In rat membranous nephropathy, complement C5b-9 induces glomerular epithelial cell (GEC) injury and proteinuria, which in some models is partially mediated by eicosanoids. Recently, it has been demonstrated that the C5b-9 complex activates a high molecular mass cytosolic phospholipase A$ sb2$ (cPLA$ sb2)$ in cultured rat GEC, which leads to release of arachidonic acid (AA) and eicosanoids, and may contribute toward plasma membrane injury. In this study, we investigated if C5b-9 can also activate other PLA$ sb2$ isoforms. In GEC stably overexpressing sPLA$ sb2$ (Type II PLA$ sb2,$ 14 kD) (produced by transfection), the C5b-9-stimulated increase in free $ sp3$H-AA, ($ sim$2 fold above basal) was not significantly different from neo (control) GEC. In contrast, in GEC overexpressing cPLA$ sb2,$ C5b-9 stimulated a $ sim$9 -fold increase in free AA, as compared to unstimulated cells. This result suggests that C5b-9 activates cPLA$ sb2,$ but not the sPLA$ sb2$ enzyme, to release free AA. In further studies, we addressed the regulation of cPLA$ sb2$ in C5b-9-stimulated GEC. The present model of cPLA$ sb2$ activation involves the combined effects of: (1) an increase in cytosolic calcium concentration, which induces translocation of cPLA$ sb2$ from the cytosol to the plasma membrane, and may be mediated by the amino terminal "CaLB" (Ca$ sp{+2}$-dependent lipid binding) domain, and (2) phosphorylation by MAP kinase. To test the role of the CaLB domain, release of AA was monitored in GEC stably transfected with three constructs; cPLA$ sb2$ wild type (wt), cPLA$ sb2 Delta$CaLB (i.e., cPLA$ sb2$-wt with deleted CaLB domain), and neo (control) GEC. In GEC overexpressing cPLA$ sb2$-wt, C5b-9 stimulated a marked increase in free AA (as above). In contrast, in GEC overexpressing the cPLA$ sb2 Delta$CaLB enzyme, the C5b-9 induced increase in free $ sp3$H-AA was comparable to neo, despite expression of cPLA$ sb2 Delta$CaLB at levels similar to cPLA$ sb2$-wt. The results indicate tha
| Identifer | oai:union.ndltd.org:LACETR/oai:collectionscanada.gc.ca:QMM.24031 |
| Date | January 1996 |
| Creators | Panesar, Mandip |
| Contributors | Cybulsky, Andrey (advisor) |
| Publisher | McGill University |
| Source Sets | Library and Archives Canada ETDs Repository / Centre d'archives des thèses électroniques de Bibliothèque et Archives Canada |
| Language | English |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Format | application/pdf |
| Coverage | Master of Science (Department of Physiology.) |
| Rights | All items in eScholarship@McGill are protected by copyright with all rights reserved unless otherwise indicated. |
| Relation | alephsysno: 001529691, proquestno: MM19841, Theses scanned by UMI/ProQuest. |
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