Protein tyrosine kinase-dependent modulation of voltage-dependent potassium channels by genistein in rat cardiac ventricular myocytes

Effects of the isoflavone protein tyrosine kinase (PTK) inhibitor genistein on voltage-dependent K(+) currents, i.e., transient outward K(+) current (I(to)), sustained K(+) current (I(ss)), and inward rectifier K(+) current (I(K1)) were studied in rat cardiac ventricular myocytes. It was found that I(to) was reversibly inhibited by genistein in a concentration-dependent manner (IC(50)=28.1 microM), while I(ss) was suppressed by genistein with IC(50) of 18.5 microM. In addition, I(K1) (at -50 mV) was significantly decreased by 36.3+/-4.4% with 25 microM genistein. The inhibition of I(to), I(ss), and I(K1) by genistein was significantly reversed by the application of the protein tyrosine phosphatase inhibitor sodium orthovanadate (1 mM). However, I(to), I(ss), and I(K1) were not affected by the non-isoflavone PTK inhibitor tyrphostin A23 (100 microM) and PP2 (1 microM). These results indicate that activation of I(to), I(ss), and I(K1) channels is modulated by genistein-sensitive PTKs in rat ventricular myocytes.     

抗心律失常药RP62719对哺乳动物心室肌K+电流的抑制

使用全细胞膜片箝技术, 研究RP62719对内向整流钾电流(IK1)、瞬时外向钾电流(Ito)和延迟外向整流钾电流(IK)的作用, 并探讨其抗心律失常作用的机制.实验结果表明, 在指令电压为-100 mV时, RP62719可显著抑制豚鼠心室肌细胞IK1, 半数抑制浓度(IC50)为5.0±1.0 μmol/L.RP62719 10 μmol/L在+40 mV时对犬心室肌细胞Ito抑制率为84.0±4.4%, IC50为1.2±0.51 μmol/L.在+40 mV时, 50 μmol/L RP62719还可使豚鼠心室肌细胞IKstep 减少50.0±8.3%, IKtail减少56.0±4.9%, IC50分别为4.2±0.8 μmol/L和3.3±0.75 μmol/L.提示RP62719抗心律失常的离子机制与其对IK1、Ito及IK的抑制有关.     

Novel regulation of Na, K-ATPase by Src tyrosine kinases in cortical neurons

The Na+, K+-ATPase or Na+, K+-pump plays a critical role in ion homeostasis and many cellular events. The Na+, K+-pump activity is regulated by serine/threonine phosphorylation, the role of tyrosine kinases in the regulation, however, is obscure. We now present novel evidence showing that tyrosine phosphorylation activates the Na+, K+-pump in cortical neurons. The electrogenic activity of the Na+, K+-pump was measured using whole-cell voltage clamp. A tonic activity was revealed by an inward current induced by the specific inhibitor ouabain or strophanthidin; an outward current due to activation of the pump was triggered by raising extracellular K+. The inward and outward currents were attenuated by the tyrosine kinase inhibitor genistein, herbimycin A, or lavendustin A, while blocking tyrosine phosphatases increased the pump current. Down-regulation of the pump current was also seen with the Src inhibitor PP1 and intracellularly applied anti-Lyn or anti-Yes antibody. Consistently, intracellular application of Lyn kinase up-regulated the pump current. Immunoprecipitation and western blotting showed tyrosine phosphorylation and a direct interaction between Lyn and the alpha3 subunit of the Na+, K+-pump. The tyrosine phosphorylation of the alpha3 subunit was reduced by serum deprivation. These data suggest that the Na+, K+-ATPase activity in central neurons is regulated by specific Src tyrosine kinases via a protein-protein mechanism and may play a role in apoptosis.     

Effects of anandamide on potassium channels in rat ventricular myocytes: a suppression of I(to) and augmentation of K(ATP) channels

Anandamide is an endocannabinoid that has antiarrhythmic effects through inhibition of L-type Ca(2+) channels in cardiomyocytes. In this study, we investigated the electrophysiological effects of anandamide on K(+) channels in rat ventricular myocytes. Whole cell patch-clamp technique was used to record K(+) currents, including transient outward potassium current (I(to)), steady-state outward potassium current (I(ss)), inward rectifier potassium current (I(K1)), and ATP-sensitive potassium current (I(KATP)) in isolated rat cardiac ventricular myocytes. Anandamide decreased I(to) while increasing I(KATP) in a concentration-dependent manner but had no effect on I(ss) and I(K1) in isolated ventricular myocytes. Furthermore, anandamide shifted steady-state inactivation curve of I(to) to the left and shifted the recovery curve of I(to) to the right. However, neither cannabinoid 1 (CB(1)) receptor antagonist AM251 nor CB(2) receptor antagonist AM630 eliminated the inhibitory effect of anandamide on I(to). In addition, blockade of CB(2) receptors, but not CB(1) receptors, eliminated the augmentation effect of anandamide on I(KATP). These data suggest that anandamide suppresses I(to) through a non-CB(1) and non-CB(2) receptor-mediated pathway while augmenting I(KATP) through CB(2) receptors in ventricular myocytes.     

Transient outward current carried by inwardly rectifying K+ channels in guinea pig ventricular myocytes dialyzed with low-K+ solution

There have been periodic reports of nonclassic (4-aminopyridine insensitive) transient outward K+ current in guinea pig ventricular myocytes, with the most recent one describing a novel voltage-gated inwardly rectifying type. In the present study, we have investigated a transient outward current that overlaps inward Ca2+ current (I(Ca,L)) in myocytes dialyzed with 10 mM K+ solution and superfused with Tyrode's solution. Although depolarizations from holding potential (Vhp) -40 to 0 mV elicited relatively small inward I(Ca,L) in these myocytes, removal of external K+ or addition of 0.2 mM Ba2+ more than doubled the amplitude of the current. The basis of the enhancement of I(Ca,L) was the suppression of a large transient outward K+ current. Similar enhancement was observed when Vhp was moved to -80 mV and test depolarizations were preceded by short prepulses to -40 mV. Investigation of the time and voltage properties of the outward K+ transient indicated that it was inwardly rectifying and unlikely to be carried by voltage-gated channels. The outward transient was attenuated in myocytes dialyzed with high-Mg2+ solution, accelerated in myocytes dialyzed with 100 microM spermine solution, and abolished with time in myocytes dialyzed with ATP-free solution. These and other findings suggest that the outward transient is a component of classic "time-independent" inwardly rectifying K+ current.     

Gender differences in ANG II levels and action on multiple K+ current modulation pathways in diabetic rats

Gender differences were studied in ventricular myocytes from insulin-deficient (Type 1) diabetic rats. Cells were obtained by enzymatic dispersion of hearts from control male and female rats and from rats made diabetic with streptozotocin (100 mg/kg) 7-14 days before experiments. ANG II content, measured by ELISA, was augmented in diabetic males but unaltered in diabetic females. In diabetic ovariectomized females, ANG II levels were augmented as in males. ANG II affects multiple cellular pathways including activation of protein kinase C (PKC) and several tyrosine kinases as well as inhibition of protein kinase A (PKA). The involvement of these pathways in modulating outward K(+) currents was studied. Transient and sustained outward K(+) currents were measured using the whole cell voltage-clamp method. In males, these currents are attenuated under diabetic conditions but are augmented by the ANG II-converting enzyme inhibitor quinapril. Activation of PKA by 8-bromo-cAMP enhanced both K(+) currents in cells from diabetic males. The augmentation of these currents by quinapril was blocked when PKA inhibition was maintained with the Rp isomer of 3',5'-cyclic monophosphorothioate. Inhibition of tyrosine kinases by genistein also augmented K(+) currents in cells from diabetic males. Action potentials were abbreviated by 8-bromo-cAMP and genistein. However, both genistein and 8-bromo-cAMP had no effect on K(+) currents in cells from diabetic females. In cells from ovariectomized diabetic females, 8-bromo-cAMP and genistein enhanced these K(+) currents as in males. Inhibition of PKC augmented the transient and sustained K(+) currents in cells from diabetic males and females. A contribution of non-ANG II-dependent activation of PKC is suggested. These results describe some of the mechanisms that may underlie gender-specific differences in the development of cardiac disease and arrhythmias.     

Block of the background K(+) channel TASK-1 contributes to arrhythmogenic effects of platelet-activating factor

Platelet-activating factor (PAF), an inflammatory phospholipid, induces ventricular arrhythmia via an unknown ionic mechanism. We can now link PAF-mediated cardiac electrophysiological effects to inhibition of a two-pore domain K(+) channel [TWIK-related acid-sensitive K(+) background channel (TASK-1)]. Superfusion of carbamyl-PAF (C-PAF), a stable analog of PAF, over murine ventricular myocytes causes abnormal automaticity, plateau phase arrest of the action potential, and early afterdepolarizations in paced and quiescent cells from wild-type but not PAF receptor knockout mice. C-PAF-dependent currents are insensitive to Cs(+) and are outwardly rectifying with biophysical properties consistent with a K(+)-selective channel. The current is blocked by TASK-1 inhibitors, including protons, Ba(2+), Zn(2+), and methanandamide, a stable analog of the endogenous lipid ligand of cannabinoid receptors. In addition, when TASK-1 is expressed in CHO cells that express an endogenous PAF receptor, superfusion of C-PAF decreases the expressed current. Like C-PAF, methanandamide evoked spontaneous activity in quiescent myocytes. C-PAF- and methanandamide-sensitive currents are blocked by a specific protein kinase C (PKC) inhibitor, implying overlapping signaling pathways. In conclusion, C-PAF blocks TASK-1 or a closely related channel, the effect is PKC dependent, and the inhibition alters the electrical activity of myocytes in ways that would be arrhythmogenic in the intact heart.     

Tyrosine kinase and protein kinase C regulate L-type Ca(2+) current cooperatively in human atrial myocytes

The effects of tyrosine protein kinases (TK) on the L-type Ca(2+) current (I(Ca)) were examined in whole cell patch-clamped human atrial myocytes. The TK inhibitors genistein (50 microM), lavendustin A (50 microM), and tyrphostin 23 (50 microM) stimulated I(Ca) by 132 +/- 18% (P < 0.001), 116 +/- 18% (P < 0.05), and 60 +/- 6% (P < 0.001), respectively. After I(Ca) stimulation by genistein, external application of isoproterenol (1 microM) caused an additional increase in I(Ca). Dialyzing the cells with a protein kinase A inhibitor suppressed the effect of isoproterenol on I(Ca) but not that of genistein. Inhibition of protein kinase C (PKC) by pretreatment of cells with 100 nM staurosporine or 100 nM calphostin C prevented the effects of genistein on I(Ca). The PKC activator phorbol 12-myristate 13-acetate (PMA), after an initial stimulation (75 +/- 17%, P < 0.05), decreased I(Ca) (-36 +/- 5%, P < 0.001). Once the inhibitory effect of PMA on I(Ca) had stabilized, genistein strongly stimulated the current (323 +/- 25%, P < 0.05). Pretreating myocytes with genistein reduced the inhibitory effect of PMA on I(Ca). We conclude that, in human atrial myocytes, TK inhibit I(Ca) via a mechanism that involves PKC.     

Both EGFR kinase and Src-related tyrosine kinases regulate human ether-à-go-go-related gene potassium channels

Human ether-à-go-go-related gene (hERG or Kv11.1) encodes the rapidly activated delayed rectifier K(+) current (I(Kr)) in the human heart. Potential regulation of hERG channel by protein tyrosine kinases (PTKs) is not understood. The present study was designed to investigate whether this channel is modulated by PTKs using whole-cell patch clamp technique, and immunoprecipitation and Western blot analysis in HEK 293 cells stably expressing hERG gene. We found that the broad-spectrum PTK inhibitor genistein (30 muM), the selective EGFR (epidermal growth factor receptor) kinase inhibitor AG556 (10 muM) and the Src-family kinase inhibitor PP2 (10 muM) remarkably inhibited hERG channel current (I(hERG)), and the effects were significantly countered by the protein tyrosine phosphatase (PTP) inhibitor orthovanadate (1 mM). Immunoprecipitation and Western blot analysis demonstrated that membrane protein tyrosine phosphorylation of hERG channels was reduced by genistein, AG556, and PP2. The reduction of hERG channel phosphorylation level by genistein, AG556 or PP2 was antagonized by orthovanadate. Single point mutation(s) of Y475A and/or Y611A dramatically attenuated the inhibitory effect of I(hERG) by PP2 and/or AG556. Our results demonstrate the novel information that I(hERG) is modulated not only by Src-family kinases, but also by EGFR kinases. Y475 and/or Y611 are likely the preferred phosphorylation sites. Regulation of hERG channels by PTKs modifies the channel activity and thus likely alters electrophysiological properties including action potential duration and cell excitability in human heart and neurons.     

Postnatal development has a marked effect on ventricular repolarization in mice

To better understand the mechanisms that underlie cardiac repolarization abnormalities in the immature heart, this study characterized and compared K(+) currents in mouse ventricular myocytes from day 1, day 7, day 20, and adult CD1 mice to determine the effects of postnatal development on ventricular repolarization. Current- and patch-clamp techniques were used to examine action potentials and the K(+) currents underlying repolarization in isolated myocytes. RT-PCR was used to quantify mRNA expression for the K(+) channels of interest. This study found that action potential duration (APD) decreased as age increased, with the shortest APDs observed in adult myocytes. This study also showed that K(+) currents and the mRNA relative abundance for the various K(+) channels were significantly greater in adult myocytes compared with day 1 myocytes. Examination of the individual components of total K(+) current revealed that the inward rectifier K(+) current (I(K1)) developed by day 7, both the Ca(2+)-independent transient outward current (I(to)) and the steady-state outward K(+) current (I(ss)) developed by day 20, and the ultrarapid delayed rectifier K(+) current (I(Kur)) did not fully develop until the mouse reached maturity. Interestingly, the increase in I(Kur) was not associated with a decrease in APD. Comparison of atrial and ventricular K(+) currents showed that I(to) and I(Kur) density were significantly greater in day 7, day 20, and adult myocytes compared with age-matched atrial cells. Overall, it appears that, in mouse ventricle, developmental changes in APD are likely attributable to increases in I(to), I(ss), and I(K1), whereas the role of I(Kur) during postnatal development appears to be less critical to APD.     

A cAMP-dependent protein kinase inhibitor modulates the blocking action of ATP and 5-hydroxydecanoate on the ATP-sensitive K+ channel.

We studied the blocking mechanism of 5-hydroxydecanoate, a novel antiarrhythmic agent, on the ATP-sensitive K+ channel in the single ventricular myocytes using the inside-out patch clamp technique. The channel activity in response to 5-hydroxydecanoate varied with each membrane patch corresponding to the sensitivity to ATP. In this condition the exogenous application of cAMP or cAMP-dependent protein kinase (PKA) obviously recovered the ATP-sensitive K+ channel activity after channel deactivation. By contrast, in membrane patches exhibited low sensitivity to ATP, endogenous cAMP-dependent protein kinase inhibitor (PKI) depressed the channel activity and restored the inhibitory action of 5-hydroxydecanoate and ATP on the channel. These results suggest that PKA-PKI system is involved in the regulatory mechanism of gating activity of the ATP-sensitive K+ channel and the blocking action of 5-hydroxydecanoate and ATP appears to be exerted by potentiating the inhibitory action of PKI on the channel.     

Direct inhibition of a PKA inhibitor, H-89 on KV channels in rabbit coronary arterial smooth muscle cells

We examined the effects of the protein kinase A (PKA) inhibitor H-89 on voltage-dependent K(+) (K(V)) currents in freshly isolated rabbit coronary arterial smooth muscle cells, using a whole-cell patch clamp technique. H-89 inhibited the K(V) current in a concentration-dependent manner, with a K(d) value of 1.02 microM. However, the PKA inhibitors KT 5720 and Rp-8-CPT-cAMPS did not significantly alter the K(V) current or the inhibitory effects of H-89 on the K(V) current. Moreover, H-85, a structurally similar but inactive analog of H-89, showed similar inhibitory effects on the K(V) channel. H-89 had no effect on the voltage-dependency of activation or inactivation, or on recovery kinetics. These results suggest that in rabbit coronary arterial smooth muscle cells, H-89 inhibits the K(V) current directly by blocking the pore cavity, an effect independent of PKA inhibition.     

氧自由基致豚鼠心室肌细胞跨膜电位变化的离子电流基础

目的：旨在提示氧自由基参与缺血／再灌注性心委失常发生的离子电流基础。方法：采用膜片钳全细胞式记录技术,观察Ｈ２Ｏ２（１ｍｍｏｌ／Ｌ）对豚鼠心室肌细胞跨膜电位和相关离子电流的影响。结果：Ｈ２Ｏ２使豚鼠心肌单细胞的静息电位（ＲＰ）降低,动作电位时程（ＡＳＤ）显著缩短,对动作电位幅度（ＡＰＡ）和超射（ＯＳ）及钠电流的峰值（ＩＮａ）均无明显影响;明显抑制内向整流钾电流（ＩＫ１）,尤其在超极化时;增强延迟外     

Evidence for a modulatory effect of external potassium ions on ionic current mediated by the cardiac Na+-Ca2+ exchanger

The aim of this study was to investigate whether or not the activity of the cardiac Na(+)-Ca(2+) exchanger might be directly sensitive to external K(+) concentration ([K(+)](e)). Measurements of whole-cell exchanger current (I(NaCa)) were made at 37 degrees C from guinea-pig isolated ventricular myocytes, using whole-cell patch clamp recording with major interfering conductances blocked. Changing [K(+)](e) from 0 to 5mM significantly reduced both outward and inward exchange currents in a time-dependent manner. Various [K(+)](e) between 1 and 15 mM were tested and the inhibitory effect was observed to be concentration-dependent. At steady-state, 5mM [K(+)](e) decreased the density of Ni(2+)-sensitive current by 52.8+/-4.3% (mean+/-S.E.M., n=6) and of 0Na0Ca-sensitive current by 39.0+/-4.4% (n=5). The possibility that the inhibitory effect of external K(+) on I(NaCa) might wholly or in part be secondary to activation of the sarcolemmal Na(+)-K(+) pump was investigated by testing the effect of K(+) addition in the presence of a high concentration of strophanthidin (500 microM). Ni(2+)-sensitive I(NaCa) was still observed to be sensitive to external K(+) (I(NaCa) decreased by 39.4+/-9.4%, n=4), suggesting that the inhibitory effect could occur independently of activation of the Na(+)-K(+) pump. The effect of external K(+) on I(NaCa) was verified using a baby hamster kidney (BHK) cell line stably expressing the cardiac Na(+)-Ca(2+) exchanger isoform, NCX1. Similar to native I(NaCa), NCX1 current was also suppressed by [K(+)](e). However, [K(+)](e) did not alter current amplitude in untransfected BHK cells. The effect of [K(+)](e) on I(NaCa) could not be attributed to simply adding any monovalent cation back to the external solution, since it was not reproduced by application of equimolar Li(+), Cs(+) and TEA(+). Rb(+), however, could mimic the effect of K(+). Collectively, these data suggest that external K(+) at physiologically and pathologically relevant concentrations might be able to modulate directly the activity of the cardiac Na(+)-Ca(2+) exchanger.     

三羟异黄酮对豚鼠心室肌细胞L-型钙通道电流的影响

本实验用全细胞膜片钳技术观察三羟异黄酮(genistein,GST)对豚鼠心室肌细胞L-钙通道电流(ICa、L)的影响。结果如下:(1)GST(10、50、100 μmol/L)可浓度依赖性地降低ICa,L(n=6,P<0.01)。GST的非活性结构类似物daidzein(100μmol/L),在同一浓度范围对ICa,L没有影响(n=5,P>0.05)。(2)GST使I-V曲线上移,但对ICa,L的电压依赖特征和最大激活电压无明显影响。(3)GST对ICa,L的激活动力学特性也无影响,但可使钙电流稳态失活曲线左移。V0.5从对照的-28.6±0.6 mV变为-32.8±1.1mV,κ值从对照的5.8±0.5 mV升至6.5±0.9 mV(n=6,P<0.05)。(4)GST明显使复活曲线右移,从而使ICa,L从失活状态下恢复明显减慢(n=7,P<0.01)。(5)酪氨酸磷酸酶抑制剂正钒酸钠(1 mmol/L)显著对抗GST引起的ICa,L抑制效应(n=6,P<0.01)。根据以上结果得出的结论是:GST抑制ICa,L加速钙通道失活和钙通道在失活状态下恢复减慢;GST对ICa,L的这种抑制作用与蛋白酪氨酸激酶(PTK)抑制有关。     

Anti-adrenergic effect of adenosine on Na(+)-Ca(2+) exchange current recorded from guinea-pig ventricular myocytes

The Na(+)-Ca(2+) exchanger is a protein present in the cell membrane of many cell types. In heart it plays important roles in Ca homeostasis and ionic current generation. Recently, it has been reported that the beta-adrenergic agonist isoprenaline (ISO) can increase directly Na(+)-Ca(2+) exchanger activity in guinea-pig ventricular myocytes. Adenosine (ADO) exerts anti-adrenergic properties that make it effective against some arrhythmias and the aim of the present study was to determine whether or not ADO can antagonize the direct modulatory effect of ISO on the exchanger.Whole-cell patch clamp measurements of Na(+)-Ca(2+) exchanger current (I(NaCa)) were made from guinea-pig ventricular myocytes, with major interfering currents inhibited. I(NaCa) was measured at 378 degrees C as current sensitive to external nickel (Ni(2+), 10 mM) during an applied descending voltage ramp. ISO (1 microM) significantly increased both inward and outward I(NaCa). This effect was abolished in the presence of ADO (200 microM). ADO alone did not significantly alter the amplitude of I(NaCa). The effect of ADO on the response of I(NaCa) to ISO was mimicked by the A(1)ADO receptor agonist N(6)-cyclopentyladenosine (CPA, 10 microM), whereas the effect of ADO on the response of I(NaCa) to ISO was inhibited by the A(1)ADO receptor antagonist 8-cyclopentyl-1,3-dipropylxanthine (DPCPX, 2 microM). These data suggest that the A(1)ADO receptor mediated the response. The anti-adrenergic effects on I(NaCa) of ADO were not affected by the protein kinase C (PKC) inhibitor, chelerythrine (CLT, 1 microM), nor by the nitric oxide (NO) synthase inhibitor, N (G)-nitro-L-arginine methyl ester((L)-NAME, 0.5 mM). Moreover, in the presence of PKC activator phorbol 12-myristate 13-acetate (PMA, 1 microM) or exogenous NO donor sodium nitroprusside (SNP, 100 microM), ISO preserved its stimulatory effect on I(NaCa). However, prior incubation of myocytes with pertussis toxin (PTX, 5 microg ml(-1) did prevent the effect of ADO. The anti-adrenergic effect of ADO on I(NaCa) was mimicked by externally applied carbachol (CCh, 10 microM), a muscarinic receptor agonist. We conclude that ADO antagonized the effect of beta-adrenergic stimulation of I(NaCa) by directly activating inhibitory G-protein (G(i))-linked A(1) receptors in guinea-pig ventricular myocytes. These findings may suggest a novel mechanism by which adenosine exerts some of its antiarrhythmic effects.     

RPTPmu and protein tyrosine phosphorylation regulate K(+) channel mRNA expression in adult cardiac myocytes

Previously, we reported that cell-cell contact regulates K(+) channel mRNA expression in cultured adult rat cardiac myocytes. Here we show that exposing cardiac myocytes to tyrosine kinase inhibitors (genistein, tyrphostin A25), but not inactive analogs, prevents downregulation of Kv1.5 mRNA and upregulation of Kv4.2 mRNA normally observed when they are cultured under low-density conditions. Furthermore, cardiac myocytes cocultured with cells that endogenously (Mv 1 Lu) or heterologously (Chinese hamster ovary cells) express the receptor-type protein tyrosine phosphatase mu (RPTPmu) display Kv1.5 mRNA levels paralleling that which was observed in myocytes cultured under high-density conditions and in intact tissue. In contrast, myocytes cocultured with control cells failed to produce this response. Finally, it is shown that Kv4.2 mRNA expression is unaffected by RPTPmu. These findings reveal that multiple tyrosine phosphorylation-dependent mechanisms control cardiac myocyte K(+) channel genes. Furthermore, we conclude that RPTPmu specifically regulates cardiac myocyte Kv1.5 mRNA expression. Thus this receptor protein tyrosine phosphatase may be important in responses to pathological conditions associated with the loss of cell-cell interactions in the heart.     

Regulation of ROMK1 channels by protein-tyrosine kinase and -tyrosine phosphatase

We have used the two-electrode voltage clamp technique and the patch clamp technique to investigate the regulation of ROMK1 channels by protein-tyrosine phosphatase (PTP) and protein-tyrosine kinase (PTK) in oocytes coexpressing ROMK1 and cSrc. Western blot analysis detected the presence of the endogenous PTP-1D isoform in the oocytes. Addition of phenylarsine oxide (PAO), an inhibitor of PTP, reversibly reduced K(+) current by 55% in oocytes coinjected with ROMK1 and cSrc. In contrast, PAO had no significant effect on K(+) current in oocytes injected with ROMK1 alone. Moreover, application of herbimycin A, an inhibitor of PTK, increased K(+) current by 120% and completely abolished the effect of PAO in oocytes coexpressing ROMK1 and cSrc. The effects of herbimycin A and PAO were absent in oocytes expressing the ROMK1 mutant R1Y337A in which the tyrosine residue at position 337 was mutated to alanine. However, addition of exogenous cSrc had no significant effect on the activity of ROMK1 channels in inside-out patches. Moreover, the effect of PAO was completely abolished by treatment of oocytes with 20% sucrose and 250 microg/ml concanavalin A, agents that inhibit the endocytosis of ROMK1 channels. Furthermore, the effect of herbimycin A is absent in the oocytes pretreated with either colchicine, an inhibitor of microtubules, or taxol, an agent that freezes microtubules. We conclude that PTP and PTK play an important role in regulating ROMK1 channels. Inhibiting PTP increases the internalization of ROMK1 channels, whereas blocking PTK stimulates the insertion of ROMK1 channels.     

三羟异黄酮对豚鼠心室肌细胞内游离钙浓度的影响

用激光共聚焦显微镜观察研究三羟异黄酮(genistein,GST)对豚鼠心室肌细胞内游离钙浓度([Ca^2 ]i)的影响。结果用相对荧光强度(FI-F0／FX0,％)表示。实验结果显示,在正常台氏液、无钙台氏液和正常台氏液中加入3mmol／L EGTA后,GST(10～40μmol／L)浓度依赖性地降低细胞内钙浓度。蛋白酪氨酸磷酸酶抑制剂正钒酸钠(sodium orthovanadate)和L-型Ca^2 通道激动剂Bay K8644可部分抑制正常台氏液时GST的效应。当细胞外液钙浓度由1mmol／L增加到10mmol／L而诱发心室肌细胞钙超载时,部分心室肌细胞产生可传播的钙波,GST(40μmol／L)可降低钙波的传播速度和持续时间,最终阻断钙波。以上结果提示,GST降低心室肌细胞内游离钙浓度,此作用与其抑制电压依赖性Ca^2 通道、减弱酪氨酸激酶抑制和豚鼠心室肌细胞肌浆网内钙释放有关。     

血管紧张素Ⅱ对缺血心肌细胞钾离子通道的作用

实验用胶原酶酶解法急性分离豚鼠心室肌细胞,利用全细胞膜片钳的方法记录心室肌细胞的延迟整流钾电流(Ik）、内向整流钾电流（Ik1）和ATP敏感钾电流（IKATP）。采用低氧、无糖、高乳酸和酸中毒综合方式模拟缺血灌流,造成细胞的模拟缺血,并在缺血的基础上继续用含100nmol/L AngⅡ灌流细胞,观察Ang Ⅱ对模拟缺血心室肌细胞钾离子通道的影响。实验结果显示：（1）模拟缺血时,Ik明显减小;Ang Ⅱ能进一步抑制Ik。（2）模拟缺血条件下,Ik1受到抑制,并且以内向电流的抑制为主;Ang Ⅱ可加强对Ik1内向电流的抑制,而对部分外向电流则有增加的作用。（3）模拟缺血使IKATP外向电流略有增加;Ang Ⅱ则明显加强IKATP外向电流,此效应能被优降糖所阻断。