulated increase of NO production through inhibition of plasma membrane Na(+)/K(+)-ATPase activity and an increase in intracellular Na(+) concentrations. The membrane was then depolarized, leading to a decline in the bradykinin-stimulated increase in [Ca(2+)](i) by forward mode Na(+)/Ca(2+) exchange to prolong the Ca(2+) signal time. From these results, we suggest that nanomolar levels of ouabain modulate [Ca(2+)](i) movements and NO production in RAEC.     

神经元型一氧化氮合酶在心脏功能调节中的作用

神经元型一氧化氮合酶(nNOS)不仅在神经细胞中表达,也存在于心肌细胞。生理状况下,nNOS源性的一氧化氮(NO)通过自分泌以负反馈的调节方式维持细胞内Ca2+浓度稳态,防止细胞内Ca2+浓度超载,对于维持正常的心功能具有重要意义。病理状况下,nNOS的活性发生明显变化,nNOS源性的NO可能参与了心脏疾病的发生和发展。     

rIL-2诱导一氧化氮产生对培养心肌细胞线粒体活性的影响

目的研究人类重组白介素2（rIL-2）对培养大鼠心肌细胞产生一氧化氮（NO）及线粒体活性的影响。方法在培养心肌细胞时分别或同时加入rIL-2、单甲基L-精氨酸（L-NMMA）以及L-精氨酸（L-Arg），并测定培养液中NO浓度，心肌细胞内的线粒体活性值。结果心肌细胞加rIL-2培养48h与对照组相比NO产生显著增加[（85±6.1）比（10±2.5）nmol·（106细胞）-1，P＜0.01]。rIL-2刺激NO产生呈时间（6～48h）和剂量（5×104～1×106IU·L-1）相关性。L-NMMA可抑制rIL-2诱导NO的产生而添加L-Arg可逆转这一作用。rIL-2组与对照组相比线粒体活性值明显受抑（0.397±0.03比0.599±0.02，P＜0.01），而添加L-NMMA可逆转（0.536±0.03，P＜0.01）。NO产生量与线粒体活性值呈负相关（P＜0.01）。结论rIL-2刺激培养大鼠心肌细胞产生NO可抑制线粒体活性。     

Store-operated calcium channels and pro-inflammatory signals

In non-excitable cells such as T lymphocytes, hepatocytes, mast cells, endothelia and epithelia, the major pathway for calcium [Ca2+] entry is through store-operated Ca2+ channels in the plasma membrane. These channels are activated by the emptying of intracellular Ca2+ stores, however, neither the gating mechanism nor the downstream targets of these channels has been clear established. Here, I review some of the proposed gating mechanisms of store-operated Ca2+ channels and the functional implications in regulating pro-inflammatory signals.     

缺氧复氧诱导的心肌细胞凋亡及一氧化氮对此过程的作用

目的　研究缺氧复氧损伤诱导体外培养的新生大鼠心肌细胞凋亡及一氧化氮 (NO)对此过程的作用。方法　取体外培养的新生大鼠心肌细胞 ,分两组 ,一组于 0 95N2 、0 0 5CO2 孵箱中培养 16h、32h、4 8h ,再恢复正常条件培养6h ,造成缺氧复氧损伤的细胞模型 ,TUNEL法观察心肌细胞凋亡形态学特征 ,流式细胞仪检测心肌细胞凋亡率 ;另一组缺氧培养 16h、32h、4 8h后 ,将NO供体亚硝基青霉胺(SNAP)加入培养基中 ,使其终浓度为 10 0 μmol·L-1,再于正常条件培养 6h ,检测心肌细胞凋亡率。结果　心肌细胞在缺氧 16h、32h和 4 8h后复氧 6h ,TUNEL法可检测到阳性的凋亡细胞 ,流式细胞仪检测其凋亡率分别为 :5 5 %±0 7% ,11 0± 1 1%和 14 2 %± 1 6 % ;心肌细胞缺氧培养16h、32h、4 8h后加入SNAP ,再复氧 6h ,流式细胞仪检测其凋亡率分别为 :3 2 %± 0 7% ,7 8%± 0 7%和 10 9%±1 0 %。结论　缺氧复氧损伤引起的心肌细胞凋亡率随着缺氧时间的延长而增高 ;NO对缺氧复氧损伤引起的心肌细胞凋亡有抑制作用     

Effects of slow, sustained, and rate-tunable nitric oxide donors on human aortic smooth muscle cells proliferation

Smooth muscle cell (SMC) proliferation has been accepted as a common event in the pathophysiology of vascular diseases, including atherogenesis and intimal hyperplasia. Delivery of the nitric oxide synthase (NOS) substrate l-arginine, pharmacological nitric oxide (NO) donors, NO gas or overexpression of NOS proteins can inhibit SMC proliferation and reduce the injury responses within the blood vessel wall. Although commercial development of NO donors that attempt to provide exogenous delivery of NO has accelerated over the last few years, none of the currently available products can provide controlled, sustained, time-tunable release of NO. Nitrosamine-based NO donors, prepared in our laboratory, present a unique and innovative alternative for possible treatments for long-term NO deficiency-related diseases, including atherosclerosis, asthma, erectile dysfunction, cancer, and neurodegenerative diseases. A family of secondary amines prepared via nucleophilic aromatic displacement reactions could be readily N-nitrosated to produce NO donors. NO release takes place in three distinct phases. During the initial phase, the release rate is extremely fast. In the second phase, the release is slower and the rate remains essentially the same during the final stage. These compounds inhibited up to 35% human aortic smooth muscle cell proliferation in a concentration-dependent manner.     

一氧化氮对大鼠实验性肝纤维化的影响

目的　研究一氧化氮 (NO)在肝纤维化发病中的作用。方法　建立大鼠肝纤维化模型 ,给予NO前体———L 精氨酸 (L Arg)及一氧化氮合酶 (NOS)抑制剂———硝基 L 精氨酸 (L NNA) ,应用病理组织学检查、放免法及生化学方法 ,观察其对肝纤维化程度、透明质酸 (HA)含量、谷 草转氨酶 (AST)及谷 丙转氨酶 (ALT)活性的影响 ,同时测定NO、一氧化氮合酶 (NOS)水平变化。结果　NO能明显降低肝纤维化程度、HA含量、AST及ALT活性。结论　NO对大鼠具有保护肝细胞和抗肝纤维化作用     

Effects of duramycin on cardiac voltage-gated ion channels

The amphipathic peptide duramycin is in clinical development for the treatment of cystic fibrosis. It is deposited in cellular membranes where it binds to phosphatidylethanolamine. Duramycin may thereby change the biophysical membrane properties and perturb the function of ion channels. If so, in heart tissue, its application carries the risk to elicit cardiac arrhythmias. In fact, premature ventricular complexes were observed in the electrocardiogram during toxicological testing in dogs. To study the arrhythmogenic potential of duramycin, we investigated its effects on currents through voltage-gated hERG potassium, sodium, and calcium channels in native cells, and using a heterologous expression system, by means of the whole-cell patch clamp technique; duramycin bath concentrations between 1 nM and 0.1 μM did not generate any effects on these currents. Concentrations ≥0.3 μM, however, reduced the amplitudes of all investigated currents. Moreover, sodium current fast inactivation kinetics was slowed in the presence of duramycin. A further rise in duramycin bath concentration (≥3.3 μM) induced a leak current consistent with pore formation. The reported effects of duramycin on ion channel function are likely to arise from a change in the biophysical properties of the membrane rather than from a specific interaction of the peptide with ion channel proteins. Under therapeutic conditions (i.e., administration via inhalation), duramycin plasma concentrations are below 0.5 nM. Thus, upon inhalation, duramycin has a large safety margin and is highly unlikely to elicit arrhythmias. Eva Zebedin, and Xaver Koenig contributed equally.     

香菇多糖对巨噬细胞一氧化氮和一氧化氮合酶活性的影响

目的研究香菇多糖(LTN)诱导巨噬细胞的一氧化氮(NO)生成和一氧化氮合酶(iNOS)的活性,探讨LTN的免疫调节作用机理.方法采用Griess反应和荧光法测定不同剂量的LTN作用小鼠腹腔巨噬细胞后NO的生成量和iNOS活性.观察mRNA转录抑制剂、蛋白质合成抑制剂和iNOS抑制剂对巨噬细胞NO的生成和iNOS活性的影响.结果LTN能使小鼠腹腔巨噬细胞NO生成增加,iNOS活性增高,并呈作用剂量依赖关系.3种抑制剂均能抑制LTN诱导的小鼠腹腔巨噬细胞N0的生成和iNOS活性.结论LTN能刺激小鼠腹腔巨噬细胞提高iNOS活性和NO的生成.提示LTN的免疫调节作用机制可能与LTN刺激巨噬细胞NO生成有关.     

红霉素对人脐静脉内皮细胞一氧化氮及钙离子水平的影响

目的　探讨红霉素对人脐静脉内皮细胞一氧化氮通路及钙离子的影响。方法　应用一氧化氮及一氧化氮合酶试剂盒测定内皮细胞NO的含量及NOS的活性 ,采用Fura 2负载荧光技术检测胞内游离钙水平。结果　红霉素能明显增加内皮细胞NO的产生和胞内游离钙水平 ,并能明显增强NOS的活性 ,具有浓度和时间效应。结论　红霉素对人脐静脉内皮细胞一氧化氮通路的影响可能通过胞内游离钙而起作用。     

Urocortin舒张自发性高血压大鼠胸主动脉与NO和K~+通道的关系

目的探讨Urocortin(Ucn)对自发性高血压大鼠(SHR)胸主动脉舒缩功能的作用及机制。方法采用体外血管灌流,观察Ucn对SHR胸主动脉的舒张作用,以及左旋硝基精氨酸甲酯(N(ω)n itro-L-argin ine methyl ester,L-NAME)、亚甲蓝(M ethylene B lue,MB)和格列本脲(G lybenc lam ide)对其舒张作用的影响。结果Ucn(1 nmol.L-1~1μmol.L-1)可明显舒张内皮完整和去内皮SHR胸主动脉(P<0.01),此作用具有剂量依赖性;一氧化氮(NO)合成酶抑制剂L-NAME(100μmol.L-1)和鸟苷酸环化酶(GC)抑制剂MB部分抑制Ucn舒张血管的作用,而且增强去甲肾上腺素(NE)产生的收缩反应。ATP敏感钾通道(KATP)阻断剂格列本脲(10μmol.L-1)可减弱Ucn的舒血管作用。结论Ucn对SHR血管具有内皮依赖性和非内皮依赖性舒张作用,此作用部分是Ucn增加血管内皮细胞NO水平实现的,并且与NO-cGMP通路和KATP通道有关。     

Inhibition of voltage-gated L-type calcium channels by labedipinedilol-A involves protein kinase C in rat cerebrovascular smooth muscle cells

Labedipinedilol-A, a novel calcium channel blocker with α/β-adrenoceptor blockade properties, inhibits L-type calcium channels (LTCCs) in rat cerebrovascular smooth muscle cells (CSMCs). We used conventional whole cell patch-clamp electrophysiology to investigate Ba²⁺ currents (I_Ba) through LTCCs in rat CSMCs enzymatically dissociated from rat cerebral arteries. Labedipinedilol-A (1, 10 µM) reversibly inhibited I_Ba in a voltage-dependent manner without modifying the I_Ba current–voltage relationship. The I_Ba was also abolished by the LTCC blocker nifedipine (1 µM), but enhanced by the LTCC activator Bay K8644 (100 nM). Labedipinedilol-A shifted the steady-state inactivation curve of I_Ba to more negative potentials. Additionally, labedipinedilol-A had greater inhibitory activity on I_Ba holding at − 40 mV than at − 80 mV. This might contribute to labedipinedilol-A's more selective effect on vascular muscles compared to cardiac muscles. The protein kinase C (PKC) activator phorbol 12-myristate 13-acetate (PMA) and norepinephrine-enhanced I_Ba were also inhibited by labedipinedilol-A. Pretreatment with the PKC inhibitor chelerythrine (5 µM) attenuated labedipinedilol-A-mediated I_Ba inhibition. However, the Rho kinase inhibitor Y-27632 (30 µM) had little effect on labedipinedilol-A inhibition of I_Ba. Labedipinedilol-A inhibition of voltage-dependent LTCCs may be, at least in part, due to its modulation of the PKC pathway.     

Catecholamine-induced cardiac hypertrophy uncouples β-adrenoceptors from slow calcium channels

Changes in the parameters of Ca²⁺-dependent slow action potentials (APs) and in their sensitivity to noradrenaline, forskolin, dibutryl-cAMP and extracellular Ca²⁺ concentration were studied and compared in left ventricular trageculae from normal control rats and rats with cardiac hypertophy. Cytochemical studies were also carried out to determine changes in the activity of membrane-bound adenylate cyclase. Hypertrophy was induced by administration of 5 mg/kg isoproterenol once daily for 7 days. In hypertrophied cardiac muscle, the overshoot of the slow APs was increased by 75%, the maximum rate of rise ( _max) increased by 76% and the AP duration at 50% repolarization (APD₅₀) prolonged by 56%. The _max, an indicator of the slow inward Ca²⁺ current, increased, in a dose-dependent manner, in response to the β-adrenoceptor agonist noradrenaline, the adenylate cyclase activator forskolin, the protein kinase aktivator cAMP and elevated Ca²⁺ concentration in normal control preparations, whereas in hypertrophied myocardium, the β-agonist noradrenaline and the adenylate cyclase activator forskolin had no effect. In cytochemical studies with ATP as substrate, adenylate cyclase activity was localized in the sarcolemma, and significantly fewer reaction products appeared on the outer side of the cell membrane in hypertrophied myocytes than in control myocytes. The results suggest that catecholamine-induced cardiac hypertrophy damages the catalytic subunit of membrane-bound adenylate cyclase, thus uncoupling β-adrenoceptors from slow Ca²⁺ channels in the transmembrane signalling process.     

海嘧啶注射剂对SGC-7901人胃癌细胞凋亡的实验研究

目的：揭示海嘧啶的抗肿瘤作用机理。方法：采用流式细胞仪测定海嘧啶对人胃癌细胞凋亡及细胞周期的影响，采用激光扫描共聚焦技术观测海嘧啶对人胃癌细胞内[Ca^2 ]i的影响及[Ca^2 ]i变化时Ca^2 的来源，结果：海嘧啶可诱导肿瘤细胞凋亡，升高肿瘤细胞内[Ca^2 ]i浓度，[Ca^2 ]i升高来源于细胞外钙内流和细胞内钙释放，结论：海嘧啶的抗肿瘤作用机理为诱导肿瘤细胞凋亡,通过开放细胞膜钙通道和引起细胞内钙释放两条途径升高肿瘤细胞内[Ca^2 ]i，启动细胞凋亡机制，从而诱导肿瘤细胞凋亡。     

Calcium binding protein-mediated regulation of voltage-gated calcium channels linked to human diseases

Calcium ion entry through voltage-gated calcium channels is essential for cellular signalling in a wide variety of cells and multiple physiological processes. Perturbations of voltage-gated calcium channel function can lead to pathophysiological consequences. Calcium binding proteins serve as calcium sensors and regulate the calcium channel properties via feedback mechanisms. This review highlights the current evidences of calcium binding protein-mediated channel regulation in human diseases.     

牛磺酸对大鼠心肌成纤维细胞增殖的影响

观察牛磺酸(taurine，Tau)在血管紧张素II(angiotensin II，Ang II)诱导新生大鼠心肌成纤维细胞(cardiac fibroblast，CFb)增殖过程中对一氧化氮(nitric oxide，NO)及蛋白激酶C alpha(p-PKCα)的影响，以探讨牛磺酸抑制CFb增殖的信号转导途径。胰酶消化法分离培养新生大鼠CFb，用Ang II诱导促进其增殖，采用MTT法检测细胞增殖；ELISA法测定I型胶原、 III型胶原的含量；流式细胞仪检测细胞周期；硝酸还原酶法检测细胞上清液NO含量；Western blotting法检测p-PKCα含量。牛磺酸(40、 80和160 mmol·L^-1)可抑制Ang II诱导的CFb增殖及I型胶原、III型胶原合成增加(P<0.05，P<0.01)，并使细胞G₀/G₁期百分率增加，S期细胞百分率降低(P<0.05，P<0.01)。牛磺酸明显抑制p-PKCα的蛋白表达， 提高CFb NO含量， 与Ang II组比较差别具有明显统计学意义(P<0.05， P<0.01)。牛磺酸通过降低p-PKCα的表达而提高CFb NO的含量，使CFb的增殖和胶原合成受到抑制。