LIGHT, D.B.*; DAHLSTROM, P.K.: EXTRACELLULAR ATP STIMULATES VOLUME DECREASE IN NECTURUS ERYTHROCYTES VIA A P2X2 RECEPTOR. Receptor
Regulatory volume decrease (RVD) in mudpuppy (Necturus maculosus) erythrocytes depends on a K+ conductance that is activated during cell swelling. In addition, hypotonic swelling causes release of ATP into the extracellular medium, which leads to increased K+ efflux (Light et al. 1999. Amer. J. Physiol. 277: C480). This study was designed to determine the nature of the P2 receptor involved in this process. The hemolytic index (HI), a measure of osmotic fragility, was determined with a spectrophotometer. This value decreased with extracellular ATP (50 ? MICROmolar), ATP-GAMMA-S (50 ? MICROmolar), and 2-MeS-ATP (50 ?MICROmolar). In contrast, adenosine (50? MICROmolar), UTP (50 ? MICROmolar) and ?,?-Me-ATP (50 MICROmolar) had no effect on HI. Furthermore, HI increased with the P2 antagonist suramin (100 ? MICROmolar) and the P2X inhibitor PPADS (10-100 MICROmolar), but not with the P2Y antagonist RB2 (10 MICROmolar). We also measured the percent volume recovery of cells following hypotonic shock (0.5X Ringer) electronically with a Coulter counter. This value increased with extracellular ATP (50 ? MICROmolar), ATP?S (50 ? MICROmolar), and 2-MeS-ATP (50 ? MICROmolar). In contrast, suramin (100 ? MICROmolar) inhibited volume recovery and this affect was reversed with gramicidin (5 MICROmolar). Adenosine (50 MICROmolar), UTP (50 MICROmolar) and ?,?-Me-ATP (50 MICROmolar) had no effect on RVD. Finally, we used the whole-cell patch clamp technique to measure ion currents directly. We found ATP (50 MICROmolar) enhanced a K+-conductance that was activated during cell swelling. This current was inhibited by suramin (100 ? MICROmolar). Conclusion: our results are most consistent with extracellular ATP activation of a P2X2 receptor during cell swelling, which in turn leads to volume decrease by enhancing K+ efflux. (Supported by NSF grant MCB-9603568 and MCB-0076006.)
