This REVISED proposal seeks to explore the role of intracellular Ca++ in the development of cell-cell contact, junctions and apical-basolateral polarity in kidney epithelia. The """"""""Ca++ switch,"""""""" an MDCK cell model for developmental events in early nephrogenesis, will be employed. Preliminary studies (NEW DATA) suggest that mechanisms regulating intracellular Ca++, as well as signalling events activated by changes in intracellular Ca++, may be important in junction formation and polarization. Based on the preliminary data, it is hypothesized that global and/or local changes in intracellular Ca++ mediate the development of cell-cell contact, assembly of junctions (tight and desmosomal) and apical-basolateral polarization in the MDCK cell Ca++ switch model. Studies proposed seek to establish: 1) the mechanism of intracellular Ca++ rise, fall and stabilization during Ca++ switch (using digital imaging, single cell spectrofluorimetric measurements); 2) whether perturbation of intracellular Ca++ levels during the Ca++ switch affects the development of cell-cell contact, junctions and polarity (immunofluorescence); and 3) the role of Ca++ activated signalling events in the development of cell-cell contact, junctions and polarity (cell fractionation, binding assays). Additional experiments are proposed to continue studies aimed at elucidating the Ca++regulatory machinery which the PI has previously identified in the rough endoplasmic reticulum (RER): calcium binding proteins (CaBPs), the IP3 receptor (IP3R) and a small molecular mass GTP binding protein (SMG). These proteins regulate the IP3- sensitive intracellular Ca++ pool and may play important roles in the marked intracellular Ca++ changes observed during the MDCK cell Ca++ switch. Proposed studies will determine: 1)whether CaBPs exist as a Ca++- dependent complex in the RER (Western blots, column chromatography, isopycnic gradients); 2) whether the IP3Rs in RER and non-RER pools are functionally and structurally distinct (Cleveland digests, 2-D gels); and 3) whether a SMG in the RER is involved in Ca++ movement between the IP3- sensitive and insensitive intracellular Ca++ pools (ADP-ribosylation, Ca++ release assays). Preliminary data is provided for all sets of experiments proposed.

Agency
National Institute of Health (NIH)
Institute
National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)
Type
Research Project (R01)
Project #
1R01DK044503-01A1
Application #
3246029
Study Section
General Medicine B Study Section (GMB)
Project Start
1992-09-30
Project End
1996-09-29
Budget Start
1992-09-30
Budget End
1993-09-29
Support Year
1
Fiscal Year
1992
Total Cost
Indirect Cost
Name
Brigham and Women's Hospital
Department
Type
DUNS #
071723621
City
Boston
State
MA
Country
United States
Zip Code
02115
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Stuart, R O; Nigam, S K (1995) Regulated assembly of tight junctions by protein kinase C. Proc Natl Acad Sci U S A 92:6072-6
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Derman, M P; Cunha, M J; Barros, E J et al. (1995) HGF-mediated chemotaxis and tubulogenesis require activation of the phosphatidylinositol 3-kinase. Am J Physiol 268:F1211-7
Cantley, L G; Barros, E J; Gandhi, M et al. (1994) Regulation of mitogenesis, motogenesis, and tubulogenesis by hepatocyte growth factor in renal collecting duct cells. Am J Physiol 267:F271-80

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