Calcium Signaling in Developing Intestinal Epithelium

描述(申请人摘要)：结肠的主要功能 上皮细胞通过保持平衡来保存水和电解质 体液分泌和吸收之间的关系。这种结肠功能特别是 对新生儿来说很重要，因为新生儿的储备很少。大多数人的分泌物 上皮细胞受C_1-转运机制控制，受秒调节。 如环核苷酸和钙等信使。钙依赖 分泌剂负责离子的每一分钟的调节。 肠道需要转运。然而，人们对此知之甚少。 哺乳动物发育过程中C1-分泌的调节。其中有耐人寻味的， 虽然在一些组织中有证据表明钙信号可能是出生后产生的 受监管的。关于C1-分泌物个体发育的研究较少，主要集中在 兔远端结肠和显示胆汁酸，如牛磺酸脱氧胆酸(TDC) 刺激成人CFs的转运，但不能促进新生儿CFs的转运。CA2和cAMP是 被牵连为贸易发展局行动的调解人。为了阐明细胞基础， 与年龄相关的C1转运调节，本实验室研究了 CAMP和Ca~(2+)依赖型促分泌剂在原代培养中的作用 从新生儿远端结肠(NBN)分离的上皮细胞(结肠细胞)， 断奶(WN)和成年(AD)兔。主要研究结果如下： 环磷酸腺苷在各年龄段均可促进CFs的转运。然而，钙依赖 促分泌剂，如神经降压素(NT)，可刺激AD患者的C1转运，但不能 在WN或NBN的结肠细胞中。AD组NT升高，WN组则无明显变化 结肠癌细胞。同样，TDC可增加钙离子浓度，但不增加cAMP，并刺激CF 在AD中转运，但不在WN或NBN结肠细胞中转运。相比之下，钙 离子载体在所有年龄段都刺激了c1-转运，这意味着远端台阶 在钙离子中，信号在幼年动物中起作用。考试的结果 近端步骤，即磷脂酶C的受体/G蛋白激活为IP3 到钙储存，显示断奶动物有足够的钙储存 刺激纤维的运输。然而，NT和TDC在AD时增加了[IP3]i，但在AD时不增加 Wn，结肠细胞，尽管在所有年龄段都存在PLCβ和伽马蛋白。 NT激活AD和WN结肠细胞的Galphaq，但TDC，即使在成人中， 不会刺激Galphaq。因此，假设多个 IP3的生成和作用途径受个体基因调控 产后结肠提供保护，防止体内液体过量流失 对不断变化的环境的反应。这项提议将检验这一假设，如 AIM I包括鉴定由NT和NT激活的PLC亚型 阿尔茨海默病的TDC及其组织特异性、时程和影响因素的测定 有助于PLC的年龄依赖性反应导致C1- 运输。AIM II是一项关于钙信号传导的其他步骤的检查， 如IP3受体和其他肌醇磷酸盐，有助于 Ca~(2+)介导的CF转运的年龄依赖性出现。AIM III是一个 TDC与结肠细胞相互作用的特征，包括年龄依赖性 以及参与NT和TDC刺激的[Ca~(2+)]i和Cf的激酶级联(S) 运输。通过描绘发育中的肠道的“保护”机制 已经进化到可以应对其环境的挑战，这些研究将 为制定抗击危及生命的 婴儿慢性腹泻。

DESCRIPTION (Applicant's Abstract): The major function of the colonic epithelium is to conserve water and electrolytes by maintaining a balance between fluid secretion and absorption. This colonic function is especially important in newborns, where reserves are small. Secretion across most epithelia is governed by C1- transport mechanisms and is regulated by second messengers such as cyclic nucleotides and calcium. Calcium-dependent secretagogues are responsible for the minute-by-minute regulation of ion transport needed in the intestine. However, not much is known about the regulation of C1- secretion in the developing mammal. There is intriguing, albeit sparse, evidence in some tissues that Ca2+ signaling may be postnatally regulated. The few studies on ontogeny of C1- secretion were conducted in the rabbit distal colon and showed that bile acids such as taurodeoxycholate (TDC) stimulate CF transport in the adult but not in the neonate. Ca2+ and cAMP were implicated as mediators of TDC action. To elucidate the cellular basis of the age-related regulation of C1- transport, this laboratory investigated the effects of cAMP- and Ca2+-dependent secretagogues in primary cultures of epithelial cells (colonocytes) isolated from the distal colon of newborn (NBN), weanling (WN) and adult (AD) rabbits. The salient findings were as follows: Cyclic AMP stimulated CF transport at all ages. However Ca2+-dependent secretagogues, such as neurotensin (NT), stimulated C1 transport in AD, but not in WN or NBN colonocytes. In parallel, NT increased [Ca2+], in AD but not in WN colonocytes. Similarly, TDC increased Ca2+, but not cAMP, and stimulated CF transport in AD, but not in WN or NBN colonocytes. In contrast, calcium ionophore stimulated C1- transport at all ages, implying that the distal steps in Ca2+ signaling are functional in the young animal. Examination of the proximal steps, i.e., receptor/G-protein activation of phospholipase C to IP3 to Ca2+ stores, revealed that the weanling animal had sufficient Ca2+ stores to stimulate CF transport. However, NT and TDC increased [IP3]i in AD, but not in WN, colonocytes despite the presence of PLCbeta and gamma proteins at all ages. NT activated Galphaq in both AD and WN colonocytes, but TDC, even in the adult, does not stimulate Galphaq. Therefore, it is hypothesized that multiple pathways in IP3 generation and action are ontogenetically regulated in the postnatal colon to provide protection against excessive loss of fluid in response to its changing milieu. This proposal will test this hypothesis as follows: Aim I involves identification of the PLC isoforms activated by NT and TDC in AD and determination of the tissue specificity, time course and factors contributing to the age-dependent responsiveness of PLC leading to C1- transport. Aim II is an examination of whether other steps in Ca2+ signaling, such as IP3 receptors and other inositol phosphates, contribute to the age-dependent appearance of Ca2+-mediated CF transport. Aim III is a characterization, including age-dependence, of TDC interaction with colonocytes and of the kinase cascade(s) involved in NT- and TDC-stimulated [Ca2+]i and CF transport. By delineating the "protective" mechanisms that the developing gut has evolved to meet the challenges of its environment, these studies will provide important insights for devising ways to combat the life-threatening chronic diarrheas of infants.