Intracellular Signaling In Endocrine Cells

内分泌细胞的细胞内信号传导

• 批准号: 7198282
• 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/7198282
• 关键词: Intracellular; Signaling; Endocrine; Cells

Studies on calcium signaling and cellular regulation are focused on the biophysical basis of pituitary cell type-specific calcium signaling-secretion coupling, the roles of G protein-coupled and tyrosine kinase receptors in control of signaling, hormone secretion and gene expression, and the molecular dissection of ATP-gated P2X receptor channels. Our earlier studies revealed that cultured pituitary cells, including lactotrophs, frequently exhibit larger membrane potential oscillations, on top of which the depolarizing plateau and bursts of action potentials are generated, with spikes that usually do not reach the reverse potential. In lactotrophs such spontaneous electrical activity and the associated voltage-gated calcium influx are sufficient to maintain high prolactin release. The finding that normal and immortalized pituitary cells express calcium-inhibitable adenylyl cyclases prompted us to examine the hypothesis that cyclic nucleotides may have a role in spontaneous pacemaking and basal prolactin release in these cells by controlling hyperpolarization-activated cation channels and/or cyclic nucleotide-gated channels. Consistent with this, we found that stimulation of adenylyl cyclases by forskolin initiates firing of action potentials in quiescent lactotrophs and increases the spiking frequency in spontaneously active cells. This in turn facilitates voltage-gated calcium influx and prolactin secretion. Inhibition of phosphodiesterases by 3-isobutyl-1-methylxanthine also stimulates cyclic nucleotide accumulation and prolactin release. Conversely, MDL-12330A inhibits basal and forskolin-stimulated cyclic nucleotide production in a concentration-dependent manner, as well as electrical activity, calcium transients, and prolactin secretion. Basal cyclic AMP production is augmented by removal of extracellular calcium and is attenuated by facilitation of voltage-gated calcium influx. These results suggest that the intrinsic activity of calcium-inhibitable adenylyl cyclases contributes to the control of spontaneous pacemaking activity. Our results further indicate that endothelin receptors inhibit voltage-gated calcium influx-dependent prolactin release. However, these receptors inhibit secretion downstream of voltage-gated calcium influx and in a phospholipase C and tyrosine kinase-independent manner. We also found that endothelin receptors are coupled to both pertussis toxin-sensitive and insensitive Gi proteins. Finally, we discovered that the coupling of endothelin receptors to the Gz signaling pathway accounts for inhibition of prolactin secretion downstream of voltage-gated calcium influx. Sustained inhibition of secretion is achieved through down-regulation of the adenylyl cyclase signaling cascade, whereas rapid inhibition also occurs at elevated cAMP levels regardless of the status of phospholipase C, tyrosine kinases, and protein kinase C. These results indicate that the coupling of seven transmembrane domain receptors to Gz proteins provides a pathway that effectively blocks hormone secretion for a prolonged time without interfering with pacemaking activity and calcium influx-dependent cellular functions. We previously found that the purinergic signaling system is operative in normal and immortalized anterior pituitary cells. These cells release ATP under resting conditions and in response to activation of calcium mobilizing receptors. However, there is no correlation between the rate of basal hormone and ATP release, suggesting that ATP is not co-secreted with hormones by regulated exocytosis. Experiments in progress are directed toward the characterization of a pathway responsible for ATP release. These cells also express ecto-nucleotidases, which hydrolyze ATP, resulting in formation of the respective nucleoside and free phosphate. The transcripts for ecto-nucleotidase eNTPDase 1-3 were found in pituitary cells. The products of this hydrolytic cascade, ADP and adenosine, also act as extracellular messengers by activating distinct plasma membrane receptors. These receptors are termed purinergic and belong to two groups: P1 and P2 receptors. P2X receptors are a family of ligand-gated cation channels composed of two transmembrane domains, N- and C-termini located intracellularly, and a large extracellular loop containing the ATP binding domain. To identify regions important for binding and gating, our experimental work with recombinant channels is focused on chimeras and point mutagenesis of conserved ectodomain residues. Mutant channels were expressed in human embryonic kidney 293 cells and mouse gonadotropin-releasing hormone-secreting GT1 neurons and analyzed using calcium imaging and patch clamp techniques. Experiments with chimeric P2XRs helped in characterization of gating and ionic conduction, deactivation of receptors, structural determinants of receptor desensitization and recovery from desensitization. To identify regions important for ATP binding we used the known sequence and secondary structure similarities between the Lys180-Lys326 ectodomain region of P2X4 and the class II aminoacyl-tRNA synthetases as a guide to generate a three-dimensional model of the receptor-binding site and to design mutants. The interplay between homology modeling and site-directed mutagenesis suggested that the Asp280 residue of P2X4R coordinates ATP binding via the magnesium ion, Phe230 coordinates the binding of the adenine ring of ATP, and Lys190, His286 and Arg278 coordinate the actions of negatively charged alpha, beta, and gamma phosphate groups, respectively. Until the crystal structure of the channel is solved, this model could provide a useful approach for future studies on identification of ATP binding domain and gating of P2XRs.

Studies on calcium signaling and cellular regulation are focused on the biophysical basis of pituitary cell type-specific calcium signaling-secretion coupling, the roles of G protein-coupled and tyrosine kinase receptors in control of signaling, hormone secretion and gene expression, and the molecular dissection of ATP-gated P2X receptor channels.我们的较早研究表明，培养的垂体细胞（包括乳营养素）经常显示出更大的膜电位振荡，其上是产生去极化的高原和动作电位爆发的，通常无法达到反向电位的尖峰。在乳营养素中，这种自发性电活动和相关的电压门控钙的流入足以维持高催乳素释放。正常和永生的垂体细胞表达可抑制钙的腺苷酸循环酶的发现促使我们研究了一个假说，即环状核苷酸可能通过控制超极化激活的阳离子阳离子阳离子通道和/或环状核透明核苷透明核苷透明核定通道在这些细胞中的自发起搏和基底催乳素释放中发挥作用。与此相一致，我们发现，通过福斯科蛋白来刺激腺苷酸环化酶在静止的乳酸营养中引发动作电位的发射，并增加了自发活性细胞中的尖峰频率。反过来，这促进了电压门控钙的流入和催乳素分泌。 3-异丁基-1-甲基黄嘌呤抑制磷酸二酯酶还刺激环状核苷酸的积累和催乳素释放。相反，MDL-12330a以浓度依赖性的方式以及电活动，钙瞬变和催乳素分泌抑制基底和福斯科蛋白刺激的环状核苷酸产生。通过去除细胞外钙来增加基础环状AMP的产生，并通过促进电压门控钙涌入来减弱。这些结果表明，可抑制钙的腺苷酸环化酶的内在活性有助于控制自发起搏活性。我们的结果进一步表明，内皮素受体抑制电压门控钙依赖性催乳素释放。但是，这些受体抑制了电压门控钙涌入以及磷脂酶C和酪氨酸激酶非依赖性的分泌。我们还发现，内皮素受体均与对百日咳毒素敏感和不敏感的胃肠道蛋白偶联。最后，我们发现内皮素受体与GZ信号通路的偶联解释了电压门控钙涌入下游催乳素分泌的抑制。通过下调腺苷酸环化酶信号传导级联的持续抑制作用，而在较高的营地水平上，无论磷脂酶C C，酪氨酸激酶和蛋白激酶的状态如何长时间的时间不干扰起搏活性和流入钙依赖性细胞功能。 我们先前发现，嘌呤能信号传导系统在正常和永生的前垂体细胞中是可操作的。这些细胞在静止条件下释放ATP，并响应钙动员受体的激活。但是，基础激素和ATP释放速率之间没有相关性，这表明ATP不是通过调节的胞吐作用与激素共归因的。正在进行的实验是针对负责ATP释放的途径的表征。这些细胞还表达了核苷酸酶，该核苷酸酶水解ATP，从而形成了相应的核苷和游离磷酸盐。在垂体细胞中发现了核苷酸酶Entpase 1-3的转录本。这种水解级联ADP和腺苷的产物也通过激活不同的质膜受体来充当细胞外信使。这些受体被称为嘌呤能，属于两组：P1和P2受体。 P2X受体是一个由两个跨膜结构域组成的配体门控阳离子通道的家族，位于n-和c-termini，位于n-和c-末端，以及一个包含ATP结合结构域的大型细胞外环。为了确定对结合和门控重要的区域，我们与重组通道的实验性工作集中在嵌合体和保守性外域残基的点诱变上。突变通道在人类胚胎肾脏293细胞中表达，小鼠促性腺激素释放激素分泌GT1神经元，并使用钙成像和斑块夹技术进行了分析。使用嵌合P2XRS进行的实验有助于表征门控和离子传导，受体失活，受体脱敏的结构决定因素以及从脱敏中恢复。为了确定对ATP结合重要的区域，我们使用了P2X4的LYS180-LYS326胞外域区域与II类氨基酰基-TRNA合成酶之间的已知序列和二级结构相似性作为产生受体结合位点和设计突变体的三维模型的指南。 The interplay between homology modeling and site-directed mutagenesis suggested that the Asp280 residue of P2X4R coordinates ATP binding via the magnesium ion, Phe230 coordinates the binding of the adenine ring of ATP, and Lys190, His286 and Arg278 coordinate the actions of negatively charged alpha, beta, and gamma phosphate groups, respectively.在解决通道的晶体结构之前，该模型可以为鉴定ATP结合域和P2XR的门控的将来的研究提供有用的方法。