Signal transduction, second messenger, and protein kinase responses during freezing exposures in wood frogs

Changes in the percentage of adenosine 3',5'-cyclic monophosphate (cAMP)-dependent protein kinase present as the active catalytic subunit (PKAc) and in the levels of the second messengers cAMP, guanosine 3',5'- cyclic monophosphate (cGMP), and D-myo-inositol 1,4,5-trisphosphate (IP3) were quantified in tissues of the freeze-tolerant wood frog Rana sylvatica over the course of freezing at -2.5°C and thawing at 5°C. Freezing exposure rapidly raised liver cAMP concentration and %PKAc (by 2- and 6-fold, respectively) within 2 min postnucleation; both peaked and stabilized between 5 and 60 min postnucleation but declined with longer freezing. Other organs also showed elevated PKAc during freezing, particularly skeletal muscle. By contrast, cGMP concentration was reduced in muscle and kidney after 24 h of freezing but rose after thawing in muscle. Liver also showed a twofold elevation of cGMP during thawing. The protein kinase C (PKC) second messenger, IP3, rose throughout freezing in liver, reaching levels 11-fold higher than control values after 24 h of freezing. IP3 was also elevated in brain after 4 and 8 h of freezing. The different patterns of cAMP, protein kinase A (PKA), and IP3 changes in liver suggest that, whereas cAMP and PKA clearly mediate the rapid activation of glucose output as a cryoprotectant, IP3 and PKC may be involved instead with metabolic responses that deal with the consequences of long-term freezing, such as ischemia resistance or cell volume control