ABSTRACT
This study examined the role of a P2 receptor and arachidonic acid (AA) in regulatory volume decrease (RVD) by American alligator red blood cells (RBCs). Osmotic fragility was determined optically, mean cell volume was measured by electronic sizing, and changes in intracellular Ca(2+) concentration were visualized using fluorescence microscopy. Gadolinium (50 µM), hexokinase (2.5 U/ml), and suramin (100 µM) increased osmotic fragility, blocked volume recovery after hypotonic shock, and prevented a rise in intracellular Ca(2+) that normally occurs during cell swelling. The P2X antagonists PPADS (50 µM) and TNP-ATP (10 µM) also increased fragility and inhibited volume recovery. In contrast, ATPγS (10 µM), α,ß-methylene-ATP (50 µM) and Bz-ATP (50 µM) had the opposite effect, whereas 2-methylthio-ATP (50 µM) and UTP (10 µM) had no effect. In addition, the phospholipase A(2) (PLA(2)) inhibitors ONO-RS-082 (10 µM), chlorpromazine (10 µM), and isotetrandrine (10 µM) increased osmotic fragility and blocked volume recovery, whereas AA (10 µM) and its nonhydrolyzable analog eicosatetraynoic acid (ETYA, 10 µM) had the reverse effect. Further, AA (10 µM), but not ATPγS (10 µM), prevented the inhibitory effect of a low Ca(2+)-EGTA Ringer on RVD, whereas both AA (10 µM) and ATPγS (10 µM) caused cell shrinkage under isosmotic conditions. In conclusion, our results are consistent with the presence of a P2-like receptor whose activation stimulated RVD. In addition, AA also was important for volume recovery.
