Contribution of spontaneous L-type Ca2+ channel activation to the genesis of Ca2+ sparks in resting cardiac myocytes

Ca²⁺ sparks are the elementary events of intracellular Ca²⁺ release from the sarcoplasmic reticulum in cardiac myocytes. In order to investigate whether spontaneous L-type Ca²⁺ channel activation contributes to the genesis of spontaneous Ca²⁺ sparks, we used confocal laser scanning microscopy and fluo-4 to visualize local Ca²⁺ sparks in intact rat ventricular myocytes. In the presence of 0.2 mmol/L CdCI₂ which inhibits spontaneous L-type Ca²⁺ channel activation, the rate of occurrence of spontaneous Ca²⁺ sparks was halved from 4.20 to 2.04 events/(100 μm · s), with temporal and spatial properties of individual Ca²⁺ sparks unchanged. Analysis of the Cd²⁺-sensitive spark production revealed an open probability of ~10 ^-5 for L-type channels at the rest membrane potentials (-80 mV). Thus, infrequent and stochastic openings of sarcolemmal L-type Ca²⁺ channels in resting heart cells contribute significantly to the production of spontaneous Ca²⁺ sparks.     

The α2δ subunit augments functional expression and modifies the pharmacology of CaV1.3 L-type channels

The auxiliary Ca_Vα₂δ-1 subunit is an important component of voltage-gated Ca²⁺ (Ca_V) channel complexes in many tissues and of great interest as a drug target. Nevertheless, its exact role in specific cell functions is still unknown. This is particularly important in the case of the neuronal L-type Ca_V channels where these proteins play a key role in the secretion of neurotransmitters and hormones, gene expression, and the activation of other ion channels. Therefore, using a combined approach of patch-clamp recordings and molecular biology, we studied the role of the Ca_Vα₂δ-1 subunit on the functional expression and the pharmacology of recombinant L-type Ca_V1.3 channels in HEK-293 cells. Co-expression of Ca_Vα₂δ-1 significantly increased macroscopic currents and conferred the Ca_V1.3α₁/Ca_Vβ₃ channels sensitivity to the antiepileptic/analgesic drugs gabapentin and AdGABA. In contrast, Ca_Vα₂δ-1 subunits harboring point mutations in N-glycosylation consensus sequences or the proteolytic site as well as in conserved cysteines in the transmembrane δ domain of the protein, reduced functionality in terms of enhancement of Ca_V1.3α₁/Ca_Vβ₃ currents. In addition, co-expression of the δ domain drastically inhibited macroscopic currents through recombinant Ca_V1.3 channels possibly by affecting channel synthesis. Together these results provide several lines of evidence that the Ca_Vα₂δ-1 auxiliary subunit may interact with Ca_V1.3 channels and regulate their functional expression.     

Role of protein kinase C in NADPH oxidase derived O2-mediated regulation of KV-LVOCC axis under U46619 induced increase in [Ca]i in pulmonary smooth muscle cells

Treatment of bovine pulmonary smooth muscle cells with the TxA₂ mimetic, U46619 stimulated [Ca²⁺]_i, which was inhibited upon pretreatment with apocynin (NADPH oxidase inhibitor). Pretreatment with cromakalim (K_V channel opener) or nifedepine (L-VOCC inhibitor) inhibited U46619 induced increase in [Ca²⁺]_i, indicating a role of K_V-LVOCC axis in this scenario. Neither cromakalim nor nifedepine inhibited U46619 induced increase in NADPH oxidase activity, suggesting that the NADPH oxidase activation is proximal to the K_V-LVOCC axis in the cells. Pretreatment with calphostin C (PKC inhibitor) markedly reduced U46619 induced increase in NADPH oxidase activity and [Ca²⁺]_i in the cells. Calphostin C pretreatment also markedly reduced p^47phox phosphorylation and translocation to the membrane and association with p^22phox, a component of Cyt.b₅₅₈ of NADPH oxidase in the membrane. Overall, PKC plays an important role in NADPH oxidase derived O₂⁻-mediated regulation of K_V-LVOCC axis leading to an increase in [Ca²⁺]_i by U46619 in the cells.     

Silencing of Cav1.2 gene in neonatal cardiomyocytes by lentiviral delivered shRNA

Cav1.2 (α_1C) and Cav1.3 (α_1D) L-type Ca channels are co-expressed in the heart. To date, there are no pharmacological or biophysical tools to separate α_1D from α_1C Ca currents (I_Ca-L) in cardiomyocytes. Here, we established a physiological model to study α_1D I_Ca-L in native myocytes using RNA interference. Transfection of rat neonatal cardiomyocytes (RNC) with α_1C specific siRNA resulted in low silencing efficiency (50-60%) at the mRNA and protein levels. The use of lentivirus shRNA resulted in 100% transfection efficiency and 92% silencing of the α_1C gene by real-time PCR and Western blot. Electrophysiological experiments showed that the total I_Ca-L was similarly reduced by 80% in lentivirus transfected cells. Both biochemical and functional data demonstrated high transfection and silencing efficiency in the cardiomyocytes using lentiviral shRNA. This novel approach allows for the assessments of the roles of α_1C and α_1D Ca channels in native myocytes and could be used to examine their roles in physiological and pathological settings.     

The role of caveolae and caveolin 1 in calcium handling in pacing and contraction of mouse intestine

In mouse intestine, caveolae and caveolin‐1 (Cav‐1) are present in smooth muscle (responsible for executing contractions) and in interstitial cells of Cajal (ICC; responsible for pacing contractions). We found that a number of calcium handling/dependent molecules are associated with caveolae, including L‐type Ca²⁺ channels, Na⁺‐Ca²⁺ exchanger type 1 (NCX1), plasma membrane Ca²⁺ pumps and neural nitric oxide synthase (nNOS), and that caveolae are close to the peripheral endo‐sarcoplasmic reticulum (ER‐SR). Also we found that this assemblage may account for recycling of calcium from caveolar domains to SR through L‐type Ca ⁺ channels to sustain pacing and contractions. Here we test this hypothesis further comparing pacing and contractions under various conditions in longitudinal muscle of Cav‐1 knockout mice (lacking caveolae) and in their genetic controls. We used a procedure in which pacing frequencies (indicative of functioning of ICC) and contraction amplitudes (indicative of functioning of smooth muscle) were studied in calcium‐free media with 100 mM ethylene glycol tetra‐acetic acid (EGTA). The absence of caveolae in ICC inhibited the ability of ICC to maintain frequencies of contraction in the calcium‐free medium by reducing recycling of calcium from caveolar plasma membrane to SR when the calcium stores were initially full. This recycling to ICC involved primarily L‐type Ca²⁺ channels; i.e. pacing frequencies were enhanced by opening and inhibited by closing these channels. However, when these stores were depleted by block of the sarco/endoplasmic reticulum Ca²⁺‐ATPase (SERCA) pump or calcium release was activated by carbachol, the absence of Cav‐1 or caveolae had little or no effect. The absence of caveolae had little impact on contraction amplitudes, indicative of recycling of calcium to SR in smooth muscle. However, the absence of caveolae slowed the rate of loss of calcium from SR under some conditions in both ICC and smooth muscle, which may reflect the loss of proximity to store operated Ca channels. We found evidence that these channels were associated with Cav‐1. These changes were all consistent with the hypothesis that a reduction of the extracellular calcium associated with caveolae in ICC of the myenteric plexus, the state of L‐type Ca²⁺ channels or an increase in the distance between caveolae and SR affected calcium handling.     

霍乱毒素对三氟氯氰菊酯抗性及敏感棉铃虫神经细胞L型钙通道的调节作用

霍乱毒素（CTX）可激活兴奋性异三聚体G蛋白（Gα_s）的α-亚基和刺激电压门控L-型钙通道,而昆虫的L-型钙通道可能是拟除虫菊酯类杀虫剂的作用靶点。为进一步探讨农业害虫对拟除虫菊酯类杀虫剂产生抗药性的作用机理,我们检测了CTX对三氟氯氰菊酯抗性及敏感棉铃虫Helicoverpa armigera中枢神经细胞电压门控L-型钙通道的调节作用。分别急性分离三氟氯氰菊酯抗性及敏感的3～4龄棉铃虫幼虫胸腹神经节细胞,并在改良的L15培养基（加入或未加入700 ng/mL的CTX）中培养12～16 h。钡离子为载流子,应用全细胞膜片钳技术记录电压门控L_型钙通道电流。结果显示,CTX可使敏感组棉铃虫神经细胞L-型钙通道的峰值电流密度增大36％、峰值电压左移5 mV,但对抗性组棉铃虫神经细胞L-型钙通道无上述作用。并且,CTX对敏感组及抗性组棉铃虫神经细胞L_型钙通道的激活电位、翻转电位、激活曲线和失活曲线等其他一些参数的影响也不明显。在无CTX作用时,所检测到的抗性组与敏感组棉铃虫神经细胞L_型钙通道的上述参数值间差异不显著。结果提示,棉铃虫神经细胞内存在Gs腺苷酸环化酶(AC)-cAMP-蛋白激酶A (PKA)-L-型钙通道信号调节系统;与敏感棉铃虫神经细胞L-型钙通道相比,三氟氯氰菊酯抗性棉铃虫神经细胞L-型钙通道的活性相对不易受到CTX调节,这可能与昆虫对拟除虫菊酯产生抗药性的机理有关。     

不同温度两类诱导剂形成细菌L型的研究

以抗生素（苯唑青霉素、羧苄青霉素、氨苄青霉素）及中药（大黄、黄连）纸片,３７,３２和２８℃不同温度分别诱导金黄色葡萄球菌、福氏痢疾杆菌和大肠埃希菌形成Ｌ型。结果３种不同温度,２类诱导剂诱导形成Ｌ型,菌落形成时间、特征、形态及染色性等均无明显差别。表明诱导细菌 Ｌ型除常规３７℃外, ３２℃和 ２８℃均可形成。即使中药也如此。为临床对疾病诊治,为探讨Ｌ型可在自然界产生和存在是否与某些传染病的流行和传播有关提供了参考依据,同时提示注意环境中Ｌ型的消毒与灭菌。     

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.     

银杏苦内酯B对缺血豚鼠心室肌动作电位、L-型钙电流和延迟整流钾电流的作用

目的:研究银杏苦内酯B对正常和缺血心室肌细胞动作电位(action potential,AP),L-型钙电流(L-type calcium current,ICa-L)、延迟整流钾电流(Delayed Rectifier Currennt,IK)的影响.方法:用常规细胞内微电极方法记录豚鼠心室肌细胞动作电位,用全细胞膜片钳技术记录游离心室肌细胞离子流.结果:①在生理条件下,银杏苦内酯B可缩短心室肌细胞动作电位时程 (action potential duration,APD),但对AP其他参数无影响,银杏苦内酯B可增大IK,呈浓度依赖性,但对ICa-L无显著作用;②在缺血条件下,APD50、APD90明显缩短,RP、APA减小,Vmax减慢,而银杏苦内酯B则可延缓和减轻缺血所引起上述参数的变化;3.在缺血条件下,IK和ICa-L均受到抑制,但加入银杏苦内酯B后可逆转缺血所造成这两种离子流的减小.结论:银杏苦内酯B可对抗心肌缺血所引起的心肌电生理的变化,提示银杏苦内酯B可预防心律失常的发生.