Intracellular Signaling In Endocrine Cells

钙信号和细胞调控的研究主要集中在垂体细胞类型特异性钙信号-分泌偶联的生物物理基础，G蛋白偶联和酪氨酸激酶受体在信号、激素分泌和基因表达中的调控作用，以及ATP门控的P2X受体通道的分子解剖。我们早期的研究表明，包括哺乳细胞在内的培养的垂体细胞经常表现出更大的膜电位振荡，在其上产生去极化平台和动作电位爆发，而动作电位的尖峰通常不会达到反向电位。在哺乳细胞中，这种自发的电活动和相关的电压门控钙内流足以维持催乳素的高释放。正常和永生化的垂体细胞表达钙抑制的腺酰环化酶的发现促使我们检验了环核苷酸可能通过控制超极化激活的阳离子通道和/或环核苷酸门控通道而在这些细胞中发挥自发起搏和基础催乳素释放的作用的假说。与此一致的是，我们发现Forsklin对腺苷环化酶的刺激启动了静止的哺乳细胞动作电位的激发，并增加了自发活动细胞的尖峰频率。这反过来又促进了电压门控钙内流和催乳素的分泌。3-异丁基-1-甲基黄嘌呤抑制磷酸二酯酶还能刺激环核苷酸积累和催乳素释放。相反，MDL-12330A以浓度依赖的方式抑制基础和Forsklin刺激的环核苷酸的产生，以及电活动、钙瞬变和催乳素分泌。基础环磷酸腺苷的产生通过细胞外钙的去除而增加，而通过促进电压门控的钙内流而减弱。这些结果表明，钙抑制的腺酰环化酶的固有活性有助于控制自发的起搏活动。我们的结果进一步表明，内皮素受体抑制电压门控钙内流依赖的催乳素的释放。然而，这些受体抑制电压门控钙内流下游的分泌，并以磷脂酶C和酪氨酸激酶不依赖的方式抑制。我们还发现内皮素受体与百日咳毒素敏感和不敏感的Gi蛋白偶联。最后，我们发现内皮素受体与Gz信号通路的偶联是电压门控钙内流下游催乳素分泌受到抑制的原因。分泌的持续抑制是通过下调腺苷环化酶信号级联来实现的，而cAMP水平升高时也会发生快速抑制，而与磷脂酶C、酪氨酸激酶和蛋白激酶C的状态无关。这些结果表明，七个跨膜域受体与Gz蛋白的偶联提供了一条有效地长时间阻止激素分泌的途径，而不干扰起搏活性和钙内流依赖的细胞功能。 我们先前发现，嘌呤能信号系统在正常和永生化的垂体前叶细胞中起作用。这些细胞在静息状态下释放三磷酸腺苷，以响应钙动员受体的激活。然而，基础激素的释放速率与ATP的释放没有相关性，这表明ATP不是通过调控的胞吐作用与激素共同分泌的。正在进行的实验旨在描述一条负责ATP释放的途径。这些细胞还表达胞外核苷酸酶，它能水解三磷酸腺苷，形成相应的核苷和游离磷酸盐。外核苷酸酶eNTPDase1-3的转录本存在于垂体细胞中。这种水解级联反应的产物，ADP和腺苷，也通过激活不同的质膜受体而充当细胞外信使。这些受体被称为嘌呤能，属于两组：P1和P2受体。P2X受体是一个配体门控的阳离子通道家族，由位于细胞内的N-末端和C-末端两个跨膜结构域和一个包含ATP结合域的胞外大环组成。为了确定对结合和门控重要的区域，我们使用重组通道的实验工作集中在保守的胞外结构域残基的嵌合体和点突变上。突变通道在人胚胎肾293细胞和小鼠促性腺激素释放激素分泌的GT1神经元中表达，并使用钙成像和膜片钳技术进行分析。使用嵌合的P2XRs的实验有助于表征门控和离子传导、受体的失活、受体脱敏的结构决定因素以及从脱敏中恢复。为了确定对ATP结合重要的区域，我们使用了P2X4的Lys180-Lys326胞外结构域和II类氨基酰-tRNA合成酶之间的已知序列和二级结构相似性作为指南，以生成受体结合位点的三维模型并设计突变体。同源模拟和定点突变之间的相互作用表明，P2X4R的Asp280残基通过镁离子与ATP结合，Phe230与ATP的腺嘌呤环结合，Lys190、His286和Arg278分别协调带负电荷的α、β和γ磷酸基团的作用。在确定该通道的晶体结构之前，该模型可以为进一步研究ATP结合域的确定和P2XRs的门控提供一种有用的方法。

Studies on calcium signaling and cellular regulation are focused on the biophysical basis of pituitary cell type-specific calcium signaling-secretion coupling, the roles of G protein-coupled and tyrosine kinase receptors in control of signaling, hormone secretion and gene expression, and the molecular dissection of ATP-gated P2X receptor channels. Our earlier studies revealed that cultured pituitary cells, including lactotrophs, frequently exhibit larger membrane potential oscillations, on top of which the depolarizing plateau and bursts of action potentials are generated, with spikes that usually do not reach the reverse potential. In lactotrophs such spontaneous electrical activity and the associated voltage-gated calcium influx are sufficient to maintain high prolactin release. The finding that normal and immortalized pituitary cells express calcium-inhibitable adenylyl cyclases prompted us to examine the hypothesis that cyclic nucleotides may have a role in spontaneous pacemaking and basal prolactin release in these cells by controlling hyperpolarization-activated cation channels and/or cyclic nucleotide-gated channels. Consistent with this, we found that stimulation of adenylyl cyclases by forskolin initiates firing of action potentials in quiescent lactotrophs and increases the spiking frequency in spontaneously active cells. This in turn facilitates voltage-gated calcium influx and prolactin secretion. Inhibition of phosphodiesterases by 3-isobutyl-1-methylxanthine also stimulates cyclic nucleotide accumulation and prolactin release. Conversely, MDL-12330A inhibits basal and forskolin-stimulated cyclic nucleotide production in a concentration-dependent manner, as well as electrical activity, calcium transients, and prolactin secretion. Basal cyclic AMP production is augmented by removal of extracellular calcium and is attenuated by facilitation of voltage-gated calcium influx. These results suggest that the intrinsic activity of calcium-inhibitable adenylyl cyclases contributes to the control of spontaneous pacemaking activity. Our results further indicate that endothelin receptors inhibit voltage-gated calcium influx-dependent prolactin release. However, these receptors inhibit secretion downstream of voltage-gated calcium influx and in a phospholipase C and tyrosine kinase-independent manner. We also found that endothelin receptors are coupled to both pertussis toxin-sensitive and insensitive Gi proteins. Finally, we discovered that the coupling of endothelin receptors to the Gz signaling pathway accounts for inhibition of prolactin secretion downstream of voltage-gated calcium influx. Sustained inhibition of secretion is achieved through down-regulation of the adenylyl cyclase signaling cascade, whereas rapid inhibition also occurs at elevated cAMP levels regardless of the status of phospholipase C, tyrosine kinases, and protein kinase C. These results indicate that the coupling of seven transmembrane domain receptors to Gz proteins provides a pathway that effectively blocks hormone secretion for a prolonged time without interfering with pacemaking activity and calcium influx-dependent cellular functions. We previously found that the purinergic signaling system is operative in normal and immortalized anterior pituitary cells. These cells release ATP under resting conditions and in response to activation of calcium mobilizing receptors. However, there is no correlation between the rate of basal hormone and ATP release, suggesting that ATP is not co-secreted with hormones by regulated exocytosis. Experiments in progress are directed toward the characterization of a pathway responsible for ATP release. These cells also express ecto-nucleotidases, which hydrolyze ATP, resulting in formation of the respective nucleoside and free phosphate. The transcripts for ecto-nucleotidase eNTPDase 1-3 were found in pituitary cells. The products of this hydrolytic cascade, ADP and adenosine, also act as extracellular messengers by activating distinct plasma membrane receptors. These receptors are termed purinergic and belong to two groups: P1 and P2 receptors. P2X receptors are a family of ligand-gated cation channels composed of two transmembrane domains, N- and C-termini located intracellularly, and a large extracellular loop containing the ATP binding domain. To identify regions important for binding and gating, our experimental work with recombinant channels is focused on chimeras and point mutagenesis of conserved ectodomain residues. Mutant channels were expressed in human embryonic kidney 293 cells and mouse gonadotropin-releasing hormone-secreting GT1 neurons and analyzed using calcium imaging and patch clamp techniques. Experiments with chimeric P2XRs helped in characterization of gating and ionic conduction, deactivation of receptors, structural determinants of receptor desensitization and recovery from desensitization. To identify regions important for ATP binding we used the known sequence and secondary structure similarities between the Lys180-Lys326 ectodomain region of P2X4 and the class II aminoacyl-tRNA synthetases as a guide to generate a three-dimensional model of the receptor-binding site and to design mutants. The interplay between homology modeling and site-directed mutagenesis suggested that the Asp280 residue of P2X4R coordinates ATP binding via the magnesium ion, Phe230 coordinates the binding of the adenine ring of ATP, and Lys190, His286 and Arg278 coordinate the actions of negatively charged alpha, beta, and gamma phosphate groups, respectively. Until the crystal structure of the channel is solved, this model could provide a useful approach for future studies on identification of ATP binding domain and gating of P2XRs.