Regulatory Volume Increase in Rana pipiens Hepatocytes: Discovery of the Cellular Transport Mechanisms and an Examination of the Effects of Pyrene on Volume Restoration.

摘要

Consistency of volume is essential to cellular homeostasis. Various osmolyte transport systems including ionic cotransporters, exchangers, and channels are responsible for regulating cell volume following an osmotic challenge. This study used isolated hepatocytes from leopard frogs, monitored changes in cell volume during an osmotic disturbance, and attempted to identify the cellular mechanisms responsible for restoration of cell volume. Furthermore, the effect of the polycyclic aromatic hydrocarbon (PAH) pyrene, on the ability of frog hepatocytes to volume regulate was investigated. Hepatocytes exposed to an increase in extracellular solution osmolality (from 230 to 290 mOsm/kg) immediately had a significant reduction in cell volume, and subsequently underwent a regulatory volume increase (RVI). The data showed that RVI is dependent on the availability of extracellular Na+ and Cl-. Inhibition of Na+/H+ exchange with amiloride, Cl-/HCO3- exchange with DIDs, and the enzyme carbonic anhydrase with acetazolamide, in separate experiments, all suggest the involvement of Na+/H+ and Cl -/HCO3- exchange during volume recovery. Inhibitors of Na+-K+-2Cl- cotransport, Na+/Cl- channels, and Ca2+ entry did not affect RVI, indicating that these mechanisms do not contribute to this process. Finally, an in vivo pyrene exposure was shown to adversely affect hepatocyte RVI when compared to volume regulation from control frogs. Although hepatocyte cell viability from control and pyrene exposed frogs was not significantly different, hepatocytes isolated from pyrene exposed frogs were significantly swollen compared to hepatocytes from control frogs. This suggests that cell volume regulation may be compromised as a result of the pyrene exposure. In fact, isolated hepatocytes from pyrene exposed frogs were incapable of restoring cell volume when osmotically challenged. These data indicate that pyrene exposure may compromise frog survival potential limiting vital cell processes.