Wood frogs are hardy little critters; they let their bodies freeze in winter and hop away just hours after the first rays of sunshine thaw them out in spring. One secret of their success is glucose; when temperatures plummet,wood frogs pack their cells with this cryoprotectant. This lowers the freezable portion of water inside their cells so they don't shrink as much from dehydration, which minimizes mechanical damage to the cell membranes. Now, Jon Costanzo and Richard Lee reveal that glucose isn't the only cryoprotectant that wood frogs use; they have discovered that urea also protects these amphibians from freezing damage().
Urea is a very unlikely candidate to play a protective role inside cells,because `it has a very bad reputation', Costanzo explains. The molecule wreaks havoc in cells, causing proteins to unfold and stop working, which perhaps explains why nobody has ever considered whether urea also plays any beneficial roles. But when Costanzo and Lee discovered that wood frogs' urea levels shoot up 25-fold during hibernation, they began to ponder whether the `disreputable'molecule might play a part in wood frogs' remarkable freeze tolerance.
To test whether urea protects wood frogs' cells from freezing damage,Costanzo froze and thawed frogs' red blood cells in the presence and absence of two well-known cryoprotectants, glucose and glycerol, and the putative cryoprotectant, urea. He gauged the degree of cell injury by measuring the amounts of haemoglobin and the intracellular enzyme lactate dehydrogenase that had escaped from damaged cells. To his surprise, the cells fared extremely well in the presence of urea; it offered as much protection from cellular damage as glycerol and was even more effective than glucose.
Encouraged by this finding, Costanzo went on to see if urea protects wood frog tissues. He first loaded different tissues with urea by bathing tissue samples of wood frog muscle, liver, heart and kidney in a urea solution; since urea is very permeable, it readily enters cells. He then froze and thawed the tissues and tested whether the cells had survived. To determine the level of cell damage, he measured lactate dehydrogenase leakage again. To confirm that the tissues were still alive, he added a blue indicator dye that turns red if cells are metabolizing. Sure enough, he discovered that tissues loaded with urea survived freezing better than tissues without urea, confirming that urea acts as a cryoprotectant.
It seems urea is much more versatile than we suspected. Urea appears to help wood frogs survive dehydration, because the animals' urea levels closely track seasonal changes in soil moisture. Costanzo and Lee now suggest that urea also plays a role as a cryoprotectant. And there is even evidence for a third role; when he loaded the frogs' liver and muscle tissues with urea,Costanzo noticed that the tissues' metabolic rates dropped, suggesting that urea could be acting as a metabolic depressant. If that's true, then there's`an awful lot for one molecule to do!' says Costanzo. Not bad for a waste product; perhaps it's time to reassess its reputation?
