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All cells need to maintain a state of homeostasis in order to perform the functions necessary to sustain life. Osmotic homeostasis is especially important in animal cells because they lack a cell wall and are often faced with water flux across the plasma membrane. When placed in a dilute (hypotonic) solution, cells swell. In order to return to their homeostatic volume, they initiate a regulatory volume decrease (RVD) response that results in selective loss of osmolytes and water (O’Connor and Kimelberg, 1993). Although volume regulation is an essential property of animal cells, the underlying mechanisms of RVD, including the signal transduction pathways involved with this process, are not well understood and appear to vary between species and even between cell types within an organism (Basavappa et al., 1998, Okada, 2004). My study focused on the specific RVD signaling pathways in Alligator mississippiensis erythrocytes. Cells were subjected to a hypotonic shock (0.5X Ringer), and thereafter volume recovery was measured electronically over 40 minutes. Extracellular calcium (Ca²⁺e) was found to play an important role in RVD, because volume recovery was inhibited when this ion was buffered to 10 or 1000 nM with EGTA. It also was determined that Ca²⁺ likely entered the cell through an ATP-gated purinoreceptor (hexokinase, an ATP scavenger, inhibited RVD), thereby leading to K⁺ efflux through a quinine-sensitive channel. In addition, studies with gramicidin showed that the efflux of K⁺ was a rate limiting step during volume recovery. Furthermore, a fluorescence microscopy protocol using a Ca²⁺-specific probe (fluo-4, AM) was investigated as a potential protocol for qualitatively examining levels of intracellular calcium during RVD.


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