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, 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 since no method exists to measure eNOS. 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 varied considerably from 5-11fmol/mg wet weight and was different between normo- and hypotensive animals (p=0.023). The variability of eNOS 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 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 itself could be an important factor in cerebrovascular regulation.