Translocation of telomerase reverse transcriptase coincided with ATP release in postnatal cochlear supporting cells

The spontaneous bursts of electrical activity in the developing auditory system are derived from the periodic release of adenosine triphosphate(ATP)by supporting cells in the Kölliker’s organ.However,the mechanisms responsible for initiating spontaneous ATP release have not been determined.Our previous study revealed that telomerase reverse transcriptase(TERT)is expressed in the basilar membrane during the first postnatal week.Its role in cochlear development remains unclear.In this study,we investigated the expression and role of TERT in postnatal cochlea supporting cells.Our results revealed that in postnatal cochlear Kölliker’s organ supporting cells,TERT shifts from the nucleus into the cytoplasm over time.We found that the TERT translocation tendency in postnatal cochlear supporting cells in vitro coincided with that observed in vivo.Further analysis showed that TERT in the cytoplasm was mainly located in mitochondria in the absence of oxidative stress or apoptosis,suggesting that TERT in mitochondria plays roles other than antioxidant or anti-apoptotic functions.We observed increased ATP synthesis,release and activation of purine signaling systems in supporting cells during the first 10 postnatal days.The phenomenon that TERT translocation coincided with changes in ATP synthesis,release and activation of the purine signaling system in postnatal cochlear supporting cells suggested that TERT may be involved in regulating ATP release and activation of the purine signaling system.Our study provides a new research direction for exploring the spontaneous electrical activity of the cochlea during the early postnatal period.

Spontaneous Neurotransmitter Release Depends on Intracellular Rather than ER Calcium Stores in Cultured Xenopus NMJ

Calcium ions are important in many vital neuron processes, including spontaneous neurotransmitter release. Extracellular calcium has long been known to be related to spontaneous neurotransmitter release, but the detailed mechanism for the effect of intracellular Ca^2＋ on synaptic release has not yet been understood. In this research, 1,2-bis-（o-aminophenoxy）-ethane-N, N, N′, N′-tetraacetic acid tetraacetoxymethyl ester （BAPTA-AM） was used to combine with cytosolic free Ca^2＋ in a calcium free medium of cultured Xenopus neuromuscular junctions （NMJ）, The spontaneous synaptic current （SSC） frequency was obviously reduced. Then, drugs were applied to interrupt and activate the Ca2＋ release channels in the endoplasmic reticulum （ER） membrane, but the SSC frequency was not affected. The results show that spontaneous neurotransmitter release depends on intracellular rather than ER calcium in cultured Xenopus NMJ without extracellular calcium.

卡维地洛抑制大鼠起搏心肌细胞钙库超载诱导的钙释放

目的:探讨非选择性β受体阻滞剂卡维地洛对起搏心肌细胞钙库超载诱导钙释放(SOICR)的作用及其机制。方法:电刺激起搏大鼠单个心肌细胞并灌注异丙肾上腺素及咖啡因诱导钙库钙超载,从而触发肌浆网钙释放通道(兰尼碱受体2,RyR2)舒张期开放引起SOICR。应用钙离子荧光成像技术记录胞内钙浓度的实时变化。实验分为对照组、卡维地洛组、美托洛尔组、酚妥拉明组和硝苯地平组。结果:(1)与基线刺激时比较,对照组细胞灌注异丙肾上腺素和咖啡因后,起搏细胞钙瞬变振幅显著增高(P<0.01),SOICR的发生率显著增加(P<0.01)。(2)在1~4 Hz起搏频率下卡维地洛组细胞SOICR发生率分别为2.00%、6.00%、10.00%和16.00%,均显著低于对照组(分别为43.59%、74.36%、87.18%和89.74%,均P<0.01);卡维地洛对心肌细胞SOICR的抑制在不同起搏频率下无明显差异(P>0.05)。酚妥拉明、美托洛尔和硝苯地平组细胞与对照组细胞比较,SOICR发生率无差异(均P>0.05)。(3)起搏细胞钙瞬变振幅的比较,各组间相比未见明显差异(P>0.05);咖啡因峰值估测钙库钙总量的比较,各组间也无明显差异(P>0.05)。结论:卡维地洛可明显抑制起搏心肌细胞SOICR的发生,其作用机制可能是直接抑制RyR2的自发性开放而非源于对α1、β1受体和L型钙通道的阻滞作用。

Dynamics of Calcium Signal and Leukotriene C_(4) Release in Mast Cells Network Induced by Mechanical Stimuli and Modulated by Interstitial Fluid Flow

Mast cells(MCs)play an important role in the immune system.Through connective tissues,mechanical stimuli activate intracellular calcium signaling pathways,induce a variety of mediators including leukotriene C4(LTC4)release,and affect MCs’microenvironment.This paper focuses on MCs’intracellular calcium dynamics and LTC4 release responding to mechanical stimuli,explores signaling pathways in MCs and the effect of interstitial fluid flow on the transport of biological messengers and feedback in the MCs network.We use a mathematical model to show that(i)mechanical stimuli including shear stress induced by interstitial fluid flow can activate mechano-sensitive(MS)ion channels on MCs’membrane and allow Ca^(2+)entry,which increases intracellular Ca^(2+)concentration and leads to LTC4 release;(ii)LTC4 in the extracellular space(ECS)acts on surface cysteinyl leukotriene receptors(LTC4R)on adjacent cells,leading to Ca^(2+)influx through Ca^(2+)release-activated Ca^(2+)(CRAC)channels.An elevated intracellular Ca^(2+)concentration further stimulates LTC4 release and creates a positive feedback in the MCs network.The findings of this study may facilitate our understanding of the mechanotransduction process in MCs induced by mechanical stimuli,contribute to understanding of interstitial flow-related mechanobiology in MCs network,and provide a methodology for quantitatively analyzing physical treatment methods including acupuncture and massage in traditional Chinese medicine(TCM).

Effect of Calcium on Spontaneous Quantal Transmitter Secretion from ACh-Loaded Myocytes

Spontaneous secretions occur in both neurons and non-neuronal cells, and calcium is important for these secretion processes. However, the detailed roles of calcium on the secretions have not yet been identified. In the present study, cultured Xenopus myocytes loaded with exogenous acetylcholine （ACh） into the cytoplasm in the absence of extracellular Ca^2＋ undergo spontaneous quantal ACh secretion as detected by the appearance of pulsatile miniature endplate currents. Analysis of the frequencies, amplitudes, and time courses of these currents suggests that similar cellular mechanisms are involved in the secretions of ACh in normal medium and Ca^2＋-free solution. Various doses of ryanodine were used to regulate the intra- cellular Ca^2＋ to different levels. The spontaneous ACh secretion from myocytes in Ca^2＋-free medium was decreased by reducing intracellular Ca^2＋ levels and enhanced by increasing cytosolic Ca^2＋ levels. These observations demonstrate that the spontaneous secretion from isolated myocytes and the effect of ryanodine on ACh-loaded cells are both independent of extracellular Ca^2＋ while Ca^2＋ in the sarcoplasmic reticulum plays a crucial role in the secretions.

雷诺嗪抑制心肌细胞自发性钙漏机制研究

目的:探讨雷诺嗪对心肌细胞钙漏的影响及其机制。方法:分离儿茶酚胺敏感性室速转基因小鼠CASQ2R33Q心室肌细胞,应用钙显像技术探讨雷诺嗪对R33Q细胞内钙转运的影响。结果:91%R33Q细胞在30nmol/L异丙基肾上腺素(Iso)刺激下可诱发自发性钙波;10μmol/L雷诺嗪预处理孵育后,仅40%R33Q细胞可被Iso诱发出自发性钙波(P<0.05)。在Iso诱发自发性钙波后,再灌流雷诺嗪,75%的细胞自发性钙波完全被抑制。雷诺嗪对基础状态下心肌细胞的钙瞬变参数无明显作用,但显著抑制Iso所致钙瞬变幅值增高、延缓Iso所致钙瞬变衰减的加速和降低Iso所致肌浆网钙库的增加。在穿孔的R33Q细胞,雷诺嗪对自发性钙火花参数无明显影响。结论:雷诺嗪可以抑制Iso所致的心肌细胞钙漏,其机制与直接阻滞兰尼丁受体无关,雷诺嗪具有β受体阻滞效应是可能机制之一。