ABSTRACT
Epoxyeicosatrienoic acids (EETs) carry out a number of functions in brain tissue, some of which include vasodilatation of cerebral vessels and endothelial tube formation. The excitatory neurotransmitter glutamate, releases EETs from cultured astrocytes. Inhib ition of E E T synthesis in the whisker barrel cortex of the rat partially blocks functional hyperemia. To better understand these functions of EETs an attempt to overexpress cytochrome P450-epoxygenases in rat brain was made. This approach has been chal lenging because P450 enzymes need specific cofactors. Also gene delivery into brain in vivo is difficult due to the high cell density in the parenchyma. Recombinant genes have been successfully expressed in rat brain by infusion of adenoviral vectors into the lateral ventricle. When introduced in this way, viral coded genes showed abundant expression in the ependymal cells. In this study expression in the choroid plexus was also observed. The next step was to introduce a bacterial cytochrome P450 epoxygenase, B M - 3 F87V, into cultured rat astrocytes. The recombinant B M - 3 F87V cloned in an adenovirus genome, synthesized 14S, 15R-EET and its corresponding dihydroxyeicosatrienoic acids (DiHETEs) after infection into cultured astrocytes. The bacterial gene also increased growth of the astrocytes after stimulation with serum, as measured by radioactive thymidine incorporation assays. Expression of Ad5/BM-3 F87V in brain tissue was monitored by infusion of the virus into the rat brain ventricle followed by the substrate, 1 4C-arachidonic acid, after 24 hours. Cellular lipids were extracted and resolved by reverse phase high performance liquid chromatography ( rpHPLC). The results showed conversion of the arachidonic acid into 14,15-EET demonstrating potential for use of recombinant P450 enzymes for research as well as gene therapy in the brain.
