Intracellular Signaling In Endocrine Cells

我们将继续研究垂体前叶细胞表达的受体和通道及其在垂体功能中的作用。乙酰胆碱（Acetylcholine, ACh）已被确定为脑垂体的副分泌因子，但介导ACh作用的受体和承载这些受体的细胞类型尚未确定。结果表明，在培养的大鼠垂体细胞中，烟碱亚基mrna的表达顺序为beta2 > beta1 = alpha9 > alpha4。在垂体细胞中也发现了M4 bb0 M3毒蕈碱受体mrna。用GnRH处理培养的垂体细胞可下调α 9和α 4 mRNA的表达，但不影响M3和M4受体mRNA的表达，ACh不改变GnRH受体mRNA的表达。我们还进行了双免疫染色，以显示β 2亚基和M4受体蛋白在促性腺激素中的表达。在单个细胞中发现了能够产生向内电流、促进电活动和钙内流的功能性尼古丁通道。M3受体介导的磷脂酶c依赖性钙动员激活了向外的阿帕胺敏感钾电流并引起超极化。乙酰胆碱激活M4受体抑制cAMP的产生和gnrh诱导的LH释放。我们认为，促性腺激素中有多种胆碱能受体表达，而乙酰胆碱能的主要分泌作用是通过m4受体介导的cAMP的下调而受到抑制。体外尼古丁受体的表达弥补了促性腺激素缺乏常规GnRH刺激。cAMP和cGMP是细胞内信使，由三磷酸核苷酸通过一系列由腺苷酸环化酶和胍基环化酶组成的酶产生。在垂体细胞中，我们之前的工作揭示了环核苷酸及其激酶在控制垂体内分泌细胞的电活动和参与这一过程的通道中起重要作用。最近，我们关注这些信使在控制钙信号传导中下游电活动的激素分泌中的作用。激素释放研究表明，在腺苷酸环化酶活化剂福斯克林和膜透性cAMP类似物dbcAMP的刺激下，离体大鼠乳养动物的泌乳素（PRL）释放表现出双相浓度依赖性。虽然在较低浓度下（2-10 M福斯克林和2.5-5 mM dbcAMP），这些药物刺激PRL释放，但在较高浓度下测量到抑制作用。通过高分辨率电容（Cm）测量，我们记录了Cm的离散增加，这代表了基本的胞吐事件。cAMP的升高使完全融合事件的频率保持不变，而增加了瞬态事件的频率。通过增加的融合孔电导和延长的融合孔停留时间来测量，这些材料表现出更宽的融合孔。dbcAMP提高了观察瞬时融合孔节律性重新打开的概率。综上所述，cAMP介导的宽融合孔的稳定阻止了囊泡进入胞吐的完全融合阶段，从而阻碍了高cAMP浓度下囊泡内容物的排出。许多内分泌干扰物影响靶细胞中的环核苷酸信号。阿特拉津是世界上最常用的除草剂之一，具有内分泌干扰物的作用，但其作用机制尚未明确。在最近的一项研究中，我们发现阿特拉津可以迅速增加培养的大鼠垂体和睾丸间质细胞中的cAMP水平，但效果不如3-异丁基-1-甲基黄嘌呤，一种竞争性的非特异性磷酸二酯酶（PDEs）抑制剂。在福斯克林和probenecid（一种环核苷酸转运蛋白抑制剂）处理的细胞中，而在3-异丁基-1-甲基黄嘌呤处理的细胞中，阿特拉津进一步增加了cAMP水平，表明PDEs的抑制作用导致了cAMP的积累。与cAMP相反，阿特拉津不改变cGMP水平，进一步表明它抑制cAMP特异性PDEs。阿特拉津诱导的cAMP水平变化足以刺激垂体细胞中PRL的释放和间质细胞中雄激素的产生，这表明它在通过胞外分泌预先储存激素的细胞和通过重新合成激素分泌的细胞中都是内分泌干扰物。在两种细胞类型中，罗利普兰消除了阿特拉嗪对cAMP释放的刺激作用，表明它是pde4的抑制剂，pde4和PDE8A的mRNA在这些细胞中占主导地位。这些结果表明，阿特拉津通过抑制camp特异性PDE4s发挥一般内分泌干扰物的作用。我们还进一步研究了从垂体细胞克隆的P2X受体（P2XR）通道的门控特性。P2X7R在乳养菌中天然表达，作为细胞溶解和凋亡受体，但也控制持续的细胞反应，包括细胞生长和增殖。然而，尚不清楚同一受体如何介导这种相反的作用。为了解决这个问题，我们结合电生理、成像和数学研究，使用野生型和突变大鼠P2X7Rs。低浓度激动剂激活神经受体引起单相缓慢脱敏电流和受体内化，而细胞形态没有其他变化，很像其他P2XRs。相反，饱和激动剂浓度诱导高振幅双相电流，反映孔隙扩张并引起细胞快速肿胀和溶解。这两种信号模式的存在是用一个修正的马尔可夫状态模型来解释的，该模型除了包括正常和敏感状态外，还包括脱敏状态。占据一个或两个中性受体的atp结合位点有利于缓慢过渡到脱敏状态，而占据第三个结合位点有利于过渡到敏化/扩张状态。与模型预测一致，非膨胀P2X7R突变体总是产生脱敏电流。这些结果表明，配体结合位点的饱和水平决定了P2X7R门控和细胞作用的性质。P2XR的激活引起受体细胞外前庭的扩大，导致跨膜孔的打开，但前庭氨基酸残基在受体激活中的具体作用尚未得到系统的评估。我们对大鼠P2X4R的V47-V61和F324- n338序列进行了丙氨酸或半胱氨酸扫描突变，结果显示V49、Y54、Q55、F324和G325突变体对ATP的反应较差。Y54F和Y54W突变挽救了受体功能，而不是Y54L突变，这表明芳香残基在该位置很重要。Y54A和Y54C受体功能被伊维菌素部分挽救，表明ATP激活P2X4R的能力降低。Q55T、Q55N、Q55E和Q55K突变导致受体无应答性，只有Q55E突变体对伊维菌素敏感。F324L、F324Y和F324W突变也恢复了受体功能，所有突变体都保留了伊维菌素对通道门控的作用，ATP响应性的变化与取代基的疏水性和侧链体积相关。G325P突变体对ATP的反应正常，表明G325是一种柔性铰链。拓扑分析显示G325和F324残基在ATP结合时破坏β -sheet。这些结果表明细胞外前庭氨基酸残基在P2X4R功能中的多重作用：V49残基在受体运输中起重要作用，Y54和Q55残基在通道门控中起关键作用，F324和G325残基引起前庭拓宽。

We continue investigations on receptors and channels expressed in anterior pituitary cells and their roles in pituitary functions. Acetylcholine (ACh) has been established as a paracrine factor in the pituitary gland, but the receptors mediating ACh action and the cell types bearing these receptors have not been identified. Our results showed that the expression of the nicotinic subunits mRNAs followed the order beta2 > beta1 = alpha9 > alpha4 in cultured rat pituitary cells. M4 > M3 muscarinic receptor mRNAs were also identified in pituitary cells. The treatment of cultured pituitary cells with GnRH down-regulated the expression of alpha9 and alpha4 mRNAs, without affecting the expression of M3 and M4 receptor mRNAs, and ACh did not alter the expression of GnRH receptor mRNA. We also performed double immunostaining to show the expression of beta2 subunit and M4 receptor proteins in gonadotrophs. Functional nicotinic channels capable of generating an inward current, facilitation of electrical activity and calcium influx were identified in single cells. The M3 receptor-mediated, phospholipase C-dependent calcium mobilization activated an outward apamin-sensitive potassium current and caused hyperpolarization. The activation of M4 receptors by ACh inhibited cAMP production and GnRH-induced LH release. We concluded that multiple cholinergic receptors are expressed in gonadotrophs and that the main secretory action of ACh is inhibitory through M4-receptor-mediated down-regulation of cAMP production. The expression of nicotinic receptors in vitro compensates for the lack of regular GnRH stimulation of gonadotrophs. cAMP and cGMP are intracellular messengers that are produced from the nucleotide triphosphates by a family of enzymes consisting of adenylyl and guanylyl cyclases. In pituitary cells, our previous work revealed that the cyclic nucleotides and their kinases play important role in the control of electrical activity of the endocrine pituitary cells and channels involved in this process. More recently, we focused on the role of these messengers in control of hormone secretion downstream on electrical activity in calcium signaling. The hormone release studies show that prolactin (PRL) release from isolated rat lactotrophs stimulated by forskolin, an activator of adenylyl cyclases, and membrane- permeable cAMP analog (dbcAMP), exhibit a biphasic concentration dependency. Although at lower concentrations (2-10 M forskolin and 2.5-5 mM dbcAMP) these agents stimulate PRL release, an inhibition is measured at higher concentrations. By using high-resolution capacitance (Cm) measurements, we recorded discrete increases in Cm, which represent elementary exocytic events. An elevation of cAMP leaves the frequency of full-fusion events unchanged, while increasing the frequency of transient events. These exhibited a wider fusion pore as measured by increased fusion-pore conductance and a prolonged fusion-pore dwell-time. The probability of observing rhythmic reopening of transient fusion pores was elevated by dbcAMP. In conclusion, cAMP-mediated stabilization of wide fusion pores prevents vesicles from proceeding to the full-fusion stage of exocytosis, which hinders vesicle content discharge at high cAMP concentrations. Many endocrine disrupters affect cyclic nucleotide signaling in target cells. Atrazine, one of the most commonly used herbicides worldwide, acts as an endocrine disruptor, but the mechanism of its action has not been characterized. In a recent study, we show that atrazine rapidly increases cAMP levels in cultured rat pituitary and testicular Leydig cells, but less effectively than 3-isobutyl-1-methylxanthine, a competitive non-specific inhibitor of phosphodiesterases (PDEs). In forskolin and probenecid (an inhibitor of cyclic nucleotide transporters)-treated cells, but not in 3-isobutyl-1-methylxanthine-treated cells, atrazine further increased cAMP levels, indicating that inhibition of PDEs accounts for accumulation of cAMP. In contrast to cAMP, atrazine did not alter cGMP levels, further indicating that it inhibits cAMP-specific PDEs. Atrazine-induced changes in cAMP levels were sufficient to stimulate PRL release in pituitary cells and androgen production in Leydig cells, indicating that it acts as an endocrine disrupter both in cells that secrete by exocytosis of prestored hormones and in cells that secrete by de novo hormone synthesis. Rolipram abolished the stimulatory effect of atrazine on cAMP release in both cell types, suggesting that it acts as an inhibitor of PDE4s, isoforms whose mRNA transcripts dominate in these cells together with mRNA for PDE8A. These results indicate that atrazine acts as a general endocrine disrupter by inhibiting cAMP-specific PDE4s.We also progressed in investigation the gating properties of P2X receptor (P2XR) channels cloned from pituitary cells. The P2X7R, natively expressed in lactotrophs, operates as a cytolytic and apoptotic receptor but also controls sustained cellular responses, including cell growth and proliferation. However, it has not been clarified how the same receptor mediates such opposing effects. To address this question, we combined electrophysiological, imaging, and mathematical studies using wild type and mutant rat P2X7Rs. Activation of nave receptors by low agonist concentrations caused monophasic slow desensitizing currents and internalization of receptors without other changes in the cellular morphology, much like other P2XRs. In contrast, saturating agonist concentrations induced high amplitude biphasic currents, reflecting pore dilation and causing rapid cell swelling and lysis. The existence of these two signaling patterns was accounted for using a revised Markov state model that included, in addition to nave and sensitized states, desensitized states. Occupancy of one or two ATP-binding sites of nave receptors favored a slow transition to desensitized states, whereas the occupancy of the third binding site favored a transition to sensitized/dilated states. Consistent with model predictions, nondilating P2X7R mutants always generated desensitizing currents. These results suggest that the level of saturation of the ligand binding sites determines the nature of the P2X7R gating and cellular actions. Activation of P2XR causes an enlargement of the receptor extracellular vestibule, leading to opening of the transmembrane pore, but specific roles of vestibule amino acid residues in receptor activation have not been evaluated systematically. Our alanine or cysteine scanning mutagenesis of V47-V61 and F324-N338 sequences of rat P2X4R revealed that V49, Y54, Q55, F324, and G325 mutants were poorly responsive to ATP. The Y54F and Y54W mutations, but not the Y54L mutation, rescued receptor function, suggesting that an aromatic residue is important at this position. The Y54A and Y54C receptor function was partially rescued by ivermectin, suggesting a decreased potency of ATP to activate P2X4R. The Q55T, Q55N, Q55E, and Q55K mutations resulted in non-responsive receptors and only the Q55E mutant was ivermectin-sensitive. The F324L, F324Y, and F324W mutations also rescued receptor function, ivermectin action on channel gating was preserved in all mutants, and changes in ATP responsiveness correlated with the hydrophobicity and side chain volume of the substituent. The G325P mutant had a normal response to ATP, suggesting that G325 is a flexible hinge. A topological analysis revealed that the G325 and F324 residues disrupt a beta-sheet upon ATP binding. These results indicate multiple roles of the extracellular vestibule amino acid residues in the P2X4R function: the V49 residue is important for receptor traffickin, the Y54 and Q55 residues play a critical role in channel gating and the F324 and G325 residues cause vestibule widening.