Contribution of paracellular transport to fluid secretion of the salivary gland.

初级唾液是通过跨细胞和细胞旁途径分泌的混合物。本项目旨在阐明细胞旁转运的控制机制。在项目期间获得了以下结果。i）在离体大鼠下颌下腺（SMG）灌注过程中由蔗糖产生的渗透压变化表明分泌速率的变化比渗透液产生理论预测的要大得多。然而，这些与涉及AQP 5控制细胞旁液体转移的理论雅阁。运输速率的变化可以预测与此腺和SMG系统的定量模型，涉及AQP 5在顶端膜的一个传感器功能的参数较早确定。用来自遗传选择的大鼠的SMG进行实验，所述遗传选择的大鼠具有如通过蛋白质印迹法测定的非常低水平的AQP 5。渗透压改变后的液体分泌和行为与正常相似。我们建议 ...更多信息 考虑到涉及AQP 5的反馈控制即使在低水平下仍然可以操作，并且可能涉及信号系统的其他元件的更大放大。然而，逆行注射Hg离子到导管中以部分抑制AQP 5导致流速的浓度依赖性降低，但高渗休克后液体分泌的减少与预期的血管平衡系统相同。结果表明，在SMG中的流体生产是不渗透的稳定状态，但涉及到一个反馈回路的控制下的AQP 5作为一个神经元的传感器。ii）控制细胞旁运输的形态学基础进行了检查，通过电子显微镜观察的深蚀刻的快速冷冻样品从隔离灌注大鼠下颌下腺无论是在休息和刺激灌注卡巴胆碱/异丙肾上腺素。形成紧密连接的整合蛋白质通过短而细的纤维与更深的肌动蛋白纤维连接。同时刺激毒蕈碱和α肾上腺素能受体增加胞质Ca和cAMP，并增加细胞旁途径的渗透性，如荧光染料渗透增加所揭示的。在这种刺激过程中，连接颗粒的链改变了它们的顺序，并且链向基底方向扩展。基底外侧膜和紧密连接下的肌动蛋白丝膜下纤维束变得致密。这表明，肌动蛋白丝结构的动态变化可以改变连接颗粒的分布，从而可能增加细胞旁通透性。（3）通过逆行注射氯化汞，从功能和形态上研究了汞对水通道蛋白的抑制作用。从1 μ M到10 μ M的汞开始抑制离体灌流大鼠下颌下腺的液体分泌，但高分辨率扫描电镜观察不到形态学变化。而进一步的浓度诱导细胞间小管表面上的细皱纹。iv）为了检查大分子在细胞旁途径通过期间的分子调节，使用牛血清白蛋白设置实验系统。少

The primary saliva is a mixture of secretes both via transcellular and paracellular pathways. This project was designed to clarify the control mechanisms for the paracellular transport. The following results were obtained during the project term.i)Osmolarity changes created by sucrose during the perfusion of isolated rat submandibular glands (SMG) in vitro show that secretion rates alter much more than predicted by the theory of osmotic fluid production. However, these are in accord with a theory involving AQP5 control of paracellular fluid transfer. The changes in transport rate can be predicted with parameters determined earlier for this gland and a quantitative model of the SMG system is presented involving an osmosensor function for AQP5 at the apical membrane. Experiments were performed with SMG from genetically selected rats that have very low levels of AQP5 as determined by Western blotting. The fluid secretion and behavior after osmolarity changes were similar to normal. We sug … More gest that the feedback control involving AQP5 still can operate even at low levels and may probably involve a greater amplification by other elements of the signalling system. While, retrograde injection of Hg ions into the duct to partially inhibit AQP5 lead to a concentration-dependent reduction in flow rates but reduction of fluid secretion after hyper osmotic shock was to that expected for an osmotically equilibrating system. The results indicate that fluid production in the SMG is not osmotic in the steady-state but involves a feedback loop under control of AQP5 acting as an osmosensor.ii)Morphological basis for control of paracellular transport was examined by electron microscope observation of the deep-etching of the rapid frozen samples from isolated perfused rat submandibular gland both in the resting and stimulated perfusion with carbachol/isoproterenol. The integrated proteins which form the tight junction, were connected with deeper actin fibers via the short and fine fibers. Simultaneous stimulation of muscarinic and alpha adrenergic receptors increases both cytosolic Ca and camp and increases the permeability of paracellular route, as revealed by increased permeation of fluorescent dye. During such stimulation the strand of junctional particles changed their order and the strand was expanded toward the basal direction. The submembranous bundles of actin filaments beneath the basolateral membrane and tight junction became dense. The observation indicates that the dynamic structural change of actin filaments could vibrate the distribution of junctional particles, thus may increase the paracellular permeability.iii)Mercurial inhibition of aquaporin was examined functionally and morphologically by retrograde-injection of merculic chloride. From 1 microM to 10 microM of Hg the fluid secretion started to be inhibited in the isolated perfused rat submandibular gland, but no morphological change was observed by High resolution SEM. Whereas further concentration induced fine wrinkles on the surface of intercellular canaluculi.iv)To examine the molecular modulation of large molecule during the passage of paracellular route, the experimental system was set using bovine serum albumine. Less