BAUMANN, N.L.*; LIGHT, D.B.: Purinergic Receptor Activation Leads to a Calcium-Sensitive Volume Decrease in Mudpuppy Erythrocytes

Regulatory volume decrease (RVD) in mudpuppy (Necturus maculosus) erythrocytes has been shown to depend on ATP-, Ca²⁺-, and phospholipase A₂ (PLA₂)-dependent pathways (Light et al. 1997. J. Membr. Biol. 158: 229). This study was designed to determine the relationship between these agents during RVD. The hemolytic index (HI) decreased with extracellular ATP (50 MICROmolar) and the Ca²⁺ ionophore A23187 (0.5 MICROmolar). In contrast, HI increased with the purinoceptor antagonist suramin (100 MICROmolar) and the PLA₂ inhibitor ONO-OS-082 (10 MICROmolar). Interestingly, A23187 (0.5 MICROmolar) reversed the inhibitory effect of suramin (100 MICROmolar), indicating the Ca²⁺-dependent step was downstream of a P2 receptor. Extracellular ATP (50 MICROmolar) did not reverse the antagonistic affect of ONO-OS-082 (10 MICROmolar), suggesting that P2 receptor activation occurred prior to production of arachidonic acid. The percent volume recovery of cells following hypotonic shock (0.5X Ringer), measured with a Coulter counter, increased with extracellular ATP (50 MICROmolar) and A23187 (0.5 MICROmolar). In contrast, suramin (100 MICROmolar) inhibited volume recovery; this inhibition was reversed with A23187 (0.5 MICROmolar). Finally, using the whole-cell patch clamp technique, we found ATP (50 MICROmolar) enhanced a K⁺-conductance that was activated during cell swelling. This current was inhibited by ONO-RS-082 (10 MICROmolar) and suramin (100 MICROmolar). Conclusion: our results are most consistent with extracellular ATP activation of a P2 receptor occurring prior to a rise in intracellular Ca²⁺. This, in turn, activates PLA₂ and subsequent K⁺ efflux through a conductive pathway. (Supported by NSF grants MCB-9603568 and MCB-0076006.)