A new and simple method for delivering clamped @entity842 concentrations in the physiological range: application to activation of guanylyl cyclase-coupled XXXX receptors.

@entity6706 @entity3542 @entity6836 @entity842 @entity26890 @entity291
The signaling molecule @entity842 (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 @entity26890 donor with NO consumption by a sink. On theoretical grounds, @entity6706 ( @entity6706 ) was selected as the sink. The mixture additionally contained urate to convert an unwanted product of the reaction ( @entity6836 ) into @entity3542 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 @entity291 lung, which is lower than estimated previously using a biological NO sink (red blood cells). The corresponding values for the alpha1beta1 and alpha2beta1 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.
