NMDA causes release of nitric oxide from rat spinal cord in vitro

Anatomic studies have localized nitric oxide synthase (NOS) activity in the rat spinal cord dorsal horn and intermediolateral cell column. Behavioral and electrophysiologic studies suggest that N-methyl-D-aspartate (NMDA) stimulates nitric oxide synthesis in the dorsal horn. This report describes a novel bioassay to determine directly in vitro whether NMDA causes release of nitric oxide from rat spinal cord. Modified Krebs-Henseleit solution at 26 degrees C was perfused over spinal cord slices from adult male rats, then dropped onto a ring of endothelium-denuded rat aorta. Following preconstriction with phenylephrine, NMDA (10(-10) to 10(-3) M) alone or with other drugs was added to the perfusion solution and vascular tension measured. NMDA-containing solutions applied directly on the preconstricted vessels without exposure to spinal cord tissue had no effect on vessel tone. In contrast, NMDA via the spinal cord perfusion caused concentration-dependent vascular relaxation, which was blocked by MK-801, hemoglobin, methylene blue, and several arginine analogues which inhibit NOS. [14C]citrulline assay suggested NOS in rat spinal cord was non-endothelial in nature. NMDA perfusion of spinal cord slices in vitro causes vascular relaxation in this bioassay due to actions on NMDA receptors and which is consistent with release of nitric oxide. These results support previous anatomical, behavioral, and electrophysiologic studies in rat spinal cord and describe a novel, sensitive, and simple bioassay for nitric oxide release from neural tissue in vitro.