INTRACELLULAR SIGNALING IN ENDOCRINE CELLS

GnRH和ET受体与磷脂酶C和D依赖性的偶联 在垂体细胞中检测细胞内信号。 GnRH诱导两种 初始和持续的IP 3/DAG产生，而ET-1仅诱导了 短暂反应，与GnRH诱导的DAG相当 在蛋白激酶C耗尽的细胞中的反应。 GnRH和ET诱导 磷脂酶C抑制剂降低了IP 3/DAG和钙反应， U 73122和新霉素。 这两种磷脂酶抑制剂也降低了 激动剂诱导的培养垂体细胞LH释放。 相比 磷脂酶C、磷脂酶D活性被激动剂刺激 促性腺激素释放激素（GnRH）而不是内皮素受体的激活。 此外，GnRH-而不是ET- 诱导的DAG形成减少，在存在的两个 磷脂酶D抑制剂，乙醇和心得安，以及DAG的下降 促性腺激素释放激素（GnRH）诱导的c-fos表达受到抑制 表情 我们还研究了这些之间的相互作用 受体介导的细胞内信号和质膜电 事件 结果表明，垂体细胞具有兴奋性， 每一个钙峰都是由在一个单一的钙离子通道中的钙离子进入产生的。 动作电位（AP）。AP和钙振荡的频率 促性腺激素细胞通过去极化/超极化脉冲来调节。 利用这些和其他数据对质膜通道表征 在前面提供的促性腺激素细胞中， 开发了预测时空钙波动的方法。 模型 包含一组Hodgkin-Huxley方程， 钙向细胞中心扩散。这表明AP诱导的 钙尖峰仅在邻近的薄壳层中突出， 细胞表面 我们还发现，在GnRH刺激的细胞中， 在持续阶段期间的去极化脉冲可以引起瞬态 钙的上升类似于内源性循环。 此外，钙进入 在一个单一的去极化脉冲期间， 随后的内源性钙振荡，而不影响其 频率. 两个连续去极化脉冲的应用表明， 第二次脉冲引起的钙离子升高的大小取决于 两个连续脉冲之间的时间流逝，表明每个脉冲 内源性或诱发性钙升高周期使钙释放 促性腺激素细胞处于不应状态的机制。 电活动 可能在磷脂酶C诱导的 钙振荡，包括重置振荡器的时钟。

Coupling of GnRH and ET receptors with phospholipase C- and D-dependent intracellular signals was examined in pituitary cells. GnRH induced both initial and sustained IP3/DAG production, while ET-1 induced only a transient response, a profile that was comparable to the GnRH-induced DAG response in protein kinase C-depleted cells. GnRH- and ET-induced IP3/DAG and calcium responses were reduced by phospholipase C inhibitors, U73122 and neomycin. Those two phospholipase inhibitors also reduced agonist-induced LH release by cultured pituitary cells. In contrast to phospholipase C, phospholipase D activity was stimulated by agonist activation of GnRH but not ET receptors. Furthermore, GnRH- but not ET- induced DAG formation was reduced in the presence of the two phospholipase D inhibitors, ethanol and propanolol, and the fall in DAG production was accompanied by the inhibition of GnRH-induced c-fos expression. We have also examined the interactions between these receptor-mediated intracellular signals and plasma membrane electrical events. The results showed that pituitary cells are excitable and that each calcium spike is produced by the calcium entry during a single action potential (AP). The frequency of APs and calcium oscillations in gonadotrophs was modulated by depolarization/hyperpolarization pulses. Using these and other data on plasma membrane channel characterization in gonadotrophs provided earlier, a quantitative mathematical model predicting spatio-temporal calcium oscillations was developed. The model contains a set of Hodgkin-Huxley-like equations and the equation for calcium diffusion towards the cell center. It suggests that AP-induced calcium spiking is prominent only in a thin shell layer neighboring the cell surface. We also found that in GnRH-stimulated cells a brief depolarization pulse during the sustained phase can elicit a transient calcium rise similar to the endogenous cycle. In addition, calcium entry during a single depolarizing pulse was found to shift the phase of subsequent endogenous calcium oscillations, without affecting their frequency. The application of two consecutive depolarizing pulses showed that the size of the calcium rise evoked by the second pulse depended on the time lapsed between two consecutive pulses, indicating that each endogenous or evoked calcium rise cycle leaves the calcium release mechanism of the gonadotroph in a refractory state. Electrical activity may play an important role in the modulation of phospholipase C-induced calcium oscillations, including resetting the clock of the oscillator.