Probing the presence of the ligand‐binding haem in cellular nitric oxide receptors

Background and purpose: Nitric oxide (NO) acts on receptors coupled to guanylyl cyclase (GC), leading to cGMP accumulation. The NO binding site is a haem group, oxidation or loss of which diminishes NO‐stimulated activity. Agonists reportedly engaging both these NO‐insensitive forms have emerged. Here we characterize the effect of a prototype compound (BAY 58‐2667) and use it to assess the haem status of cellular GC. Experimental approach: GC activity measurements were made on the purified protein and on rat platelets. Key results: Experiments on purified GC showed that the target for BAY 58‐2667 is the haem‐free GC, not the haem‐oxidized form. The efficacy of BAY 58‐2667 was about half that shown normally by NO. In platelets, BAY 58‐2667 was a potent GC activator (EC 50 ∼15 nM) but the maximum effect was only about 1% of that achievable with NO. Nevertheless, it was enough to evoke cGMP‐dependent protein phosphorylation. Profound (85 %) desensitization of NO‐evoked GC activity did not alter the effectiveness of BAY 58‐2667. Haem oxidation, however, increased the efficacy of BAY 58‐2667 by 22‐fold, implying that about half the cellular GC was then haem‐free. Oxidation appeared to enhance the rate of haem dissociation from purified GC. Conclusions and implications: Compounds such as BAY 58‐2667 are useful for probing the occupancy of the haem pocket of NO receptors in cells but not for distinguishing oxidized from reduced haem. In vivo , such compounds are likely to be particularly effective in conditions where there is deficient haem incorporation or enhanced haem loss. British Journal of Pharmacology (2008) 153 , 1495–1504; doi: 10.1038/sj.bjp.0707687 ; published online 21 January 2008