Abnormal electrolyte transport in exocrine glands is one of the hallmarks of cystic fibrosis (CF), the most common autosomal recessive disease among Caucasian populations. A consensus is emerging, based on other investigator's patch clamp studies of CF tracheal cells, that the CF defect is due to an abnormal cAMP-dependent regulation of chloride channels. Whether or not the lack of beta adrenergic sweating in CF previously reported by us as well as the well known defective ductal Na absorption are both explained by the same mechanism remains unknown. Further understanding of the defects in the CF sweat gland is hampered by our lack of knowledge on the intra- and extracellular regulation of membrane transport in the sweat duct and secretory coils. We therefore propose to study the intracellular regulation of ion transport in the sweat gland using state of the art investigative approaches. These include: the use of fluorescent probes for identifying intracellular Ca2+ , Mg2+, Na_, Cl-, and H+ in single, freshly dissociated or cultured sweat gland cells; patch clamping techniques for studying regulation of membrane channels; and electrophysiological techniques for reexamining cultured cells with respect to the regulation of pharmacological responsiveness and membrane transport. These approaches will be complemented by the conventional methodologies of micro sweat induction, ductal perfusion, microtitration of cellular electrolytes in intact sweat glands, and application of conventional electrophysiological techniques to the naive ducts and secretory coils. The specific questions to be addressed (in both ductal and secretory cells in CF and control) are: 1, what neurotransmitters are involved in regulation of ductal and secretory coil function and whether or not CF sweat glands respond differently to them?; 2, is intracellular Ca2+ the sole mediator of cholinergic stimulation or are other mediators (e.g. protein kinase C, calmodulin, cytoskeleton, inositol phosphates, cAMP, cGMP, or ATP) also involved?; 3, what are the characteristics of membrane channels and how are they regulated?; 4, how are various intracellular electrolytes regulated by various pharmacological stimulations and by what mechanisms?; and 5, how do the behaviors of cells change during the course of cell culture relative to pharmacological regulation of membrane transport?
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