LIGHT, D.B.; CAMPION, B.K.; Ripon College; Ripon College: Cell Volume Regulation by Hyla chrysoscelis Erythrocytes

Hyla chrysoscelis (Cope�s gray treefrog) is a freeze-tolerant anuran. The process of freezing and thawing of extracellular fluid generates an osmotic imbalance with intracellular fluid compartments. Accordingly, we examined regulated volume decrease (RVD) by erythrocytes of this species. Cell volume was measured with a Coulter counter (model Z2). Exposure of erythrocytes to a hypotonic (0.5X) Ringer caused cells to quickly swell, which was followed by a slower, spontaneous RVD. Surprisingly, cells slowly swelled again approximately 15 minutes after hypotonic shock . The potassium channel blocker quinine (1 mM) partially inhibited RVD and completely blocked secondary swelling. Substituting choline for sodium in the extracellular solution prevented secondary swelling, indicating it required sodium influx via a quinine-sensitive pathway. Further, the cationophore gramicidin (0.5 micromolar) potentiated volume recovery, indicating potassium efflux is a rate-limiting step for RVD. We next examined the role of calcium. Releasing calcium from intracellular stores with cyclopiazonic acid (10 micromolar) potentiated RVD. However, buffering cytosolic calcium with BAPTA (100 micromolar) had no significant effect, indicating the stimulatory effect of calcium was an epiphenomenon. In contrast, chelating extracellular calcium to 50 micromolar with EGTA or addition gadolinium (10 micromolar) blocked secondary swelling, suggesting it depended on calcium influx via a stretch-activated channel. Conclusions: RVD relies on potassium efflux, whereas the anomalous secondary swelling phasedepended on calcium-stimulated sodium influx. (Supported by a Merck/AAAS USRP award).