Lake Forest College, Biology, Lake Forest, IL 60045
A cells ability to regulate its volume is a property that has been conserved across diverse cell types and is pivotal in the maintenance of homeostasis. This is especially important for cells exposed to changing extracellular solute concentrations and for cells that experience fluctuating intracellular osmolality due to solute absorption. For instance, when a cell is exposed to a hypotonic environment, it swells as a result of water influx. To recover, it initiates a regulatory volume decrease (RVD) response whereby the cell selectively loses osmolytes and water. The control mechanisms and signal transduction processes of RVD are not well understood and appear to vary among species and cell types. The purpose of my study was to help elucidate pathways involved in RVD. Specifically, the role of calcium and purinergic (P2) receptor activation by ATP was examined using red blood cells of the American alligator (Alligator mississippiensis). A Coulter counter was used to electronically measure the changes in cell volume that occurred upon exposure to solutions of different osmolarities and solutions containing various pharmacological agents. It was found that RVD was inhibited when cells were exposed to a medium containing the calcium chelator EGTA, indicating extracellular calcium is necessary for volume recovery. In addition, inhibition of P2 receptors with the ATP scavenger hexokinase also reduced RVD. In contrast, cell recovery was greatly enhanced with the addition of the cationophore gramicidin (used with choline substituted for sodium in the extracellular medium). In conclusion, our results are most consistent with a swelling-activated, calcium-dependent potassium efflux during RVD. Furthermore, calcium influx into the cell appears to be linked with activation of P2 receptors. Future studies will elucidate the role of calcium once it has entered the cell, as well as developing a protocol using fluorescence microscopy to examine the presence of intracellular calcium.
[Abstract (DOC)]