Ethanol consumption at low to moderate levels protects the heart and vasculature from the deleterious effects of ischemia and reperfusion (I/R). Our recent work indicates that antecedent ethanol ingestion provokes the development of an anti-inflammatory phenotype in postcapillary venules such that these microvessels fail to support leukocyte/endothelial cell adhesive interactions during I/R. Surprisingly, AMP-activated kinase (AMPK) plays a necessary role in initiating this adaptive transformation to a protected phenotype in postcapillary venules. However, it is not clear how this enzyme, best known for its role as a metabolic master switch, is involved in transducing signals elicited by ethanol to effect the acquisition of tolerance to I/R in the microcirculation. Our overall hypothesis is that ethanol-induced, AMPK-dependent eNOS activation serves as an important initiating factor to trigger a cascade of signaling events that ultimately act to promote the expression of cytochrome P450 epoxygenases (CYP), which serve as an important effector of the beneficial microvascular actions of ethanol by catalyzing the formation of reaction products which exhibit powerful anti-adhesive and antioxidant properties. To address this postulate, we propose to determine whether: (1) ethanol-induced AMPK activation initiates the development of an anti-inflammatory phenotype by stimulating the formation of eNOS-derived NO; (2) NO-induced release of calcitonin gene-related peptide (CGRP) plays an essential role in the acquisition of tolerance to ischemia that is evoked by antecedent ethanol consumption; (3) the cystic fibrosis transmembrane regulator (CFTR) serves as an obligatory downstream signaling element that participates in inaugurating the development of the protected phenotype precipitated by ethanol-induced AMPK activation; and (4) ethanol-induced, AMPK-triggered increases in CYP activity during I/R prevents postischemic leukocyte adhesion in postcapillary venules by abrogating P-selectin expression. Intravital microscopic approaches will be used to quantify leukocyte rolling and adhesion in wild-type control mice and in mutant mice lacking AMPK, eNOS, CGRP, or CFTR. The influence of ethanol on l/R-induced adhesion molecule expression, AMPK and eNOS activity, CGRP release, and CYP protein expression and activity will also be investigated. Collectively, these aims address novel mechanisms whereby antecedent ethanol ingestion induces the development of an anti-inflammatory phenotype in postcapillary venules. This work will identify new links between ethanol-induced, AMPK-dependent, eNOS-derived NO formation as essential triggering elements, CGRP release and CFTR function as obligatory downstream mediators, and increased CYP activity as a major effector in the acquisition of tolerance to I/R by antecedent ethanol ingestion. ? ? ?
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