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、肌肉和脂肪组织细胞，增强葡萄糖合成，促进脂肪和肌肉蛋白质的分解。皮质醇和其他激素对血糖浓度的精确控制至关重要，因为血糖是大脑的主要能量来源。低血糖会导致脑损伤甚至死亡。异常的皮质类固醇分泌是导致库欣病和艾迪生病的原因。皮质醇分泌主要由垂体肽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。虽然已经确定了调节皮质醇分泌的生理刺激，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的Ca+进入相耦合，但特定离子通道在AZF细胞分泌中的功能尚未明确。牛AZF呼叫表达一种新的K+电流（I[AC]），当ACTH、AII和外部ATP的浓度与AZF细胞去极化和刺激皮质醇分泌的浓度相同时，它会设定静息电位。这些通道也被细胞内ATP直接激活。三种G蛋白偶联受体对I[AC]的趋同抑制及其被细胞内ATP激活表明，该通道是激素和代谢信号整合并转导到与皮质醇分泌相关的通透性和膜电位变化的控制点。对这一通道的详细描述，以及调节其活动的信号通路，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子对I[AC] K+通道的调节。该研究的目的是：(1)确定I[AC]通道门控是否与ATP水解循环耦合，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素是否也调节I[AC] K+通道；(3)确定ACTH抑制I[AC]信号通路的分子成分；(4)鉴定I[AC] K+通道cdna在真核细胞系中克隆表达后的性质和分布。

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.