The proposed research would continue investigations of passive transport of Na ion through amiloride-sensitive channels in frogskin and isolated human eccrine sweat glands. The function and localization of these channels will be compared in normal and CF sweat glands. Qualitative and quantitative characteristics of amiloride-sensitive channels will be studied by uptake and washout of 14C-amiloride and by electron spin resonance spectroscopy of a spin-labelled derivative of amiloride. The localization of amiloride binding sites will be examined by fluorescence microscopy by taking advantage of the fluorescent properties of amiloride and its derivative. We plan to compare the resistance of tight junctions of the flow of electrolytes in isolated normal and CF human eccrine sweat glands and, also, to correlate the freeze-fracture image of tight junctions with the cytochemical methods used for thin section electron microscopy to compare changes in the paracellular flow of electrolytes in resting and stiumlated sweat glands in vitro. The morphologic characteristics of water and electrolyte transport will be quantitatively related to the physiologic response in toad urinary bladders stimulated by various pharmacologic agents, particularly those related to metabolites of the arachidonic acid-ester pathway which is activated by vasopressin. The role of microtubules and calcium will also be evaluated. Stereo-electron microscopy of thick sections (0.3 micron) will be used in both toad bladder and sweat glands to characterize the role of """"""""tubulo-endoplasmic reticulum"""""""" in water and electrolyte transport. Interest will continue in the further development of ultrastructural cytochemical methods for differentiating and characterizing complex carbohydrates in situ and relating the morphological distribution of membrane components to function in both normal and abnormal physiological conditions. In addition, examination of the effect of reserpine on secretory activity in exocrine glands and the influence of this drug on the response to cholinergic and adrenergic stimulaton will be continued using recently developed autoradiographic techniques for characterizing and quantifying the binding of adrenergic and cholinergic agonists and antagonists.
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