INTRACELLULAR SIGNALING IN ENDOCRINE CELLS

内分泌细胞中的细胞内信号传导

• 批准号: 6290161
• 负责人: STANKO S. STOJILKOVIC
• 金额: --
• 依托单位: EUNICE KENNEDY SHRIVER NATIONAL INSTITUTE OF CHILD HEALTH & HUMAN DEVELOPMENT
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/6290161
• 关键词: adenosine triphosphate; biological signal transduction; calcium channel; calcium flux; gonadotropin releasing factor; hormone receptor; inositol phosphates; luteinizing hormone; nucleotidases; purinergic receptor; receptor coupling; voltage gated channel

This project addresses the cellular signaling cascade in endocrine and neuroendocrine cells and the interactions between plasma membrane electrical events and receptor-mediated signaling. Current emphasis is on the characterization of action potential (AP)-driven calcium signaling in hypothalamic neurons (GT1 cells) and pituitary somatotrophs. Our analysis revealed that most GT1 cells exhibited spontaneous, extracellular calcium-dependent APs, which was initiated by a slow pacemaker depolarization. More hyperpolarized cells fired sharp APs with limited capacity to promote calcium influx, whereas more depolarized cells fired broad APs with enhanced capacity for calcium influx. Characterization of the inward currents in these cells revealed the presence of TTX-sensitive sodium and T- and L-type calcium components. The availability of sodium and T-type calcium channels was dependent on the baseline potential, which determined the activation/inactivation status of these channels. Whereas all three channels were involved in the generation of sharp APs, L-type channels were solely responsible for the spike depolarization in cells exhibiting broad APs. Activation of GnRH receptors led to biphasic changes in intracellular calcium concentration with an early, extracellular calcium-independent peak and a sustained, extracellular calcium-dependent phase. During the peak calcium response, electrical activity was abolished due to transient hyperpolarization. This was followed by sustained depolarization of cells and resumption of firing of increased frequency with a shift from sharp to broad APs. The transient hyperpolarization was caused by the initial spike in cytosolic calcium and was mediated by SK-type potassium channels, which were also operative during the subsequent depolarization phase. Agonist-induced depolarization and increased firing were independent of cytosolic calcium and were not mediated by inhibition of potassium current, but by facilitation of a voltage-insensitive calcium-conducting inward current. Store depletion by thapsigargin, a blocker of calcium pump expressed in endoplasmic reticulum, also activated this inward depolarizing current and increased the firing frequency. These results indicate that in both unstimulated and agonist-stimulated GT1 cells, membrane depolarization limits the participation of sodium and T-type channels in firing, but facilitates AP-driven calcium influx. Furthermore, the pattern of firing in GT1 neurons is coordinately regulated by calcium-controlled SK current and the store depletion-activated calcium current. Like GT1 neurons, somatotrophs also exhibited periods of spontaneous AP firing that generated high amplitude fluctuations in cytosolic calcium. In contrast to GT1 neurons, no information was available on the expression and coupling of calcium-mobilizing receptors and their role in the control of electrical activity. We have found the message and the specific binding sites for ET-A but not ET-B receptors in mixed pituitary cells and in highly purified somatotrophs. Activation of these receptors by ET-1 led to an increase in inositol trisphosphate production and the associated rise in cytoslic calcium and GH secretion. The calcium-mobilizing action of ET-1 lasted for 2-3 minutes and was followed by an inhibition of AP-driven calcium influx and GH secretion to below the basal levels. This inhibition was accompanied by the down regulation of adenylyl cyclase activity and by the stimulation of inward rectifier potassium current. In somatotrophs treated with pertussis toxin overnight, the ET-1-induced calcium-mobilizing phase was preserved, but was immediately followed by facilitated calcium influx and GH secretion. ET-1-induced inhibition of adenylyl cyclase activity was abolished in pertussis toxin-treated cells. These results indicate that the transient cross-coupling of calcium-mobilizing ET-A receptors to Gi/Go pathway in somatotrophs provides an effective mechanism to change the rhythm of calcium signaling and GH secretion during continuous agonist stimulation. Thus, in contrast to GnRH action in GT1 cells, where calcium-mobilization phase was accompanied with facilitated voltage-gated calcium influx, these two phases in ET-stimulated somatotrophs were transiently dissociated.

该项目解决了内分泌和神经内分泌细胞中的细胞信号级联，以及质膜电气事件与受体介导的信号传导之间的相互作用。当前的重点是下丘脑神经元（GT1细胞）和垂体淋巴瘤中动作电位（AP）驱动的钙信号传导的表征。我们的分析表明，大多数GT1细胞表现出自发的细胞外钙依赖性AP，这是由缓慢的起搏器去极化引发的。更多的超极化细胞发射了锋利的AP，其能力有限，无法促进钙涌入，而更多的去极化细胞发射了宽阔的AP，具有增强的钙涌入能力。这些细胞中内流电流的表征揭示了TTX敏感的钠以及T-和L型钙成分的存在。钠和T型钙通道的可用性取决于基线电位，这确定了这些通道的激活/灭活状态。尽管所有三个通道都参与了尖锐的AP的产生，但L型通道完全负责显示宽AP的细胞中的尖峰去极化。 GnRH受体的激活导致细胞内钙浓度的双相变化，其早期的细胞外钙独立峰和持续的细胞外钙依赖性相。在峰值钙反应期间，由于短暂的超极化而消除了电活动。随后是细胞的持续去极化，并随着从尖锐的AP转移而恢复频率增加的频率。瞬态超极化是由胞质钙的初始峰值引起的，并由SK型钾通道介导，后者在随后的去极化阶段也是可操作的。激动剂诱导的去极化和发射增加与胞质钙无关，不是通过抑制钾电流而介导的，而是通过促进不敏感的钙导向电流来介导的。在内质网中表达的钙泵的阻滞剂Thapsigargin的存储耗竭也激活了这种内向的去极化电流并增加了点火频率。这些结果表明，在未刺激和激动剂刺激的GT1细胞中，膜去极化限制了钠和T型通道在发射中的参与，但促进了AP驱动的钙涌入。此外，GT1神经元中的发射模式由钙控制的SK电流和存储耗尽激活的钙电流协调调节。像GT1神经元一样，人体营养也表现出自发性AP放电时期，在胞质钙中产生了高振幅波动。与GT1神经元相反，没有关于钙振动受体的表达和耦合及其在控制电活动中的作用的信息。我们已经找到了ET-A的信息和特定结合位点，但在混合垂体细胞和高度纯化的植物营养中没有ET-B受体。 ET-1通过ET-1激活这些受体导致肌醇三磷酸盐产生的增加以及胞质钙和GH分泌的相关升高。 ET-1的钙动作持续了2-3分钟，随后抑制了AP驱动的钙流入和GH分泌至基础水平以下。这种抑制作用伴随着腺苷环化酶活性的下调和刺激内向整流钾电流的刺激。在用百日咳毒素治疗的夜间营养循环中，保留了ET-1诱导的钙液化相，但紧随其后的是促进钙的流入和GH分泌。 ET-1诱导的对百日咳毒素处理的细胞中抑制腺苷酸环化酶活性的抑制作用。这些结果表明，钙模化ET-A受体与gi/go途径的瞬时交叉偶联在持续激动剂刺激过程中更改钙信号传导和GH分泌的节奏提供了有效的机制。因此，与GT1细胞中的GnRH作用相反，GNRH的作用与钙润滑阶段伴有促进的电压门控钙涌入，这两个阶段在ET刺激的植物营养中瞬时分离。