Clark P. Holden and Kenneth B. Storey
Changes in the percentage of cyclic AMP-dependent protein kinase present as the active catalytic subunit (PKAc) and in the levels of the second messengers adenosine 3':5'-cyclic monophosphate (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] and % PKAc (by 2- and 6-fold, respectively) within 2 min post-nucleation; both peaked and stabilized between 5 and 60 min post-nucleation but declined with longer freezing. Other organs also showed elevated PKAc during freezing, particularly skeletal muscle. By contrast, [cGMP] was reduced in muscle and kidney after 24 h freezing but rose after thawing in muscle. Liver also showed a 2-fold elevation of cGMP during thawing. The protein kinase C (PKC) second messenger, IP3, rose throughout freezing in liver reaching 11-fold higher than control values after 24 h freezing. IP3 was also elevated in brain after 4 and 8 h freezing. The different patterns of cAMP, 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.