Polyamine Transport and Ornithine Decarboxylase Activity in Hypoxic Pulmonary Artery Smooth Muscle Cells

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Abstract
Hypoxia causes remodeling of the pulmonary circulation that is dependent on increases in lung polyamine contents. Mechanisms by which polyamines are regulated in hypoxic lung cells are unknown, but ornithine decarboxylase (ODC) activity, the initial enzyme in de novo biosynthesis, is depressed and polyamine transport is augmented in lungs from hypoxic rats (R.-T. Shiao et al. 1990. Am. J. Physiol. 259:L351–L358). To determine if hypoxia directly influences polyamine regulatory mechanisms in pulmonary vascular cells, we examined [14C]spermidine (SPD) transport and ODC activity in bovine main pulmonary artery smooth muscle cells (PASMCs) cultured under standard (culture medium Po2: > 100 mm Hg), “normoxic” (culture medium Po2: 50 to 70 mm Hg), or “hypoxic” (culture medium Po2: 18 to 30 mm Hg) conditions. Uptake of [14C]SPD in cells cultured under standard conditions was temperature- and concentration-dependent, exhibited saturation kinetics, and was abolished by metabolic inhibition. Modeling of transport according to Michaelis-Menten kinetics revealed that [14C]SPD uptake in cells cultured under standard conditions was characterized by Km and Vmax values of 0.78 µM and 4.5 pmol/min/106 cells, respectively. In comparison to cells cultured under standard conditions, Km was unaffected by culture under normoxic or hypoxic conditions while Vmax was increased to 18 pmol/min/106 cells in normoxic cells and to 33 pmol/min/106 cells in preparations cultured under hypoxic conditions. Inhibition of ODC with α-difluoromethylornithine (DFMO) also induced SPD transport, as evidenced by an increase in the Vmax to 65 pmol/min/106 cells. Both hypoxia- and DFMO-induced increases in [14C]SPD transport were suppressed by cycloheximide and actinomycin D, thus highlighting the importance of protein and RNA synthesis. ODC activity did not differ between cells cultured under standard and normoxic conditions (48 pmol/mg protein/60 min) but was reduced by 75% to 12 pmol/60min/mg protein in hypoxic cells. The abundance of ODC mRNA also was reduced by 80 to 85% in hypoxic cells relative to cells cultured under standard conditions. In contrast to the reduction in ODC activity, hypoxia failed to inhibit the activity of another important enzyme in polyamine synthesis, S-adenosylmethionine decarboxylase. These findings indicate that hypoxia induces polyamine transport in PASMCs, perhaps secondary to decreased ODC activity, and suggest that increases in lung polyamine contents necessary for hypoxic vascular remodeling may be ascribed in part to induction of transport in cells of the pulmonary circulation.