The long term goals of this proposed study are to delineate the mechanisms and regulation of aquaporin-2 (AQP2) trafficking in kidney collecting duct at normal and pathophysiological conditions. The antidiuretic hormone (arginine vasopressin, AVP) regulates osmotic water permeability (Pf) of kidney collecting duct, conferring the precise control of renal excretion of water. AVP stimulates Pf in collecting duct by triggering the translocation and fusion of cytoplasmic AQP2 containing vesicles to apical membrane of principal cells. By developing confocal imaging techniques using styryl dye FM1-43 as a plasma membrane marker to monitor exocytosis, fluorescien sulfonate as volume marker to monitor Pf and fluo-4 as intracellular Ca 2+ concentration ([Ca2+]i) marker in perfused rat inner medullary collecting duct (IMCD), it was found that AVP induces [Ca2+]i oscillations in individual IMCD cells, and that AVP induced apical exocytosis and increase of Pf are Ca 2+ dependent. AVP induced Ca 2+ mobilization is cAMP-dependent but is not sensitive to protein kinase A inhibitor at a dose known to inhibit AQP-2 phosphorylation. Physiological dose of atrial natriuretic factor (ANF) inhibits AVP stimulated Pf and [Ca2+]i oscillations.
The aims of this proposal are (1) to test whether AQP2 exocytosis is coupled to intracellular Ca2+ mobilization, (2) to test whether cAMP directly involved in Ca2+ mobilization and AQP2 exocytosis via cAMP-guanine-nucleotide-exchange factors (3) to elucidate the subcellular mechanisms and the physiological significance of [Ca2+]i oscillations in AVP induced exocytosis, (4) to determine the interactions between vasopressin and atrial natriuretic peptide in regulating [Ca2+]i and AQP2 exocytosis. All proposed studies will be conducted in live cells of perfused IMCD. Apical exocytosis is monitored by FM1-43 fluorescence. Pf is monitored by the emission of fluorescien sulfonate included in the luminal perfused. An UV pulse laser is used to manipulate [Ca2+]i via flash photolysis of photoactivatable intracellular Ca2+ chelators or caged signaling molecules without interrupting image acquisition. A reversible permeabilization procedure is used to delivery antibodies, inhibitory peptides, and impermeant caged compounds into IMCD cells. Complementary immunolocalization of AQP2 with fluorescence microscopy will be employed. This proposed study will provide new information on the mechanisms and regulation of the insertion and trafficking of AQP2 in intact cell of perfused IMCD. These may lead a better understand of the role of kidney collecting duct in water balance disorders.
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