Intracellular Signaling In Endocrine Cells

内分泌细胞的细胞内信号传导

• 批准号: 7333387
• 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/7333387
• 关键词: Intracellular; Signaling; Endocrine; Cells

We investigate cellular signaling cascades and secretion in neuroendocrine cells, with special emphasis on the interactions between plasma membrane electrical events and receptor-controlled pathways. The majority of neuroendocrine cells exhibit spontaneous firing of action potentials and calcium transients, and hormonal stimulation leads to up- or down-regulation of electrical activity and voltage-gated calcium influx-dependent secretion through a complex cascade of events. We are currently studying the biophysical basis of pituitary cell type-specific calcium signaling-secretion coupling, the metabolism and roles of cyclic nucleotides in regulation of electrical activity and calcium signaling, and the expression pattern and molecular properties of calcium-conducting P2X receptor channels. Our recent experiments in anterior pituitary cells indicated that both calcium influx inhibitable and insensitive adenylyl cyclase (AC) subtypes contributed to the basal cAMP production, and that soluble guanylyl cyclase (sGC) was exclusively responsible for basal cGMP production. Inhibition of basal AC activity but not sGC activity reduced prolactin (PRL) release. In contrast, forskolin stimulated cAMP and cGMP production as well as pacemaking, calcium influx, and PRL secretion. Elevation of cAMP and cGMP levels by inhibition of phosphodiesterase activity was also accompanied by increased PRL release. The AC inhibitors attenuated forskolin-stimulated cyclic nucleotide production, calcium influx, and PRL release. Cell-permeant 8-Br-cAMP stimulated firing of action potentials and PRL release, and rescued hormone secretion in cells with inhibited ACs in an extracellular calcium-dependent manner, whereas 8-Br-cGMP and 8-pCPT-2Me-cAMP were ineffective. Protein kinase A inhibitors did not change spontaneous and forskolin-stimulated pacemaking, calcium influx, and PRL release. Hyperpolarization-activated (HCN) channels are members of cyclic nucleotide-gated channels and may thus mediate the action of cAMP on pacemaking activity in pituitary cells. Consistent with this hypothesis, our RT-PCR analysis revealed the presence of mRNA transcripts for HCN2, HCN3 and HCN4 subunits in these cells. Furthermore, hyperpolarization of the membrane potential below -60 mV elicited a slowly activating voltage-dependent and caesium and ZD7288-sensitive inward current (Ih) in the majority of tested cells. Receptor- and non-receptor-mediated activation of AC and sGC, and the addition of the membrane permeant cAMP analogue, 8-Br-cAMP, did not affect Ih. Inhibition of basal AC activity, but not basal sGC activity, caused reduction of the peak amplitude and a leftward shift in the activation curve of Ih. Such inhibition of current was reversed by stimulation of AC with forskolin and by the addition of 8-Br-cAMP. Application of caesium had no significant effect on the resting membrane potential or electrical activity, whereas ZD7288 exhibited complex and Ih-independent effects on spontaneous electrical activity, calcium signalling, and prolactin release. These results indicate that HCN channels in pituitary cells are under tonic activation by the basal level of cAMP and are not critical for spontaneous firing of action potentials. Lipid membranes are virtually impermeable to cyclic nucleotides and their extrusion against a concentration gradient is consistent with operation of an ATP-dependent and probenecid-sensitive transport mediated by plasma membrane proteins, termed cyclic nucleotide efflux pumps. Such energy-dependent cyclic nucleotide cellular efflux is operative in numerous eukaryotic cells and could be mediated by the multidrug resistance proteins MRP4, MRP5, and MRP8. Our recent experiments indicated that cellular efflux of cyclic nucleotides was also detectable in normal and immortalized pituitary cells under resting conditions and was increased after concurrent stimulation of cAMP and cGMP production. In resting and stimulated cells, the efflux pumps transported the majority of de novo produced cGMP, limiting its intracellular accumulation to a concentration range of 1-2 ?M. In contrast, only a small fraction of cAMP was released and there was a time- and concentration-dependent accumulation of this messenger in the cytosol, ranging from 1 to 100 ?M. Stimulation and inhibition of cGMP production alone did not affect cAMP efflux, suggesting the operation of two different transport pathways in pituitary cells. The rates of cAMP and cGMP effluxes were comparable and both pathways were blocked by probenecid and progesterone. Normal pituitary cells expressed mRNA transcripts for MRP4, MRP5, and MRP8, whereas immortalized GH3 pituitary cells expressed only transcripts for MRP5. Down-regulation of MRP5 expression in GH3 cells decreased cGMP release without affecting cAMP efflux. These results indicate that cyclic nucleotide cellular efflux has a critical role in elimination of intracellular cGMP but not cAMP in pituitary cells and that such selectivity is achieved by expression of MRP5. P2X receptors are a family of ligand-gated cation channels composed of two transmembrane domains, with N- and C-termini located intracellularly and a large extracellular loop containing the ATP binding domain. To measure the inter-subunit interaction in living cells, the efficient transfer of bioluminescent resonance energy between luciferase and fluorescent proteins attached to the N- or C-subunit termini of these subunits was used. The constitutive interactions between the full-length C-termini of P2X2 receptor were detected by a significant increase in fluorescence/luminescence intensity ratio compared with negative controls. Moreover, interactions between C-termini, and between C- and N-termini of adjacent subunits, were significantly enhanced in homomeric and heteromeric receptors containing P2X2b or P2X2e subunits. Finally, deletion of two amino acids at the splicing junction, but not at the C-terminal end of the P2X2b receptor, caused enhancement of channel desensitization and luminescence resonance energy transfer. These results indicate that C-terminal structure has a critical role in the cytoplasmic intersubunit interactions, and suggest that the extent of subunit interactions before ATP application could contribute to the subsequent channel activity and conformation changes associated with agonist-dependent desensitization. We also progressed in characterizing the expression and electrophysiological properties of P2X receptors in pituitary gonadotrophs from embryonic, neonatal, and adult rats. In cells from all three age groups, the calcium-mobilizing agonist GnRH induced oscillatory, hyperpolarizing, non-desensitizing, and slow deactivating currents. In contrast, ATP induced non-oscillatory, depolarizing, slowly desensitizing, and rapidly deactivating current, indicating that these cells express cation-conducting P2X channels but not calcium-mobilizing P2Y receptors. The amplitudes of P2X current response and the rates of receptor desensitization were dependent on ATP concentration. The biophysical and pharmacological properties of P2X currents were consistent with the expression of the P2X2 subtype of channels in these cells. ATP-induced rapid depolarization of gonadotrophs lead to initiation of firing in quiescent cells, an increase in the frequency of action potentials in spontaneously active cells, and a transient stimulation of LH release. ATP also influenced GnRH-induced current and membrane potential oscillations, as well as LH release, in an extracellular Ca2+-dependent manner. These results indicate that P2X receptors in gonadotrophs could operate as pacemaking channels and modulators of GnRH-regulated electrical activity and secretion.

我们研究了神经内分泌细胞中细胞信号传导级联反应和分泌，特别强调了质膜电气事件与受体控制途径之间的相互作用。大多数神经内分泌细胞表现出对动作电位和钙瞬变的自发性解散，激素刺激会导致电活动的上调或下调电活动和电压门控钙依赖性依赖性分泌，并通过复杂的事件级联反应。我们目前正在研究垂体细胞类型特异性钙信号 - 分泌耦合的生物物理基础，环状核苷酸在调节电活动和钙信号传导调节中的代谢和作用，以及钙传导P2X受体通道的表达模式和分子特性。 我们最近在前垂体细胞中进行的实验表明，钙流入可抑制和不敏感的腺苷酸环化酶（AC）亚型有助于基础cAMP的产生，并且可溶性鸟叶兰氏环酶（SGC）仅负责基础CGMP产生。抑制基础AC活性，但不抑制SGC活性会降低催乳素（PRL）释放。相比之下，福斯科林刺激了CAMP和CGMP的产生以及起搏，钙涌入和PRL分泌。通过抑制磷酸二酯酶活性的cAMP和CGMP水平的升高也伴随着PRL释放的增加。 AC抑制剂减弱了孔科林刺激的环状核苷酸产生，钙涌入和PRL释放。细胞 - 磨损8-BR训练营刺激了动作电位和PRL释放的发射，并以细胞外钙依赖性方式挽救了抑制ACS的细胞中的激素分泌，而8-BR-CGMP和8-PCPT-2ME-CAMP的效果则无效。蛋白激酶A抑制剂没有改变自发性和福斯科蛋白刺激的起搏，钙流入和PRL释放。超极化激活（HCN）通道是环状核苷酸门控通道的成员，因此可以介导cAMP对垂体细胞中的起搏活性的作用。与这一假设一致，我们的RT-PCR分析揭示了这些细胞中HCN2，HCN3和HCN4亚基的mRNA转录本。此外，低于-60 mV的膜电位的超极化引起了大多数测试细胞的缓慢激活电压依赖性和剖宫产和ZD7288敏感的内电流（IH）。受体和非受体介导的AC和SGC的激活，以及8-Br-cAMP的膜笼cAMP类似物的添加不影响IH。基础AC活性的抑制，而不是基础SGC活性，导致峰幅度降低和IH激活曲线的向左移动。通过使用福斯科蛋白刺激AC并添加8-BR-cAMP，对电流的抑制作用逆转。剖宫产的应用对静息膜电位或电活动没有显着影响，而ZD7288对自发性电活动，钙信号传导和催乳素释放表现出复杂的和IH独立的影响。这些结果表明，垂体细胞中的HCN通道受cAMP的基础水平的强化激活，对于自发发射动作电位并不是至关重要的。 脂质膜实际上是对环状核苷酸的不渗透，它们针对浓度梯度的挤出与质膜蛋白介导的ATP依赖性和探针敏感的转运的运行是一致的，该转运被称为环状核苷酸外排泵。这种能量依赖性的环状核苷酸细胞外排在许多真核细胞中都是可操作的，可以通过多药耐药蛋白MRP4，MRP5和MRP8介导。我们最近的实验表明，在静止条件下，在正常和永生的垂体细胞中也可以检测到环状核苷酸的细胞外排，并在同时刺激CAMP和CGMP产生后增加。在静止和刺激的细胞中，外排泵运输了大多数从头产生的CGMP，将其细胞内积累限制为1-2？m的浓度范围。相反，仅释放了一小部分cAMP，并且该信使在细胞质中存在时间和浓度依赖性的积累，范围为1至100？m。仅刺激和抑制CGMP产生并不影响cAMP外排，这表明垂体细胞中两种不同的传输途径的运行。 CAMP和CGMP外排的速率是可比的，并且两种途径都被探针和孕酮阻塞。正常垂体细胞表达了MRP4，MRP5和MRP8的mRNA转录本，而永生的GH3垂体细胞仅表示MRP5的转录本。 GH3细胞中MRP5表达的下调降低了CGMP的释放而不会影响CAMP外排。这些结果表明，环状核苷酸细胞外排在消除垂体细胞中的细胞内CGMP而不是cAMP中具有关键作用，并且通过MRP5的表达来实现这种选择性。 P2X受体是由两个跨膜结构域组成的配体门控阳离子通道的家族，其n-和c-termini位于细胞内，一个含有ATP结合结构域的大细胞外环。为了测量活细胞中的亚基间相互作用，使用了这些亚基的N-或c-Subunit末端附着的荧光素酶和荧光蛋白之间的生物发光共振能的有效转移。与阴性对照相比，通过荧光/发光强度比显着增加，可以检测到P2X2受体的全长C末端之间的组成型相互作用。此外，在包含P2X2B或P2X2E亚基的同源和异源受体中，C-末端之间以及相邻亚基的C和N末端之间的相互作用显着增强。最后，在剪接连接处的两个氨基酸的缺失，但在P2X2B受体的C末端并非导致通道脱敏和发光共振能传递的增强。这些结果表明，C末端结构在细胞质间相互作用中具有关键作用，并表明在ATP应用之前的亚基相互作用的程度可能有助于随后的通道活性和与依赖激动剂依赖性脱敏相关的构象变化。我们还取得了表征，表征了来自胚胎，新生儿和成年大鼠的垂体促性腺营养性p2x受体的表达和电生理特性。在所有三个年龄段的细胞中，钙动力型激动剂GNRH诱导振荡，超极化，非敏感性和缓慢失活的电流。相比之下，ATP诱导非振荡，去极化，缓慢脱敏并迅速停用电流，表明这些细胞表达阳离子传统的P2X通道，但不表达钙的P2Y受体。 P2X电流反应的幅度和受体脱敏的速率取决于ATP浓度。 P2X电流的生物物理和药理特性与这些细胞中通道的P2X2亚型的表达一致。 ATP诱导的促性腺营养快速去极化导致静态细胞发射的引发，自发活性细胞中动作电位的频率增加以及LH释放的瞬时刺激。 ATP还以细胞外Ca2+依赖性方式影响了GNRH诱导的电流和膜电位振荡以及LH释放。这些结果表明，促性腺营养中的P2X受体可以作为GNRH调节的电活动和分泌的起搏通道和调节剂的作用。