Calcium Signaling in Developing Intestinal Epithelium

描述（申请人摘要）：结肠的主要功能 上皮细胞通过维持平衡来保存水和电解质 介于液体分泌和吸收之间。这种结肠功能尤其 对于新生儿很重要，因为新生儿的储备很少。大部分的分泌 上皮细胞受 C1- 转运机制控制，并受第二 环核苷酸和钙等信使。钙依赖性 促分泌剂负责离子的每分钟调节 需要在肠道内运输。然而，人们对此知之甚少 发育中哺乳动物中 C1- 分泌的调节。有一点耐人寻味， 尽管很少，但某些组织中的证据表明 Ca2 信号传导可能是出生后的 受监管。关于 C1- 分泌的个体发育的少数研究是在 兔远端结肠并显示胆汁酸如牛磺脱氧胆酸 (TDC) 刺激成人而不是新生儿的 CF 运输。 Ca2 和 cAMP 为 作为 TDC 行动的调解人。阐明细胞基础 C1-运输的年龄相关调节，该实验室研究了 cAMP 和 Ca2 依赖性促分泌剂在原代培养物中的作用 从新生儿远端结肠分离的上皮细胞（结肠细胞）（NBN）， 断奶兔 (WN) 和成年兔 (AD)。主要调查结果如下： 环磷酸腺苷刺激所有年龄段的 CF 运输。然而Ca2依赖 促分泌剂，例如神经降压素 (NT)，可以刺激 AD 中的 C1 转运，但不能 在 WN 或 NBN 结肠细胞中。同时，NT 在 AD 中增加 [Ca2]，但在 WN 中则没有 结肠细胞。类似地，TDC 增加 Ca2，但不增加 cAMP，并刺激 CF 在 AD 中转运，但在 WN 或 NBN 结肠细胞中不转运。相比之下，钙 离子载体刺激所有年龄段的 C1- 转运，这意味着远端步骤 Ca2 信号传导在幼年动物中发挥作用。的检查 近端步骤，即磷脂酶 C 的受体/G 蛋白激活至 IP3 钙储存，显示断奶动物有足够的钙储存 刺激 CF 运输。然而，NT 和 TDC 在 AD 中增加了 [IP3]i，但在 AD 中则没有增加。 WN，结肠细胞，尽管在所有年龄段都存在 PLCbeta 和 γ 蛋白。 NT 激活 AD 和 WN 结肠细胞中的 Galphaq，但 TDC，即使在成人中， 不刺激Galphaq。因此，推测多个 IP3 生成和作用的途径在个体发育中受到调节 产后结肠可提供保护，防止体内液体过度流失 对其不断变化的环境做出的反应。该提案将测试这一假设： 如下：目标 I 涉及鉴定由 NT 激活的 PLC 同工型 AD 中的 TDC 以及组织特异性、时程和因素的确定 有助于 PLC 的年龄依赖性反应能力，从而导致 C1- 运输。目标 II 是检查 Ca2 信号传导中的其他步骤是否 例如 IP3 受体和其他磷酸肌醇，有助于 Ca2 介导的 CF 运输的年龄依赖性外观。目标 III 是 TDC 与结肠细胞相互作用的表征，包括年龄依赖性 以及参与 NT 和 TDC 刺激的 [Ca2 ]i 和 CF 的激酶级联 运输。通过描述发育中的肠道的“保护”机制 已经进化到能够应对环境的挑战，这些研究将 为设计对抗威胁生命的方法提供重要见解 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.