In Vivo Study of Ethanol-Activated Brain Protein Kinase A: Manipulations of Ca2+ Distribution and Flux
The cAMP-dependent protein kinase (PKA) signaling transduction pathway has been shown to play an important role in the modulation of several ethanol (EtOH)-induced behavioral actions. In vivo, short-term exposure to EtOH up-regulates the cAMP-signaling cascade. Interestingly, different Ca2+-dependent cAMP–PKA cascade mediators play a critical role in the neurobehavioral response to EtOH, being of special relevance to the Ca2+-dependent adenylyl cyclases 1 and 8. We hypothesize an intracellular PKA activation elicited by EtOH administration, which may be regulated by a Ca2+-dependent mechanism as an early cellular response. Thus, the present work aims to explore the role of Ca2+ (internal and external) on the EtOH-activated PKA cascade.
Swiss male mice received an intraperitoneal injection of EtOH (0 or 4 g/kg), and brains were dissected following a temporal pattern (7, 15, 30, 45, 90, or 120 minutes). Either the enzymatic PKA activity or its fingerprint was analyzed on different brain areas (cortex, hypothalamus, hippocampus, and striatum). To explore the role of Ca2+ on the EtOH-activated PKA cascade, mice were pretreated with diltiazem (0 or 20 mg/kg), dantrolene (0 or 5 mg/kg), or 3,7-Dimethyl-1-(2-propynyl)xanthine (0 or 1 mg/kg) 30 minutes before EtOH (4 g/kg) administration. After 45 minutes of EtOH administration, brains were removed and dissected to measure the PKA activity or its fingerprint.
Results from these experiments showed an EtOH-dependent activation of PKA in different brain areas. Manipulations involving a disruption of intracellular Ca2+ release from the endoplasmic reticulum resulted in a decreased EtOH-induced activation of PKA. On the contrary, extracellular-to-cytoplasm Ca2+ manipulations did not prevent the PKA activation by EtOH.
Altogether, these results show the critical role of stored Ca2+ as an intracellular mediator of different neurobiological actions of EtOH and provide further evidence of a possible new target for EtOH within the central nervous system.