Inactivation of calcium-activated chloride conductance in Xenopus oocytes: roles of calcium and protein kinase C

Inactivation of Ca²⁺ -induced Cl^– currents was studied in Xenopus oocytes using the two-electrode voltage-clamp technique. In oocytes permeabilized to Ca²⁺ by treatment with the ionophore A23187, Ca²⁺ influx caused by the addition of 2.5–5 mM Ca²⁺ to the extracellular solution elicited Cl^– currents consisting of two components: a fast, transient one (I _fast) and a slow one (I _slow). In response to a subsequent application of the same dose of Ca²⁺, I _fast and I _slow were reduced (inactivation phenomenon). The inactivation did not depend on the direction of current flow, but did depend on the duration of the first exposure to Ca²⁺. The extent of inactivation of I _fast was more significant than that to I _slow. Both I _fast and I _slow fully recovered from inactivation in less than 30 min. Intracellular injections of 100–400 pmol CaCl₂ evoked large inward currents but did not reduce the amplitude of currents evoked by Ca²⁺ influx. The activator of protein kinase C, -phorbol dibutyrate, caused full inhibition of I _fast without any change in I _slow. H-7 (1,5-isoquinolinesulfonyl-1,2 methylpiperazine), an inhibitor of protein kinases, strongly reduced the extent of inactivation. Our results suggest that elevation of intracellular Ca²⁺ by Ca²⁺ influx through the plasma membrane causes inactivation of the Ca²⁺ -dependent Cl^– conductance via activation of a Ca²⁺ -dependent protein kinase, possibly protein kinase C, whereas Ca²⁺ arriving at the membrane from inside the cell does not initiate the processes leading to inactivation.     

兔左室肥厚心肌跨室壁复极不均一性及其膜离子流变化的实验研究

目的:探讨兔左心室肥厚心肌心外膜下、中层、心内膜下3层心肌细胞动作电位及膜离子流变化的不均一性。方法: 心肌肥厚组以腹主动脉缩窄术复制兔压力超负荷心肌肥厚模型,并设正常对照组以作比较。胶原酶两步消化法分离获取兔单个心室肌细胞,其中用植皮刀分离左室游离壁内膜下、中层、外膜下3层心肌。以全细胞膜片钳记录单细胞跨膜动作电位和离子电流。结果: 肥厚组3层心肌细胞动作电位时程(APD₉₀)较对照组3层心肌细胞APD₉₀均分别有明显延长,而以中层心肌细胞APD₉₀延长最为明显(延长比例:中层26.0%±2.7%,外膜14.0%±1.6%,内膜10.0%±1.1%),使肥厚心肌跨室壁复极不均一性明显大于对照组。肥厚组各层心肌细胞瞬时外向钾电流(I_to)和缓慢激活的延迟整流钾电流(I_Ks)密度均低于对照组,且均以中层细胞下降的幅度最大。肥厚组各层心肌细胞L型钙电流(I_Ca,L)与快速激活的延迟整流钾电流(I_Kr) 密度与对照组均无明显差异。肥厚组各层心肌细胞内向整流钾电流(I_K1) 均明显低于对照组,但各层变低的幅度无明显差异。 结论:兔肥厚心肌跨室壁复极不均一性明显增大,I_to及I_Ks的跨室壁不均一性下降可能是其主要原因。     

The existence of a highly tetrodotoxin sensitive Na channel in freshly dispersed smooth muscle cells of the rabbit main pulmonary artery

To characterize the inward current recorded from single smooth muscle cells of the rabbit main pulmonary artery, a voltage clamp procedure using patch pipettes filled with high Cs solution to inhibit K currents was employed. Under superfusion with normal physiological salt solution, application of a command potential to –10 mV from the holding potential of –80 mV elicited an inward current comprising fast and slow components. In Ca-free solution containing 2.5 mM Mn and 134 mM Na, the major part of the slow inwart current (I _slow) ceased, but a transient fast inward current (I _fast) remained. A reduction in the Na concentration in the bath solution inhibited the amplitude ofI _fast. Both nicardipine (30 nM) and diltiazem (1–10 M) inhibitedI _slow. but had no effect onI _fast. Application of tetrodotoxin (>1 nM) in Ca free solution inhibited the amplitude ofI _fast in a dose-dependent manner with a dissociation constant of 8.7 nM. Chloramine-T (0.3 mM) increased the peak amplitude and reduced the rate of decay ofI _fast and completely inhibitedI _slow. These results suggest that the inward curent generated in the smooth muscle cells of the rabbit main pulmonary artery is associated with activation of a voltage-dependent Ca channel and a tetrodotoxin-sensitive Na channel.     

Cellular basis of sex disparities in human cardiac electrophysiology

Aim: Sex disparities in electrocardiogram variables and dysrhythmia susceptibility exist, notably in long QT syndrome (LQTS) and Brugada syndrome, but the underlying mechanisms in man are unknown. We studied the cellular basis of sex distinctions in human cardiac electrophysiology and dysrhythmia susceptibility using mathematical models of human ventricular myocytes. Methods: We implemented sex differences in the Priebe–Beuckelmann and ten Tusscher–Noble–Noble–Panfilov human ventricular cell models by modifying densities of the L‐type Ca²⁺ current (I_Ca,L), transient outward K⁺ current (I_to), and rapid delayed rectifier K⁺ current (I_Kr), according to experimental data from male and female hearts of various species. Sex disparities in transmural repolarization were studied in transmural strands of cells with ion current densities based on canine experimental data. Results: Female cells have longer action potential duration (APD), steeper APD‐heart rate relationship, larger transmural APD heterogeneity, and a greater susceptibility to pro‐dysrhythmogenic early afterdepolarizations (EADs) than male cells. Conversely, male cells have more prominent phase‐1 repolarization and are more susceptible to all‐or‐none repolarization. Conclusion: Sex differences in I_Ca,L, I_to and I_Kr densities may explain sex disparities in human cardiac electrophysiology. Female cells exhibit a limited ‘repolarization reserve’ as demonstrated by their larger susceptibility to EADs, which, combined with their larger transmural electrical heterogeneity, renders them more vulnerable to tachydysrhythmias in LQTS. Conversely, male cells have a limited ‘depolarization reserve’, as shown by their larger susceptibility to all‐or‐none repolarization, which facilitates tachydysrhythmias in Brugada syndrome. These general principles may also apply to dysrhythmia susceptibility in common disease.     

Transfection by eukaryotic expression vector pcDNA3-HERG inhibits the cultured neonatal rabbit ventricular myocyte hypertrophy induced by phenylephrine

ObjectiveProlonged action potential and decreased outward K⁺ currents are consistent findings in hypertrophic myocardium. The relation between action potential prolongation and myocyte hypertrophy has remained unclear. The present study investigated the temporal relation between action potential prolongation and myocyte hypertrophy, and the effect of enhancing repolarization on myocyte hypertrophy induced by phenylephrine.MethodsNeonatal rabbit ventricular myocytes were cultured and treated with 10 μmol/l phenylephrine. At 6 and 48 h after phenylephrine stimulation, myocyte hypertrophic parameters (including myocyte volume, total protein content, and membrane capacitance), action potential duration (APD), and calcineurin activity were measured; meanwhile, the effect of human-ether-a-go-go–related gene (HERG; encoding the αsubunit of rapidly activating delayed rectifier potassium channel) transfection on the above parameters at 48 h of phenylephrine stimulation was also measured.ResultsAt 6 h after phenylephrine treatment, APD at 90% repolarization of neonatal rabbit ventricular myocytes was prolonged by 14.3% (P<.05), but myocyte hypertrophy was not found. At 48 h after phenylephrine stimulation, APD at 90% repolarization of neonatal rabbit ventricular myocytes was furthermore prolonged by 18.8% (P<.05); at the same time, myocyte volume, total protein content, membrane capacitance, and calcineurin activity were increased by 40.0%, 41.8%, 36.4%, and 124.1%, respectively (P<.01). Neonatal rabbit ventricular myocytes transfected by pcDNA3-HERG overexpressed I_HERG,tail current, which was about fourfold higher than I_Kr (rapidly activating delayed rectifier K⁺ current) of neonatal rabbit ventricular myocytes without transfection of HERG. HERG overexpression could accelerate repolarization and shorten APD at 90% repolarization prolonged by phenylephrine and partially inhibit myocyte hypertrophy and calcineurin activation.ConclusionsDuring the myocyte hypertrophy induced by phenylephrine, prolongation of APD at 90% repolarization is not secondary to but precedes myocyte hypertrophy. HERG overexpression could enhance the repolarization and inhibit the calcineurin activation and myocyte hypertrophy induced by phenylephrine.     

Electrophysiological determinants of hypokalaemia‐induced arrhythmogenicity in the guinea‐pig heart

Aim: Hypokalaemia is an independent risk factor contributing to arrhythmic death in cardiac patients. In the present study, we explored the mechanisms of hypokalaemia‐induced tachyarrhythmias by measuring ventricular refractoriness, spatial repolarization gradients, and ventricular conduction time in isolated, perfused guinea‐pig heart preparations. Methods: Epicardial and endocardial monophasic action potentials from distinct left ventricular (LV) and right ventricular (RV) recording sites were monitored simultaneously with volume‐conducted electrocardiogram (ECG) during steady‐state pacing and following a premature extrastimulus application at progressively reducing coupling stimulation intervals in normokalaemic and hypokalaemic conditions. Results: Hypokalaemic perfusion (2.5 mm K⁺ for 30 min) markedly increased the inducibility of tachyarrhythmias by programmed ventricular stimulation and rapid pacing, prolonged ventricular repolarization and shortened LV epicardial and endocardial effective refractory periods, thereby increasing the critical interval for LV re‐excitation. Hypokalaemia increased the RV‐to‐LV transepicardial repolarization gradients but had no effect on transmural dispersion of APD₉₀ and refractoriness across the LV wall. As determined by local activation time recordings, the LV‐to‐RV transepicardial conduction and the LV transmural (epicardial‐to‐endocardial) conduction were slowed in hypokalaemic heart preparations. This change was attributed to depressed diastolic excitability as evidenced by increased ventricular pacing thresholds. Conclusion: These findings suggest that hypokalaemia‐induced arrhythmogenicity is attributed to shortened LV refractoriness, increased critical intervals for LV re‐excitation, amplified RV‐to‐LV transepicardial repolarization gradients and slowed ventricular conduction in the guinea‐pig heart.     

Role of Ca2+‐activated Cl− current during proarrhythmic early afterdepolarizations in sheep and human ventricular myocytes

Objectives: The proarrhythmic early afterdepolarizations (EADs) during phase‐2 of the cardiac action potential (phase‐2 EADs) are associated with secondary Ca²⁺‐release of the sarcoplasmic reticulum. This makes it probable that the Ca²⁺‐activated Cl^? current [I_Cl(Ca)] is present during phase‐2 EADs. Activation of I_Cl(Ca) during phase‐2 of the action potential will result in an outwardly directed, repolarizing current and may thus be expected to prevent excessive depolarization of phase‐2 EADs. The present study was designed to test this hypothesis. Methods and Results: The contribution of I_Cl(Ca) during phase‐2 EADs was studied in enzymatically isolated sheep and human ventricular myocytes using the patch‐clamp methodology. EADs were induced by a combination of a low stimulus frequency (0.5 Hz) and exposure to 1 μm noradrenaline. In sheep myocytes, the I_Cl(Ca) blocker 4,4′‐diisothiocyanostilbene‐2,2′‐disulfonic acid (DIDS, 0.5 mm ) abolished phase‐1 repolarization of the action potential in all myocytes tested. This indicates that I_Cl(Ca) is present in all sheep myocytes. However, DIDS had no effect on phase‐2 EAD characteristics. In human myocytes, DIDS neither affected phase‐1 repolarization nor phase‐2 EAD characteristics. Conclusion: In sheep ventricular myocytes, but not in human ventricular myocytes, I_Cl(Ca) contributes to phase‐1 repolarization of the action potential. In both sheep and human myocytes, I_Cl(Ca) plays a limited role during phase‐2 EADs.     

Delayed onset and slow time course of the non-M-type muscarinic current in bullfrog sympathetic neurons

The onset and time course of the muscarinic currents induced by brief applications of acetylcholine (ACh) were examined in voltage-clamped neurons of bullfrog sympathetic ganglia bathed in a solution containing d-tubocurarine. At a potential of –40 mV, the ACh-induced current (I _ACh) appeared within 1.2 s and rapidly increased to its peak with a half-activation time of 2.2 s. This initial current was termed the fastI _ACh and was blocked by 4 mM Ba²⁺. At a potential more negative than –60 mV, the fastI _ACh disappeared and the remainingI _ACh activated with a delay of 3.9 s and slowly increased to its peak with a half-activation time of 8.2 s. This delayed current was termed the slowI _ACh and is thought to be associated with inhibition of a K⁺ current, orI _M, as well as activation of an inward current through non-M-type muscarinic cation channels. The slowI _ACh was not inhibited by Ba²⁺, but its amplitude was reduced with depolarization (the extra-polated reversal potential was +3 mV). In Na⁺-free solution, the amplitude of the slowI _ACh reduced, but its polarity did not reverse in the voltage region examined (-30 to –100 mV). The slow excitatory postsynaptic current was also recorded, and was shown to have a similar delay in onset and slow time course. The results demonstrate that ACh activates the non-M-type muscarinic current three times more slowly than it inhibitsI _M.     

Ca2+‐activated Cl− current reduces transmural electrical heterogeneity within the rabbit left ventricle

Objective: Various cationic membrane channels contribute to the heterogeneity of action potential configuration between the transmural layers of the left ventricle. The role of anionic membrane channels is less intensively studied. We investigated the role of the Ca²⁺‐activated Cl^? current, I_Cl(Ca), in transmural electrical heterogeneity. Methods and Results: We determined the density of I_Cl(Ca) and its physiological role in subepicardial and subendocardial ventricular myocytes of rabbit using the patch‐clamp technique. I_Cl(Ca) was measured as the 4,4′diisothiocyanatostilbene‐2,2′‐disulphonic acid (DIDS) sensitive current. The current–voltage relationships and the densities of I_Cl(Ca) were similar in subepicardial and subendocardial myocytes. However, the functional role of I_Cl(Ca) exhibited striking differences. In subendocardial myocytes, blockade of I_Cl(Ca) by DIDS increased action potential duration (APD) significantly at all measured stimulus frequencies (3.33–0.2 Hz). In subepicardial myocytes, I_Cl(Ca) blockade increased APD only at 3.33 Hz, but not at the lower stimulus frequencies. At 1 Hz, I_Cl(Ca) blockade in subepicardial myocytes only caused an APD increase when the transient outward K⁺ current, I_to1, was blocked. Conclusions: The densities and gating properties of I_Cl(Ca) are similar in subepicardial and subendocardial myocytes. I_Cl(Ca) contributes to APD shortening in subendocardial, but not in subepicardial myocytes except at 3.33 Hz. These differences in functional expression of I_Cl(Ca) reduce the electrical heterogeneity in rabbit left ventricle.     

Single-channel properties of I K,slow1 and I K,slow2 in mouse ventricular myocytes

I _K,slow1 and I _K,slow2 are two important voltage-gated potassium (K⁺) currents expressed in mouse ventricular myocytes. However, their properties at the single-channel level have not been characterized. In this paper, we report two new single K⁺ channels, mK1 and mK2, in myocytes isolated from mouse ventricles and their possible correlation with the macroscopic currents I _K,slow1 and I _K,slow2. The conductance of mK1 and mK2 was 24 and 17 pS, respectively. Ensemble-averaged current demonstrated an inactivation time constant of 400 to 500 ms for mK1 compared with 1,300 to 2,000 ms for mK2. The mK1 channel was more sensitive than the MK2 channel to the K channel blocker 4-AP. In myocytes isolated from Kv1DN mice with functional knock out of the Kv1.5 channel, mK1 was not detectable but mK2 was present. Our data suggest that the newly characterized K⁺ channels, mK1 and mK2, likely correspond to the macroscopic currents of I _K,slow1 and I _K,slow2, respectively.     

Expression environment determines K+ current properties: Kv1 and Kv4 α-subunit-induced K+ currents in mammalian cell lines and cardiac myocytes

 Voltage-gated K⁺ channel (Kv) pore-forming (α) subunits of the Kv1 and Kv4 subfamilies have been cloned from heart cDNA libraries, and are thought to play roles in the generation of the transient outward K⁺ current, I _to. Heterologous expression of these subunits in Xenopus oocytes, however, reveals K⁺ currents that are quite distinct from I _to. In the experiments here, the detailed time- and voltage-dependent properties of the currents expressed in mammalian cell lines and in cardiac myocytes by Kv1.4 and Kv4.2 were examined and compared to previous findings in studies of oocytes, as well as to I _to characterized in various myocardial cells. As in oocytes, expression of Kv1.4 in HEK-293, Ltk ^– or neonatal rat ventricular cells reveals rapidly activating K⁺ currents. In contrast to the currents in oocytes, however, there are two components of inactivation of the Kv1.4-induced currents in mammalian cells, and both components are significantly slower in myocytes than in either HEK-293 or Ltk ^– cells. In addition, in all three cell types, recovery of Kv1.4 from steady-state inactivation is very slow, proceeding with mean time constants in the range of 6–8 s. The properties of Kv4.2-induced currents also vary with cell type and, importantly, the rates of activation, inactivation and recovery from inactivation are significantly faster in mammalian cells than in Xenopus oocytes. In HEK-293, Chinese hamster ovary (CHO) and neonatal rat ventricular cells, for example, the currents recover from steady-state inactivation with mean (±SD) time constants of 153±32 (n=12), 245±112 (n=10) and 86±38 (n=11) ms, respectively; therefore, recovery proceeds 5–10 times faster than observed for Kv4.2 in oocytes. These results emphasize the importance of the cellular expression environment in efforts to correlate endogenous K⁺ currents with heterologously expressed K⁺ channel subunits. In addition, the finding that Kv α subunits produce distinct K⁺ currents in different cells suggests that cell-type-specific associations with endogenous Kv α or accessory β subunits and/or post-translational processing play roles in determining the properties of functional K⁺ channels. Received: 14 August 1998 / Accepted: 19 October 1998     

Effects of Mixed Herbal Extracts from Parched Puerariae Radix, Gingered Magnoliae Cortex, Glycyrrhizae Radix and Euphorbiae Radix (KIOM-79) on Cardiac Ion Channels and Action Potentials

KIOM-79, a mixture of ethanol extracts from four herbs (parched Puerariae radix, gingered Magnoliae cortex, Glycyrrhizae radix and Euphorbiae radix), has been developed for the potential therapeutic application to diabetic symptoms. Because screening of unexpected cardiac arrhythmia is compulsory for the new drug development, we investigated the effects of KIOM-79 on the action potential (AP) and various ion channel currents in cardiac myocytes. KIOM-79 decreased the upstroke velocity (V_max) and plateau potential while slightly increased the duration of action potential (APD). Consistent with the decreased V_max and plateau potential, the peak amplitude of Na⁺ current (I_Na) and Ca²⁺ current (I_Ca,L) were decreased by KIOM-79. KIOM-79 showed dual effects on hERG K⁺ current; increase of depolarization phase current (I_depol) and decreased tail current at repolarization phase (I_tail). The increase of APD was suspected due to the decreased I_tail. In computer simulation, the change of cardiac action potential could be well simulated based on the effects of KIOM-79 on various membrane currents. As a whole, the influence of KIOM-79 on cardiac ion channels are minor at concentrations effective for the diabetic models (0.1-10 µg/mL). The results suggest safety in terms of the risk of cardiac arrhythmia. Also, our study demonstrates the usefulness of the cardiac computer simulation in screening drug-induced long-QT syndrome.     

Accumulation of long-lasting inactivation in rat brain K+-channels

We studied the phenomenon of cumulative inactivation in the voltage-dependent K⁺ channels of the Shaker-related subfamily Kv1 cloned from rat brain and expressed in Xenopus oocytes. In Kv1.4, repetitive stimulations at intervals shorter than 20 s produce cumulative inactivation even for brief stimuli that elicit K⁺ currents which do not show any significant decline during the depolarising pulse. These effects are absent or greatly reduced in the clones Kv1.1, Kv1.3, Kv1.5 and Kv1.6, and in the deletion mutant Kv1.4--110, characterised by lack of fast (N-type) inactivation. We find that the inactivation caused by a single pulse increases after the pulse while the channels deactivate, and subsides with two time constants, indicating the existence of (at least) two inactivated states: I_S, with a slow recovery kinetics and I_F, with faster kinetics. In the simplest kinetic scheme accounting for our observations, I_F is coupled sequentially to the open state O, while I_S can be reached at a fast rate both from I_F and from a pre-open, activated state, A, that is in fast equilibrium with O. The accumulation of long-lasting inactivation during the repolarisation is favoured by the prolongation of the lifetime of activated states due to the presence of I_F. This explains the smaller accumulation effect observed in channels lacking fast inactivation. The physiological implications of these findings suggest how different channels of the Kv1 subfamily can affect differently the firing behaviour of neurones.     

5-羟基癸酸盐对慢性低氧大鼠肺动脉Kv1.5通道表达的影响

目的: 以大鼠为研究对象,研究线粒体ATP敏感性钾通道(mitoK_ATP)的抑制剂5-羟基癸酸盐(5-HD)对慢性低氧肺动脉高压大鼠的影响及其潜在机制。方法: 24只SD雄性大鼠随机分成对照组、低氧组、低氧+5-羟基癸酸盐干预组,每组8只。将低氧组和5-HD干预组大鼠放入常压低氧舱内 以建立低氧肺动脉高压模型。 4周后测定平均肺动脉压(mPAP)及右心室与左心室及室间隔的重量比 ,并采用RT-PCR及Western blotting技术,分析各组肺动脉Kv1.5 mRNA及蛋白表达。结果: (1) 慢性低氧组大鼠的mPAP及RV/(LV+S)显著高于正常对照组(P<0.05),5-HD干预组mPAP及RV/(LV+S)显著低于低氧组,均P<0.05。(2) 低氧组Kv1.5通道mRNA及蛋白表达显著低于正常组,5-HD组Kv1.5通道表达显著高于低氧组, 均P<0.05。结论: mitoK_ATP通道的抑制剂5-HD通过降低mPAP及RV/(LV+S),在慢性低氧肺动脉高压中起保护作用。mitoK_ATP通道的抑制及Kv1.5通道表达的上调可能与该保护作用有关。     

Intracellular calcium and electrical restitution in mammalian cardiac cells

The role of calcium current and changes in intracellular calcium concentration ([Ca²⁺]_i) in regulation of action potential duration (APD) during electrical restitution process was studied in mammalian ventricular preparations. Properly timed action potentials were recorded from multicellular preparations and isolated cardiomyocytes using conventional microelectrodes and EGTA-containing patch pipettes. APD increased monotonically in canine and guinea pig ventricular preparations with increasing diastolic interval (DI), while in rabbit papillary muscles the restitution process was biphasic: APD first lengthened, then shortened as the DI increased. When the restitution process was studied in single cardiomyocytes using EGTA-containing patch pipettes, the restitution pattern was similar in the three species studied. Similarly, no difference was observed in the recovery time constant of calcium current (I_Ca-L) measured under these conditions in voltage clamped myocytes. Loading the myocytes with the [Ca²⁺]_i-chelator BAPTA-AM had adverse effects in rabbit and canine cells. In rabbit myocytes steady-state APD lengthened and the late shortening component of restitution was abolished in BAPTA-loaded cells. In canine myocytes BAPTA-load shortened steady-state APD markedly, and during restitution, APD decreased with increasing DI. The late shortening component of restitution, observed in untreated rabbit preparations, was greatly reduced after nifedipine treatment, but remained preserved in the presence of 4-aminopyridine or nicorandil. Beat to beat changes in APD, peak I_Ca-L and [Ca²⁺]_i, measured using the fluorescent dye, Fura-2, were monitored in rabbit ventricular myocytes after a 1-min period of rest. In these cells, the shortening of APD was accompanied by a gradual reduction of the peak I_Ca-L and elevation of diastolic [Ca²⁺]_i during the initial eight post-rest action potentials. It is concluded that elevation of [Ca²⁺]_i shortens, while reduction of [Ca²⁺]_i lengthens APD in rabbit, but not in canine ventricular myocytes. These differences may probably be related to different distributions of [Ca²⁺]_i-dependent ion currents and/or to differences in calcium handling between the two species.     

Remodelling of human atrial K<Superscript>+</Superscript> currents but not ion channel expression by chronic β-blockade

Chronic β-adrenoceptor antagonist (β-blocker) treatment in patients is associated with a potentially anti-arrhythmic prolongation of the atrial action potential duration (APD), which may involve remodelling of repolarising K⁺ currents. The aim of this study was to investigate the effects of chronic β-blockade on transient outward, sustained and inward rectifier K⁺ currents (I_TO, I_KSUS and I_K1) in human atrial myocytes and on the expression of underlying ion channel subunits. Ion currents were recorded from human right atrial isolated myocytes using the whole-cell-patch clamp technique. Tissue mRNA and protein levels were measured using real time RT-PCR and Western blotting. Chronic β-blockade was associated with a 41% reduction in I_TO density: 9.3 ± 0.8 (30 myocytes, 15 patients) vs 15.7 ± 1.1 pA/pF (32, 14), p < 0.05; without affecting its voltage-, time- or rate dependence. I_K1 was reduced by 34% at −120 mV (p < 0.05). Neither I_KSUS, nor its increase by acute β-stimulation with isoprenaline, was affected by chronic β-blockade. Mathematical modelling suggested that the combination of I_TO- and I_K1-decrease could result in a 28% increase in APD₉₀. Chronic β-blockade did not alter mRNA or protein expression of the I_TO pore-forming subunit, Kv4.3, or mRNA expression of the accessory subunits KChIP2, KChAP, Kvβ1, Kvβ2 or frequenin. There was no reduction in mRNA expression of Kir2.1 or TWIK to account for the reduction in I_K1. A reduction in atrial I_TO and I_K1 associated with chronic β-blocker treatment in patients may contribute to the associated action potential prolongation, and this cannot be explained by a reduction in expression of associated ion channel subunits.     

Effects of diltiazem upon a rapidly exchanging calcium compartment related to repriming in frog skeletal muscle

Summary Following spontaneous relaxation, fast skeletal muscle must first repolarize and then undergo a first-order repriming reaction before depolarization will result in maximal tension production. ⁴⁵Ca exposure during repriming defined two Ca compartments during subsequent efflux, named Ca_fast and Ca_slow. Ca_slow had an average time constant of 112±17 min. On the basis of slow turnover and content determined by a variety of methods, I suggest Ca_slow represents Ca within the sarcoplasmic reticulum. Ca_fast contained 12 pmol Ca per fibre and resting exchange had a time constant of 5.1±0.4 min. A total of 12 pmol ⁴⁵Ca within Ca_fast was released during a maximal contracture. Most of the Ca released from Ca_fast rapidly entered the extracellular space; however, 0.39±0.15 pmol Ca per fibre transferred from Ca_fast into Ca_slow when the muscle bundle contracted. When 1–10 M diltiazem reduced contracture time-tension, release of Ca_fast was reduced proportionally. When 10 M diltiazem paralyzed excitation-contraction coupling, Ca_fast was not released. Refilling of Ca_fast was proportional to the extent of repriming during ⁴⁵Ca exposure. Although release and refilling of Ca_fast is related to contraction, its role in excitation-contraction coupling remains to be elucidated.     

家兔陈旧性心肌梗死心室肌细胞钾离子电流的变化

目的:探讨陈旧性心肌梗死(HMI)心律失常的发生机制,观察HMI非梗死区心肌细胞动作电位时程(APD)、瞬时外向钾电流(I_to)、延迟整流钾电流(I_K)和内向整流钾电流(I_K1)的变化。方法: 12只家兔随机分为2组,陈旧性心肌梗死组(HMI)开胸结扎冠状动脉左回旋支,假手术组开胸但不结扎冠状动脉。3个月后应用全细胞膜片钳技术记录非梗死区心肌细胞的APD、I_to、 I_K和I_K1。 结果: (1)HMI组心肌细胞的膜电容明显高于假手术组;(2)HMI组心肌细胞的APD显著延长,并有早期后除极(EAD)出现;(3)HMI组心肌细胞I_to 、I_K,tail和I_K1的电流密度分别为(4.03±0.33)pA/pF、(1.14±0.11)pA/pF和(17.6±2.3)pA/pF,显著低于假手术组的(6.72±0.42)pA/pF、(1.54±0.13)pA/pF和(25.6±2.6)pA/pF(P<0.01)。结论: HMI非梗死区心室肌细胞I_to 、I_K,tail、和I_K1的电流密度的降低是其APD延长和EAD出现的离子流基础,而APD延长和EAD的出现,可能在HMI恶性心律失常的发生中起着重要作用。     

Adaptative modifications of right coronary myocytes voltage-gated K+ currents in rat with hypoxic pulmonary hypertension

Chronic hypoxia (CH)-induced pulmonary hypertension (PHT) is well known to alter K+ channels in pulmonary myocytes. PHT induces right ventricle hypertrophy that increases oxygen demand; however, coronary blood flow and K+ channel adaptations of coronary myocytes during PHT remain unknown. We determined whether CH and PHT altered K+ currents and coronary reactivity and what impact they might have on right myocardial perfusion. Right ventricle perfusion, as attested by microspheres, was redistributed toward hypertrophied right ventricle [RV/LV (%)=0.59+/-0.07% in CH rats vs. 0.29+/-0.03 in control rats, P<0.05]. Whole-cell patch clamping showed a reduction of global outward current in hypoxic right coronary artery myocytes (H-RCA), whereas hypoxic left coronary artery myocytes exhibited an increase. K+ channel blockers revealed that a 4-aminopyridine (4AP)-sensitive current (Kv current) was decreased in H-RCA (14.3+/-1.1 vs. 23.4+/-2.5 pA/pF at 60 mV in control RCA, P<0.05) and increased in hypoxic left coronary artery myocytes (H-LCA; 26.4+/-3.8 vs. 11.8+/-1.6 pA/pF at 60 mV in control LCA, P<0.05). Constriction to 4AP was decreased in H-RCA when compared to normoxic control and increased in H-LCA when compared to LCA. Finally, we observed that the expression of Kv1.2 and Kv1.5 were lower in H-RCA than that in H-LCA. This study reveals that CH differentially regulates Kv channels in coronary myocytes. Hypoxia decreases Kv currents and therefore reduces vasoreactivity that contributes to an adaptative response leading to right hypertrophied ventricle perfusion enhancement at rest.     

血小板活化因子对豚鼠心室肌细胞钾电流及动作电位的影响

 目的：研究血小板活化因子(PAF)对豚鼠心室肌细胞钾电流及动作电位的影响。 方法:应用全细胞膜片钳技术，记录豚鼠心室肌细胞动作电位及钾电流(I_K 与I_K1)。 结果:当电极内液ATP浓度为5 mmol/L，1 μmol/L PAF使APD₉₀由对照的(225.8±23.3)ms延长至(352.8±29.8)ms(n=5, P<0.05)；使I_K尾电流在指令电压 +30 mV 时由对照的(173.5±16.7)pA降为(152.1±11.5)pA(P<0.05, n=4)；使I_K1在指令电压 -120 mV 时从(-6.1±1.3)nA降为(-5.6±1.1)nA(P<0.05, n=5)；当电极内液ATP 为0 mmol/L，APD₉₀明显缩短，1 μmol/L PAF使APD₉₀由对照的(153.0±24.6)ms缩短为(88.2±19.4)ms (n=5, P<0.01)，而用1 μmol/L格列本脲 ( I_KATP特异性阻滞剂)预处理后，恢复了PAF可显著延长动作电位时程的作用。 结论: PAF使缺血区K_ATP开放，动作电位时程缩短，却可抑制正常区I_K 与I_K1，使动作电位延长，从而放大了缺血区与正常区的不均一性，这可能与缺血时心律失常的发生有关。