Intracellular Signaling In Endocrine Cells

本项目研究内分泌和神经内分泌细胞中的细胞信号级联，以及质膜电事件与受体介导的细胞内信号和分泌之间的相互作用。目前的重点是天然和重组核苷酸受体（P2 R）和一氧化氮（NO）-cGMP信号通路。在垂体前叶细胞中完成了对天然P2 R的研究。我们的研究结果表明，这些细胞分泌ATP和表达G蛋白偶联受体（P2 YRs）和阳离子传导通道（P2 XRs）。ATP和ADP，但不是UTP和UDP，触发钙信号在大多数催乳素和催乳素（PRL）的释放在混合垂体细胞，和他们的行动被废除的腺苷三磷酸双磷酸酶，外核苷酸酶的存在下。Gq偶联钙动员P2 Y1 R，P2 Y2 R，P2 Y 4 R和P2 Y 6 R，以及Gi偶联P2 Y12 R的转录本，在混合垂体前叶细胞中鉴定。钙动员依赖性信号传导和PRL分泌的药理学特征表明，P2 Y1 R介导ATP和ADP的刺激作用。P2 X2 aR、P2 X2 bR、P2 X3 R、P2 X4 R和P2 X7 R也在P2 X3细胞中表达。多条证据表明P2 X4 R亚型为钙内流依赖性信号传导和PRL分泌提供了主要途径。对重组大鼠P2 XR的研究集中在这些通道的钙信号传导以及不同受体亚结构域对亚型特异性行为的贡献。重组P2 XR在持续ATP刺激过程中产生去极化电流，其脱敏顺序（从快速脱敏到非脱敏）：P2 X3 R> P2 X2 b + X4 R> P2 X2 bR> P2 X2 a + X4 R> P2 X4 R> P2 X2 aR> P2 X7 R。当在GT 1细胞中表达时，所有野生型和嵌合型P2 XR都对与全局钙信号结合的激动剂作出反应，其以与电流信号相同的顺序脱敏，但以明显较慢的方式脱敏。钙信号的全球分布是目前独立的电流脱敏率。钙信号的时间特性不受电压门控钙内流和细胞外钠清除的影响。钙信号很好地反映了ATP的受体特异性EC 50和P2 XRs的细胞外锌和pH敏感性。为了测试激动剂对结合结构域的亲和力解释配体特异性脱敏模式的假设，我们使用对ATP具有可变敏感性的受体产生嵌合体，顺序为：P2 X4 R> P2 X2 aR = P2 X2 bR>> P2 X7 R。在P2 X4 R的骨架中具有P2 X2 R的胞外域Ile 66-Tyr 310序列和P2 X7 R的Val 61-Phe 313序列的嵌合体被表达，但为无功能通道。在P2 X2 aR和P2 X2 bR的主链中具有P2 X4 R的Val 66-Tyr 315胞外域序列的P2 X2 a + X4 R和P2 X2 b + X4 R嵌合体是功能性的，并且与两种亲本受体相比表现出对配体的增加的敏感性。这些嵌合体也比亲本受体更快地脱敏，并且以配体非特异性的方式。但与亲本P2 X2 bR和P2 X2 aR一样，嵌合体P2 X2 b + X4 R比P2 X2 a + X4 R更快地脱敏，并且P2 X2 a + X4 R的Arg 371-Pro376胞内C端序列被P2 X4 R的Glu 376-Gly 381序列取代后脱敏率增加。这些结果表明胞外域和跨膜结构域周围的侧翼区之间的相互作用对配体效力和受体活化的相关性。此外，配体效力与受体脱敏率呈正相关，但不影响C-末端特异性脱敏模式。 以前已经建立了NO-cGMP信号通路和垂体催乳素释放之间的耦合。然而，信使介导的行动，这一信号通路对激素分泌和分泌机制的影响，钙依赖性或非依赖性，尚未确定。抑制组成性表达的神经元NO合酶降低NO和cGMP水平，增加基础PRL释放。缓慢释放的NO供体的添加增加cGMP水平，并以时间依赖性方式抑制基础PRL释放。诱导型NO合酶的表达也增加NO和cGMP水平，并抑制基础，去极化诱导，和TRH诱导的PRL释放，而抑制这种酶减少NO和cGMP的生产和恢复PRL释放。这些治疗均不影响自发性和刺激性电压门控钙内流。在基础NO水平下，添加可渗透的cGMP类似物并不抑制PRL分泌。在升高的NO水平，cGMP生产的抑制和促进其降解没有逆转抑制PRL分泌。这些实验表明，NO抑制钙依赖性PRL分泌的cGMP-独立的方式和下游的电压门控钙内流。

This project addresses the cellular signaling cascade in endocrine and neuroendocrine cells, and the interactions between plasma membrane electrical events and receptor-mediated intracellular signaling and secretion. Current emphasis is on native and recombinant nucleotide receptors (P2Rs) and nitric oxide (NO)-cGMP signaling pathways. Work on native P2Rs was done in anterior pituitary cells. Our results indicate that these cells secrete ATP and express G protein-coupled receptors (P2YRs) and cation-conducting channels (P2XRs). ATP and ADP, but not UTP and UDP, triggered calcium signaling in a majority of lactotrophs and prolactin (PRL) release in mixed pituitary cells, and their actions were abolished in the presence of apyrase, an ectonucleotidase. Transcripts for Gq-coupled calcium-mobilizing P2Y1R, P2Y2R, P2Y4R, and P2Y6R, as well as Gi-coupled P2Y12R, were identified in mixed anterior pituitary cells. The pharmacological profile of calcium mobilization-dependent signaling and PRL secretion indicated that P2Y1R mediates the stimulatory action of ATP and ADP. Pituitary cells also express P2X2aR, P2X2bR, P2X3R, P2X4R, and P2X7R. Several lines of evidence suggested that P2X4R subtype provides a major pathway for calcium influx-dependent signaling and PRL secretion. Work on recombinant rat P2XR was focused on calcium signaling by these channels and the contributions of distinct receptor subdomains to the subtype-specific behavior. Recombinant P2XR generated depolarizing currents during the sustained ATP stimulation, which desensitized in order (from rapidly desensitizing to non-desensitizing): P2X3R > P2X2b+X4R > P2X2bR > P2X2a+X4R > P2X4R > P2X2aR > P2X7R. When expressed in GT1 cells, all wild type and chimeric P2XRs responded to agonist binding with global calcium signals, which desensitized in the same order as current signals but in a significantly slower manner. The global distribution of calcium signals was present independently of the rate of current-desensitization. The temporal characteristics of calcium signals were not affected by voltage-gated calcium influx and removal of extracellular sodium. Calcium signals reflected well the receptor-specific EC50s for ATP and the extracellular zinc and pH sensitivities of P2XRs. To tested the hypothesis that affinity of agonists for binding domain accounts for ligand-specific desensitization pattern, we generated chimeras using receptors with variable sensitivity to ATP in order: P2X4R > P2X2aR = P2X2bR >> P2X7R. Chimeras having the ectodomain Ile66-Tyr310 sequence of P2X2R and Val61-Phe313 sequence of P2X7R in the backbone of P2X4R were expressed, but were non-functioning channels. P2X2a+X4R and P2X2b+X4R chimeras having the Val66-Tyr315 ectodomain sequence of P2X4R in the backbones of P2X2aR and P2X2bR were functional and exhibited increased sensitivity to ligands compared to both parental receptors. These chimeras also desensitized faster than parental receptors and in a ligand-nonspecific manner. However, like parental P2X2bR and P2X2aR, chimeric P2X2b+X4R desensitized more rapidly than P2X2a+X4R, and the rate of desensitization of P2X2a+X4R increased by substituting its Arg371-Pro376 intracellular C-terminal sequence with Glu376-Gly381 sequence of P2X4R. These results indicate the relevance of interaction between ectodomain and flanking regions around the transmembrane domains on ligand potency and receptor activation. Furthermore, the ligand potency positively correlates with the rates of receptor-desensitization, but does not affect the C-terminal-specific pattern of desensitization. The coupling between NO-cGMP signaling pathway and PRL release in pituitary lactotrophs has been established previously. However, the messenger that mediates the action of this signaling pathway on hormone secretion and the secretory mechanism affected, calcium-dependent or independent, have not been identified. Inhibition of constitutively expressed neuronal NO synthase decreased NO and cGMP levels and increased basal PRL release. The addition of a slowly releasable NO donor increased cGMP levels and inhibited basal PRL release in a time-dependent manner. Expression of inducible NO synthase also increased NO and cGMP levels and inhibited basal, depolarization-induced, and TRH-induced PRL release, whereas inhibition of this enzyme decreased NO and cGMP production and recovered PRL release. None of these treatments affected spontaneous and stimulated voltage-gated calcium influx. At basal NO levels, the addition of permeable cGMP analogs did not inhibit PRL secretion. At elevated NO levels, inhibition of cGMP production and facilitation of its degradation did not reverse inhibited PRL secretion. These experiments indicate that NO inhibits calcium-dependent PRL secretion in a cGMP-independent manner and downstream of voltage-gated calcium influx.