RT Journal Article
SR Electronic
T1 A New and Simple Method for Delivering Clamped Nitric Oxide Concentrations in the Physiological Range: Application to Activation of Guanylyl Cyclase-Coupled Nitric Oxide Receptors
JF Molecular Pharmacology
JO Mol Pharmacol
FD American Society for Pharmacology and Experimental Therapeutics
SP 1349
OP 1356
DO 10.1124/mol.64.6.1349
VO 64
IS 6
A1 Charmaine Griffiths
A1 Victoria Wykes
A1 Tomas C. Bellamy
A1 John Garthwaite
YR 2003
UL http://molpharm.aspetjournals.org/content/64/6/1349.abstract
AB The signaling molecule nitric oxide (NO) could engage multiple pathways to influence cellular function. Unraveling their relative biological importance has been difficult because it has not been possible to administer NO under the steady-state conditions that are normally axiomatic for analyzing ligand-receptor interactions and downstream signal transduction. To address this problem, we devised a chemical method for generating constant NO concentrations, derived from balancing NO release from a NONOate donor with NO consumption by a sink. On theoretical grounds, 2-4-carboxyphenyl-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (CPTIO) was selected as the sink. The mixture additionally contained urate to convert an unwanted product of the reaction (NO2) into nitrite ions. The method enabled NO concentrations covering the physiological range (0-100 nM) to be formed within approximately 1 s. Moreover, the concentrations were sufficiently stable over at least several minutes to be useful for biological purposes. When applied to the activation of guanylyl cyclase-coupled NO receptors, the method gave an EC50 of 1.7 nM NO for the protein purified from bovine lung, which is lower than estimated previously using a biological NO sink (red blood cells). The corresponding values for the α1β1 and α2β1 isoforms were 0.9 nM and 0.5 nM, respectively. The slopes of the concentration-response curves were more shallow than before (Hill coefficient of 1 rather than 2), questioning the need to consider the binding of more than one NO molecule for receptor activation. The discrepancies are ascribable to limitations of the earlier method. Other biological problems can readily be addressed by adaptations of the new method.