Cellular and biochemical mechanisms of intracellular putrescine pool regulation

Abstract

Three important aspects of intracellular polyamine pool regulation were investigated in this dissertation: synthesis, uptake and efflux. Ornithine decarboxylase (ODC), a key enzyme in the biosynthetic pathway of polyamines, is characterized by rapid turnover. It has been proposed that degradation of ODC involves two mechanisms, constitutive degradation pathway and antizyme-mediated degradation pathway, the latter requires binding of an ODC-inhibitory protein, antizyme. To address that ODC was degraded by a single mechanism, i.e. antizyme-mediated pathway, wildtype and mutant ODCs were compared for their stability and affinity to interact with antizyme. The data revealed a correlation of increased stability of mutant ODCs with decreased affinity to interact with antizyme, suggesting that antizyme is an essential mediator of ODC degradation. Uptake of polyamines from extracellular medium is also a highly regulated process. Antizyme has been implicated in the feedback repression of polyamine uptake, although the mode of action is unknown. In the present study, the physiological consequences of loss of regulation of ODC protein or uptake on intracellular polyamine contents and cell viability were examined. Stabilization of ODC increased intracellular putrescine contents slightly, but had no toxic effects on cells. However, loss of uptake regulation caused over-accumulation of putrescine and triggered cells undergoing apoptosis, suggesting the importance of uptake regulation in preventing polyamine toxicity. The process of putrescine export from mammalian cells was characterized by using an inside-out membrane vesicle system. It was a saturable and ATP-independent process. The specific inhibition by verapamil and reserpine suggests involvement of membrane protein. The transporter system appeared to be a diamine exporter, mediating efflux of not only putrescine, but also other diamine compounds and acetyl-polyamines in vitro. Putrescine and other physiological diamines such as, cadaverine, N1- and N8-acetylspermidine, were found accumulated in intact cells when the exporter was blocked. Spermidine and spermine did not appear to be exported by this mechanism. While optimal polyamine levels, which are essential for cell proliferation, require finely control in a combination of synthesis, uptake and efflux, regulation of uptake is of more importance in preventing the toxic effects of excessive polyamines.