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Abstract:
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A majority of cells exposed to hypotonic environments swell and consequently activate volume-sensitive ion channels that facilitate ion efflux as a means of reestablishing cell volume. The signaling pathway for this mechanism is not completely defined; but previous studies conducted on a variety of cell types propose that the swelling of a cell initiates the release of ATP which subsequently, stimulates purinergic receptors leading to the efflux of Cl- ions; thus, restoring cell volume. Many studies focus on a pharmacological approach to manipulate the purinergic signaling pathway. Because this approach is potentially non-specific we have adopted a genetic approach. The goal of our study is to determine whether the purinergic receptor plays a critical role in volume regulation. For this study we used two cell lines derived from 1321N1 cells obtained from a human astrocytoma. The first cell is the original clone of 1321N1 cells (parental) which lack P2Y receptors. And the second clone is stably transfected to express only the P2Y1 receptor. For the current study we measured cell volume changes during exposure to isotonic (290 mOsm) and hypotonic (200 mOsm or 150 mOsm) environments. Cells were maintained in culture in log-growth phase and replated weekly. To measure volumes, wells were removed from the culture dish with a brief exposure to trypsin and then incubated in isotonic solution for 30 min. Then cells were diluted into 20 ml of isotonic or hypotonic solutions. A Coulter Counter was used to measure cells volumes at various times of incubation in these solutions (0, 3, 7, 15, and 30 minutes). Cells from both clones had stable volumes in isotonic solution during the 30 min experimental period. Similarly, cells from both clones treated with 200 mOsm or 150 mOsm swelled to a similar extent within 3 min (127±6% and 142±6% of their isotonic volume, respectively). During the subsequent period in 200 mOsm solution, P2Y1 cells recovered their normal volume, while Parental cells remained swollen. It is apparent, according to the data collected that cell exposed to isotonic environments tend to maintain a relatively stable volume, as expected, while P2Y1 cells exposed to hypotonic environments tend to increase in volume and eventually return to a near homeostatic volume. In contrast, cells lacking purinergic receptors have not capacity for hypoosmotic volume regulation. We conclude that purinergic receptors are necessary for cellular volume regulation. |
