Extracellular ATP stimulates volume decrease in Necturus red blood cells

This study examined whether extracellular ATP stimulates regulatory volume decrease (RVD) in Necturus maculosus (mudpuppy) red blood cells (RBCs). The hemolytic index (a measure of osmotic fragility) decreased with extracellular ATP (50 microM). In contrast, the ATP scavenger hexokinase (2.5 U/ml, 1 mM glucose) increased osmotic fragility. In addition, the ATP-dependent K+ channel antagonist glibenclamide (100 microM) increased the hemolytic index, and this inhibition was reversed with ATP (50 microM). We also measured cell volume recovery in response to hypotonic shock electronically with a Coulter counter. Extracellular ATP (50 microM) enhanced cell volume decrease in a hypotonic (0.5x) Ringer solution. In contrast, hexokinase (2.5 U/ml) and apyrase (an ATP diphosphohydrolase, 2.5 U/ml) inhibited cell volume recovery. The inhibitory effect of hexokinase was reversed with the Ca2+ ionophore A-23187 (1 microM); it also was reversed with the cationophore gramicidin (5 microM in a choline-Ringer solution), indicating that ATP was linked to K+ efflux. In addition, glibenclamide (100 microM) and gadolinium (10 microM) inhibited cell volume decrease, and the effect of these agents was reversed with ATP (50 microM) and A-23187 (1 microM). Using the whole cell patch-clamp technique, we found that ATP (50 microM) stimulated a whole cell current under isosmotic conditions. In addition, apyrase (2.5 U/ml), glibenclamide (100 microM), and gadolinium (10 microM) inhibited whole cell currents that were activated during hypotonic swelling. The inhibitory effect of apyrase was reversed with the nonhydrolyzable analog adenosine 5'-O-(3-thiotriphosphate) (50 microM), and the effect of glibenclamide or gadolinium was reversed with ATP (50 microM). Finally, anionic whole cell currents were activated with hypotonic swelling when ATP was the only significant charge carrier, suggesting that increases in cell volume led to ATP efflux through a conductive pathway. Taken together, these results indicate that extracellular ATP stimulated cell volume decrease via a Ca2+-dependent step that led to K+ efflux.