P-type ion pumps are membrane transporters that have been classified into five subfamilies termed P₁–P₅. The ion transported by the P₅-ATPases is not known. Five genes, ATP13A (ATPase type 13A) 1–ATP13A5, that belong to the P₅-ATPase group have been identified in humans. Mutations of the human gene ATP13A2 underlie a form of PD (Parkinson's disease). Previous studies have suggested a relation between polyamines and P_5B-ATPases. We have recently shown that the cytotoxicity induced by the polyamine analogue paraquat (1,1′-dimethyl-4,4′-bipyridinium), which is an environmental agent related to PD development, was increased in ATP13A2-expressing CHO (Chinese-hamster ovary) cells. In the present study we showed that ATP13A2-expressing CHO cells exhibit a 2-fold higher accumulation of spermidine. Increasing concentrations of spermidine reduced the viability of CHO cells stably expressing ATP13A2. The higher levels of spermidine attained by the ATP13A2-expressing CHO cells were correlated with an increase in the ATP-dependent spermidine uptake in an isolated subcellular fraction containing lysosomes and late endosomes. The results of the present study support the idea that the human P_5B-ATPase ATP13A2 is involved in polyamine uptake.

Hypotonicity triggered in human hepatoma cells (Huh-7) the release of ATP and cell swelling, followed by volume regulatory decrease (RVD). We analyzed how the interaction between those processes modulates cell volume. Cells exposed to hypotonic medium swelled 1.5 times their basal volume. Swelling was followed by 41% RVD₄₀ (extent of RVD after 40 min of maximum), whereas the concentration of extracellular ATP (ATP_e) increased 10 times to a maximum value at 15 min. Exogenous apyrase (which removes di- and trinucleotides) did not alter RVD, whereas exogenous Na⁺-K⁺-ATPase (which converts ATP to ADP in the extracellular medium) enhanced RVD₄₀ by 2.6 times, suggesting that hypotonic treatment alone produced a basal RVD, whereas extracellular ADP activated RVD to achieve complete volume regulation (i.e., RVD₄₀ ≈100%). Under hypotonicity, addition of 2-(methylthio)adenosine 5′-diphosphate (2MetSADP; ADP analog) increased RVD to the same extent as exposure to Na⁺-K⁺-ATPase and the same analog did not stimulate RVD when coincubated with MRS2211, a blocker of ADP receptor P2Y₁₃. RT-PCR and Western blot analysis confirmed the presence of P2Y₁₃. Cells exhibited significant ectoATPase activity, which according to RT-PCR analysis can be assigned to ENTPDase2. Both carbenoxolone, a blocker of conductive ATP release, and brefeldin A, an inhibitor of exocytosis, were able to partially decrease ATP_e accumulation, pointing to the presence of at least two mechanisms for ATP release. Thus, in Huh-7 cells, hypotonic treatment triggered the release of ATP. Conversion of ATP_e to ADP_e by ENTPDase 2 activity facilitates the accumulated ADP_e to activate P2Y₁₃ receptors, which mediate complete RVD.