Objective: The aims were to investigate the requirement for and regulation of cellular polyamines during vascular endothelial cell proliferation.
Methods: Proliferation of cultured bovine pulmonary artery endothelial cells was determined after cellular polyamine depletion. In addition, polyamine synthesis and uptake mechanisms were examined in the presence and absence of trophic stimuli and density dependent inhibition of cell proliferation.
Results: Serum stimulated, subconfluent cells ceased cell division following the inhibition of ornithine decarboxylase (ODC), a rate limiting enzyme for polyamine biosynthesis. The addition of 10 microM putrescine, the product of the enzyme reaction catalysed by ODC, completely reversed the inhibition of cell growth. Serum deprivation reduced cytosolic ODC activity to near non-detectable levels. Readdition of 10% fetal bovine serum (FBS) resulted in transient increases in ODC activity which preceded DNA synthesis and mitosis. Basic fibroblast growth factor also stimulated ODC activity in a dose dependent manner with levels approximating the maximum obtainable with FBS. In contrast, platelet derived growth factor and epidermal growth factor did not stimulate ODC activity. Finally, mitogenic stimuli did not induce ODC activity in density arrested cells. The uptake of radiolabelled putrescine from the cell medium was time dependent and saturable. Kinetic studies from dividing cells revealed a single transport component for putrescine uptake [maximum rate of uptake (Vmax) = 11.2(SEM 2.0) pmol.10(5) cells-1.h-1; Michaelis constant (Km) = 1.1(0.3) microM]. Putrescine uptake by density arrested cells was characterised by a 57% decrease in Vmax with no change in Km. Readdition of FBS to serum deprived subconfluent cells, in the presence of ODC inhibitors, resulted in a rapid increase in the rate of putrescine uptake with Vmax increasing by 533% over FBS alone by 48 h.
Discussion: These data suggest that polyamines are essential for endothelial cell proliferation and that synthesis and uptake mechanisms are regulated according to cell growth.