Intracellular Signaling In Endocrine Cells

我们将继续研究垂体前叶中表达的受体和通道及其在信号传导、基因表达和激素分泌中的作用。在最近的一项研究中，我们检测了发育中的雄性和雌性的五种分泌细胞类型中主要垂体前叶基因的表达。促肾上腺皮质激素在两性中显示出相似的促肾上腺素原（Pomc）谱，在婴儿时期表达最高。生长激素（Gh）和催乳素（Prl）在胚胎期和幼年期也没有差异，但在青春期和青春期后，雌性的Prl和雄性的Gh表达增加。促性腺激素在两性中表现出高度同步的促黄体生成素β (Lhb)、促卵泡激素β (Fshb)、促性腺/乳营养特异性α (Cga)和促性腺激素释放激素受体（Gnrhr）基因表达，但表达高峰出现在雌性的婴儿期和雄性的青春期末期。促甲状腺激素也表现出不同的发育促甲状腺激素（Tshb）谱，在雄性中与促性腺激素基因的表达同步，而在雌性中则不同步。这些结果表明，性别对Pomc表达没有影响，其他垂体基因的表达存在两种性别二态性模式：出生后发育期间峰值表达的时间转移，很可能反映了围出生期性别特异性脑分化，以及发育后期表达幅度的调节，这是继下丘脑-垂体-性腺和-甲状腺轴的建立之后。在这一主题的进一步研究中，我们检测了在青春期后动物甲状腺功能亢进亚群中起作用的三个基因的体内和体外表达模式：Cga， Tshb和促甲状腺素释放激素受体（Trhr）。在体内，Cga和Tshb的表达较强，而Trhr的表达较低。在培养的垂体细胞中，Cga、Tshb和Trhr的表达呈进行性下降。Tshb的表达不能通过脉冲或连续应用不同浓度和治疗时间的TRH，也不能通过从血清中去除甲状腺激素和类固醇激素来逆转。与此同时，CGA和TSHB蛋白在两性垂体细胞中的表达逐渐下降。TRH对Tshb表达影响的缺失与垂体培养年龄和功能性TRHR的存在无关。在垂体片段中，这些基因的表达也迅速下降，但TRH能够诱导短暂的Tshb表达。这些观察结果表明，缺乏垂体前叶结构和/或垂体内因素的影响可能是分散的垂体细胞中基底和trh刺激的Tshb表达缺失的原因。本主题的另一个重点是垂体乳营养物，在体外没有任何激素作用的情况下分泌高水平的PRL。我们之前已经证明，这种高泌乳素血症（HPRL）是由自发电活动和伴随的钙内流驱动的。HPRL是用于治疗精神分裂症患者的抗精神病药物的常见体内不良反应。我们比较了两种非典型抗精神病药物帕利哌酮和阿立哌唑对体外雌性乳养大鼠cAMP/钙信号传导和PRL释放的影响。多巴胺抑制自发性cAMP/钙信号和PRL释放。在多巴胺存在的情况下，帕利哌酮以浓度依赖的方式挽救cAMP/钙信号和PRL释放，而阿立哌唑仅部分有效。在缺乏多巴胺的情况下，帕利哌酮刺激cAMP/钙信号和PRL释放，而阿立哌唑对信号和分泌的抑制作用比多巴胺更强，但效果不如多巴胺。帕利哌酮促进福斯克林刺激的cAMP生成，阿立哌唑抑制cAMP生成，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积累或生长激素分泌。这些数据表明，帕利哌酮在乳养菌中具有双重HPRL作用：i)在多巴胺存在时，通过保持自发电活动和PRL分泌的偶联；ii)在缺乏多巴胺的情况下，通过抑制内在多巴胺受体活性，导致钙信号和分泌增强。相反，阿立哌唑通过减弱而不是消除钙-分泌偶联作用于PRL分泌。我们研究的另一个重点是两种atp门控的P2X受体P2X2和P2X4的结构和功能表征，P2X2和P2X4分别在促性腺激素和乳养细胞中表达。P2X2激活的特点是在持续的ATP应用过程中，电流的快速增长伴随着电流的衰减，这种现象被称为受体脱敏。使用大鼠、小鼠和人类受体，我们最近发现两个过程有助于受体脱敏：浴钙独立和依赖。钙依赖性脱敏在初始激动剂应用期间是实质性的，并且在ATP和浴钙浓度依赖的方式下，在重复激动剂应用期间逐渐增加。NMDG（一种大型有机阳离子）的实验表明，与受体脱敏相比，受体孔扩张是一个不依赖钙的过程。通过改变保持电位来降低钙的驱动力进一步表明，钙通过通道孔流入至少部分地解释了受体脱敏。不同受体嵌合体的实验也表明，P2X2R的跨膜和胞内结构域是钙依赖性脱敏的必要条件，电流振幅的降低减缓了受体的脱敏。同时钙和电流记录显示钙依赖性脱敏的发展没有增加整体细胞内钙浓度。结合夹紧不同水平的小管内钙浓度的实验，这些实验表明结构域钙足以在全细胞记录实验中建立钙依赖性受体脱敏。我们还使用伊维菌素（IVM）研究了变构对P2X4孔扩张的影响，伊维菌素是该通道的一种已建立的正变构调节剂。在没有IVM的情况下，该通道快速激活和失活，不表现出从开放状态到扩张状态的转变，以中等速率完全脱敏，并且在冲洗期间仅部分恢复。IVM治疗增加了ATP激活通道的功效，减缓了持续ATP应用期间的受体脱敏和ATP冲洗后的受体失活。受体从脱敏中恢复的时间与孔扩张一致，并且扩张的通道可以在ATP冲洗后重新激活。前庭和跨膜结构域受体突变体的实验进一步证实，IVM对通道孔的打开和扩张有明显的影响，前者导致峰值电流振幅的增加，后者与EC50的变化和受体失活动力学相关。相应的动力学（马尔可夫状态）模型表明，ivm依赖的从开放到扩张状态的转变与受体敏化相耦合，这将受体从脱敏和随后的内化中拯救出来。因此，变构诱导的P2X4R敏化在长时间和重复的ATP刺激中提供持续的信号传导。

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.