Dynamic changes in NADPH-diaphorase staining reflect activity of nitric oxide synthase: evidence for a dopaminergic regulation of striatal nitric oxide release.
In fixed tissue, neuronal NADPH-diaphorase staining results from nitric oxide synthase (NOS) activity. Neuronal NOS only synthesizes nitric oxide once activated by the binding of Ca2+/calmodulin. We show here that neuronal NADPH-diaphorase staining is also dependent on Ca2+/calmodulin, implying that only activated NOS is detected. In addition, in bovine pulmonary endothelial cells, carbachol and bradykinin dramatically and rapidly increase the intensity of NADPH-diaphorase staining. Furthermore, administration of MK801, an NMDA antagonist, decreases neuronal NADPH-diaphorase staining. This suggests that the intensity of the NADPH-diaphorase staining is related to the level of enzyme activation at the moment of tissue fixation. The potential of exploiting this observation to detect cellular activation of NOS is illustrated by the observations that the intensity of NADPH-diaphorase staining in rat striatal neurones is decreased following systemic treatment with the D1-like dopamine receptor antagonist SCH23390, and increased by the D2-like antagonist eticlopride. These results therefore provide strong evidence that the NADPH-diaphorase reaction can be used to monitor NOS activity at a cellular level of resolution, and reveal a dopaminergic regulation of NOS activity in the striatum mediated by D1-like and D2-like dopamine receptors.
Morris
B J
BJ
Division of Neuroscience and Biomedical Systems, Institute of Biomedical and Life Sciences, University of Glasgow, UK.
Simpson
C S
CS
Mundell
S
S
Maceachern
K
K
Johnston
H M
HM
Nolan
A M
AM
D000818:Animals; D002478:Cells, Cultured; D004396:Coloring Agents; D004298:Dopamine / Q000502:physiology*; D018492:Dopamine Antagonists / Q000494:pharmacology; D065127:Dopamine D2 Receptor Antagonists; D004727:Endothelium / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D018691:Excitatory Amino Acid Antagonists / Q000494:pharmacology; D006651:Histocytochemistry; D008297:Male; D009252:NADPH Dehydrogenase / Q000378:metabolism*; D017072:Neostriatum / Q000201:enzymology / Q000378:metabolism / Q000502:physiology*; D009569:Nitric Oxide / Q000378:metabolism*; D019001:Nitric Oxide Synthase / Q000378:metabolism*; D051381:Rats; D017208:Rats, Wistar; D017447:Receptors, Dopamine D1 / Q000819:agonists / Q000037:antagonists & inhibitors; D017448:Receptors, Dopamine D2 / Q000819:agonists
D004396:Coloring Agents; D018492:Dopamine Antagonists; D065127:Dopamine D2 Receptor Antagonists; D018691:Excitatory Amino Acid Antagonists; D017447:Receptors, Dopamine D1; D017448:Receptors, Dopamine D2; D009569:Nitric Oxide; D019001:Nitric Oxide Synthase; D009252:NADPH Dehydrogenase; D004298:Dopamine