Polyamine transport is mediated by both endocytic and solute carrier transport mechanisms in the gastrointestinal tract

Abstract

The polyamines spermidine and spermine, and their precursor putrescine, are required for cell growth and cellular functions. The high levels of tissue polyamines are implicated in carcinogenesis. The major sources of exogenous polyamines are diet and intestinal luminal bacteria in gastrointestinal (GI) tissues. Both endocytic and solute carrier-dependent mechanisms have been described for polyamine uptake. Knocking down of caveolin-1 protein increased polyamine uptake in colon cancer-derived HCT116 cells. Dietary supplied putrescine was accumulated in GI tissues and liver in caveolin-1 knockout mice more than wild-type mice. Knocking out of nitric oxide synthase (NOS2), which has been implicated in the release of exogenous polyamines from internalized vesicles, abolished the accumulation of dietary putrescine in GI tissues. Under conditions of reduced endogenous tissue putrescine contents, caused by treatment with the polyamine synthesis inhibitor difluoromethylornithine (DFMO), small intestinal and colonic mucosal polyamine contents increased with dietary putrescine levels, even in mice lacking NOS2. Knocking down the solute carrier transporter SLC3A2 in HCT116-derived Hkh2 cells reduced the accumulation of exogenous putrescine and total polyamine contents in DFMO treated cells, relative to non-DFMO-treated cells. These data demonstrate that exogenous putrescine is transported into GI tissues by caveolin-1- and NOS2-dependent mechanisms, but that the solute carrier transporter SLC3A2 can function bidirectionally to import putrescine under conditions of low tissue polyamines.

Fig. 1.

Effect of caveolin-1 on polyamine transport. Putrescine (A), spermidine (B), and spermine (C) uptake activities in HCT116/Mock (○) and HCT116/Cav-1 A. S. cells (●) were measured as described in materials and methods. Values are means ± SE of triplicate determinations. Curve fitting was calculated by using the Microsoft Excel program. D: caveolin-1 protein levels in HCT116/Mock and HCT116/Cav-1 A. S. cells. Flotilin 1 levels are shown as a loading control. *P < 0.05 against HCT116/Mock.

Fig. 2.

Effect of caveolin-1 and NOS2 knockout on putrescine transport in vivo. Wild-type, caveolin-1 knockout, and NOS2 knockout mice were treated with 1% putrescine (hatched bar), 1% difluoromethylornithine (DFMO; solid bar), and the combination of 1% putrescine and 1% DFMO (dotted bar) for 2 wk and polyamine contents in the small intestine (A), colon (B), and liver (C) were determined. Open bar, no treatment. Values are means ± SE of determinations in 3 mice. *Statistically significant (P < 0.05) against wild type. D: protein levels of caveolin-1, NOS2, and NOS3 in the small intestine and colon. β-Tubulin levels are shown as a loading control.

Fig. 3.

Effect of SLC3A2 on putrescine transport. Hkh2 cells transfected short hairpin RNA (shRNA) for SLC3A2 (solid bar) and mock vector (open bar) were cultured with 5 mM [³H]putrescine in the presence and absence of 5 mM DFMO and 50 μM 1400W for 2 days. [³H]putrescine accumulation was measured as described in materials and methods. Values are shown as means ± SE of triplicate determinations. *P < 0.01. B: SLC3A2, caveolin-1, and NOS2 protein levels in mock and SLC3A2 shRNA transfected cells. Flotilin 1 was shown as a loading control.

Fig. 4.

Model of polyamine transport in animal cells. The regulation of cellular polyamine levels by transport is depicted. The polyamine binds to polyamine binding protein(s) and is internalized by caveolar endocytosis, which is negatively regulated by caveolin-1 (Cav-1). The nitric oxide (NO) produced by NOS2 releases polyamine from polyamine binding protein(s). Putrescine and acetylated polyamines (AcPolyamine), which produced by spermidine/spermine N-acetyltransferase (SAT1), are exported by SLC3A2. When cells are treated with the polyamine biosynthesis inhibitor DFMO, SLC3A2 can catalyze the uptake of putrescine by a reverse reaction. ODC, ornithine decarboxylase.