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P2X7R，它在多种细胞中表达，包括小胶质细胞和巨噬细胞。我们与Heegaard博士合作，分析了这些通道在骨癌疼痛中的作用。我们证明P2X7R基因敲除小鼠易患骨癌疼痛，而且与患癌的野生型小鼠相比，疼痛相关行为的发病时间更早。此外，选择性P2X7受体拮抗剂a -438079的急性治疗在星形胶质细胞激活和不激活的骨癌疼痛模型中均未能缓解疼痛相关行为，这表明星形胶质细胞P2X7R在骨癌疼痛中的作用可以忽略不计。这些结果支持了骨癌疼痛与神经性疼痛和炎性疼痛相比是一种独立的疼痛状态的假设。P2X7R是一个三聚体通道，具有三个ATP结合位点，但这些位点的占用如何影响门控仍然不清楚。至少有两种相互矛盾的假设被提出来调和这些发现：1)孔洞扩张假说表明，阳离子传导孔洞存在一个渐进的扩张过程。2)双孔假说表明，一个内源性的P2X7R孔被激活，可渗透无机阳离子，并伴随着一个称为泛联蛋白的独特通道的持续激活，该通道可渗透较大的有机阳离子和荧光染料。最近，我们解决了这两种假设。我们的研究结果表明，在激动剂浓度较低和较高的情况下，幼稚受体是单相激活和失活的。应用p2x7r特异性拮抗剂Az10606120消除了这两个反应期。在双相反应中，电流的缓慢二次增长与孔隙扩张在时间上是一致的。相同激动剂浓度的重复刺激引起受体的敏化，表现为电流振幅的逐渐增加，伴随着较慢的失活速率。一旦达到稳定的二次电流水平，在高激动剂浓度下的反应不再是双相的，而是单相的。受体的敏化不依赖于钠和钙的内流，需要大约30分钟的冲洗才能恢复初始的门控特性。T15E-和T15K-P2X7突变体对激动剂的敏感性增加，在所有激动剂浓度下均以单相电流响应，立即激活，孔扩张，缓慢失活。野生型通道所表现出的复杂门控模式可以用马尔科夫状态模型来解释，该模型包括：占据一个或两个结合位点导致激动剂与未敏化受体结合的负协同性，当两个结合位点结合时，通道孔打开到低电导状态，当三个位点被占据时，孔扩张致敏到高电导状态。Pannexins是一个新发现的在大脑和外周组织中表达的三成员蛋白家族，属于间隙连接蛋白超家族。然而，在哺乳动物中，泛联蛋白不形成间隙连接，其在脑垂体中的表达和功能尚未被研究。我们发现大鼠脑垂体表达pannexin1和2的mRNA和蛋白转录本，但不表达pannexin3。Pannexin 1在前叶表达较多，而Pannexin 2在垂体中、后叶表达较多。Pannexin 1在促肾上腺皮质激素和部分生长激素、垂体后叶s100阳性的垂体细胞和小鼠垂体促肾上腺皮质激素分泌细胞at -20以及分泌催乳素的大鼠永生化垂体细胞中被检测到，而Pannexin 2在垂体前叶s100阳性的滤泡星状细胞、中间叶的黑色素细胞以及后叶含有抗利尿激素的轴突和神经末梢中被检测到。pannexins 1和2的过表达不影响P2X7R门控，但增加了ATP在细胞外介质中的释放，这被间隙连接抑制剂卡贝诺洛酮阻断。下调内源性pannexin 1的表达，而不下调pannexin 2及其短干扰rna的表达，也抑制了At-T20细胞的基础ATP释放。这些结果表明，pannexins可能提供了一种传递ATP的途径，ATP是垂体内源性表达的多种P2X阳离子通道和G蛋白偶联的P2Y受体的天然激动剂。在与Startakis博士小组的合作中，我们还研究了垂体细胞自发和camp促进的电活动对浴钠的依赖性。我们的研究结果表明，福斯克林剂量依赖性地增加cAMP的产生，并促进约30%的大鼠和小鼠垂体细胞在其最大浓度下的钙内流。福斯可林对钙内流的刺激作用在PKA（蛋白激酶A）受到抑制的细胞和PKA主要调控亚基单倍体不足的细胞中消失，但在PKA主要催化亚基单倍体不足的细胞中保留。自发和福斯克林刺激的钙内流存在于具有抑制电压门控钠和超极化激活阳离子通道的细胞中，而不存在于浸泡在以有机阳离子取代钠的培养基中的细胞中。与钠传导非选择性阳离子通道在pka刺激的钙内流中的作用一致，cAMP诱导了一个缓慢发展的电流，其逆转电位约为0 mV。两种TRP（瞬时受体电位）通道阻滞剂SKF96365和2-APB以及氟芬那酸（一种非选择性阳离子通道抑制剂）也能抑制自发性和福斯克林刺激的电活动和钙内流。定量RT-PCR分析显示大鼠垂体细胞中TRPC1 >> TRPC6 > TRPC4 > TRPC5 > TRPC3 mRNA转录本的表达。这些实验表明，在垂体细胞中，PKA进一步刺激了构成活性的阳离子通道，并通过以钠依赖的方式间接控制起搏去极化和直接通过传导钙来促进钙信号。最后，在我们与克莱因博士小组的合作中，我们通过穿孔膜片钳记录来研究去甲肾上腺素对大鼠松果体细胞膜电位（Vm）和自发电活动的控制。去甲肾上腺素没有改变尖峰频率。然而，它被发现通过α 1-肾上腺素受体以浓度依赖的方式作用于Vm，产生双相变化。最初的反应是由瞬时向外钾电流引起的超极化。这种电流似乎是由细胞内储存的钙释放引起的，根据观察，在缺乏钙的培养基中也可以看到这种电流。此外，药理学研究表明，该电流依赖于磷脂酶C （PLC）的激活，并部分由双库克林和阿帕胺敏感的钙控制钾通道介导。由于向内电流的作用，初始的瞬态超极化之后是持续的去极化。这种反应依赖于plc依赖的钠/钙内流激活，但不涉及硝苯地平敏感的电压门控钙通道。总之，这些结果首次表明，α 1肾上腺素受体的激活在松果体细胞Vm中启动plc依赖的双相变化，其特征是由钙激活的钾通道混合物介导的初始瞬时超极化，然后是持续的去极化。

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 P2X7R, which is expressed in a large variety of cells, including microglia and macrophages. In collaboration with Dr. Heegaard, we analyzed the role of these channels in bone cancer pain. We demonstrated that P2X7R knockout mice were susceptible to bone cancer pain and moreover had an earlier onset of pain-related behaviors compared with cancer-bearing, wild type mice. Furthermore, acute treatment with the selective P2X7 receptor antagonist, A-438079, failed to alleviate pain-related behaviors in models of bone cancer pain with and without astrocyte activation, suggesting that astrocytic P2X7R play a negligible role in bone cancer pain. The results support the hypothesis that bone cancer pain is a separate pain state compared with those of neuropathic and inflammatory pain. The P2X7R is a trimeric channel with three binding sites for ATP, but how the occupancy of these sites affects gating is still not understood. At least two conflicting hypotheses have been postulated to reconcile these findings: 1) The pore-dilation hypothesis suggests that there is a progressive dilation of the cation-conducting pore. 2) The two-pore hypothesis implies the activation of an endogenous P2X7R pore permeable for inorganic cations, accompanied by sustained activation of a distinct channel called pannexin, which is permeable to larger organic cations and fluorescent dyes. Recently, we addressed both hypotheses. Our results indicate that naive receptors activated and deactivated monophasically at low and biphasically at higher agonist concentrations. Both phases of response were abolished by application of Az10606120, a P2X7R-specific antagonist. The slow secondary growth of current in the biphasic response coincided temporally with pore dilation. Repetitive stimulation with the same agonist concentration caused sensitization of receptors, which manifested as a progressive increase in the current amplitude, accompanied by a slower deactivation rate. Once a steady level of the secondary current was reached, responses at high agonist concentrations were no longer biphasic but monophasic. Sensitization of receptors was independent of sodium and calcium influx and about 30 min washout was needed to reestablish the initial gating properties. T15E- and T15K-P2X7 mutants showed increased sensitivity for agonists, responded with monophasic currents at all agonist concentrations, activated immediately with dilated pores, and deactivated slowly. The complex pattern of gating exhibited by wild-type channels can be accounted for by a Markov state model that includes negative cooperativity of agonist binding to unsensitized receptors caused by the occupancy of one or two binding sites, opening of the channel pore to a low conductance state when two sites are bound, and sensitization with pore dilation to a high conductance state when three sites are occupied. Pannexins are a newly discovered three-member family of proteins expressed in the brain and peripheral tissues that belong to the superfamily of gap junction proteins. However, in mammals pannexins do not form gap junctions, and their expression and function in the pituitary gland have not been studied. Here we show that the rat pituitary gland expresses mRNA and protein transcripts of pannexins 1 and 2 but not pannexin 3. Pannexin 1 was more abundantly expressed in the anterior lobe, whereas pannexin 2 was more abundantly expressed in the intermediate and posterior pituitary. Pannexin 1 was identified in corticotrophs and a fraction of somatotrophs, the S100-positive pituicytes of the posterior pituitary and AtT-20 (mouse pituitary adrenocorticotropin-secreting cells) and rat immortalized pituitary cells secreting prolactin, whereas pannexin 2 was detected in the S100-positive folliculostellate cells of the anterior pituitary, melanotrophs of the intermediate lobe, and vasopressin-containing axons and nerve endings in the posterior lobe. Overexpression of pannexins 1 and 2 did not affected P2X7R gating but enhanced the release of ATP in the extracellular medium, which was blocked by the gap junction inhibitor carbenoxolone. Basal ATP release in At-T20 cells was also suppressed by down-regulating the expression of endogenous pannexin 1 but not pannexin 2 with their short interfering RNAs. These results indicate that pannexins may provide a pathway for delivery of ATP, which is a native agonist for numerous P2X cationic channels and G protein-coupled P2Y receptors endogenously expressed in the pituitary gland. In collaboration with Dr. Startakis group, we also studied dependence of spontaneous and cAMP-facilitated electrical activity of pituitary cells on bath sodium. Our results indicate that forskolin dose-dependently increases cAMP production and facilitates calcium influx in about 30% of rat and mouse pituitary cells at its maximal concentration. The stimulatory effect of forskolin on calcium influx was lost in cells with inhibited PKA (protein kinase A) and in cells that were haploinsufficient for the main PKA regulatory subunit but was preserved in cells that were also haploinsufficient for the main PKA catalytic subunit. Spontaneous and forskolin-stimulated calcium influx was present in cells with inhibited voltage-gated sodium and hyperpolarization-activated cation channels but not in cells bathed in medium, in which sodium was replaced with organic cations. Consistent with the role of sodium-conducting nonselective cation channels in PKA-stimulated calcium influx, cAMP induced a slowly developing current with a reversal potential of about 0 mV. Two TRP (transient receptor potential) channel blockers, SKF96365 and 2-APB, as well as flufenamic acid, an inhibitor of nonselective cation channels, also inhibited spontaneous and forskolin-stimulated electrical activity and calcium influx. Quantitative RT-PCR analysis indicated the expression of mRNA transcripts for TRPC1 >> TRPC6 > TRPC4 > TRPC5 > TRPC3 in rat pituitary cells. These experiments suggest that in pituitary cells constitutively active cation channels are stimulated further by PKA and contribute to calcium signaling indirectly by controlling the pacemaking depolarization in a sodium-dependent manner and directly by conducting calcium. Finally, in our collaborative work with Dr. Kleins group, we perforated patch clamp recording to study the control of membrane potential (Vm) and spontaneous electrical activity in the rat pinealocyte by norepinephrine. Norepinephrine did not alter spiking frequency. However, it was found to act through alpha1-adrenoreceptors in a concentration-dependent manner to produce a biphasic change in Vm. The initial response was a hyperpolarization due to a transient outward potassium current . This current appears to be triggered by calcium released from intracellular stores, based on the observation that it was also seen in cells bathed in calcium-deficient medium. In addition, pharmacological studies indicate that this current was dependent on phospholipase C (PLC) activation and was in part mediated by bicuculline methiodide and apamin-sensitive calcium-controlled potassium channels. The initial transient hyperpolarization was followed by a sustained depolarization due to an inward current. This response was dependent on PLC-dependent activation of sodium/calcium influx but did not involve nifedipine-sensitive voltage-gated calcium channels. Together, these results indicate for the first time that activation of alpha1 adrenoreceptors initiates a PLC-dependent biphasic change in pinealocyte Vm characterized by an initial transient hyperpolarizzation mediated by a mixture of calcium-activated potassium channels followed by a sustained depolarization.