ATP steal between cation pumps: a mechanism linking Na+ influx to the onset of necrotic Ca2+ overload.

We set out to identify molecular mechanisms underlying the onset of necrotic Ca(2+) overload, triggered in two epithelial cell lines by oxidative stress or metabolic depletion. As reported earlier, the overload was inhibited by extracellular Ca(2+) chelation and the cation channel blocker gadolinium. However, the surface permeability to Ca(2+) was reduced by 60%, thus discarding a role for Ca(2+) channel/carrier activation. Instead, we registered a collapse of the plasma membrane Ca(2+) ATPase (PMCA). Remarkably, inhibition of the Na(+)/K(+) ATPase rescued the PMCA and reverted the Ca(2+) rise. Thermodynamic considerations suggest that the Ca(2+) overload develops when the Na(+)/K(+) ATPase, by virtue of the Na(+) overload, clamps the ATP phosphorylation potential below the minimum required by the PMCA. In addition to providing the mechanism for the onset of Ca(2+) overload, the crosstalk between cation pumps offers a novel explanation for the role of Na(+) in cell death.

Cytosolic [Ca(2+)] modulates basal GLUT1 activity and plays a permissive role in its activation by metabolic stress and insulin in rat epithelial cells.

The aim of this work was to investigate the role of cytosolic free calcium ([Ca(2+)]c) in the stimulation of GLUT1 by metabolic stress and insulin. Chelation of [Ca(2+)]c with bapta, introduced in rat liver epithelial Clone 9 cells in the acetoxymethyl (AM) form, decreased their basal rate of 2-deoxyglucose uptake in a dose-dependent fashion. Maximal inhibition at 75 microM bapta was by 38 +/- 8% (n = 8). The effect was partially reversed by ionomycin. Basal sugar uptake was also decreased by lowering extracellular [Ca(2+)] in ionomycin-permeabilized cells. Increasing [Ca(2+)]c over its resting level of 168 +/- 32 (n = 27) had no affect on sugar uptake. Chelation of [Ca(2+)]c did not change the abundance of surface GLUT1 and had no significant effect on the affinity of GLUT1 for sugars. In addition, calcium chelation abolished the activation of GLUT1 by azide, arsenate, 2,4-dinitrophenol and insulin. However, [Ca(2+)]c did not increase in the presence of azide. We conclude that [Ca(2+)]c, near or below its resting level, modulates GLUT1 activity over a considerable range and plays a permissive role in the activation of the carrier by metabolic stress and insulin.

Hyperosmotic shock induces both activation and translocation of glucose transporters in mammalian cells.

The effect of osmotic stress on sugar transport was investigated in Clone 9 epithelial cells, which express the glucose uniporter GLUT1, and in 3T3-L1 adipocytes, which express both GLUT1 and GLUT4. An acute hyperosmotic shock increased the uptake of sugars in both cell types. In Clone 9 cells, this was followed by a regulatory volume increase (RVI) response. Stimulation of transport was rapid and reversible, with half-lives (t 1/2) for stimulation of 2-deoxy-D-glucose uptake of 5.6 +/- 0.9 (n=6) and 22.7 +/- 1.5 (n=4) min for Clone 9 cells and adipocytes respectively. The effect was dose dependent, reaching a maximum at 1.1 osM of 2.9 +/- 0.1-fold (n=3) for Clone 9 cells and 8.2 +/- 0.8-fold (n=3) for adipocytes. In the latter, this stimulation correlated with translocation of the glucose transporter isoform GLUT4 to the cell surface and was not significantly different from that elicited by 160 nM insulin (7.6 +/- 1.2-fold, n=3). The effect of osmotic shock was not, however, influenced by inhibitors of either phosphoinositide 3-kinase (PI 3-kinase) (wortmannin, 250 nM) or of p38 mitogen-activated protein kinase (p38 MAP kinase) (SB203580, 20 microM), which reportedly prevent GLUT4 translocation and/or activation by insulin respectively. These inhibitors also had no effect on the stimulation of transport by osmotic shock in Clone 9 cells. However, in contrast to adipocytes, the effect of osmotic shock on glucose transport in Clone 9 cells reflected primarily a change in the intrinsic activity of cell surface transporters and there was only a minor change in their subcellular distribution as assessed by cell immunostaining or no change as assessed by surface biotinylation. These results indicate that the response of cells to osmotic shock can involve changes both in transporter activity and location. The signal transduction pathways involved include neither PI 3-kinase nor the classical, osmotically-activated component, p38 MAP kinase.