Tight Junctional Permeability in Living Cells: Dynamic Changes Directly Visualized by Confocal Laser Microscopy

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Abstract
Confocal microscopy was used to study the tight junctional permeability in living rat parotid and submandibular glands. The interstitial space of the tissue was perfused with medium containing fluorescent tracers Lucifer Yellow (anionlc: MW 457), Propidium Iodide (cationic: MW 668) and dextrans labeled with FITC or RTTC (anionic and neutral: MW 3K, 10 K, 40 K, 70 K and 500 K) to monitor whether or not these tracers permeate into the lumen across the junction. In the acini of normal glands, fluorescence was detected in the basolateral space but not in the luminal space up to 30 min. However, when secretion was induced by isoproterenol or carbachol, fluorescence appeared in the luminal space within 2 to 5 min. This did not involve the disruptive changes in tight junction ultrastructure, nor was it irreversible; the luminal fluorescence disappeared again when the secretagogues were removed. Tracers up to MW 40 K for isoproterenol and MW 10 K for carbachol revealed the luminal fluorescence in parotid acini, with little indications of the charge preference characteristics. The luminal fluoresence also appeared by anoxia, enzymatic cell dissociation and the cytochalasin D treatment It was suggested that the tight junctions in salivary acini dynamically alter their permeability and modulate the passage of large molecules through the paracellular pathway. Oxygen supply, extracellular matrices and cytoskeletons were suggested to influence these regulations.

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