Modulation of membrane potential by an acetylcholine-activated potassium current in trout atrial myocytes

Application of the current-clamp technique in rainbow trout atrial myocytes has yielded resting membrane potentials that are incompatible with normal atrial function. To investigate this paradox, we recorded the whole membrane current (I(m)) and compared membrane potentials recorded in isolated cardiac myocytes and multicellular preparations. Atrial tissue and ventricular myocytes had stable resting potentials of -87 +/- 2 mV and -83.9 +/- 0.4 mV, respectively. In contrast, 50 out of 59 atrial myocytes had unstable depolarized membrane potentials that were sensitive to the holding current. We hypothesized that this is at least partly due to a small slope conductance of I(m) around the resting membrane potential in atrial myocytes. In accordance with this hypothesis, the slope conductance of I(m) was about sevenfold smaller in atrial than in ventricular myocytes. Interestingly, ACh increased I(m) at -120 mV from 4.3 pA/pF to 27 pA/pF with an EC(50) of 45 nM in atrial myocytes. Moreover, 3 nM ACh increased the slope conductance of I(m) fourfold, shifted its reversal potential from -78 +/- 3 to -84 +/- 3 mV, and stabilized the resting membrane potential at -92 +/- 4 mV. ACh also shortened the action potential in both atrial myocytes and tissue, and this effect was antagonized by atropine. When applied alone, atropine prolonged the action potential in atrial tissue but had no effect on membrane potential, action potential, or I(m) in isolated atrial myocytes. This suggests that ACh-mediated activation of an inwardly rectifying K(+) current can modulate the membrane potential in the trout atrial myocytes and stabilize the resting membrane potential.     

腺苷抗豚鼠室性心律失常的电生理研究

实验用全细胞膜片钳技术在单个豚鼠心室肌细胞上研究了腺苷 (Ado)对正常及异丙肾上腺素 (Iso)致豚鼠心室肌细胞动作电位、迟后除极 (DAD)、L 型钙电流 (ICa.L)和短暂内向电流 (Iti)的作用。结果表明 :(1)Ado在2 0～ 10 0 μmol/L时对豚鼠心室肌细胞动作电位和ICa .L无明显直接作用 ,但却可明显降低Iso所致的动作电位时程(APD)延长和ICa .L峰值增大 ,Iso (10nmol/L)使细胞APD50 从 3 40± 2 1ms延长到 486± 2 8ms (P <0 0 1) ,APD90从 3 61± 17ms延长至 5 0 1± 2 9ms (P <0 0 1) ;ICa .L峰值从 - 6 5 3± 1 4pA/pF增大到 - 18 2 8± 2 4pA/pF (P <0 0 1) ,电流电压曲线明显左移和下移 ;Ado (5 0 μmol/L)使APD50 和APD90 降至 40 3± 19ms和 419± 2 6ms ,但并不影响动作电位其它参数 ,使ICa.L峰值降低至 - 10 2± 1 5pA/pF (P <0 0 1)。 (2 )Iso (3 0nmol/L)可诱发心室肌细胞产生DADs,其发生率为 10 0 % ;Ado (5 0 μmol/L)可完全抑制Iso引发DADs;细胞经 - 40～ +2 0mV、时程 2s的除极电压 ,Iso (3 0nmol/L)诱导出Iti,其发生率为 10 0 % ;Ado (5 0 μmol/L)可明显抑制Iso致Iti的发生 ,其发生率降为 14 3 %。研究结果提示 ,Ado对豚鼠心室肌细胞动作电位和ICa.L无明显直接作用 ,但却可显著降低Is     

L-type Ca2+ currents in ventricular myocytes from neonatal and adult rats

Postnatal changes in the slow Ca2+ current (I(Ca)(L)) were investigated in freshly isolated ventricular myocytes from neonatal (1-7 days old) and adult (2-4 months old) rats, using whole-cell voltage clamp and single-channel recordings. The membrane capacitance (mean+/-SEM) averaged 23.2+/-0.5 pF in neonates (n = 163) and 140+/-4.1 pF in adults (n = 143). I(Ca)(L) was measured as the peak inward current at a test potential of +10 mV (or +20 mV) by applying a 300-ms pulse from a holding potential of -40 mV; 1.8 mM Ca2+ was used as charge carrier. The basal ICa(L) density was 6.7+/-0.2 pA/pF in neonatal and 7.8+/-0.2 pA/pF in adult cells (p < 0.05). The time course of inactivation of the fast component (at +10 ms) was significantly longer in the neonatal (10.7+/-1.4 ms) than in the adult (6.6+/-0.4 ms) cells (p < 0.05). Ryanodine (10+/-M) significantly increased this value to 18.0+/-1.9 in neonate (n = 8) and to 17.7+/-2.0 in adult (n = 9). For steady-state inactivation, the half-inactivation potential (Vh) was not changed in either group. For steady-state activation, Vh was 5.1 mV in the neonatal (n = 6) and -7.9 mV in the adult cells (n = 7). Single-channel recordings revealed that long openings (mode-2 behavior) were occasionally observed in the neonatal cells (11 events from 1080 traces/11 cells), but not in the adult cells (400 traces/4 cells). Slope conductance was 24 pS in both the neonatal and adult cells. Results in rat ventricular myocytes suggest the following: (i) the peak Ca2+ current density is already well developed in the neonatal period (being about 85% of the adult value); (ii) the fast component of inactivation is slower in neonates than in adults; and (iii) naturally occurring long openings are occasionally observed in the neonatal stage but not in the adult. Thus, the L-type Ca2+ channels of the neonate were slightly lower in density, were inactivated more slowly, and occasionally exhibited mode-2 behavior as compared with those of the adult.     

Transient outward potassium current in rabbit atrium is depressed after short-time rapid atrial pacing but recovers after a longer pacing period

In rabbit, after short-time rapid atrial pacing (RAP), atrial ion currents are reduced similarly as in human chronic atrial fibrillation (AF). Using the rabbit model, time-course of transient outward potassium current (I(to)) remodeling due to RAP was studied. RAP (600 bpm) was applied via an atrial lead for 0 (control), 24 and 120 h, n = 4 animals/group. Using patch clamp technique in whole-cell mode, current densities and biophysical properties were measured in isolated atrial myocytes. After 24 h of RAP, a reduction of peak I(to) (mean +/- SEM, test potential +50 mV, +37 degrees C) was observed (60.3 +/- 5.4 pA/pF (control, n = 20) vs. 28.0 +/- 2.5 pA/pF (24 h, n = 21)). Inactivation of I(to) was slower after 24 h, other biophysical properties were unaltered. However, I(to) recovered after 120 h: 51.7 +/- 4.5 pA/pF (n = 26, p = n.s. vs. control). Inactivation tended to also recover to initial values but was still different to control. Early I(to) remodeling due to RAP in rabbits seems to be more complex than previously thought: a time course of I(to) remodeling with swayings has to be considered when using the rabbit model of RAP in order to study early remodeling or rather its therapeutic manipulation.     

一氧化氮增加常氧和缺氧豚鼠心室肌细胞持续性钠电流

运用全细胞膜片钳记录缺氧条件下豚鼠心室肌持续性钠电流(INa.P)的变化及施加药物对其的影响,以探讨 INa.P 的本质及缺氧增大 INa.P 的机制。结果显示:(1)在常氧条件下,一氧化氮(NO)前体 L- 精氨酸(L-Arg)和供体硝普钠(SNP)浓度依赖性地增大INa.P; (2)INa.P 随缺氧时间延长而增大, 缺氧15 min 后施加 NO 合酶(NOS)抑制剂L- 硝基精氨酸甲酯(L-NAME), 不能使增大的INa.P 明显回复[(1.344 ±0.320) vs (1.301 ±0.317) pA/pF, P>0.05, n=5]; (3)缺氧时含L-NAME 的灌流液可使INa.P 明显减小,与单纯缺氧相比有显著差异[(0.914 ± 0.263), n=5 vs (1.344 ± 0.320) pA/pF, n=6, P<0.05], 但仍比常氧条件下增大[(0.914 ±0.263) vs (0.497 ±0.149) pA/pF, P<0.05, n=5]; (4)还原剂1,4-二硫代苏糖醇(DTT)不但可使L-Arg 及缺氧后施加SNP 增大的 INa.P 回复[(1.449 ± 0.522) vs (0.414 ± 0.067) pA/pF, P<0.01, n = 6 和(0.436 ± 0.141) vs (1.786 ± 0.636) pA/pF,P<0.01, n=5],而且使正常的 INa.P 减小[(0.396 ± 0.057) pA/pF vs (0.442 ± 0.056) pA/pF, P<0.01, n=6]。本实验结果表明缺氧可增大心室肌细胞的INa.P, 其作用机制可能是缺氧时心肌产生的NO 通过氧化细胞膜上钠通道蛋白所致,正常INa.P 的产生     

Effects of carvedilol on transient outward and ultra-rapid delayed rectifier potassium currents in human atrial myocytes

Carvedilol is a beta- and alpha(1)-adrenoceptor antagonist. It is widely used in the treatment of cardiovascular diseases including atrial arrhythmias. However, it is unclear whether carvedilol may affect the repolarization currents, transient outward K(+) current (I(to)) and ultra-rapid delayed rectifier K(+) current (I(Kur)) in the human atrium. The present study evaluated effects of carvedilol on I(to) and I(Kur) in isolated human atrial myocytes by whole-cell patch-clamp recording technique. We found that carvedilol reversibly inhibited I(to) and I(Kur) in a concentration-dependent manner. Carvedilol (0.3 microM) suppressed I(to) from 9.2+/-0.5 pA/pF to 4.8+/-0.5 pA/pF (P<0.01) and I(Kur) from 3.6+/-0.5 pA/pF to 1.9+/-0.3 pA/pF (P<0.01) at +50 mV. I(to) was inhibited in a voltage-dependent manner, being significantly attenuated at test potentials from +10 to +50 mV, whereas the inhibition of I(Kur) was independent. The concentration giving a 50% inhibition was 0.50 microM for I(to) and 0.39 microM for I(Kur). Voltage-dependence of activation, inactivation and time-dependent recovery from inactivation of I(to) were not altered by carvedilol. However, time to peak and time-dependent inactivation of I(to) were significantly accelerated, indicating an open channel blocking action. The findings indicate that carvedilol significantly inhibits the major repolarization K(+) currents I(to) and I(Kur) in human atrial myocytes.     

Pituitary adenylate cyclase-activating polypeptide activates K(ATP) current in rat atrial myocytes

Because the electrophysiological effects of pituitary adenylate cyclase-activating polypeptide (PACAP) on the heart are little known, we studied the regulation of the atrial ATP-sensitive K(+) (K(ATP)) current by PACAP on primary cultured neonatal rat atrial myocytes. PACAP-38 stimulates cAMP production with EC(50) = 0.28 nmol/l (r = 0.92, P < 0.02). PACAP-38 and PACAP-27 (10 nmol/l) have similar maximal effects, whereas 100 nmol/l vasoactive intestinal polypeptide (VIP) is 2.7 times less effective (P < 0.05). RT-PCR shows the presence of cloned PACAP receptors PAC(1) (> or =2 isoforms), VPAC(1), and VPAC(2). PACAP-38 dose dependently activates the whole cell atrial K(ATP) current with EC(50) = 1-3 nmol/l (n = 44). Maximal effects occur at 10 nmol/l (91 +/- 15 pA/pF, n = 18). Diazoxide further increases the PACAP-activated current by 78% (P < 0.05; n = 6). H(89) (500 nmol/l), a protein kinase A (PKA) inhibitor, reduces the PACAP-activated K(ATP) current to 17.8 +/- 9.6% (n = 5) of the maximal diazoxide-induced current and totally inhibits the cAMP-induced K(ATP) current. A protein kinase C (PKC) inhibitor peptide (50 micromol/l) in the pipette reduces the PACAP-38-induced K(ATP) current to 33 +/- 17 pA/pF (P < 0.05, n = 6) without significantly affecting the currents induced by cAMP or VIP. The results suggest that: 1) PAC(1), VPAC(1), and VPAC(2) are present in atrial myocytes; and 2) PACAP-38 activates the atrial K(ATP) channels through both PKA and PKC pathways.     

激活δ阿片受体可抑制大鼠心室肌细胞L-型钙电流和瞬时外向钾电流

本研究采用全细胞膜片钳技术观察了SNCl62(一种选择性δ阿片受体激动剂)对人鼠心室肌细胞L型钙电流(L-type Ca2 current,ICa-L)和瞬时外向钾电流(transient outward K current,Ito)的影响.结果显示,SNCl62明显抑制大鼠心室肌细胞,Ica L和Ica L,对Ica L.和k的最大抑制率分别为(46.13±4.12)%和(36.53±10.57)%.1x10-4mol/L SNCl62使,Ica L的甲均电流密度从(8.98±0.40)pA/pF下降到(4.84±0.44)pA/pF(P<0.01,n=5),Ito的平均电流密度从(18.69±2.42)pA/pF降低到(11.73±1.67)pA/pF(P<0.01,n=5).单独应用naltrindole(一种选择性δ阿片受体拮抗剂)对大鼠心室肌细胞Ica L和Ito无显著作用,但预先应用naltrindole可以消除SNCl62对Ica L和Ito的抑制作用.结果表明,通过δ阿片受体,SNCl62(1x10-6～1x10-4mol/L)浓度依赖性地抑制人鼠心室肌细胞Ica L和Ito这可能是激动δ阿片受体产生抗心律失常效应的重要机制.     

Effects of adenosine on the contractility of normoxic rainbow trout heart

Temperature strongly affects oxygen solubility in water, oxygen convection in the blood and locomotor activity of the fish. Since oxygen supply and demand are temperature dependent, it was hypothesized that the purinergic control of the heart, one of the most important mediators in oxygen-limited conditions, might also show temperature dependence. Therefore, the present study examines the effects of adenosine (Ado), a purinergic agonist, on the contractile and electrical activity of the thermally acclimated trout ( Oncorhynchus mykiss Walbaum) heart. The fish were acclimated to either 4 degrees C or 17 degrees C and the experiments were conducted at the acclimation temperatures of the animals. In spontaneously beating hearts, Ado had a negative chronotropic and a positive inotropic effect in warm-acclimated rainbow trout while no response was detected in cold-acclimated trout. In paced atrial and ventricular preparations, Ado had a negative inotropic effect in both warm- and cold-acclimated fish, and the response was strongest in the atria of warm-acclimated trout. Ado shortened the duration of contraction 12-14% in atrial preparations but had no effect in ventricular muscle. Ado (10(-4) mol l(-1)) increased the density of the inwardly rectifying K(+) current from -3.5+/-0.6 pA pF(-1) to -8.4+/-1.4 pA pF(-1) (at -120 mV) in atrial myocytes of warm-acclimated trout but was without effect in atrial myocytes of cold-acclimated trout (-2.4+/-0.8 pA pF(-1) vs. -2.1+/-0.9 pA pF(-1)). Ado had no effect on K(+) currents of ventricular cells in either acclimation group. These results indicate that the effects of Ado on cardiac contractility and electrical activity are stronger in warm-acclimated than in cold-acclimated trout when measured at the physiological body temperatures of the fish. The balance between oxygen demand and supply of the heart might be better in the cold where more environmental oxygen is available and the power of the muscles is weaker thereby reducing the need for the purinergic control of the heart. Temperature-dependence of Ado response in the trout heart warrants that temperature should be taken into consideration when the purinergic system of the ectotherms is studied.     

Electrophysiological properties of the L-type Ca(2+) current in cardiomyocytes from bluefin tuna and Pacific mackerel

Tunas are capable of exceptionally high maximum metabolic rates; such capability requires rapid delivery of oxygen and metabolic substrate to the tissues. This requirement is met, in part, by exceptionally high maximum cardiac outputs, opening the possibility that myocardial Ca(2+) delivery is enhanced in myocytes from tuna compared with those from other fish. In this study, we investigated the electrophysiological properties of the cardiac L-type Ca(2+) channel current (I(Ca)) to test the hypothesis that Ca(2+) influx would be large and have faster kinetics in cardiomyocytes from Pacific bluefin tuna (Thunnus orientalis) than in those from its sister taxon, the Pacific mackerel (Scombe japonicus). In accordance with this hypothesis, I(Ca) in atrial myocytes from bluefin tuna had significantly greater peak current amplitudes and faster fast inactivation kinetics (-4.4 +/- 0.2 pA/pF and 25.9 +/- 1.6 ms, respectively) than those from mackerel (-2.7 +/- 0.5 pA/pF and 32.3 +/- 3.8 ms, respectively). Steady-state activation, inactivation, and recovery from inactivation were also faster in atrial myocytes from tuna than from mackerel. In ventricular myocytes, current amplitude and activation and inactivation rates were similar in both species but elevated compared with those of other teleosts. These results indicate enhanced I(Ca) in atrial myocytes from bluefin tuna compared with Pacific mackerel; this enhanced I(Ca) may be associated with elevated cardiac performance, because I(Ca) delivers the majority of Ca(2+) involved in excitation-contraction coupling in most fish hearts. Similarly, I(Ca) is enhanced in the ventricle of both species compared with other teleosts and may play a role in the robust cardiac performance of fishes of the family Scombridae.     

氨甲酰胆碱通过M2胆碱能受体对大鼠心肌细胞发挥正性肌力作用

为研究氨甲酰胆碱（carbachol,CCh）对大鼠心肌细胞的正性肌力作用机制,利用电压钳方法观察CCh对急性分离的单个大鼠心肌细胞L-型钙电流（足扎）和钠,钙交换电流（INa/Ca）的影响。细胞负载Fura-2／AM后,用离子成像系统测定场刺激下单个大鼠心肌细胞的钙瞬变和细胞缩短。结果表明,100ILmol／LCCh使正向INa/Ca从（1．18±0．57）pA／pF增加到（1．65±0．52）pA/pF（P〈O．01）,反向,INa/Ca从（1．11±0．49）pA/pF增加到（1．53±0．52）pA/pF（P〈O．01）,但不影响ICa,L。阿托品（非选择性M胆碱受体拮抗剂）和methoctramine（选择性M2胆碱受体拮抗剂）可阻断这种增加作用。100μmol／LCCh使钙瞬变从对照组的203．8±50．0增加到234．8±64.3,使细胞缩短从对照组的（3．00±0．67）μm增加到（3．55±1．21）μm。KB-R7943（选择性反向INa/Ca抑制剂）不影响钙瞬变和细胞缩短的基础水平,却完全阻断CCh引起的钙瞬变和细胞缩短的增加。尼卡地平（ICa,L抑制剂）抑制钙瞬变和细胞缩短。CCh在尼卡地平存在下仍可增加钙瞬变和细胞缩短值,提示其正性肌力作用是通过刺激钠,钙交换实现的。CCh不改变钙敏感性。阿托品和methoctramine阻断CCh的这种激动作用,说明CCh的正性肌力作用是通过M2受体实现的。以上结果提示,CCh对大鼠心肌细胞有正性肌力作用,这种作用是通过激动反向钠／钙交换实现,由M2受体介导。     

神经肽Y对心室肌细胞离子通道的影响

采用全细胞膜片钳技术观察神经肽Y(neuropeptide Y,NPY)对心室肌细胞离子通道的影响。结果如下：(1)NPY浓度在1．0～100nmol/L范围内剂量依赖性抑制大鼠心室肌细胞I_(Ca-L),IC_(50)值为1．86nmol/L。NPY对I_(Ca-L)的I-V曲线的最大峰值电位、激活和失活电位均无显著影响。NPY对去甲肾上腺素(norepinephrine,NE)增加的I_(Ca-L)有显著抑制作用。(2)NPY对人鼠心室肌细胞I_(Na/Ca)有显著抑制作用。10nmol/L NPY使前向I__(Na/Ca)由(0．27±0．11)pA/pF减小为(0．06±0．01)pA/pF;反向I__(Na/Ca)由(0．45±0．12)pA/pF降为(0．27±0．09)pA/pF(P＜0．05,n=4)。(3)NPY对大鼠心室肌细胞I_(to)有显著增强作用。10 nmol/L NPY使I_(to)由(12．5±0．70)pA/pF增加至(14．7±0．59)pA/pF(P＜0．05,n=4)。(4)10nmol/L NPY对大鼠心室肌细胞I_(Na)没有显著影响。(5)10nmol/L NPY对豚鼠心室肌细胞I_K无明显影响。研究结果证实,NPY抑制大鼠心室肌细胞I_(Ca-L)和I_(Na/Ca),增强I_(to)对I_Na和豚鼠心审肌细胞I_K没有显著作用,表明NPY对上述主要离子通道的效应与NE的效应相拮抗。     

Transmural gradients in Na/K pump activity and [Na+]I in canine ventricle

There are well-documented differences in ion channel activity and action potential shape between epicardial (EPI), midmyocardial (MID), and endocardial (ENDO) ventricular myocytes. The purpose of this study was to determine if differences exist in Na/K pump activity. The whole cell patch-clamp was used to measure Na/K pump current (I(P)) and inward background Na(+)-current (I(inb)) in cells isolated from canine left ventricle. All currents were normalized to membrane capacitance. I(P) was measured as the current blocked by a saturating concentration of dihydro-ouabain. [Na(+)](i) was measured using SBFI-AM. I(P)(ENDO) (0.34 +/- 0.04 pA/pF, n = 17) was smaller than I(P)(EPI) (0.68 +/- 0.09 pA/pF, n = 38); the ratio was 0.50 with I(P)(MID) being intermediate (0.53 +/- 0.13 pA/pF, n = 19). The dependence of I(P) on [Na(+)](i) or voltage was essentially identical in EPI and ENDO (half-maximal activation at 9-10 mM [Na(+)](i) or approximately -90 mV). Increasing [K(+)](o) from 5.4 to 15 mM caused both I(P)(ENDO) and I(P)(EPI) to increase, but the ratio remained approximately 0.5. I(inb) in EPI and ENDO were nearly identical ( approximately 0.6 pA/pF). Physiological [Na(+)](i) was lower in EPI (7 +/- 2 mM, n = 31) than ENDO (12 +/- 3 mM, n = 29), with MID being intermediate (9 +/- 3 mM, n = 22). When cells were paced at 2 Hz, [Na(+)](i) increased but the differences persisted (ENDO 14 +/- 3 mM, n = 10; EPI 9 +/- 2 mM, n = 10; and MID intermediate, 11 +/- 2 mM, n = 9). Based on these results, the larger I(P) in EPI appears to reflect a higher maximum turnover rate, which implies either a larger number of active pumps or a higher turnover rate per pump protein. The transmural gradient in [Na(+)](i) means physiological I(P) is approximately uniform across the ventricular wall, whereas transporters that utilize the transmembrane electrochemical gradient for Na(+), such as Na/Ca exchange, have a larger driving force in EPI than ENDO.     

细胞外pH值降低可增强豚鼠心室肌细胞持续性钠电流

应用全细胞和单通道(贴附式)膜片钳技术观察胞外pH值降低对心室肌细胞持续性钠电流(persistent sodium current,ⅠNa.P)的影响,探讨其作用机制。结果显示:全细胞记录模式下,细胞外pH值降低可明显增大ⅠNa.P,且呈H+浓度依赖性增强。当细胞外pH值从对照值的7．4降低为6．5时,ⅠNa.P的电流密度从(0．347±0．067)pAJpF增加到(0．817±0．137)pA/pF(P< 0．01,n=6),而加入还原剂1,4-二硫甙苏糖醇(dithiothreitiol,DTT,1 mmol／L)后可使,ⅠNa.P的电流密度回落到(0．233±0．078)pA／pF (P<0．01 vs pH 6．5,n=6)。单通道记录模式中,当细胞外pH值从对照值的7．4降低为6．5时,持续性钠通道的开放概率和开放时间分别从0．021±0．007和(0．899±0．074)ms增加到0．205±0．023和(1．593±0．158)ms(P<0．叭,n=6),再加入还原剂DTT(1 mmol／L)使开放概率和开放时间分别回落到0．019±0．005和(0．868±0．190)ms(P<0．01 vs pH 6．5,n=6);加入蛋白激酶C(protein kinase C,PKC)抑制剂bisindolylmaleimide(BIM,5μmol/L)可使pH 6．5时增大的,ⅠNa.P明显减小,开放概率和开放时间分别从0．214±0．024和(1．634±0．137)ms回落到0．025±0．006和(0．914±0．070)ms(P<0．01 vs pH 6．5,n=6)。结果表明,细胞外pH值降低可诱发心室肌细胞ⅠNa.P增大,其机制可能与PKC的激活有关。     

Chronic verapamil treatment remodels ICa,L in mouse ventricle

In this study we tested the hypothesis that ventricular homeostasis of L-type Ca(2+) current (I(Ca,L)) minimally involves regulation of the main pore-forming alpha-subunit (Ca(V)1.2) and auxiliary proteins that serve as positive or negative regulators of I(Ca,L). We treated animals for 24 h with verapamil (Ver, 3.6 mg.kg(-1).day(-1)), isoproterenol (Iso, 30 mg.kg(-1).day(-1)), or Iso + Ver via osmotic minipumps. To test for alterations of Ca(2+) channel complex components we performed real-time PCR and Western blot analysis on ventricle. In addition, cardiac myocytes (CMs) were dispersed and current was recorded in the whole cell configuration to evaluate I(Ca,L). Surprisingly, 24- to 48-h Ver increased Ca(V)1.2 mRNA and protein and I(Ca,L) current (Ver 11 +/- 1pA/pF vs. control 7 +/- 0.5pA/pF; P < 0.01). I(Ca,L) from CMs in Ver mice showed no change in whole cell capacitance. To examine the in vivo effects of a physiologically relevant Ca(2+) channel agonist, we treated mice with Iso. Twenty-four-hour Iso infusion increased heart rate; Ca(V)1.2- and Ca(V)beta(2) mRNA levels were constant, but the Ca(2+) channel subunit mRNA Rem was increased twofold. Cells isolated from 24-h Iso hearts showed no change in basal I(Ca,L) density and diminished responsiveness to acute 1 muM Iso. To further examine the homeostatic regulation of the Ca(2+) channel, we treated animals for 24 h with Iso + Ver. The influence of Iso + Ver was similar that of to Iso alone on Ca(2+) channel mRNAs and I(Ca,L), with the exception that it prevented the increase in Rem seen with Iso treatment. Long-term Ca(2+) channel blockade induces an increase of Ca(V)1.2 mRNA and protein and significantly increases I(Ca,L).     

Myocardial infarction in rat eliminates regional heterogeneity of AP profiles,I(to) K(+) currents,and [Ca(2+)](i) transients

Transient outward K(+) current density (I(to)) has been shown to vary between different regions of the normal myocardium and to be reduced in heart disease. In this study, we measured regional changes in action potential duration (APD), I(to), and intracellular Ca(2+) concentration ([Ca(2+)](i)) transients of ventricular myocytes derived from the right ventricular free wall (RVW) and interventricular septum (SEP) 8 wk after myocardial infarction (MI). At +40 mV, I(to) density in sham-operated hearts was significantly higher (P < 0.01) in the RVW (15.0 +/- 0.8 pA/pF, n = 47) compared with the SEP (7.0 +/- 1.1 pA/pF, n = 18). After MI, I(to) density was not reduced in SEP myocytes but was reduced (P < 0.01) in RVW myocytes (8.7 +/- 1.0 pA/pF, n = 26) to levels indistinguishable from post-MI SEP myocytes. These changes in I(to) density correlated with Kv4.2 (but not Kv4.3) protein expression. By contrast, Kv1.4 expression was significantly higher in the RVW compared with the SEP and increased significantly after MI in RVW. APD measured at 50% or 90% repolarization was prolonged, whereas peak [Ca(2+)](i) transients amplitude was higher in the SEP compared with the RVW in sham myocytes. These regional differences in APD and [Ca(2+)](i) transients were eliminated by MI. Our results demonstrate that the significant regional differences in I(to) density, APD, and [Ca(2+)](i) between RVW and SEP are linked to a variation in Kv4.2 expression, which largely disappears after MI.     

L-type and T-type Ca2+ current in cultured ventricular guinea pig myocytes

The aim of this investigation was to study L-type and T-type Ca(2+) current (I(CaL) and I(CaT)) in short-term cultured adult guinea pig ventricular myocytes. The isolated myocytes were suspended in serum-supplemented medium up to 5 days. Using whole-cell patch clamp techniques ICaL and ICaT were studied by applying voltage protocols from different holding potentials (-40 and -90 mV). After 5 days in culture the myocytes still showed their typical rod shaped morphology but a decline in cell membrane capacitance (26 %). The peak density of ICaT was reduced significantly between day 0 (-1.6+/-0.37 pA/pF, n=9) and day 5 (-0.4+/-0.13 pA/pF, n=11), whereas peak ICaL density revealed no significant differences during culturing. The I(CaT)/I(CaL) ratio dropped from 0.13 at day 0 to 0.05 at day 5. Compared with day 0 I(CaL) the steady state inactivation curve of day 1, day 3 and day 5 myocytes was slightly shifted to more negative potentials. Our data indicate that guinea pig ventricular L-type and T-type Ca(2+) channels are differently regulated in culture.     

Reverse engineering the L-type Ca2+ channel alpha1c subunit in adult cardiac myocytes using novel adenoviral vectors

The alpha(1c) subunit of the cardiac L-type Ca(2+) channel, which contains the channel pore, voltage- and Ca(2+)-dependent gating structures, and drug binding sites, has been well studied in heterologous expression systems, but many aspects of L-type Ca(2+) channel behavior in intact cardiomyocytes remain poorly characterized. Here, we develop adenoviral constructs with E1, E3 and fiber gene deletions, to allow incorporation of full-length alpha(1c) gene cassettes into the adenovirus backbone. Wild-type (alpha(1c-wt)) and mutant (alpha(1c-D-)) Ca(2+) channel adenoviruses were constructed. The alpha(1c-D-) contained four point substitutions at amino acid residues known to be critical for dihydropyridine binding. Both alpha(1c-wt) and alpha(1c-D-) expressed robustly in A549 cells (peak L-type Ca(2+) current (I(CaL)) at 0 mV: alpha(1c-wt) -9.94+/-1.00pA/pF, n=9; alpha(1c-D-) -10.30pA/pF, n=12). I(CaL) carried by alpha(1c-D-) was markedly less sensitive to nitrendipine (IC(50) 17.1 microM) than alpha(1c-wt) (IC(50) 88 nM); a feature exploited to discriminate between engineered and native currents in transduced guinea-pig myocytes. 10 microM nitrendipine blocked only 51+/-5% (n=9) of I(CaL) in alpha(1c-D-)-expressing myocytes, in comparison to 86+/-8% (n=9) of I(CaL) in control myocytes. Moreover, in 20 microM nitrendipine, calcium transients could still be evoked in alpha(1c-D-)-transduced cells, but were largely blocked in control myocytes, indicating that the engineered channels were coupled to sarcoplasmic reticular Ca(2+) release. These alpha(1c) adenoviruses provide an unprecedented tool for structure-function studies of cardiac excitation-contraction coupling and L-type Ca(2+) channel regulation in the native myocyte background.     

大鼠肥厚心肌细胞钙电流及 Na~ /Ca~(2 )交换电流的研究(英文)

本文观察了心肌肥厚对大鼠心肌细胞Na ／Ca2 交换电流的影响。我们采用Goldblatt两肾一夹方法诱发大鼠心肌肥厚,应用全细胞膜片钳技术记录电流。结果表明：肥厚心肌细胞的Ni2 -敏感Na ／Ca2 交换电流密度大于正常细胞。在钳制电压为＋50mV时,正常细胞的外向交换电流密度为1．53±0．31pA／pF,而肥厚细胞则为2．62±0．53pA／pF（P＜0．01）;钳制电压为-100mV时,正常细胞的内向交换电流密度为0．42±0．14pA／pF,肥厚细胞达1．12±0．33pA／pF（P＜0．001）。这些结果提示,肥厚心肌细胞的Na ／Ca2 交换电流发生了改变,其意义有待进一步探讨。     

小剂量辣椒素对豚鼠心室肌细胞L-型钙电流的影响

应用全细胞膜片钳技术研究低浓度辣椒素(capsaicin,CAP)对单个豚鼠心室肌细胞L-型钙电流的影响及其作用机制.CAP(1～25 nmol/L)可浓度依赖性增加电压依赖性的ICa-L的峰值并下移I-V曲线.CAPl,10,25 nmol/L使ICa-L最大峰值分别由-9.67±0.7pA/pF增至-10.21±0.8pA/pF(P＞0.05),-11.37±0.8pA/pF和-12.84±0.9pA/pF(P＜0.05).CAP25nmol/L可明显使稳态激活曲线左移,激活中点电压(V0.5)由-20.76±2.0mV变至-26.71±3.0mV(P＜0.05),表明低浓度CAP改变了钙通道激活的电压依赖性.CAP25nmol/L对电压依赖性稳态失活曲线和ICa-L从失活状态下复活过程无明显影响.辣椒素受体(VR1)阻断剂钌红(RR,10μmol/L)可阻断低浓度辣椒素的效应.以上结果表明,低浓度辣椒素使钙通道稳态激活曲线左移,增加ICa-L,这一效应可能由VRl介导.