The goals of the proposal are to define the biological role of nitric oxide (N0) in the regulation of glomerular endothelial cell function. The hypothesis is that expression of nitric oxide synthase (N0S) and subsequent release of N0 by human glomerular endothelial (HGE) cells play a key role in their function in both health and disease. The initial aims will focus on investigating the physiological factors which regulate N0 enzyme activation and molecular expression of N0S in primary cultures of HGE cells. Due to the finite proliferation of HGE cells and limited expression of N0 in vitro, the major thrust of the project will be directed at the development and testing of appropriate vector systems which will allow for conditional immortalization of HGE cells. Conditional immortalization will be achieved using a recombinant retroviral construct encoding the temperature - sensitive mutant of SV4 large T antigen. This thermolabile transforming protein is active at permissive temperature (33 degrees C) but degrades at temperatures greater than 37 degrees C. Developing HGE cell lines is a crucial first step in deciphering the functional role of N0 in these cells. To characterize which particular isoform exist in both primary HGE cells and in HGE cell lines studies will be designed to activate the calcium dependent (constitutive) and the cytokine inducible isoforms of N0S. N0 activity will be measured utilizing the following biochemical assays l) citrulline forming assay and 2) activation of guanylate cyclase in reporter rat fibroblast cell line, sensitive index of the formation of NO. Molecular expression of N0S will be characterized by Western and Northern blot analysis. Both biochemical and molecular studies will be directed at defining the regulator mechanisms of N0 activation. Subsequently, we will develop HGE cell line which over or under - express NOS and examine the physiological effects on HGE cell function.
