FUNCTIONAL DEVELOPMENT OF THE SALIVARY GLANDS

这个项目的长期目标是阐明不同的 刺激-反应耦合的功能要素在 大鼠生后发育过程中的唾液腺泡细胞。我们的发现 在当前的赠款期间，显示出与年龄和剂量有关的 中间体(IP3形成、钙动员)步骤的差异 胆碱能刺激后信号转导通路的改变。他们 也显示了IP3/Ca~(2+)反应和 更多的远端事件(钾和氯外流)的分泌途径 出生后发育的早期阶段。基于这些观察，我们的 新授权期的目标是进一步调查 可能有助于解释这些功能的信号转导过程 出生后腺体发育过程中的差异。和之前的研究一样， 我们将使用1天、7天、14天和21天大的成人的下颌下细胞 比较以下各项：1)信令中的近端事件， 包括对乙酰胆碱的受体亲和力；存在和受体 G蛋白偶联；磷脂酶C的活性及其异构体； 膜磷脂；lP3激酶、IP3磷酸酶、 磷脂酶C与磷脂酶C的功能偶联 M受体、P物质和α-肾上腺素能受体。2)钙离子储存 细胞内钙离子变化的部位和定位 在刺激下发生。将使用多种方法来实现此目的 目的，包括x射线微探针分析，定量 ~(35)S-硫代-IP3结合，荧光探针和~(45)Ca或 亚细胞组份中蛋白质的Ru红色标记。3) 改变了钙离子和单价离子转运之间的耦合。细胞内钙离子 将用离子霉素卡住，而86 Rb和36Cl的外排将是 在不同的外部钙离子下测量。单元格体积将在 这些条件使用同位素、成像和光散射方法以及 K通道将用~(125)I白僵菌毒素测定。离子通道将 也可以通过膜片钳来研究。X-射线微探针分析和电子显微镜 将进行膜片钳制 与教授合作。G.Roomans(乌普萨拉大学)和O.H. 彼得森(利物浦大学)。我们的工作假设是 未成熟的唾液细胞在以下方面有差异：a) 磷脂/磷脂酶复合体或其偶联到 乙酰胆碱受体；和b)细胞内钙池，其 定位或区隔或钙离子调节的通道。这个 拟议的研究应该有助于我们对功能 唾液腺的发育及其信号传递机制 在这个过程中进化。

The long-term objective of this project is to elucidate how the various functional elements of stimulus-response coupling become established in rat salivary acinar cells during postnatal development. Our findings during the current grant period have shown age-related and dose-related differences in the intermediate (IP3 formation, Ca2+ mobilization) steps of the signal transduction pathway after cholinergic stimulation. They have also shown an apparent dissociation between IP3/Ca2+ responses and more distal events (K and Cl efflux) of the secretory pathway in the early stages of postnatal development. Based on these observations, our goal for the new grant period is to further investigate aspects of the signal transduction process that may help explain these functional differences during Postnatal gland development. As in previous studies, we will use submandibular cells of 1, 7, 14 and 21-day old and of adult rats to compare the following: 1) Proximal events in signalling, including receptor affinity for acetylcholine; the presence and receptor coupling of G proteins; the activity and isoforms of phospholipase C; membrane phosphoinositides;c activities of lP3 Kinase, IP3 phosphatase, PI Kinase and PIP Kinase; the functional coupling of phospholipase C to muscarinic, substance P and alpha-adrenergic receptors. 2) Ca 2+ storage sites and the localization of the changes in intracellular Ca2+ that occur upon stimulation. A combination of methods will be used for this purpose, including x-ray microprobe analysis, quantitation of 35S-thio-IP3 binding, imaging with fluorescent probes and 45 Ca or ruthenium-red labeling of proteins in subcellular fractions. 3) The altered coupling between Ca2+ and monovalent ion transport. Cell Ca 2+ will be 'clamped' with ionomycin and the efflux of 86 Rb and 36Cl will be measured at different external Ca2+. Cell volume will be measured under these conditions using isotopic, imaging and light scattering methods and K channels will be evaluated with 125I charybdotoxin. Ion channels will also be studied by patch clamping. The x-ray microprobe analysis and the patch clamping will be done in collaboration with Profs. G. Roomans (University of Uppsala) and O.H. Petersen (University of Liverpool). Our working hypothesis is that immature salivary cells have differences in: a) the phospholipid/phospholipase complex or in their coupling to the acetylcholine receptor; and b) intracellular Ca 2+ pools, their localization or compartmentalization or Ca2+-regulated channels. The proposed studies should contribute to our understanding of the functional development of salivary glands and of how the signalling mechanism evolves during this process.