1999 European Regional Conference Proceeding
May 12 - 16, 1999
| Conference: | 1999 European Regional Conference |
|---|---|
| Title: | Functional investigations of the vasopressin regulated antidiuretic machinery in single inner medullary collecting duct (IMCD) cells |
| Authors: | Maric, Kenan; Storm, Robert; Lorenz, Dorothea; Wiesner, Burkhard; Klussmann, Enno; Rosenthal, Walter |
| Institutions: | German Federal Ministry of Health and Social Security, Forschunginstitut fur Molekulare Pharmakologie, Forschungsinstitut fur Molekulare Pharmakologie, Forschunginstitut fur Molekulare Pharmakologie (FMP), Charite - Universitatsmedizin Berlin |
Vasopressin regulates water reabsorption in renal collecting duct principal cells by inducing a cAMP-dependent translocation of water channels (aquaporin-2, AQP-2) from intracellular vesicles into the apical membranes. Last year we reported on a new in-vitro primary cell culture model of rat inner medullary collecting duct (IMCD) cells that maintains AQP-2 expression at the protein level without transfection. As a preliminary result, subcellular analysis at a high resolution confirmed the distribution of the AQP-2 protein predominantly to the basolateral cell membrane compartment in the majority of the AQP-2 expressing primary cultured principal cells. Only a small percentage of the AQP-2 expressing cells (< 1%) redistribute AQP-2 to the apical cell membrane compartment. Considering these circumstances, functional investigations of the AQP-2 protein using a modified dynamic cell height measurement method were carried out on subconfluent IMCD cell monolayers. Dynamic cell height changes of unstained, living IMCD cells were visualized in Z-scans by laser scanning confocal microscopy in the reflection mode. The analysis of the obtained Z-scan series was improved by the development of an area determining software macro. Time-dependent cell height changes of swelling and shrinking cells were fitted to exponential equations revealing time constants indicative of accelerated volume changes after vasopressin or forskolin stimulation. In summary, this assay will allow an evaluation of the possible therapeutic significance of newly developed drugs for the treatment of congenital and acquired diuretic and antidiuretic states already at the cellular level.
An assay is a test that determines the amount of a particular constituent of a mixture, or that determines the biological or pharmacological potency of a drug. Maric, et al., have developed an assay that will enable researchers to evaluate the effectiveness of drugs developed to treat the congenital and acquired forms of nephrogenic diabetes insipidus (NDI).
First, these researchers developed a new in-vitro primary cell culture model of rat inner medullary collecting duct (IMCD) cells that naturally maintains AQP2 expression at the protein level. Although subcellular analysis of the IMCD cell culture showed that in most of the cells the AQP2s were located in the cells' lateral membrane, removal of neighboring cells enabled them to conduct experiments investigating drugs that act on the water channel function, i.e. water permeability of AQP2.
Maric, et al., used a modified dynamic cell height measurement method to determine how the AQP2s in their cell culture were functioning. Dynamic cell height changes of unstained, living IMCD cells were visualized in Z-scans by laser scanning methods. The researchers developed exponential equations that expressed the time-dependent cell height changes of swelling and shrinking IMCD cells. These reflected accelerated volume changes in the cells after they were stimulated by either of the hormones, vasopressin or forskolin. The dynamics (the speed) of the volume changes indicated the effectiveness of vasopressin and forskolin. The intention of the study is to find or to develop drugs that will be able to increase the water permeability of these cells via other pathways than the hormone vasopressin.