香烟提取物活化大鼠支气管平滑肌细胞蛋白激酶C亚型及下调钾通道BK_(Ca)、Kv1.5表达(英文)

大电导的钙活化钾通道（large—conductance calcium—activated potassium channel,BKCa）和电压依赖性钾通道Kv1．5在气道高反应性的发生机制中具有重要作用。已知吸烟可致气道高反应,但钾通道的变化在其发病中的作用尚需进一步阐明。本文旨在研究香烟提取物（cigarette smoke extract,CSE）对培养的大鼠支气管平滑肌细胞（bronchial smooth muscle cells,BSMCs）钾通道BKCa和Kv1．5表达的直接作用,以及蛋白激酶C（protein kinaseC,PKC）在其中的作用。实验采用原代培养大鼠BSMCs,用5％CSE刺激,免疫印迹检测PKC亚型的表达和转位,半定量RT—PCR、免疫印迹实验检测BKCa和Kv1．5的mRNA和蛋白表达,然后用PKC抑制剂BIM和G6e6983与CSE共作用,检测其对BKCa和Kv1．5的mRNA和蛋白表达的影响。结果显示,5％CSE使PKCε、η、θ发生明显的膜转位,并使BKCa。和Kv1．5的蛋白和mRNA表达明显降低;选择性PKC抑制剂BIM或G6e6983与CSE共同作用,均可使BKCa和Kv1．5的蛋白和mRNA表达部分恢复。上述结果提示,CSE可引起BSMCs的BKCa和Kv1．5表达下调,PKCε、η、θ参与其信号转导。     

电针大鼠的血清中淋巴细胞转化抑制因子的作用机制分析

本室以前的工作表明:电针(2H_z,3V,30min/d)刺激 SD 大鼠双侧足三里-三阴交,5d后,大鼠血清中产生出淋巴细胞转化抑制因子,本工作对此抑制因子的作用机制进行了初步研究,主要结果如下:(1)电针大鼠的血清不仅显著抑制 Con A 刺激的小鼠淋巴结 T 淋巴细胞转化,还可显著抑制 Con A 刺激的小鼠胸腺细胞和脾脏 T 淋巴细胞转化;同时也发现电针大鼠的血清能显著抑制脂多糖(LPS)刺激的小鼠淋巴结 B 淋巴细胞转化。提示此淋巴细胞转化抑制因子对不同淋巴器官及不同类型的淋巴细胞无选择性作用。(2)将电针大鼠的血清同小鼠淋巴结细胞培养1h,电针大鼠的血清就可显著抑制 Con A 刺激的 T 淋巴细胞转化;将小鼠淋巴结细胞同 Con A 预培养30min,电针大鼠的血清的抑制作用便消失,提示电针大鼠血清中淋巴细胞转化抑制因子作用于 Con A 刺激 T 淋巴细胞活化的早期阶段,同时也排除了此抑制因子的细胞毒作用。(3)电针大鼠的血清显著抑制蛋白激酶 C(PKC)激活剂 PMA和 PMA 加 ca~(2+)通道 A23187刺激的小鼠淋巴结细胞转化,提示淋巴细胞转化抑制因子通过抑制 PKC 的活性或抑制 PKC 介导的细胞活化通路,抑制有丝分裂原刺激的淋巴细胞转化。     

甲状腺素对大鼠心脏细胞蛋白激酶C信号途径的影响

目的 :探讨甲状腺素对新生大鼠心脏细胞中蛋白激酶C(proteinkinaseC ,PKC)信号途径的影响。 方法 :培养新生大鼠心肌细胞及成纤维细胞 ,用 1%血清培养基或血管紧张素Ⅱ(angiotensinⅡ ,AngⅡ)处理细胞 2 4h后 ,加入甲状腺素(三碘甲状腺素原氨酸 ,triiodothyronine,T3 )继续培养 4 8h后 ,用PKC活性检测试剂盒检测细胞中PKC活性 ,用West ernblot的方法检测细胞中PKCα及PKCε的表达。结果 :在 1%血清培养基中 ,T3 能明显抑制心肌细胞中PKC活性 ,使心肌细胞中PKCε表达下降 ,对PKCα的表达却没有显著的影响 ;在心肌成纤维细胞中 ,无论是PKC活性还是PKCα及PKCε的表达均未观察到T3 的调控作用。预先用AngⅡ处理 2 4h后 ,心肌细胞及心肌成纤维细胞中PKC活性明显增加 ,PKCε的表达显著增加 ,随后用T3 处理后 ,心肌细胞中PKC活性及PKCε的表达明显降低 ;而心肌成纤维细胞中PKC活性没有发生显著性的变化。结论 :甲状腺素能明显抑制心肌细胞中PKC活性及PKCε亚型的表达 ,其对心肌细胞中PKC信号途径的调控作用可能在心肌的多种病理生理过程中起着重要的作用。     

慢性吸烟大鼠气道平滑肌大电导的钙激活钾通道和Kv1.5表达的变化

复制大鼠的慢性吸烟模型,采用气道反应性的测定、HE染色、免疫组织化学染色、原位杂交和免疫印迹实验等方法,观察吸烟对大鼠支气管平滑肌大电导的钙激活的钾通道(BKca)和电压依赖性延迟整流钾通道Kv1．5蛋白和mRNA表达的影响,以阐明吸烟引起的气道高反应性发病机制中钾通道表达变化的作用。结果显示：(1)慢性吸烟可降低大鼠大气道和小气道BKca和Kv1．5蛋白和mRNA表达;(2)大气道BKca的降低程度大于Kv1．5,小气道BKca和Kv1．5的降低程度无明显差异：(3)吸烟对全肺组织BKca和Kv1．5的蛋白表达无明显影响。上述结果提示,慢性吸烟可下调大鼠气道平滑肌钾通道BKca和Kv1．5的表达水平,是导致气道高反应的机制之一。     

人乳腺MCF10A细胞系K~+通道的表达和特性及其与增殖的关系

近年来发现,K+通道与乳腺癌细胞的增殖和转化密切相关,但机制尚不清楚。本研究室前期报道了K+通道阻断剂4-氨基吡啶(4-aminopyridine,4-AP)能够抑制人乳腺上皮细胞的增殖,本文则进一步检测几种电压门控K+通道(voltage-gatedK+channel,Kv)在人乳腺上皮细胞系MCF10A中的表达,运用全细胞膜片钳技术,初步研究了该细胞K+通道的特性,观察K+通道阻断剂对细胞增殖以及信号通路蛋白活性的影响。结果显示,MCF10A细胞均有Kv1.1、Kv1.2、Kv1.3和Kv1.5基因mRNA的表达,其中Kv1.5表达量明显高于乳腺癌细胞MCF7。全细胞膜片钳钳制细胞于-60mV,给予持续时间800ms、范围从-60mV到+60mV的去极化刺激电压,步幅为10mV,然后给予持续150ms的-60mV的刺激,刺激频率为1Hz,可记录到一种跨膜电流,该电流具有电压依赖、外向整流的特性,并且能被Kv通道阻断剂4-AP阻断,证实该细胞膜存在Kv通道。此外,4-AP阻断K+通道10min后,与增殖相关的有丝分裂原活化蛋白激酶(mitogen-activated protein kinases,MAPK)信号通路ERK1/2蛋白活性增强而p38蛋白活性减弱;5mmol/L4-AP处理细胞48h后,MCF10A的生长抑制率为25.29%。以上结果提示,在人乳腺上皮细胞系MCF10A细胞膜上存在不同亚型的Kv通道,该通道可被4-AP阻断,并且4-AP能够抑制MCF10A细胞的增殖,其机制可能与细胞增殖信号通路不同成员的活性调节有关。     

敬钊毒素-V对Kv4.3通道的抑制作用

K+通道亚型Kv4.3在调节心肌细胞动作电位的幅度与时程方面具有重要作用,是治疗心律失常的有效作用靶点,但目前世界上该通道的特异性抑制剂非常缺乏。敬钊毒素-V(Jingzhaotoxin-V,JZTX-V)是从敬钊缨毛蜘蛛粗毒中纯化到的一种新型肽类神经毒素,能够部分抑制大鼠背根神经节细胞上的瞬时外向K+电流,其半数有效抑制浓度(IC50值)为52.3nmol/L。为了研究JZTX-V对Kv4.3通道的作用,本实验通过多肽固相化学合成的方法得到JZTX-V,并用双电极杆电压钳技术检测JZTX-V对表达在非洲爪蟾卵母细胞上的Kv4.3通道电流的作用。结果显示,JZTX-V能够完全抑制Kv4.3通道电流,并且这种抑制作用具有浓度依赖性和时间依赖性,其IC50值为425.1nmol/L,JZTX-V还能够使通道的电流-电压关系曲线和稳态失活曲线分别向去极化方向漂移大约29mV和10mV,改变Kv4.3通道的动力学特征,因此我们推测JZTX-V是一种Kv4.3通道门控调制毒素。以上研究结果对于开发心肌Kv4.3通道的分子探针及以Kv4.3通道为靶点的药物设计具有借鉴作用。     

脊髓蛋白激酶Cα和γ亚型在吗啡依赖和戒断反应中的不同作用

在大鼠吗啡依赖和戒断模型上,采用行为学、免疫组织化学和Western blot方法观察鞘内应用蛋白激酶C(protien kinase C,PKC)抑制剂chelerythrine chloride(CHE)对吗啡依赖大鼠纳洛酮催促成断反应、脊髓Fos蛋白表达和脊髓神经元胞膜和胞浆PKCα、γ表达的影响,以探讨不同亚型PKC在吗啡依赖和戒断反应中的作用。结果表明,鞘内注射CHE能明显减轻吗啡成断症状的评分和吗啡戒断引起的痛觉异常,抑制吗啡成断期间脊髓Fos蛋白表达的增加;吗啡依赖可引起脊髓神经元PKCα和γ表达的上调和转位：吗啡戒断期间存在明显的且可被鞘内注射CHE抑制的PKCα转位,但未观察到明显的PKCγ转位。上述结果表明,脊髓PKC表达上调和转何可能参与吗啡依赖的形成和戒断反应的表达,且PKCα和γ亚型在吗啡依赖和戒断反应中的作用存在差异。     

一氧化氮在血管紧张素Ⅱ激活蛋白激酶C中的作用

实验在培养新生大鼠心肌细胞中检测NO前体L－精氨酸（L－Arg）和NO供体硝普钠（SNP）对血管紧张素Ⅱ（AngⅡ）激活蛋白激酶C（PKC）的作用,以探讨心肌细胞PKC水平的信号转导途径,实验结果如下：（1）无血清DMEM培养心肌细胞24h后加入AngⅡ,PKC活性呈剂量依赖性增高;（2）培养基中加入L－Arg,PKC活性呈剂量依赖性降低;（3）用L－Arg100μmol/L进行预处理,30min后分别加入AngⅡ0.1μmol/L或PMA10μmol/L,PKC活性均明显降低,与单纯AngⅡ组和单纯PMA组相比均有显著性差异;用NOS抑制剂L－NAME预处理后,再加入L－Arg,可明显阻断L－Arg对上述两个效应的影响;（4）培养液中加入NO供体SNP,PKC活性呈剂量依赖性地降低;（5）用SNP10μmol／L预处理心肌细胞,5min后分别加入AngⅡ或PMA,PKC活性分别与单纯AngⅡ和单纯PMA组相比均明显降低。以上结果表明,AngⅡ能剂量依赖性激活PKC,而NO可剂量依赖性抑制PKC活性;NOS参与L－Arg抑制AngⅡ或PMA激活PKC的作用。这些观察提示,NO抑制AngⅡ对心肌细胞的作用可能是通过抑制PKC活性实现的,PKC可能是NO和AngⅡ在心肌细胞内信号转导的交汇点（cross talk）。     

蛋白激酶C对大鼠离体肺动脉环张力及其反应性的调节作用

目的 :观察蛋白激酶C(PKC)对大鼠离体肺动脉环张力及反应性的调节作用。方法 :取Wistar大鼠肺动脉 ,观察在离体情况下PKC激活剂PMA及PKC抑制剂RO3 182 2 0对肺动脉环张力的直接作用 ;对氯化钾 (KCl)、5 羟色胺 (5 HT)和缺氧引起的收缩反应的影响 ;以及PMA对乙酰胆碱 (ACh)介导的内皮依赖性舒张 (EDR)和硝普钠(SNP)介导的内皮非依赖性舒张 (EIDR)反应的影响。结果 :①PMA(5 0 0nmol/L)使肺动脉环产生缓慢增强、持久的收缩 ,随PMA浓度增加而增强 ,RO3 182 2 0 (5 μmol/L)可完全阻断PMA的上述作用 ;②PMA可增强肺动脉对KCl、5 HT的收缩反应 ,该作用随PMA浓度增加而增强 ;③RO3 182 2 0 (5 μmol/L)几乎可以完全阻断离体肺动脉环对缺氧的第二相收缩反应 ;④PMA(10nmol/L)在 10min内完全逆转ACh(10 μmol/L)介导的EDR ,PMA(10nmol/L)还可使ACh的浓度一反应显著减弱 ,达到最大舒张反应的一半时对应的ACh浓度 (EC50 )显著增加 ,最大舒张反应明显减小 ;而PMA对SNP介导的EIDR无显著影响。结论 :PKC在与肺动脉张力及反应性的调节有关的细胞内生物信号传递过程中具有重要作用。     

蛋白激酶C在血管紧张素Ⅱ抑制心肌细胞一氧化氮合成中的作用

实验用硝酸还原酶法测定培养新生大鼠心肌细胞亚硝酸盐 (NO 2 )和硝酸盐 (NO 3)总量 (NO 2 /NO 3) ,反映心肌细胞一氧化氮 (NO)生成情况 ,观察血管紧张素Ⅱ (AngⅡ )对心肌细胞NO生成的影响及其蛋白激酶C (PKC)在该效应中的作用。结果显示 :AngⅡ可减少心肌细胞NO的含量 ,并具有明显的剂量 效应关系 ;AngⅡ受体拮抗剂saralasin可明显抑制AngⅡ对NO生成的影响 ;L 精氨酸 (L Arg)明显增加心肌细胞NO的浓度 ,此效应可被一氧化氮合酶 (NOS)抑制剂L NAME所抑制 ,L Arg未能消除AngⅡ抑制NO的作用 ;用佛波酯 (PMA)处理心肌细胞 ,其NO的生成明显减少 ,L NAME可加强此抑制效应 ;PKC抑制剂staurosporine (Stau)可明显削弱AngⅡ抑制心肌细胞NO生成的效应。结果提示 :AngⅡ具有抑制心肌细胞NO生成的作用 ,此作用可能是通过抑制心肌细胞NOS的活性而实现的 ;AngⅡ受体介导AngⅡ抑制心肌细胞NO生成的作用 ;激活PKC可使新生大鼠心肌细胞NO生成减少 ,NOS参与此抑制效应 ,新生大鼠心肌细胞NO生成过程的信号转导通路可能与PKC有关 ;PKC参与AngⅡ抑制心肌细胞NO的生成。     

Protein kinase C inhibits Kv1.1 potassium channel function

The regulation by protein kinase C (PKC) of recombinantvoltage-gated potassium (K) channels in frog oocytes was studied. Phorbol 12-myristate 13-acetate (PMA; 500 nM), an activator of PKC,caused persistent and large (up to 90%) inhibition of mouse, rat, andfly Shaker K currents. K currentinhibition by PMA was blocked by inhibitors of PKC, and inhibition wasnot observed in control experiments with PMA analogs that do notactivate PKC. However, site-directed substitution of potential PKCphosphorylation sites in the Kv1.1 protein did not prevent currentinhibition by PMA. Kv1.1 current inhibition was also not accompanied bychanges in macroscopic activation kinetics or in theconductance-voltage relationship. In Western blots, Kv1.1 membraneprotein was not significantly reduced by PKC activation. The injectionof oocytes with botulinum toxin C3 exoenzyme blocked the PMA inhibitionof Kv1.1 currents. These data are consistent with the hypothesis thatPKC-mediated inhibition of Kv1.1 channel function occurs by a novelmechanism that requires a C3 exoenzyme substrate but does not alterchannel activation gating or promote internalization of the channel protein.

     

Phorbol 12-myristate 13-acetate upregulates cyclooxygenase-2 expression in human pulmonary epithelial cells via Ras, Raf-1, ERK, and NF-kappaB, but not p38 MAPK, pathways

In this study, we investigated the signaling pathway involved in cyclooxygenase-2 (COX-2) expression and prostaglandin E2 (PGE2) release by phorbol 12-myristate 13-acetate (PMA), a protein kinase C (PKC) activator, in human pulmonary epithelial cells (A549). PMA-induced COX-2 expression was attenuated by PKC inhibitors (Go 6976 and Ro 31-8220), a Ras inhibitor (manumycin A), a Raf-1 inhibitor (GW 5074), a MEK inhibitor (PD 098059), and an NF-kappaB inhibitor (PDTC), but not by a tyrosine kinase inhibitor (genistein) or a p38 MAPK inhibitor (SB 203580). PMA also caused the activation of Ras, Raf-1, and ERK1/2. PMA-induced activation of Ras and Raf-1 was inhibited by Ro 31-8220 and manumycin A. PMA-mediated activation of ERK1/2 was inhibited by Ro 31-8220, manumycin A, GW 5074, and PD 098059. Stimulation of cells with PMA caused IkappaBalpha phosphorylation, IkappaBalpha degradation, and the formation of a NF-kappaB-specific DNA-protein complex. The PMA-mediated increase in kappaB-luciferase activity was inhibited by Ro 31-8220, manumycin A, GW5074, PD 098059, and PDTC. Taken together, these results indicate that PMA might activate PKC to elicit activation of the Ras/Raf-1/ERK1/2 pathway, which in turn initiates NF-kappaB activation, and finally induces COX-2 expression and PGE2 release in A549 cells.     

Induction of nitric oxide synthase in raw 264.7 macrophages by lipoteichoic acid from<Emphasis Type="Italic">Staphylococcus aureus:</Emphasis> Involvement of protein kinase C- and nuclear factor-kB-dependent mechanisms

This study investigates the signaling pathway involved in inducible nitric oxide synthase (iNOS) expression and nitric oxide (NO) release caused by Staphylococcus aureus lipoteichoic acid (LTA) in RAW 264.7 macrophages. A phosphatidylcholine-phospholipase C (PC-PLC) inhibitor (D-609) and a phosphatidylinositol-phospholipase C (PI-PLC) inhibitor (U-73122) attenuated LTA-induced iNOS expression and NO release. Two PKC inhibitors (Go 6976 and Ro 31-8220), an NF-kappaB inhibitor (pyrrolidine dithiocarbamate; PDTC), and long-term (24 h) 12-phorbol-13-myristate acetate (PMA) treatment each also inhibited LTA-induced iNOS expression and NO release. Treatment of cells with LTA caused an increase in PKC activity; this stimulatory effect was inhibited by D-609, U-73122, or Ro 31-8220. Stimulation of cells with LTA caused IkappaB-alpha phosphorylation and IkappaB-alpha degradation in the cytosol, and translocation of p65 and p50 NF-kappaB from the cytosol to the nucleus. Treatment of cells with LTA caused NF-kappaB activation by detecting the formation of NF-kappaB-specific DNA-protein complexes in the nucleus; this effect was inhibited by Go 6976, Ro 31-8220, long-term PMA treatment, PDTC, L-1-tosylamido-2-phenylethyl chloromethyl ketone (TPCK), and calpain inhibitor I. These results suggest that LTA might activate PC-PLC and PI-PLC to induce PKC activation, which in turn initiates NF-kappaB activation, and finally induces iNOS expression and NO release in RAW 264.7 macrophages.     

Arginine vasopressin inhibits Kir6.1/SUR2B channel and constricts the mesenteric artery via V1a receptor and protein kinase C

Kir6.1/SUR2B channel is the major isoform of K(ATP) channels in the vascular smooth muscle. Genetic disruption of either subunit leads to dysregulation of vascular tone and regional blood flows. To test the hypothesis that the Kir6.1/SUR2B channel is a target molecule of arginine vasopressin (AVP), we performed studies on the cloned Kir6.1/SUR2B channel and cell-endogenous K(ATP) channel in rat mesenteric arteries. The Kir6.1/SUR2B channel was expressed together with V1a receptor in the HEK-293 cell line. Whole cell currents of the transfected HEK cells were activated by K(ATP) channel opener pinacidil and inhibited by K(ATP) channel inhibitor glibenclamide. AVP produced a concentration-dependent inhibition of the pinacidil-activated currents with IC(50) 2.0 nM. The current inhibition was mediated by a suppression of the open-state probability without effect on single-channel conductance. An exposure to 100 nM PMA, a potent PKC activator, inhibited the pinacidil-activated currents, and abolished the channel inhibition by AVP. Such an effect was not seen with inactive phorbol ester. A pretreatment of the cells with selective PKC blocker significantly diminished the inhibitory effect of AVP. In acutely dissociated vascular smooth myocytes, AVP strongly inhibited the cell-endogenous K(ATP) channel. In isolated mesenteric artery rings, AVP produced concentration-dependent vasoconstrictions with EC(50) 6.5 nM. At the maximum effect, pinacidil completely relaxed vasoconstriction in the continuing exposure to AVP. The magnitude of the AVP-induced vasoconstriction was significantly reduced by calphostin-C. These results therefore indicate that the Kir6.1/SUR2B channel is a target molecule of AVP, and the channel inhibition involves G(q)-coupled V1a receptor and PKC.     

PKC pathway associated with the expression of an A-type K+ channel induced by TGF-beta1 in rat vascular myofibroblasts

Our previous study indicated that TGF-beta1 induced the expression of a transient outward K+ channel (A-type) during the phenotypic transformation of vascular fibroblasts to myofibroblasts. Here, we studied the relevant signal transduction pathway using whole cell recording and a quantitative RT-PCR technique. Results indicate that the protein kinase C (PKC) agonist phorbol-12-myristate-13-acetate (PMA, 1 microM) could mimic the effect of TGF-beta1 (20 ng/ml) on the expression of an A-type K+ channel and induced a similar A-type K+ current. Moreover, a PKC inhibitor, bisindolylmaleimide I (1 microM), could abrogate the effect of TGF-beta1 on K(V)4.2 expression. This result suggests that a PKC pathway may be involved in the expression of an A-type K+ channel induced by TGF-beta1 in rat vascular myofibroblasts.     

Selective effects of the PKC inhibitors Ro 31-8220 and CGP 41 251 on PMN locomotion,cell polarity,and pinocytosis

Using two newly synthesized inhibitors, Ro 31-8220 and CGP 41 251, of protein kinase C (PKC), we analyzed: (1) how distinct PMN functions (shape changes, locomotion, pinocytosis) are regulated, and (2) the role of protein phosphorylation and PKC in this process. We were able to transform: (1) resting PMNs into locomoting cells using fNLPNTL, (2) locomoting cells into non-locomoting highly pinocytic cells using PMA, and (3) PMA-stimulated cells showing marked pinocytosis into locomoting or into resting cells using Ro 31-8220. It is thus possible to selectively manipulate PMN function (resting state, locomotion, marked pinocytosis), indicating that there are different regulatory pathways. It was not possible to induce locomotion and marked pinocytosis simultaneously, indicating crosstalk between pathways. Ro 31-8220 inhibited PMA-induced shape changes (nonpolar cells) and pinocytosis, but not fNLPNTL-induced shape changes (polarity) and pinocytosis. At higher concentrations, Ro 31-8220 alone elicited cell polarity and chemokinesis, indicating that a constitutively active protein kinase is involved in maintaining the spherical shape of resting PMNs. Functional effects of another PKC inhibitor, CGP 41 251, on neutrophil function were strikingly different. CGP 41 251 selectively inhibited fNLPNTL-induced polarity and locomotion (but not colchicine or Ro 31-8220-induced polarity), and it failed to inhibit PMA-induced, stimulated pinocytosis and shape changes. Although the effects of Ro 31-8220 vs. CGP 41 251 on PMN function were strikingly different, the inhibition of profiles for constitutive and for fNLPNTL- or PMA-induced protein phosphorylation in intact PMNs showed only small differences, which could not yet be conclusively related to cell function. © 1994 Wiley-Liss, Inc.     

Inhibition of voltage-gated K(+) currents by endothelin-1 in human pulmonary arterial myocytes

Recent studies demonstrate that endothelin-1 (ET-1) constricts human pulmonary arteries (PA). In this study, we examined possible mechanisms by which ET-1 might constrict human PA. In smooth muscle cells freshly isolated from these arteries, whole cell patch-clamp techniques were used to examine voltage-gated K(+) (K(V)) currents. K(V) currents were isolated by addition of 100 nM charybdotoxin and were identified by current characteristics and inhibition by 4-aminopyridine (10 mM). ET-1 (10(-8) M) caused significant inhibition of K(V) current. Staurosporine (1 nM), a protein kinase C (PKC) inhibitor, abolished the effect of ET-1. Rings of human intrapulmonary arteries (0.8-2 mm OD) were suspended in tissue baths for isometric tension recording. ET-1-induced contraction was maximal at 10(-8) M, equal to that induced by K(V) channel inhibition with 4-aminopyridine, and attenuated by PKC inhibitors. These data suggest that ET-1 constricts human PA, possibly because of myocyte depolarization via PKC-dependent inhibition of K(V). Our results are consistent with data we reported previously in the rat, suggesting similar mechanisms may be operative in both species.     

Inhibition of voltage-dependent sodium channels by Ro 31-8220, a 'specific' protein kinase C inhibitor

We find that several protein kinase C (PKC) inhibitors, previously considered to be specific, directly inhibit voltage-dependent Na(+) channels at their useful concentrations. Bisindolylmaleimide I (GF 1092037), IX (Ro 31-8220) and V (an inactive analogue), but not H7 (a non-selective isoquinolinesulfonamide protein kinase inhibitor), inhibited Na(+) channels assessed by several independent criteria: Na(+) channel-dependent glutamate release and [(3)H]batrachotoxinin-A 20-alpha-benzoate binding in rat cortical synaptosomes, veratridine-stimulated 22Na(+) influx in CHO cells expressing rat CNaIIa Na(+) channels and Na(+) currents measured in isolated rat dorsal root ganglion neurons by whole cell patch-clamp recording. These findings limit the usefulness of the bisindolylmaleimide class PKC inhibitors in excitable cells.     

Modulation of cardiac Ca2+ channels in Xenopus oocytes by protein kinase C.

L-Type calcium channel was expressed in Xenopus laevis oocytes injected with RNAs coding for different cardiac Ca2+ channel subunits, or with total heart RNA. The effects of activation of protein kinase C (PKC) by the phorbol ester PMA (4 beta-phorbol 12-myristate 13-acetate) were studied. Currents through channels composed of the main (alpha 1) subunit alone were initially increased and then decreased by PMA. A similar biphasic modulation was observed when the alpha 1 subunit was expressed in combination with alpha 2/delta, beta and/or gamma subunits, and when the channels were expressed following injection of total rat heart RNA. No effects on the voltage dependence of activation were observed. The effects of PMA were blocked by staurosporine, a protein kinase inhibitor. beta subunit moderate the enhancement caused by PMA. We conclude that both enhancement and inhibition of cardiac L-type Ca2+ currents by PKC are mediated via an effect on the alpha 1 subunit, while the beta subunit may play a mild modulatory role.     

Staurosporine,a non-specific PKC inhibitor,induces keratinocyte differentiation and raises intracellular calcium,but Ro31–8220, a specific inhibitor,does not

The responsiveness of normal human keratinocytes to different modulators of protein kinase C (PKC) was investigated. The PKC agonist TPA, staurosporine (a non-specific inhibitor), and Ro31–8220 (a specific inhibitor) were studied for effect on cell morphology, growth rate, involucrin expression, and intracellular calcium levels. Surprisingly the response to nanomolar concentrations of staurosporine was similar to TPA and induced a fusiform morphology, inhibited growth, increased involucrin levels, and raised intracellular calcium. Staurosporine also increased the number of cornified envelopes, and its action therefore appeared identical to TPA. In contrast, Ro31–8220 had little effect on morphology or growth and blocked both the TPA-induced growth inhibition and calcium rise. Ro31–8220 had no effect on staurosporine-induced growth inhibition but partially reduced its associated calcium rise. These results suggest PKC activation is required for keratinocyte differentiation and that staurosporine acts like a PKC agonist to give a similar effect as TPA. Specific inhibition of PKC by Ro31–8220 inhibits TPA-induced differentiation. © 1994 wiley-Liss, Inc.