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The Wolfson Institute for Biomedical Research, University College London, London, United Kingdom (C.G., V.W., J.G.); and Division of Neurophysiology, National Institute for Medical Research, Mill Hill, London, United Kingdom (T.B.)
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 
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1 and 21 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.
Address correspondence to: Dr. John Garthwaite, The Wolfson Institute for Biomedical Research, University College London, Gower Street, London, WC1E 6BT, United Kingdom. E-mail: john.garthwaite{at}ucl.ac.uk
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  B. Roy, E. J. Halvey, and J. Garthwaite An Enzyme-linked Receptor Mechanism for Nitric Oxide-activated Guanylyl Cyclase J. Biol. Chem., July 4, 2008; 283(27): 18841 - 18851. [Abstract] [Full Text] [PDF]
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 J. Clasadonte, P. Poulain, J.-C. Beauvillain, and V. Prevot Activation of Neuronal Nitric Oxide Release Inhibits Spontaneous Firing in Adult Gonadotropin-Releasing Hormone Neurons: A Possible Local Synchronizing Signal Endocrinology, February 1, 2008; 149(2): 587 - 596. [Abstract] [Full Text] [PDF]
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 C. N. Hall and J. Garthwaite Inactivation of nitric oxide by rat cerebellar slices J. Physiol., December 1, 2006; 577(2): 549 - 567. [Abstract] [Full Text] [PDF]
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 B. Roy and J. Garthwaite Nitric oxide activation of guanylyl cyclase in cells revisited PNAS, August 8, 2006; 103(32): 12185 - 12190. [Abstract] [Full Text] [PDF]
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 M. Kavdia and A. S. Popel Contribution of nNOS- and eNOS-derived NO to microvascular smooth muscle NO exposure J Appl Physiol, July 1, 2004; 97(1): 293 - 301. [Abstract] [Full Text] [PDF]
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E. Mo, H. Amin, I. H. Bianco, and J. Garthwaite Kinetics of a Cellular Nitric Oxide/cGMP/Phosphodiesterase-5 Pathway J. Biol. Chem., June 18, 2004; 279(25): 26149 - 26158. [Abstract] [Full Text] [PDF]
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