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

初级唾液是通过跨细胞和旁细胞途径的分泌物的混合物。该项目旨在阐明细胞旁运输的控制机制。I)体外灌流大鼠颌下腺(SMG)时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，这与AQP5控制细胞旁液体转运的理论是一致的。转运速率的变化可以用先前为该腺体确定的参数来预测，并且提出了SMG系统的定量模型，该模型包含AQP5在顶膜的渗透传感器功能。实验是用来自基因选择的大鼠的SMG进行的，根据Western blotting，这些大鼠的AQP5水平非常低。渗透压改变后的液体分泌和行为与正常相似。我们建议…更重要的是，涉及AQP5的反馈控制即使在低水平下仍然可以操作，并且可能涉及信令系统的其他元件的更大放大。而向导管逆行注射汞离子部分抑制AQP5可引起流量的浓度依赖性降低，但高渗休克后液体分泌减少到渗透平衡系统的预期水平。结果表明，在稳态状态下，SMG的液体产生不是渗透的，而是由AQP5作为渗透感受器控制的一个反馈环。ii)通过对快速冷冻的大鼠颌下腺标本在静息状态和卡巴胆碱/异丙肾上腺素刺激灌流下的深度酸蚀的电子显微镜观察，探讨了细胞旁运输控制的形态基础。形成紧密连接的整合蛋白通过短而细的纤维与更深的肌动蛋白纤维相连。同时刺激M受体和α肾上腺素能受体可增加胞内钙和cAMP，并增加细胞旁途径的通透性，这表现为荧光染料的渗透性增加。在这种刺激过程中，连接粒子链改变了它们的顺序，并朝着基准面方向膨胀。基底膜和紧密连接下的肌动蛋白细丝亚膜下束致密。观察表明，肌动蛋白细丝的动态结构变化可以改变连接颗粒的分布，从而增加细胞旁的通透性。iii)通过逆行注射氯化汞，从功能和形态上考察了水通道蛋白的汞抑制作用。从1微米到10微米汞，大鼠离体颌下腺的液体分泌开始受到抑制，但高分辨扫描电子显微镜观察未见其形态改变。由于进一步的浓度会在细胞间通道表面产生细小的皱纹。iv)为了研究大分子在细胞旁途径中的分子调控，实验系统以牛血清白蛋白为基础。较少

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