INTRACELLULAR SIGNALING IN ENDOCRINE CELLS

本计画探讨内分泌及神经内分泌细胞的细胞信号级联反应，以及质膜电性事件与受体介导的信号传导之间的相互作用。目前的重点是表征动作电位（AP）驱动的钙信号在下丘脑神经元（GT1细胞）和垂体生长激素。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，这是由一个缓慢的起搏器去极化。更多的超极化细胞发射尖锐的AP有限的能力，以促进钙离子内流，而更多的去极化细胞发射广泛的AP增强能力的钙离子内流。在这些细胞的内向电流的表征揭示了TTX敏感的钠和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，其决定了这些通道的激活/失活状态。尽管所有三个通道都参与了尖锐AP的生成，但L型通道仅负责表现出宽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，早期，细胞外钙非依赖性峰值和持续的细胞外钙依赖性阶段。在峰值钙反应期间，由于瞬时超极化，电活动被取消。随后是持续的细胞去极化和恢复发射的频率增加，从尖锐的广泛AP的转变。瞬时超极化是由胞质钙离子的初始尖峰引起的，并由SK型钾通道介导，在随后的去极化阶段也起作用。激动剂诱导的去极化和增加射击是独立的胞浆钙，并不介导的抑制钾电流，但通过促进电压不敏感的钙传导内向电流。内质网表达的钙泵阻断剂毒胡萝卜素消耗钙库也激活了这种内向去极化电流，增加了放电频率。这些结果表明，在未受刺激和激动剂刺激的GT1细胞，膜去极化限制参与钠和T型通道在射击，但促进AP驱动的钙离子内流。此外，GT1神经元的放电模式是由钙控制的SK电流和钙池耗尽激活的钙电流协调调节的。像GT1神经元一样，生长激素细胞也表现出自发AP放电的周期，产生细胞溶质钙的高幅度波动。与GT1神经元相反，没有关于钙动员受体的表达和偶联及其在电活动控制中的作用的信息。我们在垂体混合细胞和高纯度的生长激素细胞中发现了ET-A受体的信息和特异性结合位点，但未发现ET-B受体。ET-1对这些受体的激活导致三磷酸肌醇的产生增加，并导致胞浆钙和GH分泌增加。ET-1的钙动员作用持续2 - 3分钟，随后抑制AP驱动的钙内流和GH分泌至低于基础水平。这种抑制伴随着腺苷酸环化酶活性的下调和内向整流钾电流的刺激。在生长激素细胞与百日咳毒素处理过夜，ET-1诱导的钙动员期被保存，但随后立即促进钙内流和GH分泌。ET-1诱导的腺苷酸环化酶活性的抑制被废除在百日咳毒素处理的细胞。这些结果表明，钙动员ET-A受体的瞬时交叉偶联生长激素细胞的Gi/Go通路提供了一个有效的机制，改变钙信号和GH分泌的节奏在连续激动剂刺激。因此，在GT1细胞，钙动员相伴随着促进电压门控钙内流的GnRH作用相反，这两个阶段在ET刺激的生长激素细胞被短暂分离。

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.