Intracellular Signaling In Endocrine Cells

内分泌细胞的细胞内信号传导

• 批准号: 9150047
• 负责人: STANKO S. STOJILKOVIC
• 金额: $ 102.8万
• 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/9150047
• 关键词: Accounting; Address; Adolescent; Affect; Age; Agonist; Animals; Anterior Pituitary Gland; Antipsychotic Agents; Architecture; Attenuated; Bathing; Brain; Calcium; Calcium Signaling; Cations; Cell membrane; Cells; Chimera organism; Communities; Coupled; Coupling; Cyclic AMP; Data; Development; Dopamine; Dopamine Receptor; Embryo; Endocrine; Event; Excision; Exhibits; Female; Follicle Stimulating Hormone; Forskolin; Gene Expression; Genes; Gonadotropin-Releasing Hormone Receptor; Growth; Hormones; Human; Hyperprolactinemia; Hypothalamic structure; In Vitro; Investigation; Ivermectin; Kinetics; Luteinizing Hormone; Modeling; Molecular; Mus; National Institute of Child Health and Human Development; Neuroendocrine Cell; Neurosciences; Neurosecretory Systems; Neurotransmitters; P2X-receptor; Pathway interactions; Patients; Pattern; Perinatal; Pharmaceutical Preparations; Physiological; Pituitary Gland; Pro-Opiomelanocortin; Process; Production; Prolactin; Proteins; Rattus; Receptor Gene; Recombinants; Research Personnel; Research Project Grants; Role; Schizophrenia; Secondary to; Secretory Cell; Serum; Signal Pathway; Signal Transduction; Somatotropin; System; TRH gene; Thyroid Gland; Thyroid Hormones; Thyrotropin; Thyrotropin-Releasing Hormone Receptors; Time; Transcript; Transmembrane Domain; Whole-Cell Recordings; aripiprazole; atypical antipsychotic; cell type; desensitization; driving force; in vivo; infancy; lactotroph; male; multidisciplinary; mutant; postnatal; programs; receptor; research study; sex; sexual dimorphism; steroid hormone; treatment duration

We continue investigations on receptors and channels expressed in the anterior pituitary gland and their roles in signaling, gene expression and hormone secretion. In a recent study, we examined the expression of major anterior pituitary genes in five secretory cell types of developing males and females. Corticotrophs show comparable proopiomelanocortin (Pomc) profiles in both sexes, with the highest expression occurring during the infantile period. Somatotrophs and lactotrophs also exhibit no difference in growth hormone (Gh) and prolactin (Prl) profiles during embryonic-to-juvenile age but show the amplification of Prl expression in females and Gh expression in males during peripubertal and postpubertal ages. Gonadotrophs exhibit highly synchronized luteinizing hormone beta (Lhb), follicle-stimulating hormone beta (Fshb) gonadotroph/lactotroph-specific alpha (Cga), and gonadotropin-releasing hormone receptor (Gnrhr) gene expression in both sexes, but the peak of expression occurs during the infantile period in females and at the end of the juvenile period in males. Thyrotrophs also show different developmental thyroid-stimulating hormone beta (Tshb) profiles, which are synchronized with the expression of gonadotroph genes in males but not in females. These results indicate the lack of influence of sex on Pomc expression and the presence of two patterns of sexual dimorphism in the expression of other pituitary genes: a time shift in the peak expression during postnatal development, most likely reflecting the perinatal sex-specific brain differentiation, and modulation of the amplitude of expression during late development, which is secondary to the establishment of the hypothalamic-pituitary-gonadal and -thyroid axes. In further study on this topic, we examined the in vivo and in vitro expression pattern of three genes that are operative in the thyrotroph subpopulation from postpubertal animals: Cga, Tshb, and thyrotropin-releasing hormone receptor (Trhr). In vivo, the expression of Cga and Tshb was robust, whereas the expression of Trhr was low. In cultured pituitary cells, there was a progressive decline in the expression of Cga, Tshb and Trhr. The expression of Tshb could not be reversed via pulsatile or continuous TRH application in variable concentrations and treatment duration, or by the removal of thyroid and steroid hormones from the sera. In parallel, the expression of CGA and TSHB proteins declined progressively in pituitary cells from both sexes. The lack of the effect of TRH on Tshb expression was not related to the age of pituitary cultures and the presence of functional TRHR. In pituitary fragments, there was also a rapid decline in expression of these genes but TRH was able to induce transient Tshb expression. These observations suggest that the lack of influence of anterior pituitary architecture and/or intrapituitary factors probably accounts for the loss of basal and TRH-stimulated Tshb expression in dispersed pituitary cells. The other focus in this topic was on pituitary lactotrophs, which secrete high levels of PRL in the absence of any hormone action in vitro. We have previously shown that such hyperprolactinemia (HPRL) is driven by spontaneous electrical activity and the accompanying calcium influx. HPRL is a common adverse in vivo effect of antipsychotic medications that are used in the treatment of patients with schizophrenia. We compared the effects of two atypical antipsychotics, paliperidone and aripiprazole, on cAMP/calcium signaling and PRL release in female rat lactotrophs in vitro. Dopamine inhibits spontaneous cAMP/calcium signaling and PRL release. In the presence of dopamine, paliperidone rescues cAMP/calcium signaling and PRL release in a concentration-dependent manner, whereas aripiprazole is only partially effective. In the absence of dopamine, paliperidone stimulates cAMP/calcium signaling and PRL release, whereas aripiprazole inhibits signaling and secretion more potently but less effectively than dopamine. Forskolin-stimulated cAMP production is facilitated by paliperidone and inhibited by aripiprazole, although the latter is not as effective as dopamine. None of the compounds affects Prl transcript activity, intracellular PRL accumulation, or GH secretion. These data indicate that paliperidone has dual HPRL actions in lactotrophs i) by preserving the coupling of spontaneous electrical activity and PRL secretion in the presence of dopamine and ii) by inhibiting intrinsic dopamine receptor activity in the absence of dopamine, leading to enhanced calcium signaling and secretion. In contrast, aripiprazole acts on PRL secretion by attenuating, but not abolishing, calcium-secretion coupling. The other main focus in our investigations is on structural and functional characterization of two ATP-gated P2X receptors, P2X2 and P2X4 expressed in gonadotrophs and lactotrophs, respectively. Activation of P2X2 is characterized by a rapid current growth accompanied with a decay of current during sustained ATP application, a phenomenon known as receptor desensitization. Using, rat, mouse and human receptors, we show recently that two processes contribute to receptor desensitization: bath calcium-independent and -dependent. Calcium-dependent desensitization is substantial during initial agonist application and progressively increases during repetitive agonist application in ATP- and bath calcium-concentration dependent manner. Experiments with NMDG, a large organic cation, indicate that receptor pore dilation is a calcium-independent process in contrast to receptor desensitization. A decrease in the driving force for calcium by changing the holding potential further indicates that calcium influx through the channels pore at least partially accounts for receptor desensitization. Experiments with various receptor chimeras also indicate that the transmembrane and intracellular domains of P2X2R are required for development of calcium-dependent desensitization and that decrease in the amplitude of current slows receptor desensitization. Simultaneous calcium and current recording show development of calcium-dependent desensitization without increase in global intracellular calcium concentrations. Combined with experiments with clamping intrapipette concentrations of calcium at various levels, these experiments indicate that domain calcium is sufficient to establish calcium-dependent receptor desensitization in experiments with whole-cell recordings. We also studied the influence of allostery on P2X4 pore dilation, using ivermectin (IVM), an established positive allosteric regulator of this channel. In the absence of IVM, this channel activates and deactivates rapidly, does not show transition from open to dilated states, desensitizes completely with a moderate rate, and recovers only fractionally during washout. IVM treatment increases the efficacy of ATP to activate the channel and slows receptor desensitization during sustained ATP application and receptor deactivation after ATP washout. The rescue of the receptor from desensitization temporally coincides with pore dilation, and the dilated channel can be reactivated after washout of ATP. Experiments with vestibular and transmembrane domain receptor mutants further established that IVM has distinct effects on opening and dilation of the channel pore, the first accounting for increased peak current amplitude and the latter correlating with changes in the EC50 and kinetics of receptor deactivation. The corresponding kinetic (Markov state) model indicates that the IVM-dependent transition from open to dilated state is coupled to receptor sensitization, which rescues the receptor from desensitization and subsequent internalization. Allosterically-induced sensitization of P2X4R thus provides sustained signaling during prolonged and repetitive ATP stimulation.

我们继续研究在垂体前腺体中表达的受体和通道及其在信号，基因表达和激素分泌中的作用。在最近的一项研究中，我们检查了五种发育中的雄性和女性分泌类型的垂体主要基因的表达。皮质营养噬菌体在两性中表现出可比的促蛋白酶皮质素（POMC）特征，在婴儿期间表达最高。在胚胎到生年期间，嗜营养素和乳营养素在胚胎到生的年龄期间的生长激素（GH）（GH）和催乳素（PRL）谱也没有差异，但在贝里伯贝尔贝尔伯属和后伯伯质年龄期间男性和男性中PRL表达的扩增。促性腺营养性表现出高度同步的葡萄糖激素β（LHB），卵泡刺激激素β（FSHB）促性腺激素/乳腺营养性特异性α（CGA）和促性腺激素释放的激素受体（GNRHR）基因的表达，但在两种表达中表达，但在两种表达中表达了峰值，但是在男性的少年时期结束时。甲状腺营养还显示出不同的发育性甲状腺刺激激素β（TSHB）谱，它们与雄性（但未在雌性中却没有的促性腺营养基因的表达）同步。 These results indicate the lack of influence of sex on Pomc expression and the presence of two patterns of sexual dimorphism in the expression of other pituitary genes: a time shift in the peak expression during postnatal development, most likely reflecting the perinatal sex-specific brain differentiation, and modulation of the amplitude of expression during late development, which is secondary to the establishment of the hypothalamic-pituitary-gonadal and -thyroid axes.在有关此主题的进一步研究中，我们研究了三种基因的体内和体外表达模式，这些基因在甲状腺纤维杆菌亚群中的可操作症中可起作用：CGA，TSHB和甲状腺激素释放激素受体（TRHR）。在体内，CGA和TSHB的表达很强，而TRHR的表达较低。在培养的垂体细胞中，CGA，TSHB和TRHR的表达逐渐下降。 TSHB的表达无法通过脉冲或连续的TRH施加可变浓度和治疗持续时间，或通过从血清中去除甲状腺和类固醇激素的表达。同时，CGA和TSHB蛋白的表达在两性的垂体细胞中逐渐下降。缺乏TRH对TSHB表达的影响与垂体培养的时代和功能性TRHR的存在无关。在垂体片段中，这些基因的表达也很快下降，但TRH能够诱导瞬时TSHB表达。这些观察结果表明，垂体前结构和/或膜间内因子缺乏影响可能是分散垂体细胞中基础和TRH刺激的TSHB表达的丧失。该主题的另一个重点是垂体乳营养素，在体外没有任何激素作用的情况下，它分泌了高水平的PRL。我们先前已经表明，这种高乳酸血症（HPRL）是由自发性电活动和随附的钙流入驱动的。 HPRL是用于治疗精神分裂症患者的抗精神病药物的常见不良体内影响。我们比较了两种非典型抗精神病药，帕皮酮和阿立哌唑，对雌性大鼠乳营养素在体外的CAMP/CARCIUM信号传导和PRL释放的影响。多巴胺抑制自发的营地/钙信号传导和PRL释放。在存在多巴胺的情况下，帕利培酮以浓度依赖性的方式营救了营地/钙信号传导和PRL释放，而阿立哌唑仅是部分有效的。在没有多巴胺的情况下，帕利吡啶酮刺激CAMP/钙信号传导和PRL释放，而Aripiprazole比多巴胺更有效地抑制信号传导和分泌。帕皮酮促进了福斯科林刺激的营地生产并被阿立哌唑抑制，尽管后者不如多巴胺有效。这些化合物都不影响PRL转录活性，细胞内PRL积累或GH分泌。这些数据表明，棕榈酮在乳糖营养中具有双重HPRL作用i）通过在多巴胺存在下保留自发性电活动和PRL分泌的偶联，而II）通过抑制内在的多巴胺受体受体活性，在缺乏多巴胺的情况下，从而增强了钙信号和分泌。相比之下，阿立哌唑通过衰减但不取消钙溶剂耦合来对PRL分泌。我们研究的另一个主要重点是分别在促性腺营养和乳营养营养物中表达的两个ATP门控P2X受体的结构和功能表征。 P2X2的激活的特征是快速电流生长伴随着持续ATP应用过程中电流的衰减，这是一种称为受体脱敏的现象。使用大鼠，小鼠和人体受体，我们最近表明，两个过程有助于受体脱敏：浴钙独立且依赖性。在初始激动剂施用期间，钙依赖性脱敏是很大的，并且在重复性激动剂在ATP和浴钙浓度依赖性方式中逐渐增加。 NMDG（一种大的有机阳离子）的实验表明，与受体脱敏相反，受体孔隙扩张是一个独立的钙独立过程。通过改变保持电势的钙驱动力降低进一步表明，通过通道孔隙孔隙至少部分解释受体脱敏的钙涌入。具有各种受体嵌合体的实验还表明，P2X2R的跨膜和细胞内结构域是开发钙依赖性脱敏的，并且当前当前幅度减少受体脱敏的幅度降低。同时钙和当前记录显示钙依赖性脱敏的发展，而没有增加全球细胞内钙浓度。这些实验与夹紧钙的钙内钙浓度结合在一起，这些实验表明，域钙足以在全细胞记录的实验中建立依赖性钙依赖性受体脱敏。我们还研究了伊维菌素（IVM）（IVM），这是该通道的阳性变构调节剂，研究了变构对P2X4孔扩张的影响。在没有IVM的情况下，该通道会迅速激活和停用，不会显示从开放状态向扩张状态的过渡，以中等的速率完全脱敏，并且在洗涤过程中仅恢复。 IVM治疗在ATP冲洗后持续的ATP应用和受体停用期间，在持续的ATP应用和受体失活期间，激活通道的功效增加了ATP的疗效。从脱敏中拯救受体在时间上与孔隙扩张相吻合，并且可以在ATP冲洗后重新激活扩张的通道。前庭和跨膜结构域受体突变体进行的实验进一步确定，IVM对通道孔的开放和扩张具有不同的影响，这是第一个考虑到峰值电流振幅增加的第一，而后者与EC50和动力学的变化相关。相应的动力学（马尔可夫状态）模型表明，从开放态到扩张状态的IVM依赖性过渡与受体敏化耦合，从而使受体免于脱敏和随后的内在化。因此，在延长和重复的ATP刺激期间，P2X4R的变构诱导的敏化提供了持续的信号传导。