On the selectivity of neuronal NOS inhibitors.
OBJECTIVE
Isoform-selective inhibitors of NOS enzymes are desirable as research tools and for potential therapeutic purposes. Vinyl-l-N-5-(1-imino-3-butenyl)-l-ornithine (l-VNIO) and N(ω) -propyl-l-arginine (NPA) purportedly have good selectivity for neuronal over endothelial NOS under cell-free conditions, as does N-[(3-aminomethyl)benzyl]acetamidine (1400W), which is primarily an inducible NOS inhibitor. Although used in numerous investigations in vitro and in vivo, there have been surprisingly few tests of the potency and selectivity of these compounds in cells. This study addresses this deficiency and evaluates the activity of new and potentially better pyrrolidine-based compounds.


METHODS
The inhibitors were evaluated by measuring their effect on NMDA-evoked cGMP accumulation in rodent hippocampal slices, a response dependent on neuronal NOS, and ACh-evoked cGMP synthesis in aortic rings of the same animals, an endothelial NOS-dependent phenomenon.


RESULTS
l-VNIO, NPA and 1400W inhibited responses in both tissues but all showed less than fivefold higher potency in the hippocampus than in the aorta, implying useless selectivity for neuronal over endothelial NOS at the tissue level. In addition, the inhibitors had a 25-fold lower potency in the hippocampus than reported previously, the IC(50) values being approximately 1 μM for l-VNIO and NPA, and 150 μM for 1400W. Pyrrolidine-based inhibitors were similarly weak and nonselective.


CONCLUSIONS
The results suggest that l-VNIO, NPA and 1400W, as well as the newer pyrrolidine-type inhibitors, cannot be used as neuronal NOS inhibitors in cells without stringent verification. The identification of inhibitors with useable selectivity in cells and tissues remains an important goal.
Wolfson Institute for Biomedical Research, University College London, London, UK.
B
B
Pigott
K
K
Bartus
J
J
Garthwaite
D000578:Amidines / Q000494:pharmacology*; D000818:Animals; D001013:Aorta, Thoracic / Q000187:drug effects / Q000378:metabolism; D001120:Arginine / Q000031:analogs & derivatives* / Q000494:pharmacology; D001596:Benzylamines / Q000494:pharmacology*; D002531:Cerebellum / Q000187:drug effects / Q000378:metabolism; D006152:Cyclic GMP / Q000378:metabolism; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006624:Hippocampus / Q000187:drug effects / Q000378:metabolism; D008297:Male; D051379:Mice; D008810:Mice, Inbred C57BL; D052248:Nitric Oxide Synthase Type I / Q000037:antagonists & inhibitors*; D009952:Ornithine / Q000031:analogs & derivatives* / Q000494:pharmacology; D017207:Rats, Sprague-Dawley
D000578:Amidines; D001596:Benzylamines; D004791:Enzyme Inhibitors; C111862:N(5)-(1-imino-3-butenyl)ornithine; C109599:N(omega)-propylarginine; C104820:N-(3-(aminomethyl)benzyl)acetamidine; D001120:Arginine; D009952:Ornithine; D052248:Nitric Oxide Synthase Type I; D006152:Cyclic GMP