Intracellular Signaling In Endocrine Cells

内分泌细胞的细胞内信号传导

基本信息

批准号：
7734676
负责人：
STANKO S. STOJILKOVIC
金额：
$ 113.37万
依托单位：
EUNICE KENNEDY SHRIVER NATIONAL INSTITUTE OF CHILD HEALTH & HUMAN DEVELOPMENT
依托单位国家：
美国
项目类别：
财政年份：
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/7734676
关键词：
Address Affect Agonist Allosteric Regulation Amino Acids Anterior Pituitary Gland Architecture Atrial Natriuretic Factor Attenuated Baclofen Bicuculline Binding Butyric Acid Butyric Acids Calcium Calcium Channel Calcium Signaling Cations Cell membrane Cells Chloride Ion Chlorides Collaborations Communities Condition Confocal Microscopy Coupling Cyclic AMP Cysteine Diazepam Dopamine Dopamine Agonists Dopamine D2 Receptor Dopamine Receptor Dose Dyes Electric Capacitance Endocrine Event Exocytosis FM 4-64 Face Fluorescent Probes Forskolin Frequencies Glycogen Synthase Kinase 3 Gramicidin Helix (Snails)Hormones Hypothalamic structure Investigation Ion Channel Ivermectin Label Lactation Lactones Lipids Lithium Maps Mediating Membrane Membrane Potentials Mental Depression Molecular Molecular Conformation Monitor Muscimol Mutagenesis Mutation Nature Neuroendocrine Cell Neurosecretory Systems Neurotransmitters Osmoregulation P2X-receptor PRL gene Pathway interactions Pattern Pertussis Pertussis Toxin Phorbol Esters Phosphatidylinositide 3-Kinase Inhibitor Physiological Picrotoxin Pituitary Gland Pituitary Hormones Play Potassium Probability Process Production Prolactin Protein Kinase C Protein Subunits Proteins Purpose Radioimmunoassay Range Rattus Recombinants Research Personnel Research Project Grants Reverse Transcriptase Polymerase Chain Reaction Role Running Scanning Schistosoma mansoni Secretory Cell Side Signal Pathway Signal Transduction System Tetradecanoylphorbol Acetate Time Transmembrane Domain Vesicle Work cell type cellular imaging dimer ear helix inhibitor/antagonist inward rectifier potassium channel lactotroph multidisciplinary mutant receptor receptor function receptor sensitivity response voltage voltage clamp wortmannin zolpidem

项目摘要

Our recent work was focused on secretion of prolactin, a hormone that controls lactation, by pituitary lactotrophs and the role of dopamine D2 receptors and hypotonicity in this process. We also investigated the expression and role of gamma-amino butyric acid (GABA)-A receptor-channels in lactotrophs and gonadotrophs and provided structural and functional characterization of recombinant ATP-gated P2X4 receptor-channels that we cloned from pituitary cells. The work with dopamine D2 receptors revealed that their activation in pituitary lactotrophs leads to inhibition of prolactin release. It has been suggested by others that this inhibition occurs through the Gi/o-alpha protein-mediated inhibition of cAMP production and/or Gi/o-beta/gamma dimer-mediated activation of inward rectifier potassium channels and inhibition of voltage-gated calcium channels. We show that the dopamine agonist-induced inhibition of spontaneous calcium influx and release of pre-stored PRL was preserved when cAMP levels were elevated by forskolin treatment. We further observed that dopamine agonists inhibited both spontaneous and depolarization-induced calcium influx in untreated but not in pertussis toxin-treated cells. This inhibition was also observed in cells with blocked inward rectifier potassium channels, suggesting that dopamine effects on voltage-gated calcium channel gating are sufficient to inhibit spontaneous calcium influx. However, agonist-induced inhibition of prolactin release was only partially relieved in pertussis-treated cells, indicating that dopamine receptors also inhibit exocytosis downstream of voltage-gated calcium influx. The pertussis toxin-insensitive step in agonist-induced inhibition of prlactin release was not affected by the addition of wortmannin, an inhibitor of PI3-kinase, and lithium, an inhibitor of GSK-3, but was attenuated in the presence of phorbol ester PMA, which inhibits Gz signaling pathway in a protein kinase C-dependent manner. These results indicate for the first time that dopamine inhibits basal prolactin release not only by blocking voltage-gated calcium influx through the pertussis toxin-sensitive-signaling pathway but also by desensitizing calcium-secretion coupling through the pertussis toxin-insensitive and protein kinase C-sensitive signaling pathway. In collaboration with Dr. Zorecs group, we studied the release of the pituitary hormone prolactin by hypotonicity, because this hormone also contributes to osmoregulation. In perifused rat lactotrophs, hypotonicity resulted in a transient increase followed by a sustained depression of prolactin release, as monitored by radioimmunoassay. In single cells imaged by confocal microscopy, hypotonicity elicited discharge of the fluorescently-labeled atrial natriuretic peptide cargo from 2% of vesicles/cell, which synchronously loaded the styryl dye FM 4-64 through the same fusion pores. In contrast, high potassium-induced depolarization resulted in a response of 10% of vesicles/cell, with different unloading/loading time-course of the two fluorescent probes. In cell-attached studies, discrete changes in the membrane capacitance were recorded in both unstimulated and stimulated conditions, reflecting single vesicle fusion/fissions with the plasma membrane. In stimulated cells, the probability of occurrence of full fusion events was low and unchanged, whereas over 95% of fusion events were transient, with the open fusion pore probability, the average pore dwell-time, the frequency of occurrence, and the fusion pore conductance increased. Hypotonicity only rarely elicited new fusion events in silent membrane patches. The results indicate that, in hypotonicity-stimulated lactotrophs, rapidly releasable vesicles appear pre-fused and release hormone in a kiss-and-run mode. During the last year, we also studied the expression of GABA-A receptor-channels in pituitary cells, their distribution within the secretory anterior pituitary cell types, and nature (stimulatory or inhibitory) of actions. Our results show that mRNAs for all GABAA receptor subunits are expressed in pituitary cells and that alpha1/beta1 subunit proteins are present in all secretory cells. In voltage-clamped gramicidin-perforated cells, GABA induced dose-dependent increases in current amplitude that were inhibited by bicuculline and picrotoxin and facilitated by diazepam and zolpidem in a concentration-dependent manner. In intact cells, GABA and the GABA-A receptor agonist muscimol caused a rapid and transient increase in intracellular calcium, whereas the GABA-B receptor agonist baclofen was ineffective, suggesting that chloride-mediated depolarization activates voltage-gated calcium channels. Consistent with this finding, RT-PCR analysis indicated high expression of NKCC1, but not KCC2 cation/chloride transporter mRNAs in pituitary cells. Furthermore, the GABA-A channel reversal potential for chloride ions was positive to the baseline membrane potential in most cells and the activation of ion channels by GABA resulted in depolarization of cells and modulation of spontaneous electrical activity. These results indicate that secretory pituitary cells express functional GABA-A receptor-channels that are depolarizing. In a work on structural-functional characterization of recombinant P2X4 receptor that was cloned from pituitary cells, the focus on investigations was on identification of residues contributing to allosteric regulation of these channels by ivermectin, a large macrocyclic lactone that specifically enhances P2X4 receptor-channel function by interacting with residues of transmembrane helices in the open conformation state. The sensitivity of this receptor to iveremectin was also used as an indicator in identifying residues in transmembrane regions that face the pore of the channel. For these purposes, we used cysteine-scanning mutagenesis of rat P2X4 transmembrane regions. The receptor function was unchanged by mutations in 29 different residues, and among them the ivermectin effects were altered in Gln36, Leu40, Val43, Val47, Trp50, Asn338, Gly342, Leu346, Ala349, and Ile356 mutants. The substitution-sensitive Arg33 and Cys353 mutants could also be considered as ivermectin-sensitive hits. The pattern of these 12 residues was consistent with helical topology of both transmembrane regions, with every third or fourth amino acid affected by substitution. These predominantly hydrophobic-nonpolar residues are also present in the ivermectin-sensitive Schistosoma mansoni P2X subunit. They lie on the same side of their helices, and could face lipids in the open conformation state and provide the binding pocket for IVM. In contrast, the IVM-independent hits Met31, Tyr42, Gly45, Val49, Gly340, Leu343, Ala344, Gly347, Thr350, Asp354, and Val357 map on the opposite side of their helices, probably facing the pore of receptor or protein and playing important roles in gating. Once the receptor architecture becomes available from crystal studies, we will know the real topology of these functionally important residues.

我们最近的工作集中在催乳素的分泌上，催乳素是一种通过垂体乳腺营养控制泌乳的激素以及多巴胺D2受体和低音性在此过程中的作用。我们还研究了乳腺营养和促性腺营养中γ-氨基丁酸（GABA）-a受体通道的表达和作用，并为我们从垂体细胞（垂体细胞都克隆）提供了重组ATP ATP-GET-GET-GENTP-GEL-GINES的结构和功能表征。多巴胺D2受体的工作表明，它们在垂体乳腺营养中的激活导致抑制催乳素释放。其他人已经提出，这种抑制是通过GI/O-Alpha蛋白介导的营地产量抑制和/或GI/或GI/O-BETA/GAMMA二聚体介导的内向整流性钾通道的激活和电压门控钙通道的抑制。我们表明，当通过Forskolin治疗升高cAMP水平时，多巴胺激动剂诱导的自发钙涌入并释放了预存储的PRL。我们进一步观察到，多巴胺激动剂抑制了未经处理的自发性和去极化诱导的钙涌入，但在百日咳毒素治疗的细胞中均未抑制。在内向整流性钾通道的细胞中还观察到了这种抑制作用，这表明多巴胺对电压门控钙通道门控的影响足以抑制自发性钙的流入。然而，激动剂诱导的对催乳素释放的抑制仅在百日咳治疗的细胞中部分缓解，这表明多巴胺受体还抑制了电压门控钙涌入下游的胞吐作用。 The pertussis toxin-insensitive step in agonist-induced inhibition of prlactin release was not affected by the addition of wortmannin, an inhibitor of PI3-kinase, and lithium, an inhibitor of GSK-3, but was attenuated in the presence of phorbol ester PMA, which inhibits Gz signaling pathway in a protein kinase C-dependent manner.这些结果首次表明，多巴胺不仅通过通过百日咳毒素敏感敏感的信号途径来阻止电压门控钙涌入，而且还通过脱敏钙 - 分泌耦合通过百日咳毒素毒素敏感性和蛋白质激酶C-敏感性信号通道来抑制基底催乳素的释放。与Zorecs Group合作，我们研究了垂体激素催乳素的释放，因为这种激素也有助于渗透压。在periffed的大鼠乳突中，低渗性导致短暂增加，然后通过放射免疫测定法监测，随后持续的催乳素释放抑制作用。在通过共聚焦显微镜成像的单个细胞中，低音性从2％的囊泡/细胞中引起了荧光标记的心房纳他肽货物的排放，后者通过同一融合孔同步加载了Styryl Dye FM 4-64。相比之下，高钾诱导的去极化导致10％的囊泡/细胞的反应，两种荧光探针的卸载/加载时间不同。在细胞跟踪研究中，在未刺激和刺激条件下记录了膜电容的离散变化，反映了与质膜的单囊泡融合/裂缝。在受刺激的细胞中，发生全融合事件的发生概率较低且没有变化，而超过95％的融合事件是短暂的，开放式融合孔的概率，平均孔位停留，发生频率，融合孔电导率增加。低音性仅在静音膜斑块中很少引起新的融合事件。结果表明，在低音刺激的乳酸营养物中，迅速释放的囊泡在亲吻和跑步模式下释放了激素。在过去的一年中，我们还研究了垂体细胞中GABA-A受体通道的表达，它们在垂体前垂体细胞类型中的分布以及作用的性质（刺激性或抑制性）。我们的结果表明，所有GABAA受体亚基的mRNA均在垂体细胞中表达，并且所有分泌细胞中都存在alpha1/beta1亚基蛋白。在电压钳制的谷霉素治疗细胞中，GABA诱导的剂量依赖性在当前振幅中抑制了双核和picrotoxin抑制，并以浓度依赖性的方式通过地西epa和Zolpidem促进。在完整的细胞中，GABA和GABA-A受体激动剂Muscimol导致细胞内钙的快速和短暂增加，而GABA-B受体激动剂baclofen无效，这表明氯化物介导的去极化激活了电压基因通道。与这一发现一致，RT-PCR分析表明垂体细胞中NKCC1的高表达，但不表达KCC2阳离子/氯化物转运蛋白mRNA。此外，在大多数细胞中，GABA-A通道的逆转电位对基线膜电位呈阳性，而GABA通过GABA激活离子通道导致细胞去极化和自发性电活动的调节。这些结果表明，分泌性垂体细胞表达去极化的功能性GABA-A受体通道。在从垂体细胞中克隆的重组P2X4受体的结构功能表征的工作中，重点是研究鉴定鉴定出对这些通道的残基，这是通过ivermectin（ivermectin，ivermectin，ivermectin，ivermectin，ivermectin，ivermectin，ivermectin是一种大型巨型乳酸酮，一种通过互动的构型增强了与跨性别的互动式构型相互作用的构型构型。该受体对息肉素的敏感性也被用作识别面对通道孔的跨膜区域的残基的指标。为了这些目的，我们使用了大鼠p2x4跨膜区域的半胱氨酸扫描诱变。在29个不同残基中的突变使受体功能不变，其中伊维菌素的作用在GLN36，LEU40，Val43，Val43，Val47，TRP50，ASN338，Gly342，Leu346，Leu346，Ala349，Ala349，Ala349和Ile356突变体中改变了。替代敏感的ARG33和CYS353突变体也可以被视为伊维菌素敏感的命中率。这12个残基的模式与两个跨膜区域的螺旋拓扑一致，每三个或第四个氨基酸都受替代的影响。这些主要是疏水性非极性残基也存在于伊维菌素敏感的分析瘤P2X亚基中。他们躺在螺旋的同一侧，可以在开放构象状态下面对脂质，并为IVM提供绑定的口袋。相比之下，IVM独立的命中MET31，Tyr42，Gly45，Val49，Gly340，Leu343，Ala344，Gly347，Gly347，Thr350，Asp354，Asp354和Val357地图在其螺旋螺旋的另一侧，可能面对受体或蛋白质孔和蛋白质的孔和蛋白质的孔和蛋白质的孔。一旦受体结构从晶体研究中获得，我们将知道这些功能重要的残基的真正拓扑。

项目成果

期刊论文数量（54）

专著数量（0）

科研奖励数量（0）

会议论文数量（0）

专利数量（0）

Expression of purinergic P2X2 receptor-channels and their role in calcium signaling in pituitary cells.

嘌呤能 P2X2 受体通道的表达及其在垂体细胞钙信号传导中的作用。

DOI：
发表时间：
2000
期刊：
Biochemistry and cell biology = Biochimie et biologie cellulaire
影响因子：
0
作者：
Stojilkovic,SS;Tomic,M;VanGoor,F;Koshimizu,T
通讯作者：
Koshimizu,T

Molecular cloning and characterization of alpha1-soluble guanylyl cyclase gene promoter in rat pituitary cells.

大鼠垂体细胞中α1可溶性鸟苷酸环化酶基因启动子的分子克隆和表征。

DOI：
10.1677/jme.1.02180
发表时间：
2006
期刊：
Journal of molecular endocrinology
影响因子：
3.5
作者：
Jiang,Yonghua;Stojilkovic,StankoS
通讯作者：
Stojilkovic,StankoS

Pituitary cell type-specific electrical activity, calcium signaling and secretion.

垂体细胞类型特异性电活动、钙信号传导和分泌。

DOI：
10.4067/s0716-97602006000300004
发表时间：
2006
期刊：
Biological research
影响因子：
6.7
作者：
Stojilkovic,StankoS
通讯作者：
Stojilkovic,StankoS

Characterization of calcium-mobilizing, purinergic P2Y(2) receptors in human ovarian cancer cells.

DOI：
10.1093/molehr/6.5.435
发表时间：
2000-05
期刊：
Molecular human reproduction
影响因子：
4
作者：
A. Schultze-Mosgau;A. C. Katzur;K. Arora;S. Stojilkovic;K. Diedrich;O. Ortmann
通讯作者：
A. Schultze-Mosgau;A. C. Katzur;K. Arora;S. Stojilkovic;K. Diedrich;O. Ortmann

Spontaneous and receptor-controlled soluble guanylyl cyclase activity in anterior pituitary cells.

垂体前叶细胞中自发的和受体控制的可溶性鸟苷酸环化酶活性。