Intracellular Signaling In Endocrine Cells

我们最近的工作集中在垂体乳营养细胞分泌催乳素（一种控制泌乳的激素）以及多巴胺D2受体和低张力在这一过程中的作用。我们还研究了γ -氨基丁酸(GABA)-A受体通道在乳养动物和促性腺激素中的表达和作用，并提供了从垂体细胞中克隆的重组atp门控P2X4受体通道的结构和功能表征。多巴胺D2受体的研究表明，它们在垂体乳营养物中的激活导致催乳素释放的抑制。其他人已经提出，这种抑制是通过Gi/o- α蛋白介导的cAMP产生的抑制和/或Gi/o- β / γ二聚体介导的内向整流钾通道的激活和电压门控钙通道的抑制发生的。我们发现，多巴胺激动剂诱导的自发钙内流和预先储存的PRL释放的抑制作用在福斯克林处理后cAMP水平升高时得以保留。我们进一步观察到，多巴胺激动剂在未治疗的细胞中抑制自发和去极化诱导的钙内流，而在百日咳毒素处理的细胞中则没有。在向内整流钾通道被阻断的细胞中也观察到这种抑制作用，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钙内流。然而，在百日咳处理的细胞中，激动剂诱导的催乳素释放抑制仅部分缓解，这表明多巴胺受体也抑制了电压门控钙内流下游的胞吐。在激动剂诱导的泌乳素释放抑制中，百日咳毒素不敏感的步骤不受添加wortmannin （pi3激酶抑制剂）和锂（GSK-3抑制剂）的影响，但在phorbol酯PMA存在下减弱，PMA以蛋白激酶c依赖的方式抑制Gz信号通路。这些结果首次表明，多巴胺抑制基础泌乳素释放不仅通过阻断百日咳毒素敏感信号通路的电压门控钙内流，而且通过百日咳毒素不敏感和蛋白激酶c敏感信号通路脱敏钙分泌偶联。在与Zorecs博士小组的合作中，我们研究了垂体激素催乳素的低渗释放，因为这种激素也有助于渗透调节。放射免疫法监测到，在经灌注的大鼠乳营养细胞中，低张力导致催乳素释放短暂增加，随后持续抑制。在共聚焦显微镜成像的单细胞中，低张力引起2%的囊泡/细胞排出荧光标记的心房利钠肽，它们通过相同的融合孔同步装载苯乙烯染料fm4 -64。相比之下，高钾诱导的去极化导致10%的囊泡/细胞响应，两种荧光探针卸载/加载时间过程不同。在细胞附着的研究中，在未受刺激和受刺激的条件下，膜电容的离散变化都被记录下来，反映了单个囊泡与质膜的融合/分裂。在受刺激的细胞中，完全融合事件发生的概率较低且保持不变，95%以上的融合事件是短暂的，随着开放融合孔概率、平均孔隙停留时间、发生频率和融合孔电导的增加。低渗性仅在静默膜斑块中很少引起新的融合事件。结果表明，在低张力刺激下的乳养菌中，快速释放的囊泡出现了预融合，并以一种“吻而跑”的方式释放激素。在过去的一年里，我们还研究了GABA-A受体通道在垂体细胞中的表达，它们在垂体前叶分泌细胞类型中的分布，以及作用的性质（刺激或抑制）。我们的研究结果表明，所有GABAA受体亚基的mrna都在垂体细胞中表达，α 1/ β 1亚基蛋白存在于所有分泌细胞中。在电压箝位的革兰西丁穿孔细胞中，GABA诱导电流振幅呈剂量依赖性增加，这种增加被二环霉素和微毒素抑制，而地被地西泮和唑吡坦以浓度依赖性方式促进。在完整的细胞中，GABA和GABA- a受体激动剂muscimol引起细胞内钙的快速和短暂增加，而GABA- b受体激动剂巴氯芬无效，这表明氯离子介导的去极化激活了电压门控钙通道。与此发现一致，RT-PCR分析显示垂体细胞中NKCC1高表达，而KCC2阳离子/氯转运体mrna不表达。此外，在大多数细胞中，氯离子的GABA- a通道逆转电位正于基线膜电位，GABA激活离子通道导致细胞去极化和调节自发电活动。这些结果表明，垂体分泌细胞表达功能性GABA-A受体通道是去极化的。在从垂体细胞中克隆的重组P2X4受体的结构-功能表征工作中，重点研究了伊维菌素对这些通道进行变构调节的残基，伊维菌素是一种大环内酯，通过与开放构象状态的跨膜螺旋残基相互作用，特异性增强P2X4受体通道功能。该受体对伊维菌素的敏感性也被用作识别面向通道孔的跨膜区域残基的指标。为此，我们对大鼠P2X4跨膜区域进行了半胱氨酸扫描诱变。29个不同残基的突变改变了受体功能，其中在Gln36、Leu40、Val43、Val47、Trp50、Asn338、Gly342、Leu346、Ala349和Ile356突变体中改变了伊维菌素的作用。替换敏感的Arg33和Cys353突变体也可以被认为是伊维菌素敏感的。这12个残基的模式与两个跨膜区域的螺旋拓扑结构一致，每3或4个氨基酸受到取代的影响。这些主要的疏水性非极性残基也存在于伊维菌素敏感的曼氏血吸虫P2X亚基中。它们位于螺旋的同一侧，可以在开放构象状态下面对脂质，并为IVM提供结合袋。而与ivm无关的hit Met31、Tyr42、Gly45、Val49、Gly340、Leu343、Ala344、Gly347、Thr350、Asp354和Val357则位于其螺旋的另一侧，可能面向受体或蛋白的孔，在门控制中起重要作用。一旦从晶体研究中获得受体结构，我们将知道这些功能重要残基的真实拓扑结构。

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.