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控制信号、激素分泌和基因表达中的作用，以及atp门控P2X受体通道的分子解剖。我们早期的研究表明，培养的垂体细胞，包括乳养细胞，经常表现出较大的膜电位振荡，在此之上产生去极化平台和动作电位爆发，其峰值通常不会达到反向电位。在乳养动物中，这种自发的电活动和相关的电压门控钙内流足以维持高催乳素释放。正常和永生化垂体细胞表达钙抑制腺苷酸环化酶的发现促使我们研究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在这些细胞中自发的起搏和基础催乳素释放中起作用的假设。与此一致的是，我们发现福斯克林刺激腺苷酸环化酶可以激活静止的乳养细胞的动作电位，并增加自发活性细胞的峰值频率。这反过来又促进了电压门控钙内流和催乳素分泌。3-异丁基-1-甲基黄嘌呤对磷酸二酯酶的抑制也刺激环核苷酸的积累和催乳素的释放。相反，MDL-12330A以浓度依赖的方式抑制基础和福斯克林刺激的环核苷酸产生，以及电活动、钙瞬变和催乳素分泌。基础环AMP的产生通过细胞外钙的去除而增强，并通过促进电压门控钙内流而减弱。这些结果表明，钙抑制腺苷酸环化酶的内在活性有助于控制自发起搏活动。我们的研究结果进一步表明，内皮素受体抑制电压门控钙流入依赖性催乳素释放。然而，这些受体以不依赖于磷脂酶C和酪氨酸激酶的方式抑制电压门控钙内流下游的分泌。我们还发现内皮素受体与百日咳毒素敏感和不敏感的Gi蛋白都偶联。最后，我们发现内皮素受体与Gz信号通路的偶联可以抑制电压门控钙内流下游的催乳素分泌。持续抑制分泌是通过下调腺苷酸环化酶信号级联来实现的，而快速抑制也发生在cAMP水平升高时，无论磷脂酶C、酪氨酸激酶、这些结果表明，七种跨膜结构域受体与Gz蛋白的偶联提供了一种途径，可以有效地长时间阻断激素分泌，而不会干扰起搏活动和钙流入依赖的细胞功能。

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.