Multiple effects of caffeine on calcium current in rat ventricular myocytes

Caffeine exerts a number of different effects on L-type calcium current in rat ventricular myocytes. These include: (1) a slowing of inactivation that is comparable to, but not additive to, that produced by prior treatment of the cells with ryanodine (a selective sarcoplasmic reticulum Ca²⁺ releaser) or high concentrations of intracellular 1,2-bis[2-aminophenoxy]ethane-N,N,N,N-tetraacetic acid (BAPTA) (a fast Ca²⁺ chelator), (2) a stimulation of peak I _Ca that is comparable to, but not additive to that produced by prior treatment with isobutylmethylxanthine (a selective phosphodiesterase inhibitor), and (3) a dose-dependent decrease of peak I _Ca that is not prevented by pretreatment with any of these agents. None of the caffeine actions could be mimicked or prevented by administration of 8-phenyltheophylline, a specific adenosine receptor antagonist. We conclude that only the slowing of I _Ca inactivation is due to caffeine's ability to deplete the sarcoplasmic reticulum of calcium. The stimulatory effect of caffeine on peak I _Ca is probably due to phosphodiesterase inhibition, while caffeine's inhibitory effect on I _Ca is independent of these processes and could be a direct effect on the channel. The multiplicity of caffeine actions independent of its effects on the sarcoplasmic reticulum lead to the conclusion that ryanodine, though slower acting and essentially irreversible, is a more selective agent than caffeine for probing sarcoplasmic reticulum function and its effects on other processes.The experimental part of this work was published during the postdoctoral stay of I. Zahradník in the Department of Physiology and Biophysics, The University of Texas Medical Branch, Galveston, TX 77555, USA     

水杨酸钠对大鼠下丘神经元L-型钙通道的影响

目的 了解L-型钙通道在水杨酸钠导致耳鸣的机制中所起的作用.方法 利用全细胞膜片钳技术研究水杨酸钠对急性分离的大鼠下丘神经元L-型钙通道的影响.结果 水杨酸钠抑制L-型钙通道电流(ICa,L),且具有浓度依赖性.水杨酸钠抑制ICa,L的半抑制浓度值为1.99 mmol/L.水杨酸钠不影响ICa,L的电导-电压曲线和稳态激活曲线,水杨酸钠将ICa,L的稳态失活曲线向超极化方向移动8 mV,并且延长ICa,L失活后恢复的时间.结论 水杨酸钠对ICa,L的抑制作用,可能通过减少下丘部位γ-氨基丁酸的释放,从而导致耳鸣.     

吗啡处理对大鼠延髓头端腹内侧区的L-型电压门控钙通道蛋白与电流的影响

[目的] 探讨在吗啡引起的痛觉超敏状态下,SD大鼠延髓头端腹内侧区电压门控钙通道(voltage-gated calcium channels, VGCCs)蛋白的表达变化和功能变化.[方法] 采用western blot方法检测α1C和α1H亚基蛋白在大鼠延髓头端腹内侧区(the rostral ventromedial medulla,RVM)的表达,采用脑片全细胞膜片钳技术记录大鼠RVM神经元的VGCCs电流.[结果] 在吗啡诱导的痛觉超敏状态下,SD大鼠RVM的α1C亚单位蛋白表达显著增加,同时神经元的VGCCs总电流和L-型VGCCs电流也显著增加.[结论] 延髓RVM的L-型电压门控钙通道的可塑性变化,可能是导致吗啡痛觉超敏的重要原因.     

Alterations in properties of L-type Ca channels in aging rat heart

Previous studies of whole-cell L-type Ca currents in aging heart have demonstrated an increase in the peak Ca current magnitude in proportion to the increase in membrane area, and a slowing of the time course for inactivation. However, the single-channel mechanisms underlying this upregulation, and for the slowed inactivation are not known. We have therefore compared the properties of single L-type Ca channel currents recorded from ventricular myocytes obtained from young adult (3 month), adult (6-8 month) and aging (24 month) Wistar rats, using 5 m m Ba ions as the permeant ion. We report that the peak ensemble-averaged single Ca channel currents from aging heart (-280+/-57 fA) were enhanced compared to those from young adult (-137+/-16 fA), or adult hearts (-144+/-38 fA). This surprising result was related, in part, to an apparent increase in the number of active Ca channels per patch in aging (1.90+/-0.23) v young adult (1.33+/-0.19) or adult heart (1.50+/-0.2). Moreover, there was an increase in the time constant for inactivation of the ensemble-averaged Ca currents of aging (471+/-169 ms), compared with young adult (198+/-43 ms), or adult heart (196+/-32 ms). The aging-related changes were also traced to alterations in single Ca channel gating, including an increase in the average probability of being open, and an increase in the availability of single Ca currents in aging heart. In contrast, the unitary Ca current amplitude was unchanged with aging. These novel findings suggest that the compensatory increase in the L-type Ca currents during aging is a consequence of an apparent increase in both the number, and the activity of individual L-type Ca channels.     

The late component of L-type calcium current during guinea-pig cardiac action potentials and its contribution to contraction

 L-Type Ca²⁺ current (I _Ca,L) elicited during the action potential (AP) of guinea-pig ventricular myocytes exhibits an early and a late component. The whole-cell patch-clamp technique was used to characterize the process regulating the late I _Ca,L component and to assess its contribution to excitation-contraction coupling. A stepwise decrease in repolarization rate of AP-like voltage-clamp pulses led to an exponential increase in Ca²⁺ charge carried by I _Ca,L. This saturation behaviour was significantly reduced or absent when Ba²⁺ or monovalent cations were used as charge carriers, which suggests that the late component of I _Ca,L is controlled mainly by Ca²⁺-dependent processes. Simultaneously recording I _Ca,L and zero-load shortening or the internal Ca²⁺ concentration (fura-2) revealed that Ca²⁺ carried by the late component of I _Ca,L markedly contributes to the Ca²⁺ content of the sarcoplasmic reticulum (SR). Reducing the charge transfer by late I _Ca,L during a series of AP-like conditioning clamp pulses by 48% reduced the shortening amplitude during a subsequent test stimulation by 56%. This relationship was absent during long rectangular depolarizing conditioning clamps, during which Na⁺/Ca²⁺ exchange increased its influence on SR Ca²⁺ loading. The late component of I _Ca,L developed only a minor direct influence on the simultaneous cell shortening. Thus, the main contribution of the late I _Ca,L component is to supply Ca²⁺ for SR loading. Received: 5 November 1997 / Received after revision: 12 June 1998 / Accepted: 15 June 1998     

卡维地洛对氧自由基所致心肌细胞L型钙电流异常的保护作用

目的　研究卡维地洛对氧自由基引起的豚鼠单个心室肌细胞L型钙电流异常的保护作用。方法　采用全细胞膜片钳技术 ,观察 0 5mmol/L的H2 O2 引起单个豚鼠心室肌细胞L型钙电流改变及预先应用 0 5 μmol/L卡维地洛对这种改变的影响。结果　 0 5 μmol/L卡维地洛对正常豚鼠心室肌细胞L型钙电流及其通道动力学影响不显著。 0 5mmol/LH2 O2 作用下 ,豚鼠心室肌细胞L型钙电流峰值明显降低 (P <0 0 0 1) ,电流 电压曲线上移 ,通道稳态激活曲线和稳态失活曲线左移 ,通道恢复时间明显延长 (P <0 0 0 1)。预先给予 0 5 μmol/L卡维地洛 ,明显减轻H2 O2 对L型钙电流的抑制作用 (P <0 0 1) ;并且可减轻H2 O2 对L型钙通道动力学的异常影响。结论　卡维地洛可减轻氧化应激对心肌细胞L型钙电流的影响 ,这可能是其治疗心力衰竭的机制之一     

Effect of didrovaltrate on I-calcium current in rabbit ventricular myocytes

Objective

To investigate the effect of didrovaltrate on L-type calcium current (I_ca-L) in rabbit ventricular myocytes.

Methods

We used the whole cell patch clamp recording technique.

Results

Didrovaltrate at concentrations of 30 μg/L and 100 μg/L significantly decreased peak ICa-L (ICa-Lmax) from (6.01 ± 0.48) pA/pF to (3.45±0.27) pA/pF and (2.16 ± 0.19) pA/pF (42.6% and 64.1%, n=8, P<0.01), respectively. Didrovaltrate shifted upwards the current-voltage curves of ICa-L without changing their active, peak and reverse potentials. Didrovaltrate affected the steady-state inactivation of ICa-L. The half activation potential (V_1/2) was significantly shifted from (−26 ± 2) to (−36 ± 3) mV (n=6, P<0.05), with a significant change in the slope factor (k) (from 8.8 ± 0.8 to 11.1 ± 0.9, n=6, P<0.05). Didrovaltrate did not affect the activation curve.

Conclusion

Didrovaltrate blocks ICa-L in a concentration-dependent manner and probably inhibits ICa-L in its inactive state, which may contribute to its cardiovascular effect.     

抑郁及糖尿病对大鼠心室肌离子通道表达的影响

目的:探讨抑郁及糖尿病对大鼠心室肌离子通道表达的影响。方法:将雄性SD大鼠随机分为健康对照组、抑郁组、糖尿病组及抑郁合并糖尿病组。抑郁动物模型通过持续4周的慢性温和应激获得。糖尿病动物模型通过皮下注射四氧嘧啶获得。4周后,分别取各大鼠左室心肌,采用实时荧光定量PCR方法对其L型钙通道、钠钙交换体(NCX)、瞬时外向钾通道的表达进行定量分析。结果:与健康对照组比较,抑郁组L型钙通道和NCX的表达增加,糖尿病组瞬时外向钾通道的表达减少,抑郁合并糖尿病组NCX的表达增加、瞬时外向钾通道的表达减少;抑郁合并糖尿病组瞬时外向钾通道的表达较抑郁组减少;抑郁合并糖尿病组NCX的表达较糖尿病组增加。结论:抑郁及糖尿病可引起大鼠心室肌离子通道表达的改变。抑郁与糖尿病对大鼠心室肌离子通道表达的影响有一定的累积作用。     

Functional roles of the gamma subunit of the skeletal muscle DHP-receptor

In excitation–contraction coupling (EC coupling) of skeletal muscle, large and rapid changes of the myoplasmic Ca²⁺ concentration mediate the activation and termination of force. The L-type Ca²⁺ channel (dihydropyridine receptor, DHP receptor) is a central component of the EC coupling process. Its predominant role is to provide the Ca²⁺ release channels of the sarcoplasmic reticulum (SR) with the sensitivity to cell membrane voltage. The DHP receptor consists of five different proteins (α_1S, β₁, γ₁, δ and α₂) whose tasks and functional characteristics are still incompletely understood. This short review summarizes progress made in studying the physiology of the γ₁ subunit, a membrane polypeptide that is highly specific for skeletal muscle. The focus is on recent results obtained from muscle of γ₁-deficient mice.     

Enhancement of an L-type calcium current in AtT-20 cells; a novel effect of the m4 muscarinic receptor

Activation of muscarinic receptors has been shown to inhibit L-type calcium conductances by mechanisms sensitive to pertussis toxin (PTX). In this study we show that agonist stimulation of the m4 muscarinic receptor leads to an increase in an L-type calcium conductance in the AtT-20 pituitary cell line, by a PTX-sensitive mechanism. The amplitude of the dihydropyridine (DHP)-sensitive or L-type calcium current was increased by acetylcholine (ACh), with no shift in the voltage dependence. This action of ACh was completely inhibited by PTX pre-treatment. Forskolin, cAMP and phorbol 12,13-dibutyrate reduced, while RpcAMPs, an inhibitor of cAMP-dependent protein kinase (PKA), increased the L-type calcium conductance. We propose that the m4 muscarinic receptor activates the L-type calcium channel by inhibition of adenylyl cyclase resulting in reduced cAMP levels and, hence, reduced PKA activity. This novel increase in calcium current via the m4 muscarinic receptor appears to reflect the coupling with an L-type channel of the D class, due to the sensitivity of the L-type calcium conductance to both DHPs and -conotoxin, and, thus, is distinct from the skeletal muscle and cardiac L-type channels of the C class previously studied.