Intracellular Signaling In Endocrine Cells

我们从脑垂体中克隆了5个atp门控的P2X受体通道（P2XRs）： P2X2R、P2X3R、P2X4R、P2X6R和P2X7R。我们正在进行的工作重点是表征这些通道在垂体分泌细胞的信号传导和分泌中的作用，以及重组通道的结构功能表征。最近，我们通过定量RT-PCR发现P2X4亚基的mRNA转录物在大鼠垂体前叶组织中最丰富，并通过Western blot分析证实了P2X4R蛋白的表达。单细胞膜片钳记录显示，在大多数促甲状腺激素释放激素应答的垂体细胞中，细胞外ATP诱导向内去极化电流，这与重组P2X4R产生的电流相似。通道以剂量依赖的方式激活和脱敏，并迅速失活。这些通道的激活导致电活动的刺激和促进电压门控和电压不敏感的钙内流。在伊维菌素（P2X4Rs的一种特异性变弹性调节剂）的存在下，ATP诱导的向内电流的最大振幅增加了大约四倍，同时受体对ATP的敏感性增加，受体失活减慢，ATP诱导的催乳素释放增强。这些结果表明，促甲状腺激素释放激素应答细胞，包括乳营养细胞，表达同质和/或异质P2X4R，促进钙内流和激素分泌。哺乳动物P2X受体的外结构域含有10个保守的半胱氨酸残基，形成5个二硫键（SS1-5）。通过与Zemkova博士合作，我们分析了这些SS对在大鼠P2X4受体功能中的相关性，方法是用丙氨酸或苏氨酸取代一种或两种半胱氨酸，在HEK293细胞中表达受体，并研究它们在缺乏和存在伊维菌素时对ATP的反应。当形成SS3键（C132-C159）的两种半胱氨酸被苏氨酸取代时，对ATP的反应没有改变。用苏氨酸替代SS1 （C116-C165）、SS2 （C126-C149）和SS4 (C217-C227)，而不是SS5 (C261-C270)，半胱氨酸对ATP的敏感性降低，失活时间缩短。在SS2、SS4和SS5双突变体中，最大电流幅值降低，在SS2和SS5双突变体中，伊维菌素可以部分拯救电流幅值。在许多单残基突变体中也观察到这种反应模式，但当217-半胱氨酸被苏氨酸或精氨酸取代或261-半胱氨酸被丙氨酸取代时，受体功能不受影响。这些结果表明，SS1、SS2和SS4键对配体结合袋的结构有很大贡献，而SS5键位于跨膜结构域，对受体门控有很大贡献。我们与智利天主教大学合作，重点研究了几种化合物对P2X2R的变构调节，主要作用于受体外结构域。像铜一样，汞，一种在细胞中引起氧化应激的金属，也会刺激P2X2R的活性并抑制P2X4R的活性。然而，汞调制与对铜调制至关重要的细胞外残基无关。为了确定汞作用位点，我们使用全尺寸P2X2亚基（称为P2X2a）和c端缺乏69个残基片段的剪接变体（称为P2X2b）作为嵌合体的细胞内和跨膜片段的供体，并使用P2X4亚基作为嵌合体的外结构域片段的供体，生成了两个嵌合体。汞对atp诱导电流的增强作用在表达P2X4/2a嵌合体的爪蟾卵母细胞中保留，而在表达P2X4/2b嵌合体的卵母细胞中不存在。位点诱变实验表明，Cys-430残基介导汞对P2X2aR活性的影响。由于汞可以作为氧化应激诱导剂，我们还测试了过氧化氢和线粒体应激诱导剂粘噻唑和鱼tenone是否模拟汞的作用。这些实验表明，这些化合物增强了atp引起的P2X2aR和P2X4/2aR电流，但对P2X2bR、P2X2a-C430A和P2X2a-C430S突变电流没有作用，而抗氧化剂二硫代苏硝基和n -乙酰半胱氨酸则阻止了过氧化氢的增强。Cys-430残基与甲基甲烷-硫代磺酸盐的烷基化反应也消除了汞和过氧化氢的增强作用。总之，这些结果与Cys-430残基是细胞内P2X2aR氧化还原传感器的假设是一致的。与Ambudkars博士小组合作，我们还研究了多药耐药蛋白介导的环核苷酸外泄对垂体细胞背景钠电导的依赖性。这些细胞自发地或响应激动剂刺激释放动作电位并释放环核苷酸，但电活动与环核苷酸外排之间的关系尚未被研究。我们发现河豚毒素抗性背景钠电导对动作电位的激发至关重要，多药耐药蛋白（MRPs） MRP4和MRP5参与环核苷酸外排。我们还发现，通过用有机阳离子或蔗糖完全或部分替代细胞外钠来消除大鼠垂体细胞中的背景钠电导，可诱导细胞膜的快速可逆超极化和动作电位放电的抑制，并伴随环状核苷酸外排的快速抑制。缬霉素诱导的质膜超极化也抑制环核苷酸外排，而革兰西丁抑制钙内流或刺激钠内流诱导的细胞膜去极化伴随着环核苷酸外排的促进。相反，苯丙酸抑制环核苷酸外排不影响背景钠电导。在稳定转染人MRP4或MRP5的人胚胎肾293细胞中，用有机阳离子替代浴钠也能使细胞膜超极化并抑制环核苷酸外排。在这些细胞中，钠/氢反转运蛋白莫能菌素不影响膜电位，在改变环核苷酸外排方面几乎无效。在垂体和MRP4-和mrp5表达细胞中，3-3-2-（7-氯喹啉-2-基）乙烯基苯基-（2-二甲基氨基乙基磺酰）甲基磺酰丙酸（MK571）抑制环核苷酸外排。这些结果表明，mrp4 /5介导的环核苷酸外排可以通过钠背景电导决定的膜电位快速调节。最后，我们与Naors博士的团队合作，研究了促性腺激素释放激素（GnRH）和前列腺素受体之间相互串扰的机制。这些研究结果表明，GnRH通过钙依赖性磷脂酶A2刺激花生四烯酸释放，而不是通过更常见的钙依赖性细胞质磷脂酶A2。GnRH通过蛋白激酶C-Src/磷脂酰肌醇3-激酶/MAPK途径刺激COX-1和COX-2的表达。COX-2的转录显然是由核因子-b和CCAAT/增强子结合蛋白位点介导的。PGF2和PGI2抑制GnRH受体表达的自调节表现为钙升高、cAMP形成和MAPK激活。

We have cloned five ATP-gated P2X receptor channels (P2XRs) from the pituitary gland: P2X2R, P2X3R, P2X4R, P2X6R, and P2X7R. Our ongoing work is focused on characterization of the roles of these channels in signaling and secretion in secretory pituitary cells and on the structural-functional characterization of recombinant channels. Recently, we showed by quantitative RT-PCR that mRNA transcripts for the P2X4 subunit are the most abundant in rat anterior pituitary tissue and confirm the P2X4R protein expression by Western blot analysis. Single-cell patch-clamp recordings show that extracellular ATP induced an inward depolarizing current in a majority of thyrotropin-releasing hormone-responsive pituitary cells, which resembled the current profile generated by recombinant P2X4R. The channels were activated and desensitized in a dose-dependent manner and deactivated rapidly. Activation of these channels led to stimulation of electrical activity and promotion of voltage-gated and voltage-insensitive calcium influx. In the presence of ivermectin, a specific allosteric modulator of P2X4Rs, there was an approximately fourfold increase in the maximum amplitude of the ATP-induced inward current, accompanied by an increase in the sensitivity of receptors for ATP, slowed deactivation of receptors, and enhanced ATP-induced prolactin release. These results indicate that thyrotropin-releasing hormone-responsive cells, including lactotrophs, express homomeric and/or heteromeric P2X4R, which facilitate calcium influx and hormone secretion. Mammalian P2X receptors contain ten conserved cysteine residues in their ectodomains, which form five disulfide bonds (SS1-5). In collaboration with Dr. Zemkova, we analyzed the relevance of these SS pairs in rat P2X4 receptor function by replacing one or both cysteines with alanine or threonine, expressing receptors in HEK293 cells and studying their responsiveness to ATP in the absence and presence of ivermectin. Response to ATP was not altered when both cysteines forming the SS3 bond (C132-C159) were replaced with threonines. Replacement of SS1 (C116-C165), SS2 (C126-C149) and SS4 (C217-C227), but not SS5 (C261-C270), cysteine pairs with threonines resulted in decreased sensitivity to ATP and faster deactivation times. The maximum current amplitude was reduced in SS2, SS4 and SS5 double mutants and could be partially rescued by ivermectin in SS2 and SS5 double mutants. This response pattern was also observed in numerous single residue mutants, but receptor function was not affected when the 217-cysteine was replaced with threonine or arginine or when the 261-cysteine was replaced with alanine. These results suggest that the SS1, SS2 and SS4 bonds contribute substantially to the structure of the ligand binding pocket, while the SS5 bond located towards the transmembrane domain contributes to receptor gating. Our collaborative work with P. Catholic University of Chile, was focused on allosteric modulation of P2X2R by several compounds, mainly acting at receptors ectodomain. Like copper, mercury, a metal that induces oxidative stress in cells, also stimulates the activity of P2X2R and inhibits the activity of P2X4R. However, the mercury modulation is not related to the extracellular residues critical for copper modulation. To identify the site(s) for mercury action, we generated two chimeras using the full size P2X2 subunit, termed P2X2a, and a splice variant lacking a 69-residue segment in the C-terminal, termed P2X2b, as donors for intracellular and transmembrane segments and the P2X4 subunit as the donor for ectodomain segment of chimeras. The potentiating effect of mercury on ATP-induced current was preserved in Xenopus oocytes expressing P2X4/2a chimera, but was absent in oocytes expressing P2X4/2b chimera. Site directed mutagenesis experiments revealed that the Cys-430 residue mediates effects of mercury on the P2X2aR activity. Because mercury could act as an oxidative stress inducer, we also tested whether hydrogen peroxide and mitochondrial stress inducers myxothiazol, and rotenone mimicked mercury effects. These experiments revealed that these compounds potentiated the ATP-evoked P2X2aR and P2X4/2aR currents, but not P2X2bR and P2X2a-C430A and P2X2a-C430S mutant currents, whereas antioxidants dithiothreitrol and N-acetylcysteine prevented the hydrogen peroxide-potentiation. Alkylation of Cys-430 residue with methylmethane-thiosulfonate also abolished the mercury and hydrogen peroxide potentiation. Altogether, these results are consistent with the hypothesis that the Cys-430 residue is an intracellular P2X2aR redox sensor. In collaboration with Dr. Ambudkars group, we also studied dependence of multidrug resistance protein-mediated cyclic nucleotide efflux on the background sodium conductance in pituitary cells. These cells fire action potentials and release cyclic nucleotides both spontaneously and in response to agonist stimulation, but the relationship between electrical activity and cyclic nucleotide efflux has not been studied previously. We showed that a tetrodotoxin-resistant background sodium conductance was critical for firing of action potentials and that multidrug resistance proteins (MRPs) MRP4 and MRP5 contribute to cyclic nucleotide efflux. We also showed that abolition of the background sodium conductance in rat pituitary cells by complete or partial replacement of extracellular sodium with organic cations or sucrose induced a rapid and reversible hyperpolarization of cell membranes and inhibition of action potential firing, accompanied by a rapid inhibition of cyclic nucleotide efflux. Valinomycin-induced hyperpolarization of plasma membranes also inhibited cyclic nucleotide efflux, whereas depolarization of cell membranes induced by the inhibition of calcium influx or stimulation of sodium influx by gramicidin was accompanied by a facilitation of cyclic nucleotide efflux. In contrast, inhibition of cyclic nucleotide efflux by probenecid did not affect the background sodium conductance. In human embryonic kidney 293 cells stably transfected with human MRP4 or MRP5, replacement of bath sodium with organic cations also hyperpolarized the cell membranes and inhibited cyclic nucleotide efflux. In these cells, the sodium/hydrogen antiporter monensin did not affect the membrane potential and was practically ineffective in altering cyclic nucleotide efflux. In both pituitary and MRP4- and MRP5-expressing cells, 3-3-2-(7-chloroquinolin-2-yl)vinylphenyl-(2-dimethylcarbamoylethylsulfanyl)methylsulfanyl propionic acid (MK571) inhibited cyclic nucleotide efflux. These results indicate that the MRP4/5-mediated cyclic nucleotide efflux can be rapidly modulated by membrane potential determined by the background sodium conductance. Finally, in collaboration with Dr. Naors group we studied mechanisms of the reciprocal cross talk between gonadotropin-releasing hormone (GnRH) and prostaglandin receptors. Results of these investigations indicate that GnRH stimulates arachidonic acid release via the calcium-independent phospholipase A2 and not via the more common calcium-dependent cytosolic phospholipase A2. GnRH stimulates COX-1 and COX-2 expression via the protein kinase C-Src/phosphatidylinositol 3-kinase/MAPK pathway. COX-2 transcription is mediated apparently by the nuclear factor-B and the CCAAT/enhancer-binding protein sites. The documented inhibition by PGF2 and PGI2 of the autoregulation of GnRH receptor expression is calcium elevation, cAMP formation, and MAPK activation.