渗透压对大鼠心室肌细胞L型Ca2+电流的影响

目的 :观察细胞胞外渗透压的改变对大鼠心室肌 L型 Ca2 +（ICa,L）电流的影响。方法 :采用全细胞膜片钳技术记录大鼠心室肌细胞 ICa,L。结果 :细胞外的渗透压由 3 10 m Osm / kg H2 O降低至 2 2 0 m Osm/ kg H2 O可使 ICa,L的 rundown明显变慢 ,电流大小无统计学意义上的差异 ;而当其中细胞外的渗透压由 3 10 m Osm/ kg H2 O升高至 410m Osm/ kg H2 O时可使 ICa,L减小 （5 1.4± 7.9） % （n=4,P<0 .0 1）。结论 :细胞外渗透压变化对大鼠心室肌基础 ICa,L有明显影响     

U69 593对家兔心室肌细胞L型Ca2+电流的影响

目的 :研究 U695 93对家兔心室肌细胞 L 型 Ca2 +电流 （ ICa· L）的影响。方法 :采用全细胞膜片钳技术记录家兔心室肌细胞 IC a· L。结果 :在 -2 0～ +4 0 m V测试电压范围内 ,10 0 nmol/ L U695 93显著增加 IC a· L,以 +10 m V为最大激活电压 ,其峰值 ICa· L由加药前 -7.1± 0 .4p A/ p F增至 -8.3± 0 .5 p A/ p F （ P<0 .0 1,n=8）。 10 0 nmol/ LU 695 93不影响 IC a· L电压依赖性激活与失活特性。结论 :U695 93可增加家兔心室肌细胞 ICa· L。     

肥厚心肌细胞Na+/Ca2+交换活性对β-肾上腺素能药物的反应性降低

目的探讨肥厚心肌细胞Na+/Ca2+交换对β-类肾上腺素能药物刺激的反应及发生这种改变的可能机制.方法采用胶原酶消化的高血压大鼠单个心室肌细胞及全细胞膜片钳技术,记录Na+/Ca2+交换电流并观察药物对它的影响.结果 (1)异丙肾上腺素可浓度依赖性地增加正常及肥厚大鼠心室肌细胞的Na+/Ca2+交换电流,但其对肥厚心室肌细胞Na+/Ca2+交换电流的增强作用要弱于正常心室肌细胞(P<0.05).(2)cAMP可浓度依赖性地增加正常及肥厚大鼠心室肌细胞的Na+/Ca2+交换电流,其对正常及肥厚大鼠心室肌细胞Na+/Ca2+交换电流的增强作用无差别(P>0.05).结论高血压大鼠心室肌细胞Na+/Ca2+交换对β-类肾上腺素能药物的反应性降低,可能与肥厚心肌细胞的β-受体数目及功能、G蛋白及腺苷酸环化酶的活性改变等环节有关,此种反应可能是肥厚心肌收缩及舒张功能障碍的机制之一.     

雌激素抑制大鼠心室肌细胞L-型钙电流机制的研究

目的 :探讨雌激素抑制大鼠心肌细胞 L -型钙电流 （ICa,L）的可能途径。方法 :通过胶原酶消化得到单个大鼠心室肌细胞 ,膜片钳全细胞电压钳方法记录 ICa,L。结果 :雌激素 （1～ 30μmol/ L ）可剂量依赖性地抑制 ICa,L,雌激素对ICa,L的抑制作用并不能被雌激素受体的阻断剂 Tamoxifen和 ICI 182 780所阻断。细胞外灌流耦联了牛血清白蛋白（BSA）的雌激素 [β- estradiol6 - （o- carboxy- m ethyl） oxime:BSA（EST- BSA） ],不能透过细胞膜 ]也能抑制 ICa,L,但同样浓度的 EST- BSA细胞内灌流对 ICa,L并没有明显的抑制作用。结论 :雌激素可能是通过膜表面上特异的受体介导了其对 L-型钙电流的抑制作用     

黄芪总黄酮对急性心肌梗死大鼠心室肌细胞L-型钙电流及钠电流的作用

目的研究黄芪总黄酮（TFA）对急性心肌梗死（Am）大鼠心室肌细胞L-型钙电流（L—ICa）及钠电流（INa）的作用。方法设正常对照组、AMI组及TFA实验组。大鼠开胸左前降支结扎造成AMI,开胸前5min实验组大鼠舌静脉注射剂量为20mg／kg的TFA溶液50ul。用胶原酶酶解法急性分离AMI模型大鼠心室肌细胞,采用全细胞膜片钳记录技术记录左前降支供血区心外膜细胞的L—ICa及INa的作用。结果①应用TFA（20mg／kg）后,L—ICa从（0．313±0．14）nA增加到（0．402±0．27）nA,差异有统计学意义（P〈0．01,n=6）。②应用TFA（20mg／kg）后与对照组比较,峰值从（-8．43±2,03）nA下降到（-6．37±1．58）nA,差异有统计学意义（P〈0．01,n=6）。结论TFA可以增加AMI大鼠心室肌细胞L—ICa的幅值,TFA可显著降低AMI大鼠心室肌细胞INa的幅值。     

溶血磷脂酰胆碱对心肌细胞动作电位的影响及其机制研究

目的:探讨溶血磷脂酰胆碱(LPC)对心肌细胞动作电位的影响及内在机制。方法:以胶原酶消化法获取1~3d新生Wistar大鼠心室肌细胞(共5批,每批8只),以及特异性人类心脏T型钙通道α1亚基在HEK293细胞上稳定表达的细胞株(HEK293-Cav3.1和HEK293-Cav3.2),孵育传代培养进行全细胞膜片钳记录。结果:LPC(10μmol/L)或佛波酯(PMA,1μmol/L))显著增快了新生大鼠心室肌细胞自发跳动。LPC显著增大了新生大鼠心室肌T型Ca2+通道电流(ICa,T),且这种增大作用能被特异的心肌T型钙离子通道α1亚基Cav3.2阻断剂Ni 2+显著抑制。Ni 2+显著抑制了心室肌细胞自发动作电位的频率,特别是对LPC作用下的自律性提高,但是Ni 2+没有完全抑制PMA的促进作用。结论:LPC主要通过调控Cav3.2亚基而增大ICa,T,促进心肌自律性增高,从而致心律失常发生。     

异丙肾上腺素对犬心房肌细胞动作电位及L型钙电流的影响

目的研究两种浓度的异丙肾上腺素(Isoproterenol,ISO)对犬心房肌细胞(AP)及L型钙电流(ICa,L)的作用。方法采用离体灌注和消化的方法获取心房肌细胞,用全细胞记录技术记录单个心房肌细胞AP以及ICa,L。结果低浓度ISO(10nmol/L)可延长APD,可使90%AP时程(APD90)延长34.4%,并降低AP平台期水平。高浓度ISO(1μmol/L)可减少APD,APD90减少32.1%。两种浓度的ISO均可诱发AP后除极及触发活动。10nmol/L和1μmol/LISO分别增加ICa,L36.7%和49.3%。结论两种浓度的ISO对心肌细胞ICa,L均有促进作用,Ca2+内流引起的肌浆网Ca2+释放可能是房性心律失常的发生机制之一。     

兴奋β3受体对快速心房起搏家兔心房肌细胞ICa,L的影响

目的:观察兴奋β3肾上腺素能受体（β3受体）对快速心房起搏(rapid atrial pacing,RAP)家兔心房肌细胞L型钙电流（ICa,L）的影响,为探索心房颤动（AF）的发病机制及治疗提供新的思路。方法: 35只20~25 kg家兔随机分组:1.离体部分:①假手术组（Sham组,n=7）:单纯开胸、安装起搏器不予起搏,1周后分离心房肌细胞,记录ICa,L（Sham组）。②手术组(n=7):以600次/min RAP 1周后,分离心房肌细胞,记录ICa,L（RAP组）;向细胞中加入β1、β2受体阻滞剂Nadolol及β3受体激动剂BRL 37344（BRL）,记录ICa,L（RAP+BRL组）;再加入β3受体特异性阻滞剂SR 59230A（SR）,记录ICa,L（RAP+BRL+SR组）。2.在体部分:①RAP组（n=7）:单纯RAP 1周;②RAP+BRL组（n=7）:RAP后给予Nadolol及BRL 1周;③RAP+BRL+SR组（n=7）:RAP后给予Nadolol、BRL及SR 1周。测定各组心房肌一氧化氮（NO）、环-磷酸鸟苷（cGMP）的含量及cGMP依赖性蛋白激酶（PKG）蛋白的表达。结果: ①与Sham组相比,RAP组ICa,L明显减小（P<001）;②与RAP组相比,RAP+BRL组ICa,L明显减小（P<001）,NO、cGMP的含量及PKG蛋白的表达明显增加（均为P<005）。③与RAP+BRL组相比,RAP+BRL+SR组ICa,L明显增大（P<001）,NO、cGMP的含量及PKG蛋白的表达明显减少（P<001,P<001,P<005）。结论: 兴奋β3受体可激活NO cGMP PKG通路并同时减小RAP家兔心房肌细胞ICa,L。     

卡维地洛对大鼠心室肌细胞L型钙电流的影响

目的研究卡维地洛对大鼠心室肌细胞L型钙电流(ICa,L)的影响,探讨卡维地洛在离子通道水平的药理机制。方法用急性酶解法获得大鼠单个心室肌细胞,用标准全细胞膜片钳技术记录ICa,L,观察不同浓度卡维地洛对ICa,L的影响。结果①卡维地洛呈浓度依赖性抑制ICa,L,1μmol/L卡维地洛能使心肌细胞ICa,L电流-电压关系曲线明显上移,峰电流密度减少(7.80±0.65pA/pFvs4.89±0.52pA/pF,n=10,P<0.05),但激活电位、峰电位和翻转电位无明显改变。②卡维地洛能使ICa,L失活曲线明显左移,但对ICa,L激活和复活曲线无明显影响。③0.1μmol/L卡维地洛对钙电流无明显影响,但可以明显阻断异丙肾上腺素增加钙电流效应。同时,卡维地洛对钙电流的抑制效应不能被哌唑嗪和普奈洛尔阻断。结论卡维地洛呈浓度依赖性地抑制心室肌细胞L型钙通道。     

肥大和衰竭心肌L型Ca2+通道研究进展

心肌细胞 L 型钙电流 （ IC a· L）触发了 Ca2 +诱导的 Ca2 +释放 （ CICR） ,调节着细胞内瞬时 Ca2 +动力学 ,从而在兴奋—收缩耦联过程中起关键性作用。ICa· L密度和通道功能的改变参与了心力衰竭的发生。作者综述了 :1L型 Ca2 +通道的结构和生理作用 ;2肥大和衰竭的心肌 ICa· L变化及其频率依赖性调节机制 ;3 Ca2 + 通道和心力衰竭的治疗关系 ;4L型 Ca2 +通道未来研究方向     

Evidence for participation of vicinal dithiols in the activation sequence of the respiratory burst of human neutrophils

Phenylarsine oxide (PAO) specifically forms a stable ring complex with vicinal dithiols that can be reversed with 2,3-dimercaptopropanol (DMP). Pretreatment of human neutrophils with micromolar concentrations of PAO inhibited release of superoxide anion (O2-) stimulated by N- formyl-methionyl-leucyl-phenylalanine (FMLP) or phorbol 12-myristate 13- acetate (PMA); the inhibition was reversed with DMP, but not with 2- mercaptoethanol. PAO did not affect O2- release in previously stimulated cells. PAO did not affect the FMLP-induced Ca2+ response, suggesting that PAO affects a postreceptor event that does not modulate the Ca2+ transient. Treatment of isolated membrane or cytosolic fractions with PAO did not change the rates of arachidonate-stimulated O2- production in a cell-free system. Pretreatment of unstimulated neutrophils with PAO inactivated cytosolic protein kinase C (PKC); the inactivation was reversed with DMP. However, PAO did not affect PMA- induced translocation of beta-PKC protein or reduce the PKC activity translocated to the membrane. PAO had no effect on tyrosine kinase activity but inactivated phosphotyrosine phosphatase; stimulus-induced tyrosine phosphorylation of several proteins was markedly enhanced. These results suggest that vicinal dithiols play an essential role in activation of the respiratory burst oxidase. Possible sites for the activity of these essential vicinal dithiols include PKC and the regulatory balance of tyrosine phosphatase activity and tyrosine phosphorylation.     

Ca(2+)-independent reduction of N-methyl-D-aspartate channel activity by protein tyrosine phosphatase.

Regulation of ion channel function by intracellular processes is fundamental for controlling synaptic signaling and integration in the nervous system. Currents mediated by N-methyl-D-aspartate (NMDA) receptors decline during whole-cell recordings and this may be prevented by ATP. We show here that phosphorylation is necessary to maintain NMDA currents and that the decline is not dependent upon Ca2+. A protein tyrosine phosphatase or a peptide inhibitor of protein tyrosine kinase applied intracellularly caused a decrease in NMDA currents even when ATP was included. On the other hand, pretreating the neurons with a membrane-permeant tyrosine kinase inhibitor occluded the decline in NMDA currents when ATP was omitted. In inside-out patches, applying a protein tyrosine phosphatase to the cytoplasmic face of the patch caused a decrease in probability of opening of NMDA channels. Conversely, open probability was increased by a protein tyrosine phosphatase inhibitor. These results indicate that NMDA channel activity is reduced by a protein tyrosine phosphatase associated with the channel complex.     

Phenylarsine oxide augments tyrosine phosphorylation in hematopoietic cells.

Tyrosine phosphorylation and dephosphorylation are implicated in the regulation of cell growth and differentiation. A diverse identification of key regulatory proteins by their content of phosphotyrosine has been hampered by the very low level of tyrosine phosphorylation. This is presumably caused by the relative preponderance of phosphotyrosine phosphatase activity in many cells. We report that treatment of hematopoietic cells with phenylarsine oxide (PAO), a membrane-permeable phosphotyrosine phosphatase inhibitor, induced a dramatic accumulation of phosphotyrosine in a number of cellular proteins. No changes in serine or threonine phosphorylation were detected. The PAO-induced accumulation of phosphotyrosine occurred well before any signs of toxicity or irreversible damage to the cells were seen. Addition of dithiothreitol reversed the effect of PAO. Our data demonstrate that phosphotyrosine phosphatase activity has a major impact on the level of phosphotyrosine in cellular proteins, even in cells with high protein tyrosine kinase activity. Cells with constitutively elevated tyrosine kinase activity are easily detected following treatment with PAO and substrates with an otherwise too low phosphotyrosine content or too rapid phosphate turnover can be studied. This effect of PAO allows determinations of tyrosine phosphorylation-dependent complex formation between proteins.     

Protein-tyrosine phosphorylation regulates apoptosis in human eosinophils and neutrophils.

Early signaling events that control the process of programmed cell death are largely unknown. Tyrosine phosphorylation plays a major role in transmembrane signal transduction through most cell surface receptors. Granulocyte/macrophage colony-stimulating factor (GM-CSF), a cytokine released by activated T cells, has been shown to increase tyrosine phosphorylation in several cells and to inhibit granulocyte cell death in vitro. In this study, we demonstrate that the effect of GM-CSF on granulocyte cell death can be blocked by the tyrosine kinase inhibitor genistein, suggesting that increases in tyrosine phosphorylation are essential to inhibit cell death. To analyze the role of tyrosine phosphorylation for the regulation of granulocyte cell death more precisely, we increased levels of tyrosine phosphorylation using the protein-tyrosine phosphatase inhibitor phenylarsine oxide (PAO). Similar to GM-CSF, treatment of the cells with PAO was followed by high increases in tyrosine phosphorylation and inhibition of programmed cell death in human eosinophils and neutrophils. Strikingly, at low concentrations of the inhibitor and low induction of tyrosine phosphorylation, acceleration of apoptosis was observed. Genistein and herbimycin A reversed the effects of PAO on tyrosine phosphorylation and granulocyte apoptosis. These results suggest that programmed eosinophil and neutrophil death is regulated by early events of signal transduction pathways such as tyrosine phosphorylation.     

Hypoxia alters the sensitivity of the L-type Ca(2+) channel to alpha-adrenergic receptor stimulation in the presence of beta-adrenergic receptor stimulation

The effects of alpha-adrenergic receptor (alpha-AR) stimulation alone and the effects in the presence of beta-adrenergic receptor (beta-AR) stimulation were examined on L-type Ca(2+) currents (I(Ca-L)) in the absence and presence of hypoxia. The alpha-AR agonist methoxamine either had no effect or had a slight inhibitory effect on basal I(Ca-L) in the absence and presence of hypoxia. Hypoxia significantly decreased the K(0.5) for activation of I(Ca-L) by norepinephrine from 79.8+/-6.6 to 13.3+/-0.7 nmol/L. To determine whether hypoxia specifically altered the sensitivity of the channel to alpha-AR stimulation, cells were exposed to increasing concentrations of methoxamine in the presence of 100 nmol/L isoproterenol (Iso). In the absence of hypoxia, methoxamine inhibited the Iso-activated I(Ca-L) in a concentration-dependent manner with an EC(50) of 86.9+/-9.9 micromol/L. However, in the presence of hypoxia, the EC(50) for inhibition of I(Ca-L) by methoxamine was significantly increased to 266.7+/-10.8 micromol/L. Methoxamine had little effect on I(Ca-L) activated by forskolin or histamine in the absence or presence of hypoxia. In addition, inhibition of protein kinase C by bisindolylmaleimide 1 or protein kinase C beta peptide inhibitor had no effect on the methoxamine-induced antagonism of I(Ca-L) in the absence or presence of hypoxia. The tyrosine kinase inhibitor genistein attenuated the methoxamine response in nonhypoxic cells only. However, during hypoxia it was attenuated with the phospholipase A(2) inhibitors mepacrine and indomethacin. These findings represent a novel regulation of the L-type Ca(2+) channel by the phospholipase A(2) pathway and illustrate the complexity of regulation of the channel under hypoxic conditions.     

Inhibitory effects of berberine on ion channels of rat hepatocytes

AIM: To examine the effects of berberine, an isoquinoline alkaloid with a long history used as a tonic remedy for liver and heart, on ion channels of isolated rat hepatocytes.METHODS: Tight-seal whole-cell patch-clamp techniques were performed to investigate the effects of berberine on the delayed outward potassium currents (Ik), inward rectifier potassium currents (Ik1) and Ca^2+ release-activated Ca^+ currents (ICRAC) in enzymatically isolated rat hepatocytes.RESULTS: Berbenne 1-300 μmol/L reduced/K in a concentration-dependent manner with EC50 of 38.86=1=5.37 μmol/L and nH of 0.82&#177;0.05 (n = 8). When the bath solution was changed to tetraethylammonium (TEA) 8 retool/L,IK was inhibited.Berberine 30 μmol/L reduced/K at all examined membranepote ntials, especially at potentials positive to +60 mV (n = 8,P&lt;0.05 or P&lt;0.01 vs control). Berberine had mild inhibitory effects on IK1 in rat hepatocytes. Berberine 1-300 μmol/L also inhibited ICRAC in a concentration-dependent fashion. The fitting parameters were EC50 = 47.20&#177;10.86 μmol/L,nH= 0.71&#177;0.09 (n = 8). The peak value of/CRAC in the I-Vrelationship was decreased by berberine 30 μmol/L at potentialnegative to -80 mV (n = 8, P&lt;0.05 vscontrol). But the reverse potential of/CRAC occurred at voltage 0 mV in all cells.CONCLUSION: Berberine has inhibitory effects on potassium and calcium currents in isolated rat hepatocytes, which may be involved in hepatoprotection.     

Rapid stimulation causes electrical remodeling in cultured atrial myocytes

OBJECTIVE: Rapid stimulation causes electrical remodeling in the intact atrium, with shortening of action potential duration (APD), down-regulation of L-type Ca2+ currents (I(Ca,L)), and increased vulnerability to atrial fibrillation (AF). The essential elements required for this process are currently unknown. We tested the hypothesis that rapid stimulation of cardiomyocytes in vitro is sufficient to recapitulate the remodeling process, and that atrial cells subjected to rapid pacing in culture would display changes similar to those that occur in vivo. METHODS: Atrial (HL-1) cells were cultured in the presence of rapid field stimulation (300 beats per min) for 24 h. Action potentials and ionic currents were recorded from stimulated cells, as well as control cells cultured in parallel, using whole-cell voltage-clamp techniques. RESULTS: Rapid stimulation of atrial cells for 24 h significantly shortened APD. HL-1 cells displayed both I(Ca,L) blocked by nimodipine, and T-type Ca2+ currents (I(Ca,T)) sensitive to mibefradil. Rapid activation in culture caused down-regulation of I(Ca,L), while I(Ca,T) was similarly reduced. Multiple outward currents were present in response to a depolarizing voltage-clamp protocol, and rapid pacing resulted in up-regulation of the rapidly-activating delayed rectifier K+ current, I(Kr). CONCLUSIONS: Rapid stimulation of atrial cells in culture produces electrical remodeling, recapitulating principal phenotypic features of atrial tachycardia remodeling in vivo. Our results demonstrate that an important component of this process is cell autonomous, given that in vivo conditions are not required for the development of electrical remodeling.     

Effect of genistein on voltage-gated potassium channels in guinea pig proximal colon smooth muscle cells

AIM:To investigate the action of genistein(GST),abroad spectrum tyrosine kinase inhibitor,on voltage-gated potassium channels in guinea pig proximal colonsmooth muscle cells.METHODS:Smooth muscle cells in guinea pig proximalcolon were enzymatically isolated.Nystatin-perforatedwhole cell patch clamp technique was used to recordpotassium currents including fast transient outwardcurrent(I_(Kto))and delayed rectifier current(I_(Kdr)),two ofwhich were isolated pharmacologically with 10 mmol/Ltetraethylammonium or 5 mmol/L 4-aminopyridine.Contamination of calcium-dependent potassium currentswas minimized with no calcium and 0.2 mmol/L CdCl_2 inan external solution.RESULTS:GST(10-100μmol/L)reversibly and dose-dependently reduced the peak amplitude of I_(Kto)with anICso value of 22.0 6.9μmol/L.To a lesser extent,I_(Kdr)wasalso inhibited in both peak current and sustained current.GST could not totally block the outward potassiumcurrent as a fraction of the outWard potassium current,which was insensitive to GST.GST had no effect on thesteady-state activation(n=6)and inactivation kinetics(n=6)of I_(Kto).Sodium orthovanadate(1 mmol/L),apotent inhibitor of tyrosine phosphatase,significantlyinhibited GST-induced inhibition(P<0.05).CONCLUSION:GST can dose-dependently andreversibly block voltage-gated potassium channels inguinea pig proximal colon smooth muscle cells.     

L-type Ca2+ channel density and regulation are altered in failing human ventricular myocytes and recover after support with mechanical assist devices

Ca2+ influx through the L-type calcium channel (LTCC) induces Ca2+ release from the sarcoplasmic reticulum (SR) and maintains SR Ca2+ loading. Alterations in LTCC properties, their contribution to the blunted adrenergic responsiveness in failing hearts and their recovery after support with LV assist devices (LVAD) were studied. L-type Ca2+ current (I(Ca,L)) was measured under basal conditions and in the presence of isoproterenol (ISO), dibutyryl-cAMP (db-cAMP), Bay K 8644 (BayK), Okadaic acid (OA, a phosphatase inhibitor), and phosphatase 2A (PP2A) in nonfailing (NF), failing (F), and LVAD-supported human left ventricular myocytes (HVMs). Basal I(Ca,L) density was not different in the 3 groups but I(Ca,L) was activated at more negative voltages in F- and LVAD- versus NF-HVMs (V(0.5): -7.18+/-1.4 and -7.0+/-0.9 versus 0.46+/-1.1 mV). Both ISO and db-cAMP increased I(Ca,L) in NF- and LVAD- significantly more than in F-HVMs (NF >LVAD> F: ISO: 90+/-15% versus 77+/-19% versus 24+/-12%; db-cAMP: 235%>172%>90%). ISO caused a significant leftward shift of the I(Ca,L) activation curve in NF- and LVAD- but not in F-HVMs. After ISO and db-cAMP, the I(Ca,L) activation was not significantly different between groups. BayK also increased I(Ca,L) more in NF- (81+/-30%) and LVAD- (70+/-15%) than in F- (51+/-8%) HVMs. OA increased I(Ca, L) by 85.6% in NF-HVMs but had no effect in F-HVMs, while PP2A decreased I(Ca, L) in F-HVMs by 35% but had no effect in NF-HVMs. These results suggest that the density of LTCC is reduced in F-HVMs but basal I(Ca,L) density is maintained by increasing in LTCC phosphorylation.