ATTWOOD, A.J.; SIEGEL, Corryn; LIGHT, D.B.*: Calcium Stimulates Volume Decrease in Necturus Erythrocytes
This study examined whether cytosolic calcium regulates cell volume decrease in Necturus maculosus (mudpuppy) erythrocytes. Hypotonic (0.5X Ringer) swelling stimulated an increase in intracellular calcium levels (detected with Fluo-4 and epifluorescence microscopy). A similar increase in calcium did not occur under isosmotic conditions. Although the calcium chelator EGTA (5 µM) had no effect, BAPTA-AM (100 µM, used to buffer intracellular calcium), gadolinium (10 µM), hexokinase (2.5 U/ml), and suramin (100 µM, a P2 antagonist) each prevented the swelling induced increase in calcium. Consistent with these studies, the percent volume recovery following hypotonic shock (measured with a Coulter counter) increased with the calcium ionophore A23187 (2 µM), but was unaffected by EGTA (5 µM). In contrast, BAPTA-AM (100 µM), gadolinium (10 µM), and hexokinase (2.5 U/ml) blocked volume recovery. The inhibitory effect of these three agents was reversed with the cationophore gramicidin (2 µM, used to maintain a high potassium permeability) and the latter two with A23187 (2 µM). Finally, suramin (100 µM) blocked a potassium conductance that was activated during cell-swelling (measured with the whole-cell patch clamp technique). Conclusions: regulated volume decrease was dependent, at least in part, on a rise in cytosolic calcium in response to cell swelling. Intracellular stores were the main source of this ion. Further, an increase in cytosolic calcium during hypotonic stress was linked to activation of a P2 receptor with extracellular ATP. (Supported by NSF grant MCB-0076006).
