Nitric oxide synthase activity and isoforms in rat renal vasculature

Experiments were performed to quantify nitric oxide synthase (NOS) activity and identify the NOS isoforms present in the Sprague-Dawley rat renal vasculature. NOS enzymatic activity was measured by adding [³H]arginine to microdissected renal blood vessels and quantifying the conversion to [³H]citrulline by reverse-phase high-performance liquid chromatography. Total NOS activity was greatest in microdissected vasa recta (123±41 pmol·mg^-1·h^-1, n=5) and significantly less in glomeruli (46±9 pmol·mg^-1·h^-1, n=6) and afferent arterioles (42±10 pmol·mg^-1·h^{- 1}, n=6) and averaged <5 pmol·mg^-1·h^-1 in arcuate (n=8) and interlobular (n=9) arteries. Addition of 1.0 mmol/L EDTA to the reaction decreased NOS activity to <5 pmol·mg^-1·h^-1 in afferent arterioles, glomeruli, and vasa recta (n=5 each), indicating that the NOS enzymatic activity in these segments is primarily a result of constitutive NOS. Both neuronal and endothelial NOS mRNA were identified in each vascular segment by reverse transcription polymerase chain reaction, but inducible NOS mRNA was detected only in microdissected arcuate arteries. The present experiments indicate that the vasa recta, glomeruli, and afferent arterioles contain large amounts of calcium-dependent NOS enzymatic activity and that neuronal NOS and endothelial NOS mRNA are present in these segments.