PROPERTIES OF A K+ CURRENT THAT CONTROLS SECRECTION

在哺乳动物中，肾上腺束状带 (AZF) 细胞分泌皮质醇，皮质醇作用于肝脏、肌肉和脂肪组织的细胞，以增强葡萄糖合成并促进脂肪和肌肉蛋白质的分解。通过皮质醇和其他激素精确控制血糖浓度至关重要，因为这种糖是大脑的主要能量来源。低血糖会导致脑损伤或死亡。异常的皮质类固醇分泌是库欣病和艾迪生病的原因。皮质醇的分泌主要由垂体肽 ACTH 控制。然而，许多其他生理因素，包括血管紧张素 II、瘦素、核苷酸、肾上腺素和葡萄糖，作用于 AZF 细胞来调节这种激素的分泌。虽然调节皮质醇分泌的生理刺激已被识别，但所涉及的信号传导途径尚不清楚。尽管许多激素的释放与去极化依赖性 Ca2+ 进入相关，但 AZF 细胞分泌中特定离子通道的功能尚未阐明。牛 AZF 表达一种新的 K 电流 (I[AC])，该电流设定静息电位，同时受到 ACTH、AII 和外部 ATP 的抑制，其浓度与去极化 AZF 细胞和刺激皮质醇分泌的浓度相同。这些通道也直接被细胞内 ATP 激活。三个 G 蛋白偶联受体对 I[AC] 的收敛抑制及其被细胞内 ATP 的激活表明，该通道是一个控制点，激素和代谢信号在此整合并转导为与皮质醇分泌相关的通透性和膜电位变化。该通道的详细特征以及调节其活性的信号通路对于理解肾上腺皮质生理学至关重要。全细胞和单通道膜片钳将用于描述激素和代谢因素对 I[AC] K 通道的调节。拟议研究的目的是：（1）确定 I[AC] 通道门控是否与 ATP 水解循环耦合，从而允许这些通道充当葡萄糖传感器； (2)确定生理上调节皮质醇分泌的其他激素和旁分泌因子是否也调节I[AC]K通道； (3) 鉴定ACTH抑制I[AC]信号通路的分子成分； (4) 表征 I[AC] K 通道 cDNA 在真核细胞系中克隆和表达后的特性和分布。

In mammals, adrenal zona fasciculata (AZF) cells secrete cortisol which acts on cells of the liver, muscle, and adipose tissue to enhance glucose synthesis and to promote the breakdown of fat and muscle proteins. Precise control of blood glucose concentration by cortisol and other hormones is critical because this sugar is the primary energy source for the brain. Hypoglycemia can cause brain damage or death. Aberrant corticosteroid secretion is responsible for Cushing's and Addison's diseases. Cortisol secretion is controlled primarily by the pituitary peptide ACTH. However, a number of other physiological factors, including Angiotensin II, leptin, nucleotides, epinephrine, and glucose act on AZF cells to regulate secretion of this hormone. While physiological stimuli that regulate cortisol secretion have been identified, the signalling pathways involved are not understood. Although, the release of many hormones is coupled to depolarization-dependent Ca+ entry, the function of specific ion channels in AZF cell secretion has not been clarified. Bovine AZF calls express a novel K+ current (I[AC]) that sets the resting potential while it is inhibited by ACTH, AII, and external ATP at concentrations identical to those that depolarize AZF cells and stimulate cortisol secretion. These channels are also directly activated by intracellular ATP. The Convergent inhibition of I[AC] by three G protein- coupled receptors and its activation by intracellular ATP suggest that this channel is a control point where hormonal and metabolic signals are integrated and transduced to permeability and membrane potential changes associated with cortisol secretion. A detailed characterization of this channel, and the signalling pathways that regulate its activity will be essential to an understanding of adrenal cortical physiology. Whole-cell and single channel patch clamp will be used to describe the modulation of I[AC] K+ channels by hormones and metabolic factors. The aims of the proposed research will be: (1) to determine whether I[AC] channel gating is coupled to an ATP hydrolysis cycle, allowing these channels to act as glucose sensors; (2) to determine whether other hormonal and paracrine factors which physiologically regulate cortisol secretion, also modulate I[AC] K+ channels; (3) to identify the molecular components of the signalling pathway by which ACTH inhibits I[AC]; and (4) to characterize the properties and distribution of I[AC] K+ channel cDNAs after cloning and expression in a eucaryotic cell line.