Calcium Signaling in Developing Intestinal Epithelium

描述（申请人的摘要）： 上皮细胞是通过维持平衡来保存水和电解质 液体分泌和吸收之间的联系这种结肠功能尤其 这在新生儿中很重要，因为储备很少。大多数的分泌物 上皮细胞由C1-转运机制控制，并受第二 信使如环核苷酸和钙。钙依赖 促分泌素负责每分钟的离子调节 需要在肠道内运输。然而，人们对它知之甚少。 调节发育中哺乳动物的C1分泌。有一个有趣的， 尽管很少，但在某些组织中的证据表明，Ca 2+信号可能在出生后 监管.关于C1-分泌的个体发育的研究很少在 兔远端结肠，并显示胆汁酸，如牛磺脱氧胆酸盐（TDC） 刺激成年人的CF运输，但不刺激新生儿的CF运输。Ca 2+和cAMP为 作为TDC作用的介体。为了阐明细胞的基础， 与年龄相关的C1运输调节，本实验室研究了 cAMP和Ca 2+依赖性促分泌素在原代培养的 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。主要调查结果如下： 环磷酸腺苷刺激CF运输在所有年龄。然而，钙依赖 促分泌素，如神经降压素（NT），刺激AD中的C1转运，但不 在WN或NBN结肠细胞中。与此同时，AD组NT升高[Ca ~（2+）]，而WN组则无 结肠细胞同样，TDC增加Ca 2+，但不增加cAMP，并刺激CF 在AD中，但不是在WN或NBN结肠细胞中的转运。相反，钙 离子载体刺激C1-运输在所有年龄，这意味着远端步骤， Ca 2+信号传导在年轻动物中起作用。审查 近端台阶，即，磷脂酶C受体/G蛋白活化为IP 3 显示断奶动物有足够的Ca 2+商店， 刺激CF运输。然而，NT和TDC增加AD的[IP 3]i，而不是 WN，结肠细胞，尽管在所有年龄段存在PLC β和γ蛋白。 NT在AD和WN结肠细胞中都激活了Galphaq，但TDC，即使在成人中， 不会刺激Galphaq因此，假设多个 IP 3产生和作用的途径是个体遗传学调节的， 出生后的结肠，以提供保护，防止过度流失的液体， 应对环境的变化。本提案将检验这一假设， 目的I涉及鉴定由NT激活的PLC同种型， AD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- 运输目的II是检查Ca 2+信号传导中的其他步骤， 如IP 3受体和其他肌醇磷酸，有助于 钙离子介导的CF转运的年龄依赖性外观。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的激酶级联反应 运输通过描述发育中的肠道的“保护”机制， 为了应对环境的挑战，这些研究将 为制定抗击威胁生命的疾病的方法提供了重要的见解， 婴儿的慢性腹泻。

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.