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

Abstract

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.

Fig. 1

Dilutional linearity: eNOS concentration plotted against the dilution for the “spike control”, the brain tissue lysate buffer spiked with eNOS (solid red triangles) and four brain cortex samples from rat #3: three spiked samples, one at 1:20 dilution (purple squares), the other Cleanascite-treated (green diamonds), the spiked (blue circles), and neat (magenta triangles) samples. Note the high dilutional linearity for all types of samples, with coefficients of determination, R² > 0.92 for all samples. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this paper.)

Fig. 2

Kit vs. ko brain buffer standards: the effect of matrix on standard curve data is shown by plotting absorbance values for the eNOS calibration standards prepared in the kit buffer matrix (manufacturers' recommendation) on the left ordinate, and for these same standards prepared in an “eNOS-free” matrix from a mutant eNOS-ko mouse brain tissue homogenate, ko brain buffer, on the right ordinate against their known values of [eNOS] on the abscissa. Both calibration curves show high linearity with R² > 0.99.

Fig. 3

[eNOS]_br differences between normo- and hypotensive rats: the [eNOS]_br (mean ± SD) is different (p = 0.023) between rats with normal blood pressure (rats #1–3, n = 3) and those that are moderately hypotensive (rats # 4–9, n = 6).