Intracellular sources of ornithine for polyamine synthesis in endothelial cells.

Abstract

Polyamines are essential for proliferation of endothelial cells (EC)and angiogenesis. This study was conducted to identify the metabolic source(s) of ornithine for polyamine synthesis in EC, using N(ω)-hydroxy-nor-L-arginine (Nor-NOHA, an inhibitor of arginase) and gabaculine (an inhibitor of ornithine aminotransferase; OAT). Nor-NOHA inhibited arginase with an IC50 value of 10µM for intact EC. Nor-NOHA (0.5mM) alone inhibited arginase activity in EC by 98%, increased total cellular concentrations of arginine by 14%, and decreased total cellular concentrations of ornithine, putrescine and spermidine by 17, 65 and 74%, respectively. Arginine and glutamine contributed to 73 and 26% of the ornithine produced by EC, respectively. Gabaculine (1mM) alone decreased the total cellular concentrations of arginine, ornithine, putrescine, and spermidine by 14, 96, 32, and 42%, respectively. A combination of both Nor-NOHA and gabaculine completely blocked ornithine production in EC, resulting in no detectable cellular ornithine and almost complete depletion of cellular putrescine and spermidine. Addition of 0.5mM ornithine restored the intracellular concentrations of polyamines in EC treated with Nor-NOHA plus gabaculine, indicating that Nor-NOHA and gabaculine did not inhibit ornithine decarboxylase activity. Our results suggest that the arginase and OAT pathways are the exclusive sources of ornithine in EC when there is little extracellular ornithine and that there is intracellular compartmentalization of arginine and ornithine for endothelial synthesis of polyamines. These novel findings may have important implications for improving placental vascular growth, wound healing, and cancer therapy.