INTRACELLULAR SIGNALING IN ENDOCRINE CELLS

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，以及质膜电事件和受体介导的信号之间的相互作用。目前的重点是下丘脑神经元(GT1细胞)和垂体促生长激素细胞中动作电位(AP)驱动的钙信号的特征。我们的分析表明，大多数GT1细胞表现出自发的、细胞外钙依赖的AP，这是由缓慢的起搏器去极化启动的。更多的超极化细胞发出尖锐的AP，但促进钙内流的能力有限，而更多的去极化细胞发出更广泛的AP，促进钙内流的能力增强。对这些细胞的内向电流的表征表明，存在TTX敏感的钠以及T和L类型的钙成分。钠通道和T型钙通道的可用性取决于基线电位，而基线电位决定了这些通道的激活/失活状态。这三种通道都参与了尖锐AP的产生，而L型通道仅负责表现为宽幅AP的细胞的峰去极化。GnRH受体的激活导致细胞内钙浓度的双相变化，出现一个早期的细胞外钙非依赖性峰值和一个持续的细胞外钙依赖性阶段。在钙反应高峰期间，由于短暂的超极化，电活动被取消。随后，细胞持续去极化，放电频率增加，从尖锐的AP转变为广泛的AP。短暂的超极化是由细胞内钙离子的初始尖峰引起的，并由SK-型钾通道介导，该通道在随后的去极化阶段也起作用。激动剂诱导的去极化和放电增加不依赖于细胞内钙离子，也不是通过抑制钾电流，而是通过促进电压不敏感的钙传导内向电流。内质网中表达的钙泵阻滞剂thapsigargin的存储耗竭也激活了这一内向去极化电流，并增加了放电频率。这些结果表明，在未经刺激和激动剂刺激的GT1细胞中，膜去极化限制了钠通道和T型通道对放电的参与，但促进了AP驱动的钙内流。此外，GT1神经元的放电模式受钙控制的SK电流和储存耗竭激活的钙电流的协调调节。与GT1神经元一样，促生长激素细胞也表现出自发AP放电的周期，从而产生胞浆钙的高幅度波动。与GT1神经元相比，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。我们已经在混合的垂体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部位，但没有发现ET-B受体的信号和结合部位。这些受体被ET-1激活，导致三磷酸肌醇的产生增加，细胞内钙和GH分泌增加。ET-1的钙动员作用持续2-3分钟，随后AP驱动的钙内流和GH分泌被抑制到基础水平以下。这种抑制伴随着腺酰环化酶活性的下调和内向整流钾电流的刺激。在经百日咳毒素处理的生长激素细胞中，ET-1诱导的钙动员相被保留下来，但紧随其后的是促进钙内流和GH分泌。在经百日咳毒素处理的细胞中，ET-1对腺苷环化酶活性的抑制作用被取消。这些结果表明，促生长激素细胞内钙激活的ET-A受体与GI/GO通路的短暂交叉偶联是在持续激动剂刺激下改变钙信号和GH分泌节律的有效机制。因此，与GT1细胞中GnRH的作用不同，在GT1细胞中，钙动员阶段伴随着促进电压门控钙内流，而在ET刺激的促生长激素细胞中，这两个阶段是瞬时分离的。

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.