2002 Global Researcher Conference Proceeding

April 26 - 28, 2002

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Conference: 2002 Global Researcher Conference
Title: Regulation of Aquaporin-2 trafficking by Vasopressin in Renal Collecting Duct: Roles of Ryanodine-Sensitive Ca2+ Stores and Calmodulin
Author: Knepper, Mark
Institution: National Institutes of Health
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Knepper In the renal collecting duct, vasopressin increases osmotic water permeability (Pf) by triggering trafficking of aquaporin-2 vesicles to the apical plasma membrane. We investigated the role of vasopressin-induced intracellular Ca2+ mobilization in this process. In isolated inner medullary collecting ducts (IMCDs), vasopressin (0.1 nM) and CPT-cAMP (0.1 mM) elicited marked increases in [Ca2+]i (fluo-4). Vasopressin-induced Ca2+ mobilization was completely blocked by preloading with the Ca2+ chelator BAPTA. In parallel experiments, BAPTA completely blocked the vasopressin-induced increase in Pf , without affecting adenosine 3',5'-cyclic monophosphate (cAMP) production. Previously, we demonstrated the lack of activation of the phosphoinositide signaling pathway in IMCD, suggesting an IP3 -independent mechanism of Ca2+ release. Evidence for expression of the type 1 ryanodine receptor (RyR1) in IMCD was obtained by immunofluorescence, immunoblotting and RT-PCR. Ryanodine (100 mM), a ryanodine receptor antagonist, blocked the AVP-mediated increase in Pf and blocked vasopressin-stimulated redistribution of aquaporin-2 to the plasma membrane domain in primary cultures of IMCD cells, as assessed by immunofluorescence immunocytochemistry. Calmodulin inhibitors, W7 and trifluoperazine, blocked the Pf response to vasopressin and the vasopressin-stimulated redistribution of aquaporin-2. The results suggest that Ca2+ release from ryanodine-sensitive stores plays an essential role in vasopressin-mediated aquaporin-2 trafficking via a calmodulin-dependent mechanism.

Cells within a portion of the kidney called the collecting duct help the kidney regulate water by allowing water to be reabsorbed from urine into the blood when the body is dehydrated. This reabsorption process is initiated when the hormone, vasopressin, triggers a molecular sequence that moves the protein, aquaporin-2, from within the IMCD cells to the cells' outer membrane.

Knepper, et al., investigated the role of calcium, specifically Ca2+, in this water reabsorption process. They found that vasopressin induces an increase in Ca2+ within the cells of microdissected kidney collecting ducts. This increase was blocked when the researchers added BAPTA, a molecular compound which restricts the movement of Ca2+ by binding to it, to microdissected collecting ducts. BAPTA also blocked the vasopressin-induced increase in water flow through the cells.

The Ryadodine receptor is a molecular structure which plays a role in Ca2+ regulation in cells. When Knepper, et al., prevented it releasing calcium into the cell interior, vasopressin lost its ability to increase water movement through the cells. Similar results were seen when the researchers inhibited the action of calmodulin, a protein in cells which responds to increases in intracellular calcium by altering cell function.

These results indicate that Ca2+ plays an essential role in helping water pass through collecting duct cells by helping move aquaporin-2 from within the cell to the cell outer membrane.