Chronic consumption of alcohol can lead to pathological developments in multiple organ systems, and much morbidity and mortality. While these pathological changes are well recognized, the mechanisms by which they occur remain elusive. In the liver, chronic alcohol consumption leads to changes in hormone signal transduction pathways, which may play a role in the etiology of alcoholic liver disease (ALD). The goal of this project is to understand the role of changes in cyclic-AMP (cAMP) signaling in the development of mitochondrial changes that may participate in the progression to ALD. We will pursue the following specific aims 1) cAMP signaling is increased in hepatocytes from animals chronically fed alcohol and that this increase leads to an increase in Protein Kinase A (PKA) activity, 2) increased PKA activity, potentially coupled with altered PKA localization, is responsible for mitochondrial localization of the Cytochrome P450 2E1 (CYP2E1) observed during chronic alcohol consumption, and 3) increased PKA activity is responsible for increased calcium mobilization and subsequent increased mitochondrial calcium loading in response to inositol 1,4,5-trisphosphate (IPS) -dependent hormones. These factors are proposed to act together to cause mitochondrial damage and decreased cell viability. Our central hypothesis is that increased mitochondrial CYP2E1, coupled with altered mitochondrial calcium homeostasis, may be largely responsible for the mitochondrial damage and dysfunction seen in livers of chronic abusers of alcohol.
These aims will be assessed using single-cell fluorescence imaging, with calcium sensitive dyes and fluorescence energy resonance transfer (FRET) probes for mitochondrial calcium and cAMP production, immunocytochemistry to assess PKA localization, western blots to determine CYP2E1 localization and expression, and molecular-based kits for cAMP production and PKA activity. Information gained from these experiments will contribute to a better understanding of alterations in mitochondrial functioning in ALD, and may ultimately contribute to improved therapeutic treatments. Relevance: Diseases associated with alcohol abuse are major public health concerns in the United States due to a number of well-documented pathological conditions. Alcohol-induced liver disease is responsible for much of the morbidity and mortality associated with alcohol abuse, yet the exact mechanisms by which such conditions occur remain elusive. The goal of this project is to understand how changes in cellular signaling can lead to mitochondrial dysfunction and ultimately tissue damage and disease progression. ? ? ?
