FUNCTIONAL DEVELOPMENT OF THE SALIVARY GLANDS

本项目的长期目标是阐明各种 刺激-反应偶联的功能元件在 大鼠生后发育过程中的腺泡细胞。 我们的研究结果 在目前的补助期内，显示出与年龄和剂量有关的 中间体（IP 3形成，Ca 2+动员）步骤的差异 胆碱能刺激后的信号转导通路。 他们 也显示出IP 3/Ca 2+反应之间的明显解离， 更远的事件（钾和氯流出）的分泌途径中， 出生后发育的早期阶段。 根据这些观察，我们 新赠款期的目标是进一步调查 信号转导过程可能有助于解释这些功能 在产后腺体发育过程中的差异。 与以前的研究一样， 我们将使用1、7、14和21日龄的下颌下细胞和成年的下颌下细胞， 大鼠比较以下内容：1）信号传导中的近端事件， 包括对乙酰胆碱的受体亲和力; 磷脂酶C的活性和亚型; 膜磷酸肌醇;c IP 3激酶，IP 3磷酸酶， PI激酶和PIP激酶;磷脂酶C的功能性偶联， 毒蕈碱、P物质和α-肾上腺素能受体。2)Ca 2+贮存 位点和细胞内Ca 2+变化的定位， 在刺激下发生。 为此，将使用多种方法 目的，包括X射线微探针分析， 35 S-硫代-IP 3结合，用荧光探针和45 Ca或 亚细胞组分中蛋白质的荧光标记。3)的 改变Ca 2+和单价离子转运之间的偶联。 细胞Ca 2+ 将被离子霉素“夹住”，86 Rb和36 Cl的流出将被抑制。 在不同的外部Ca ~（2+）条件下测定。 细胞体积将在 这些条件使用同位素，成像和光散射方法， K通道将用125 I Charybdotoxin进行评价。 离子通道将 也可以通过膜片钳技术进行研究。 X射线微探针分析和 将进行膜片钳术 与教授合作。 G. Roomans（乌普萨拉大学）和O.H. 彼得森（利物浦大学）。 我们的假设是 未成熟的唾液细胞在以下方面存在差异：a） 磷脂/磷脂酶复合物或它们与磷脂/磷脂酶复合物的偶联 乙酰胆碱受体;和B）细胞内Ca 2+池，其 定位或区室化或Ca 2+调节通道。 的 建议的研究应该有助于我们理解功能性 唾液腺的发育以及信号机制 在这个过程中演变。

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.