2002 Global Researcher Conference Proceeding
April 26 - 28, 2002
| Conference: | 2002 Global Researcher Conference |
|---|---|
| Title: | Vasopressin-induced / cyclic AMP-mediated aquaporin 2 translocation is a Ca2+-independent, slow exocytotic process |
| Authors: | Maric, Kenan; Lorenz, Dorothea; Hahm, Daniel; Storm, Robert; Klussmann, Enno; Pohl, Peter; Rosenthal, Walter |
| Institutions: | German Federal Ministry of Health and Social Security, Forschungsinstitut fur Molekulare Pharmakologie, Forschunginstitut fur Molekulare Pharmakologie, Charite - Universitatsmedizin Berlin |
Epithelial cells of the renal collecting duct (principal cells) represent the prime target of the antidiuretic hormone vasopressin in the kidney. In these cells, vasopressin causes an increase in intracellular cyclic adenosine monophosphate (cAMP), followed by the translocation of the water channel aquaporin 2 (AQP2) from intracellular vesicles into the plasma membrane. At present, the mechanism of this exocytic event is poorly understood. A crucial step is the phosphorylation of AQP2 by the cAMP-dependent protein kinase (PKA). Recently we found that not only the enzymatic activity of PKA but also its anchoring to a PKA-anchoring protein (AKAP) is a prerequisite for AQP2 translocation. In addition, we observed that the small GTPase Rho plays an inhibitory role in this process.
To clarify whether cAMP acts by increasing intracellular Ca2+, membrane capacitance measurements were carried out in the whole-cell patch clamp configuration. Under control conditions (2 mM extracelluar Ca2+, intracellular Ca2+ not buffered), vasopressin, forskolin, and membrane-permeable cAMP analogs caused a slow increase in membrane capacitance, indicating exocytic fusion of vesicles with the plasma membrane. The responses were observed after a delay (in the order of seconds), even if cells were loaded with a new type of a photolabile, caged cAMP analog which was uncaged by UV light within less than a microsecond. Both the delayed capacitance response and the absence of AQP2-bearing vesicles in the vicinity of the plasma membrane (analyzed by immunogold electron microscopy) point towards a lack of a pool of readily releasable vesicles. A change in the rate of endocytic events in response to a stimulus was not observed. Blocking Ca2+ transients by buffering intracellular Ca2+ did not influence the seize or kinetics of the vasopressin-induced membrane capacitance increases. The same results were obtained in immunofluorescence microscopy experiments with AQP2 antibodies. In these experiments, cells were treated with ionophore ionomycin to control intracellular Ca2+ concentrations. However, shuttling was inhibited by lowering the intracellular Ca2+ concentration below 25 nM. Exocytic insertion of AQP2 into the plasma membrane was accompanied by an equally slow increase in transepithelial water permeability as revealed by space and time-resolved microelectrode-aided measurements of K+-dilution adjacent to the cell monolayer.
Our results show that vasopressin increases water reabsorption in the kidney by triggering a slow exocytic process, which requires elevation of cAMP but not of intracellular Ca2+.
Researchers know that in kidney collecting duct cells the hormone vasopressin induces an increase in the level of cyclic adenosine monophosphate (cAMP) within the cell. This increase is followed by the movement of Aquaporin 2 (AQP2) from the cell interior to the cell wall. However, there is much about this molecular sequence that remains to be clarified. Rosenthal, et al., sought to clarify a piece of the puzzle by determining whether or not cAMP induces AQP2 movement by increasing the amount of a specific form of calcium, Ca2+, in the cell.
By performing experiments in laboratory cell cultures where the researchers controlled for intracellular Ca2+ concentrations, they were able to determine that though the movement of AQP2 to the cell membrane does require elevated levels of cAMP, it does not require increases of Ca2+.