Variations of brain endothelial nitric oxide synthase concentration in rat and mouse cortex

No information exists on the differences of eNOS concentration in brain tissue, [eNOS](br), between animals during normal and hypotensive blood pressure and both between and within animals during moderate hypotension. To address these questions, we modified a commercially available enzyme-linked immunosorbent assay (ELISA) kit for determining murine [eNOS](br) since no method exists to measure [eNOS](br). Optimization of the kit ELISA procedure using brain cortex homogenates from 3 normotensive rats and 1 wild-type and 1 eNOS(-/-) (ko) mouse included recovery evaluation for each sample and the use of an "eNOS-free" homogenate calibrator diluent obtained from a mutant eNOS-ko mouse. Initial spike-and-recovery values of 12.5-27% suggesting a substantial sample matrix effect were improved with lipid removal treatment to 37.3% and to 70% with 1:20 dilution of the sample. Calibration standards prepared using eNOS-free buffer increased recovery values to 78% in micro-punch samples. The optimized ELISA was used in micro-punch (<1mg) brain cortex samples from 6 hypotensive rats. Whole brain [eNOS](br) varied considerably from 5-11fmol/mg wet weight and was different between normo- and hypotensive animals (p=0.023). The variability of [eNOS](br) due to moderate hypotension in micro-punch rat brain cortex samples was composed of both between (24%) and within (76%) animal components. The differences and variability of [eNOS](br) between normo- and hypotensive animals, and between and within hypotensive animals suggests the potential utility of its measurement for investigations of cerebrovascular physiology and that [eNOS](br) itself could be an important factor in cerebrovascular regulation.