Histochemical localization of NADPH-dependent diaphorase (nitric oxide synthase) activity in vascular endothelial cells in the rat brain

This study investigated the localization of NADPH-dependent diaphorase activity within vascular endothelial cells in the rat brain. Light microscope observations showed that in addition to neurons and neuronal processes stained histochemically for NADPH-dependent diaphorase activity, endothelial cells in many medium to large diameter (20-100 microns) blood vessels were also stained. These vessels were either attached to the pial surface or contained within the substance of the tissue. In vascular endothelia, the formazan end-product of the diaphorase reaction was deposited as discrete clusters of darkly stained punctae that were located around the nucleus of these cells. Correlated light- and electron-microscopical examination revealed that the sites of formazan deposition occurred in regions of endothelial cytoplasm devoid of smooth and rough endoplasmic reticulum and of mitochondria. Since endothelial NADPH dependent diaphorase activity co-localizes with the activity of nitric oxide synthase (the synthetic enzyme for nitric oxide) these observations suggest that in vascular endothelial cells nitric oxide synthase may be a highly localized soluble cytosolic enzyme not structurally associated with any subcellular organelle. In addition, specific regions of the smooth muscle cells encircling the larger diameter blood vessels clearly demonstrated NADPH dependent diaphorase activity. Unmyelinated fibres and fibre-plexi surrounding blood vessels on the pial surface were also stained. The results of this study show specific NADPH dependent diaphorase activity in vascular endothelial cells in the rat brain. Therefore, together with neurons, endothelial cells may control nitric oxide-dependent vasodilation thereby regulating local blood flow in the brain.