缬草提取物8-羟基松脂醇苷对Kv1.5钾通道电流的影响

目的 探讨缬草提取物8-羟基松脂醇苷对Kv1.5钾通道电流的影响. 方法 以转基因Kv1.5HEK293细胞为受试对象,采用膜片钳技术观察10,30 μmol.L^-18-羟基松脂醇苷对转基因Kv1.5HEK293细胞电流的影响. 结果 8-羟基松脂醇苷浓度依赖性减少Kv1.5HEK293细胞电流,电流值从(148.3±13.0)PA/PF分别降至(109.4±4.4)PA/PF和(78.6±11.3)PA/PF,抑制率分别为28.5% 和37.1%. 结论 8-羟基松脂醇苷对转基因Kv1.5 HEK293细胞电流有抑制作用,且呈浓度依赖性,可以部分解释缬草抗心律失常作用机制.     

PKA和PKG信号通道对平滑肌细胞 Kv亚型1.5表达的影响

目的 研究PKA(蛋白激酶A)和PKG(蛋白激酶G)信号通道对人气道平滑肌细胞(HASMCs)电压依赖延迟整流钾通道(Kv)亚型Kv1.5表达的影响.方法 采用逆转录聚合酶链反应(RT-PCR)和Western blotting技术,观察PKA激活剂8-brom-cAMP和阻断剂Rp-cAMP及PKG激活剂8-brom-cGMP对HASMCsKv1.5 mRNA和蛋白质表达的影响.结果 PKA激活剂8-brom-cAMP显著增强Kv1.5亚型的表达,该效应可被PKA阻断剂Rp-cAMP显著逆转.PKG激活剂8-brom-cGMP显著增强Kv1.5亚型的表达.结论 活化PKA和PKG促进Kv1.5亚型的表达,抑制HASMCs兴奋性.推测可能为这两条信号通道引起HASMCs舒张反应的作用机制之一.     

多巴胺激活Kv通道抑制胰岛素分泌的电生理机制

目的探讨多巴胺激活电压依赖性钾(Kv)通道抑制葡萄糖刺激胰岛素分泌的电生理机制。方法分离雄性SD大鼠胰岛和β细胞,根据实验使用多巴胺(DA)、D;样受体激动剂(SKF38393)和D;样受体激动剂(Quinpirole)及阻断剂(Eticlopride)进行干预。酶联放射免疫实验测定胰岛素含量;膜片钳电压钳记录β细胞膜上Kv通道电流的变化,电流钳记录动作电位时程的长短;DiBAC4(3)染色观察INS-1细胞膜电位的变化。结果 SKF38393对胰岛素分泌和Kv通道均没有明显影响;Quinpirole明显抑制胰岛素分泌,并且增加Kv通道电流;多巴胺明显抑制胰岛素分泌,增加Kv通道电流,缩短β细胞动作电位时程,且均可被Eticlopride逆转;此外,多巴胺降低INS-1细胞膜电位。结论多巴胺作用于D;样受体,活化Kv通道,缩短动作电位时程,降低β细胞膜电位,从而发挥抑制葡萄糖促进胰岛素分泌的作用。     

SiRNA沉默FOXF2基因对宫颈癌Hela细胞迁移与增殖的影响

目的研究siRNA沉默FOXF2基因对宫颈癌Hela细胞迁移与增殖的影响。方法采用Real TimePCR、Western Blot法检测siRNA沉默FOXF2后,Hela细胞中FOXF2基因mRNA、蛋白表达的变化;用划痕试验检测siRNA沉默FOXF2后细胞的迁移能力,CCK8检测siRNA沉默FOXF2后细胞的增殖能力。结果 Hela细胞内FOXF2基因沉默后,Real Time-PCR、Western Blot检测显示siRNA沉默FOXF2组FOXF2基因mRNA、蛋白的表达均明显下降(P<0.01);划痕试验显示细胞迁移能力增强(P<0.01);CCK8检测显示siRNA沉默FOXF2组细胞增殖能力增强(P<0.01)。结论 SiRNA沉默FOXF2基因促进Hela细胞的迁移与增殖能力。     

钾通道阻滞剂四乙基铵敏感性钾电流在肺高压大鼠肺动脉平滑肌细胞中的变化

目的研究大鼠肺动脉平滑肌细胞(pulmonary arterial smooth muscle cells,PASMCs)中电压依赖性钾通道(voltagedepending potassium channel,K_V)电流在肺高压(pulmonary hypertension,PH)中的变化,及钾通道阻滞剂四乙基铵(tetraethylammonium,TEA)对K_V电流的影响。方法采用全细胞膜片钳技术记录PASMCs的K_V电流,观察PH大鼠中K_V电流的变化,以及TEA对K_V的影响。结果野百合碱(monocrotaline,MCT)、慢性低氧(chronic hypoxia,CH)明显减小了大鼠PASMCs上记录到的K_V电流密度;TEA干预能够明显降低正常和PH大鼠PASMCs的K_V电流,并且TEA对K_V电流的抑制作用在PH大鼠中明显减小。结论在CH、MCT诱导的两种PH大鼠模型中,PASMCs的K_V开放程度被明显抑制,而且其对应的TEA敏感性K_V电流明显减小。     

慢性孵育β淀粉样肽25-35对培养大鼠海马神经元电压依赖性外向钾通道亚型mRNA表达的影响

目的研究慢性孵育β淀粉样肽_25-35(β-AP_25-35)对海马神经元电压依赖性外向钾通道亚型mRNA表达的影响。方法用RT-PCR方法检测mRNA的表达,用光密度扫描法半定量测定表达变化。结果在正常培养的海马神经元上延迟整流(Kv2.1,Kv1.5),瞬间外向(A型)(Kv4.2,Kv1.4),钙激活的大电导(rSlo)钾通道亚型均有表达。β-AP_25-35 3 μmol·L^-1孵育细胞24 h后,Kv2.1 mRNA的表达明显上调,其它亚型则无显著性变化;β-AP_25-35上调Kv2.1 mRNA的作用主要发生在β-AP_25-35应用后48 h内;60 h后Kv2.1 mRNA表达水平显著下调。结论Kv2.1转录水平的上调可能参与β-AP_25-35选择性地增加海马神经元上延迟整流钾电流(I_K)的作用。     

靶向Akt1小干扰RNA对人喉癌细胞增殖和凋亡的影响

目的：研究Akt1基因沉默对人喉癌细胞系（Hep-2）增殖和凋亡的影响。方法：设计并合成针对Akt1的特异性小干扰RNA（siRNA）序列,利用转染试剂转入体外培养人Hep-2细胞,48h后收集细胞,RT-PCR和Westernblot验证siRNA的沉默效应,MTT法检测细胞增殖活性、台盼蓝细胞染色法计数细胞存活率、AnnexinV/PI检测细胞凋亡和坏死。结果：设计的Akt1siRNA能够有效抑制体外培养Hep-2细胞内Akt1表达实现其基因沉默效应;Akt1siRNA干扰组Akt1mRNA转录和蛋白表达水平均低于空白和阴性对照组;Akt1siRNA干扰组细胞增殖活性下降,细胞存活率下降,Akt1siRNA干扰后细胞凋亡率和坏死率增加。结论：RNA干扰Akt1基因沉默具有抑制喉癌细胞增殖,促进细胞凋亡的作用,Akt1可作为喉癌基因治疗的后选新靶点。     

东莨菪碱致记忆障碍大鼠中枢电压依赖性钾通道亚型的表达

目的研究东莨菪碱致记忆障碍大鼠模型中枢电压依赖性钾通道亚型mRNA表达的差异。方法Morris水迷宫实验检验大鼠空间学习记忆能力,用RT-PCR方法检测大脑皮层和海马中5种电压依赖性钾通道Kv1.4,Kv1.5,Kv2.1,Kv4.2及Kv4.3 mRNA的表达。结果注射东莨菪碱大鼠的学习记忆能力明显下降,大脑皮层中Kv4.2的表达比对照组降低28.8%;海马中Kv1.4的表达升高111.7%,Kv2.1的表达升高64.3%,而Kv4.2的表达降低33.9%。其它电压依赖性钾通道表达的变化不大。结论东莨菪碱致大鼠学习记忆障碍的同时可诱发中枢电压依赖性钾通道亚型的表达改变。     

吡那地尔对人肺动脉平滑肌细胞内Ca2+浓度的影响

目的 探讨ATP敏感性钾(KATP)通道开放剂吡那地尔(Pin)对内皮素1(ET-1)诱导的人肺动脉平滑肌细胞(PASMCs)增殖及细胞内[Ca2+]i的影响.方法 体外培养人PASMCs,用ET-1诱导其增殖,应用MTT法、Fluo-3和激光扫描共聚焦显微镜技术评价Pin对ET-1诱导的人PASMCs增殖及PASMCs[Ca2+]i调节的作用.结果 Pin显著抑制ET-1诱导的人PASMCs增殖.呈浓度依赖效应,KATP通道拮抗剂格列本脲呈浓度依赖性阻断Pin的作用;ET-1诱导人PASMCs内[Ca2+]i显著增加,Pin(10 μmol/L)拈抗ET-1诱导的人PASMCs内[Ca2+]i升高.结论 KATP通道开放剂Pin可明显抑制ET-1诱导的人PASMCs增殖作用,抑制细胞内Ca2+浓度增加.     

靶向沉默SSBP1基因对肝癌HepG2细胞增殖、侵袭转移的影响

目的 观察靶向沉默线粒体单链DNA结合蛋白(SSBP1)基因对肝癌HepG2细胞增殖、侵袭转移的影响.方法 设计并构建靶向SSBP1 基因的特异性siRNA,采用脂质体介导瞬时转染肝癌HepG2细胞,细胞分3组:对照组、空白转染组、转染组.Real time-PCR和Western-blot检测靶向干扰后SSBP1 mRNA和蛋白表达变化.CCK-8法检测细胞增殖.流式细胞仪检测细胞周期、凋亡率及线粒体膜电位.划痕实验及Transwell 侵袭实验检测细胞侵袭转移能力.Western-blot检测增殖、侵袭转移相关基因蛋白表达状况.结果 SSBP1 siRNA能够显著抑制SSBP1 mRNA和蛋白表达.与对照组和空白转染组比较,转染SSBP1 siRNA后HepG2细胞增殖能力、G2期和S期细胞比例、线粒体膜电位明显降低,G1期细胞比例、细胞凋亡率明显升高(P<0.05);同时细胞增殖相关基因PCNA、凋亡抑制基因Bcl-2、转移相关基因MMP-9蛋白表达显著下调,凋亡诱导基因Bax蛋白表达显著上调(P<0.05).结论 靶向沉默SSBP1基因能够通过线粒体途径抑制肝癌HepG2细胞增殖、侵袭转移,并诱导其凋亡.     

Kv通道亚型在15-HETE收缩肺动脉过程中的作用

目的从组织功能及细胞分子水平研究电压门控型钾通道(Kv通道)亚型在15-羟化二十碳四烯酸(15-HETE)致大鼠肺动脉收缩过程中的作用。方法采用组织浴槽血管环法，使用Kv通道阻断剂，确定受15-HETE调控大鼠肺动脉平滑肌细胞(PASMCs)膜上Kv亚型；使用RT-PCR和Western blotting技术观察受15-HETE调控PASMCs 膜上Kv亚型。结果阻断Kv1.1，Kv1.2，Kv1.3和Kv1.6通道并不影响15-HETE诱导肺动脉血管收缩；15-HETE不影响PASMCs膜上Kv1.1和Kv1.2通道蛋白质表达；15-HETE下调PASMCs膜上Kv1.5和Kv2.1通道mRNA和蛋白质表达。结论缺氧可能是通过15-HETE这一介导因子抑制Kv1.5和Kv2.1通道，减少PASMCs膜上功能性Kv1.5和Kv2.1通道数量，导致PASMCs收缩。     

Disappearance of Hypoxic Pulmonary Vasoconstriction and O2-Sensitive Nonselective Cationic Current in Arterial Myocytes of Rats Under Ambient Hypoxia

Acute hypoxia induces contraction of pulmonary artery (PA) to protect ventilation/perfusion mismatch in lungs. As for the cellular mechanism of hypoxic pulmonary vasoconstriction (HPV), hypoxic inhibition of voltage-gated K⁺ channel (Kv) in PA smooth muscle cell (PASMC) has been suggested. In addition, our recent study showed that thromboxane A₂ (TXA₂) and hypoxia-activated nonselective cation channel (I_NSC) is also essential for HPV. However, it is not well understood whether HPV is maintained in the animals exposed to ambient hypoxia for two days (2d-H). Specifically, the associated electrophysiological changes in PASMCs have not been studied. Here we investigate the effects of 2d-H on HPV in isolated ventilated/perfused lungs (V/P lungs) from rats. HPV was almost abolished without structural remodeling of PA in 2d-H rats, and the lost HPV was not recovered by Kv inhibitor, 4-aminopyridine. Patch clamp study showed that the hypoxic inhibition of Kv current in PASMC was similar between 2d-H and control. In contrast, hypoxia and TXA₂-activated I_NSC was not observed in PASMCs of 2d-H. From above results, it is suggested that the decreased I_NSC might be the primary functional cause of HPV disappearance in the relatively early period (2 d) of hypoxia.     

Sildenafil inhibits human pulmonary artery smooth muscle cell proliferation by decreasing capacitative Ca2+ entry

Ca(2+) is a pivotal signal in human pulmonary artery smooth muscle cells (PASMCs) proliferation. Capacitative Ca(2+) entry (CCE) via the store-operated channel (SOC), which encoded by the transient receptor potential (TRP) gene, is an important mechanism for regulating intracellular Ca(2+) concentration ([Ca(2+)](i)) in PASMCs. Sildenafil, a potent type 5 nucleotide-dependent phosphodiesterase (PDE) inhibitor, has been proposed as a therapeutic tool to treat or prevent pulmonary arterial hypertension (PAH); however, the mechanism of its antiproliferative effect on PASMCs remains unclear. This study was designed to investigate the possible antiproliferative mechanism of sildenafil on human PASMCs, namely, its effect on the Ca(2+)-signal pathway. Cultured normal PASMCs were treated with endothelin-1 (ET-1) or ET-1 plus sildenafil separately. Cell number and viability were determined with a hemocytometer or MTT assay. [Ca(2+)](i) was measured by loading PASMCs with fura 2-AM. Expression of the TRPC1 gene and protein was detected by RT-PCR and Western blot, respectively. The results show that sildenafil dose-dependently inhibited the proliferation of PASMCs, the enhancement of basal [Ca(2+)](i) level, increase of CCE, and upregulation of TRPC expression induced by ET-1. These results suggest that sildenafil potently inhibits ET-1-induced PASMCs proliferation and downregulation of CCE and TRPC expression may be responsible for its antiproliferative effect.     

Inhibition of cGMP phosphodiesterase 5 suppresses serotonin signalling in pulmonary artery smooth muscles cells

cGMP-specific phosphodiesterase 5 (PDE5) inhibition has been shown to be effective in improving pulmonary haemodynamics in both animal models and clinic patients with pulmonary hypertension. Here, we reveal a novel mechanism whereby PDE5 inhibition suppresses serotonin signalling and consequent cellular proliferation in primary cultured pulmonary artery smooth muscle cells (PASMCs). 1 μM serotonin induced 4.15-fold increases in DNA synthesis compared with control; this was accompanied by significant Ras homolog gene family member A (RhoA) activation and ERK1/2 MAP kinase phosphorylation/nucleus translocation. Furthermore, inhibition of Rho-associated kinase (ROCK) abolished serotonin-triggered DNA synthesis and ERK1/2 nucleus translocation without changing ERK1/2 phosphorylation, indicating that ERK1/2 phosphorylation and nucleus translocation are coupled to different upstream effectors. Pre-exposure of cells to sildenafil dose-dependently blocked the activation of these signalling pathways and elevated DNA synthesis. The inhibitive effects of sildenafil, however, were fully reversed by concomitant presence of PKG inhibitor in cells. These data suggest that RhoA and ERK1/2 pathways are partially cross-talked and concordantly mediate serotonin-stimulated PASMCs proliferation thereby vascular remodelling leading to the occurrence of pulmonary hypertension. Meanwhile, these two pathways are also separately modulated by enhanced cGMP–PKG signalling derived from inhibition of PDE5 with sildenafil to confer the overall roles of sildenafil against pulmonary hypertension.     

Effect of diabetes on the mechanisms of intrathecal antinociception of sildenafil in rats

The mechanism of intrathecal antinociceptive action of the phosphodiesterase 5 inhibitor sildenafil was assessed in diabetic rats using the formalin test. Intrathecal administration of sildenafil (12.5-50 microg) produced a dose-related antinociception during both phases of the formalin test in non-diabetic and diabetic rats. Intrathecal pretreatment with N-L-nitro-arginine methyl ester (L-NAME, nitric oxide (NO) synthase inhibitor, 1-50 microg), 1H-(1,2,4)-oxadiazolo(4,2-a)quinoxalin-1-one (ODQ, guanylyl cyclase inhibitor, 1-10 microg), KT5823 (protein kinase G (PKG) inhibitor, 5-500 ng), charybdotoxin (large-conductance Ca2+-activated K+ channel blocker, 0.01-1 ng), apamin (small-conductance Ca2+-activated K+ channel blocker, 0.1-3 ng) and glibenclamide (ATP-sensitive K+ channel blocker, 12.5-50 microg), but not N-D-nitro-arginine methyl ester (D-NAME, 50 microg) or saline, significantly diminished sildenafil (50 microg)-induced antinociception in non-diabetic rats. Intrathecal administration of ODQ, KT5823, apamin and glibenclamide, but not L-NAME nor charybdotoxin, reversed intrathecal antinociception induced by sildenafil in diabetic rats. Results suggest that sildenafil produces its intrathecal antinociceptive effect via activation of NO-cyclic GMP-PKG-K+ channels pathway in non-diabetic rats. Data suggest that diabetes leads to a dysfunction in NO and large-conductance Ca2+-activated K+ channels. Sildenafil could have a role in the pharmacotherapy of diabetes-associated pain.     

Subacute hypoxia suppresses Kv3.4 channel expression and whole-cell K+ currents through endogenous 15-hydroxyeicosatetraenoic acid in pulmonary arterial smooth muscle cells

We have previously reported that subacute hypoxia activates lung 15-lipoxygenase (15-LOX), which catalyzes arachidonic acid to produce 15-HETE, leading to constriction of neonatal rabbit pulmonary arteries. Subacute hypoxia suppresses Kv3.4 channel expression and results in an inhibition of whole-cell K(+) currents (I(K)). Although the Kv channel inhibition is likely to be mediated through 15-HETE, direct evidence is still lacking. To reveal the role of the 15-LOX/15-HETE pathway in the hypoxia-induced down-regulation of Kv3.4 channel expression and inhibition of I(K), we performed studies using 15-LOX blockers, whole-cell patch-clamp, semi-quantitative PCR, ELISA and Western blot analysis. We found that Kv3.4 channel expression at the mRNA and protein levels was greatly up-regulated in pulmonary arterial smooth muscle cells after blockade of 15-LOX by CDC or NDGA. The 15-LOX blockade also partially restored I(K). In comparison, 15-HETE had a stronger effect than 12-HETE on the expression of Kv3.4 channels. 5-HETE had no noticeable effect on Kv3.4 channel expression. These data indicate that the 15-LOX pathway via its metabolite, 15-HETE, seems to play a role in the down-regulation of Kv3.4 expression and I(K) inhibition after subacute hypoxia.     

Inhibition of cGMP-dependent protein kinase by the cell-permeable peptide DT-2 reveals a novel mechanism of vasoregulation

Cyclic GMP-dependent protein kinase (PKG) serves as an important physiological regulator of vascular reactivity and tone. However, available inhibitors of PKG have exhibited variable effects in intact tissue, hindering the elucidation of the functional role of PKG in blood vessels. In this study, we have determined the effects of our previously engineered potent and selective PKG Ialpha inhibitor DT-2 on basal and cGMP-stimulated purified recombinant PKG, and compared DT-2 with commonly used PKG inhibitors (8R,9S,11S)-(-)-9-methoxy-carbamyl-8-methyl-2,3,9,10-tetrahydro-8,11-epoxy-1H,8H,11H-2,7b,11a-trizadibenzo-(a,g)-cycloocta-(c,d,e)-trinden-1-one (KT-5823), Rp-8-(4-chlorophenylthio)-guanosine-3',5'-cyclic monophosphorothioate (Rp-8-pCPT-cGMPS), and (beta-phenyl-1,N2-etheno-8-bromoguanosine-3',5'-cyclic monophosphorothioate, Rp-isomer (Rp-8-Br-PET-cGMPS). As expected, all inhibitors reduced cGMP-stimulated PKG activity. However, only DT-2 decreased cGMP-independent or basal PKG activity, whereas KT5823 showed no effect and the Rp-compounds actually had partial agonist activity. To evaluate the potential functional impact of this unique inhibition by DT-2 under physiologically relevant conditions, we analyzed the inhibitors in isolated pressurized cerebral arteries. KT-5823 and Rp-8-pCPT-cGMPS demonstrated marginal reversal of vasodilation induced by 8-Br-cGMP. By comparison, DT-2 completely reversed 8-Br-cGMP induced dilations with comparable potency to Rp-8-Br-PET-cGMPS. In fact, DT-2 constricted arteries beyond their starting (pre-8-Br-cGMP) diameters and caused constriction even in the absence of exogenous 8-Br-cGMP, an effect that was not observed with any other inhibitor. The direct constricting effect of DT-2 was essentially abolished in cultured arteries, where PKG expression was reduced by approximately 90%. These findings indicate that DT-2 not only effectively inhibits cGMP-stimulated PKG activity but also reduces basal PKG activity both in vitro and in vivo. Moreover, these distinctive inhibitory properties of DT-2 suggest an important role for constitutive PKG activity in the continuous regulation of cerebral artery tone.     

Inhibitory effect of calcitonin gene-related peptide on hypoxia-induced rat pulmonary artery smooth muscle cells proliferation: role of ERK1/2 and p27

Calcitonin gene-related peptide (CGRP) inhibits angiotensin II-induced proliferation of aortic smooth muscle cells via inactivation of extracellular signal-regulated protein kinase 1/2 (ERK1/2). ERK1/2 is necessary for the degradation or down-regulation of the cell cycle inhibitor p27, and is also crucial in mediating proliferation of pulmonary artery smooth muscle cells (PASMCs). Whether ERK1/2/p27 signal pathway is involved in CGRP-mediated pathogenesis of pulmonary hypertension and vascular remodeling remains unknown. Pulmonary hypertension was induced by hypoxia in rats, and capsaicin (50 mg/kg, s.c.) was used to deplete endogenous CGRP. Proliferation of cultured PASMCs was determined by BrdU incorporation method and flow cytometry. The expression/level of CGRP, p27, ERK1/2, c-fos and c-myc was analyzed by radioimmunoassay, immunohistochemistry, real-time PCR or Western blot. Sensory CGRP depletion by capsaicin exacerbated hypoxia-induced pulmonary hypertension in rats, as shown by an increase in right ventricle systolic pressure, mean pulmonary artery pressure and vascular hypertrophy, accompanied with decreased p27 expression and increased expression of phosphorylated ERK1/2, c-fos and c-myc. Exogenous application of CGRP significantly inhibited hypoxia-induced proliferation of PASMCs concomitantly with increased p27 expression and decreased expression of phosphorylated ERK1/2, c-fos and c-myc. These effects of CGRP were abolished in the presence of CGRP(8-37). Knockdown of p27 also reversed the inhibitory effect of CGRP on proliferation of PASMCs and expression of c-fos and c-myc, but not on ERK1/2 phosphorylation. These results suggest that CGRP inhibits hypoxia-induced proliferation of PASMCs via ERK1/2/p27/c-fos/c-myc pathway. Down-regulation of CGRP may contribute to remodeling of pulmonary arteries in hypoxia-induced pulmonary hypertension.     

Role of K+ and Ca2+ fluxes in the cerebroarterial vasoactive effects of sildenafil

The aim of this study was to assess the role of K(+) and Ca(2+) fluxes in the cerebroarterial vasoactive effects of the phosphodiesterase-5 inhibitor sildenafil. We used isolated rabbit basilar arteries to assess the effects of extracellular K(+) raising on sildenafil-induced vasodilatation, and studied the pharmacological interaction of sildenafil with selective modulators of membrane K(+) and Ca(2+) channels. Expression of Kv1 subunits of K(+) channels was assessed at messenger and protein levels. Parallel experiments were carried out with zaprinast for comparison. Sildenafil (10 nM-0.1 mM) induced concentration-dependent relaxation of endothelin-1 (10 nM)-precontracted arteries, which was partially inhibited by depolarization with KCl (50 mM), 3 mM tetraethylammonium (non-selective K(+) channel blocker) or 1 mM aminopyridine (inhibitor of K(v) channels), but not by 1 microM glibenclamide (inhibitor of K(ATP) channels) or 50 nM iberiotoxin (inhibitor of K(Ca) channels). Arterial smooth muscle expressed messengers for Kv1.2, Kv1.3, Kv1.4, Kv1.5 and Kv1.6, and proteins of Kv1.1, Kv1.2 and Kv1.4. CaCl(2) (10 microM- 10 mM) induced concentration-dependent contraction in Ca(2+)-free, depolarizing (50 mM KCl) medium. Sildenafil (0.1-100 microM) produced reversible concentration-dependent inhibition of the response to CaCl(2), which was completely abolished by the highest sildenafil concentration. By contrast, only 100 microM zaprinast inhibited the response to CaCl(2). The L-type Ca(2+) channel activator Bay K 8644 (0.1 nM-1 microM) induced concentration-dependent potentiation of the response to CaCl(2) inhibited by 100 microM sildenafil. Moreover, Bay K 8644 (0.1 nM-1 microM) induced concentration-dependent contraction in slightly depolarizing (15 mM) medium, which was inhibited to the same extent and in a concentration-dependent way by sildenafil (0.1-100 microM) and zaprinast (1 or 100 microM). These results show that sildenafil relaxes the rabbit basilar artery by increasing K(+) efflux through K(v) channels, which in turn may affect Ca(2+) signalling. Expression of Kv1 subunits involved in this pharmacological effect occurs at the messenger and, in some cases, at the protein level. In addition to this phosphodiesterase-5-related effect, sildenafil and zaprinast inhibit cerebroarterial vasoconstriction at least in part by directly blocking L-type Ca(2+) channels, although a decrease in the sensitivity of the contractile apparatus to Ca(2+) can not be discarded.