Intracellular Signaling In Endocrine Cells

内分泌细胞的细胞内信号传导

• 批准号: 8553829
• 负责人: STANKO S. STOJILKOVIC
• 金额: $ 90.44万
• 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/8553829
• 关键词: Accounting; Action Potentials; Address; Adenylate Cyclase; Affect; Agonist; Alternative Splicing; Attenuated; Bathing; Behavior; Binding; Biotin; C-terminal; Calcium; Calcium Signaling; Cations; Cell Surface Receptors; Cell membrane; Cells; Collaborations; Communities; Complex; Confocal Microscopy; Cyclic AMP; Cyclic GMP; Cyclic Nucleotides; Cysteine; Development; Dominant-Negative Mutation; Endocrine; Event; Exhibits; Exons; Family; Family member; Flufenamic Acid; Frequencies; Gadolinium; Growth; Hormones; Hypothalamic structure; Immunoblot Analysis; Investigation; Kinetics; Label; Mediating; Membrane; Membrane Potentials; Messenger RNA; Modeling; Molecular; Molecular Weight; Mutagenesis; National Institute of Child Health and Human Development; Neuroendocrine Cell; Neurosciences; Neurosecretory Systems; Neurotransmitters; P2X-receptor; Pathway interactions; Pattern; Phospholipase C Signaling Pathway; Physiological; Pituitary Gland; Process; Prolactin; Protein Family; Protein Isoforms; Proteins; RNA Splicing; Rattus; Recombinants; Reporting; Research Personnel; Research Project Grants; Rest; Reverse Transcriptase Polymerase Chain Reaction; Role; Signal Pathway; Signal Transduction; Site; Sodium; System; Threonine; Transcript; Western Blotting; Whole-Cell Recordings; Work; cell type; cyclic-nucleotide gated ion channels; desensitization; disulfide bond; extracellular; gadolinium oxide; inhibitor/antagonist; lactotroph; loss of function; mRNA Expression; multidisciplinary; mutant; novel; programs; receptor; receptor expression; research study; trafficking; voltage

Channels contributing to the spike depolarization and repolarization in spontaneously firing pituitary cells have been identified. In contrast, very little is known about channels controlling resting membrane potential and initiation of firing of action potentials. During the last year, we focused on the role of two non-selective cation channels in electrical activity, calcium signaling, and hormone secretion: hyperpolarization-activated and cyclic nucleotide-gated (HCN) channels and classic transient receptor potential (TRPC) channels. The role of HCN channels was studied in cultured rat pituitary cells. Quantitative RT-PCR analysis showed higher level of expression of mRNA transcripts for HCN2 and HCN3 subunits and lower expression of HCN1 and HCN4 subunits in these cells. Western immunoblot analysis of lysates from pituitary cells showed bands with appropriate molecular weights for HCN2, HCN3 and HCN4. Electrophysiological experiments showed the presence of HCN current in gonadotrophs, thyrotrophs, somatotrophs and a fraction of lactotrophs, as well as in other unidentified pituitary cell types. Stimulation of adenylyl cyclase and addition of 8-Br-cAMP enhanced this current and depolarized the cell membrane, whereas 8-Br-cGMP did not alter the current and hyperpolarized the cell membrane. Both inhibition of basal adenylyl cyclase activity and stimulation of phospholipase C signaling pathway inhibited this current. However, inhibition of HCN channels affected the frequency of firing but did not abolish spontaneous electrical activity, indicating that other channels are critical for spontaneous pacemaking activity. In further experiments with cultured lactotrophs and immortalized GH3 cells, we found that replacement of extracellular sodium with large organic cations, but not blockade of voltage-gated sodium influx, led to an instantaneous hyperpolarization of cell membranes that was associated with a cessation of spontaneous firing. When cells were clamped at -50 mV, which was close to the resting membrane potential in these cells, replacement of bath sodium with organic cations resulted in an outward-like current, reflecting an inhibition of the inward holding membrane current and indicating loss of a background-depolarizing conductance. Quantitative RT-PCR analysis revealed the high expression of mRNA transcripts for TRPC1 and much lower expression of TRPC6 in both lactotrophs and GH3 cells. Very low expression of TRPC3, TRPC4, and TRPC5 mRNA transcripts were also present in pituitary but not GH3 cells. 2-APB and SKF-96365, relatively selective blockers of TRPC channels, inhibited electrical activity, calcium influx and prolactin release in a concentration-dependent manner. Gadolinium, and flufenamic acid, inhibitors of non-selective cation channels, also inhibited electrical activity, calcium influx and prolactin release. These results indicate that nonselective cation channels, presumably belonging to the TRPC family, contribute to the background depolarizing conductance and firing of action potentials in these cells. Our ongoing work is also focused on structural and functional characterization of ATP-gated P2X2 and P2X7 receptor-channels, which are expressed in pituitary cells. In collaboration with Dr. Sherman, we recently found that P2X2Rs exhibit two opposite activation-dependent changes, pore dilation and pore closing (desensitization), through a process that is incompletely understood. To address this issue and to clarify the roles of calcium and the C-terminal domain in gating, we combined biophysical and mathematical approaches. This receptor developed conductivity for N-methyl-D-glucamine within 2-6 s of ATP application. However, pore dilation was accompanied with a decrease rather than an increase in the total conductance, which temporally coincided with rapid and partial desensitization. During sustained agonist application, receptors continued to desensitize in calcium-independent and calcium-dependent modes. In whole-cell recording, we also observed use-dependent facilitation of desensitization of both receptors. Such behavior was accounted for by a 16-state Markov kinetic model describing ATP binding/unbinding and activation/desensitization. The model assumes that nave receptors open when two to three ATP molecules bind and undergo calcium-independent desensitization, causing a decrease in the total conductance, or pore dilation, causing a shift in the reversal potential. In calcium-containing media, receptor desensitization is facilitated and the use-dependent desensitization can be modeled by a calcium-dependent toggle switch. The experiments and the model together provide a rationale for the lack of sustained current growth in dilating P2X2Rs and show that receptors in the dilated state can also desensitize in the presence of calcium. In collaboration with Dr. Zemkova, we also studied the role of conserved ectodomain cysteine residues in P2X7R function. Single- and double-point threonine mutants of C119-C168, C129-C152, C135-C162, C216-C226, and C260-C269 cysteine pairs were expressed in HEK293 cells and studied using whole-cell current recording. All mutants other than C119T-P2X7R responded to initial and subsequent application of 300 M BzATP and ATP with small amplitude monophasic currents or were practically non-functional. The mutagenesis-induced loss of function was due to decreased cell-surface receptor expression, as revealed by assessing levels of biotinylated mutants. Coexpression of all double mutants with the wild type receptor had a transient or, in the case of C119T/C168T double mutant, sustained inhibitory effect on receptor trafficking. The C119T-P2X7R mutant was expressed on the plasma membrane and was fully functional with a slight decrease in the sensitivity for BzATP, indicating that interaction of liberated Cys168 with another residue rescues the trafficking of receptor. Thus, in contrast to other P2XRs, all disulfide bonds of P2X7R are individually essential for the proper receptor trafficking. We also studied the expression pattern and role of Pannexins, a newly discovered three-member family of proteins, in pituitary cells. The last year, we reported that Pannexin 1 (Panx1) is expressed in the pituitary gland and provides a pathway for delivery of ATP release. Recent experiments revealed that, in addition to the full size isoform of Panx1, hereafter referred to as Panx1a, pituitary cells also express two novel splice isoforms, termed Panx1c and Panx1d, which formation reflects the existence of alternative splicing sites in exons 2 and 4. Panx1c is lacking the Phe108-Gln180 sequence and P2X1d is missing the Val307-Cys426 C-terminal end sequence. Confocal microscopy and biotin labeling revealed that Panx1a is expressed in the plasma membrane, whereas Panx1c and Panx1d show the cytoplasmic localization when expressed as homomeric proteins. In co-expression studies, we further investigated the interactions of Panx1a with its two splice forms, the effect of expression of these short splice isoforms on the ATP release functions of full-size Panx1a channels, and their association with P2XRs. The three Panx1 isoforms and Panx2 form homomeric and heteromeric complexes in any combination. These splice forms can also physically associate with ATP-gated P2X2, P2X3, P2X4, and P2X7 receptor channels. The Panx1a-mediated ATP release in AtT-20 immortalized pituitary cells is attenuated when co-expressed with Panx1c or Panx1d. These results suggest that Panx1c and Panx1d may serve as dominant-negative effectors to modulate the functions of Panx1a through formation of heteromeric channels. The complex patterns of Panx1 expression and association could also define the P2X-dependent roles of these channels in cell types co-expressing both proteins.

已经确定了自发放电垂体细胞的尖峰去极化和重极化的通道。相比之下，关于控制静止膜电位和动作电位发射的启动的通道知之甚少。在过去的一年中，我们专注于两个非选择性阳离子通道在电活动，钙信号和激素分泌中的作用：超极化激活和环状核苷酸门控通道（HCN）通道以及经典的瞬态受体电位（TRPC）通道。在培养的大鼠垂体细胞中研究了HCN通道的作用。定量RT-PCR分析显示，在这些细胞中，HCN2和HCN3亚基的mRNA转录物表达更高，HCN1和HCN4亚基的表达较低。对垂体细胞的裂解物的西部免疫印迹分析显示带有适当的HCN2，HCN3和HCN4的分子量的条带。电生理实验表明，促性腺营养性，甲状腺营养，嗜营养素和一小部分乳营养素以及其他未识别的垂体细胞类型中存在HCN电流。 刺激腺苷酸环化酶并添加8-Br-cAMP增强了这种电流并将细胞膜去极化，而8-BR-CGMP并未改变电流并超极化细胞膜。抑制基底腺苷酸环化酶活性和磷脂酶C信号通路的刺激都抑制了这一电流。但是，HCN通道的抑制会影响射击频率，但没有废除自发的电活动，这表明其他通道对于自发的起搏活动至关重要。在用培养的乳腺营养和永生的GH3细胞进行的进一步实验中，我们发现用大的有机阳离子替换细胞外钠，但没有阻断电压门控钠涌入，导致瞬时的细胞膜超极化，与自发性燃料的凝结相关。当细胞以-50 mV夹紧（接近这些细胞的静息膜电位）时，用有机阳离子替换浴钠会导致外向电流，这反映了对内向保持膜电流的抑制，并表明损失了背景 - 偏振电导。定量RT-PCR分析表明，在乳营养素和GH3细胞中，TRPC1的mRNA转录物的高表达和TRPC6的表达较低。 TRPC3，TRPC4和TRPC5 mRNA转录本的表达非常低，但在垂体中也存在于GH3细胞中。 2-APB和SKF-96365，相对选择性的TRPC通道的阻滞剂，以浓度依赖性方式抑制电活动，钙涌入和催乳素释放。非选择性阳离子通道的抑制剂Gadolinium和Flufenamic Acid，还抑制了电活动，流入钙和催乳素释放。这些结果表明，非选择性的阳离子通道（可能属于TRPC家族）有助于背景去极化电导和这些细胞中作用电位的发射。我们正在进行的工作还集中在ATP门控P2X2和P2X7受体通道的结构和功能表征上，这些p2x2和P2X7受体通道在垂体细胞中表达。与谢尔曼博士合作，我们最近发现，P2X2RS通过未完全理解的过程表现出两个相反的激活依赖性变化，孔隙扩张和孔隙闭合（脱敏）。为了解决这个问题并阐明钙和C末端结构域在门控中的作用，我们结合了生物物理和数学方法。该受体在ATP应用2-6 s内为N-甲基-D-葡萄糖胺开发了电导率。然而，孔隙扩张伴随着减少而不是总电导率的增加，这在时间上与快速和部分脱敏相吻合。在持续的激动剂应用过程中，受体在钙依赖性和钙依赖性模式下继续脱敏。在全细胞记录中，我们还观察到了两种受体脱敏的使用依赖性促进。这种行为是由16个国家的马尔可夫动力学模型描述的，该模型描述了ATP结合/解开和激活/脱敏。该模型假设当两到三个ATP分子结合并经历钙独立的脱敏时，中殿受体开放，导致总导电率降低或孔隙扩张，从而导致反转电位的变化。在含钙的培养基中，受体脱敏是促进的，并且可以通过依赖钙依赖性的切换开关对使用依赖性脱敏。实验和模型共同为缺乏持续的P2X2RS生长提供了基本原理，并表明在钙存在的情况下，扩张状态下的受体也会降低。 与Zemkova博士合作，我们还研究了保守的外生域半胱氨酸残基在P2X7R功能中的作用。在HEK293细胞中表达了C119-C168，C129-C152，C129-C152，C135-C162，C135-C162，C216-C226和C260-C269半胱氨酸对的单点和双点苏氨酸突变体，并使用全细胞电流记录进行了研究。除C119T-P2X7R以外的所有突变体均对300 m BZATP和ATP的初始和随后的ATP做出了响应，幅度很小或实际上是非功能性的。诱变诱导的功能丧失是由于细胞表面受体表达降低所致，如评估生物素化突变体的水平所示。所有双重突变体与野生型受体的共表达具有短暂性，或者在C119T/C168T双突变体的情况下，对受体运输持续抑制作用。 C119T-P2X7R突变体在质膜上表达，并且完全功能齐全，对BZATP的敏感性略有降低，表明释放的Cys168与另一个残基的相互作用拯救了受体的运输。因此，与其他P2XR相反，P2X7R的所有二硫键对于适当的受体运输都是必不可少的。我们还研究了垂体细胞中新发现的三个成员的蛋白质家族Pannexin的表达模式和作用。去年，我们报道了pannexin 1（Panx1）在垂体中表达，并为ATP释放提供了途径。最近的实验表明，除了PANX1的全尺寸同工型外，以下称为panx1a，垂体细胞还表达了两个新型的剪接同工型，称为panx1c和panx1d，形成反映了exons 2和4 sequent is exos 2和4。 Val307-Cys426 C末端序列。共聚焦显微镜和生物素标记表明，panx1a在质膜中表达，而Panx1c和Panx1D表示为同源蛋白时的细胞质定位。在共表达研究中，我们进一步研究了Panx1a及其两种剪接形式的相互作用，这些短剪接同工型表达对全尺寸PANX1A通道ATP释放函数的影响以及它们与P2XR的关联。在任何组合中，三个PANX1同工型和PANX2形成同源物和异类络合物。这些剪接形式还可以与ATP门控的P2X2，P2X3，P2X4和P2X7受体通道物理相关。与PANX1C或PANX1D共表达时，PANX1A介导的ATT-20永生垂体细胞中的ATP释放会减弱。这些结果表明，PANX1C和PANX1D可以用作主导性效应子，以通过形成杂体通道来调节PANX1A的功能。 PANX1表达和关联的复杂模式还可以定义这些通道在共表达这两种蛋白的细胞类型中的P2X依赖性作用。