Nitric oxide activation of guanylyl cyclase in cells revisited.

Nitric oxide (NO) elicits physiological effects in cells largely by activating guanylyl cyclase (GC)-coupled receptors, leading to cGMP accumulation. Like other receptor-coupled effector mechanisms, NO stimulation of GC activity was previously considered to be a graded, concentration-dependent response, with deactivation following swiftly once the agonist disappeared. Recently, a new and unconventional mechanism has been proposed from experiments on purified protein [Cary, S. P. L., Winger, J. A. & Marletta, M. A. (2005) Proc. Natl. Acad. Sci. USA 102, 13064-13069]. It was concluded that GC in vivo will display a dual regulation by NO: a long-lasting tonic activity (10-20% of maximum) due to persistent occupation by NO of the heme binding site and phasic activity due to engagement of another unidentified, lower affinity site. The hypothesis was first tested by monitoring GC activity in rat platelets maintained in vitro and exposed to calibrated NO transients. The kinetics was as expected for a single binding site for NO (EC(50) = 10 nM), with activation and deactivation of enzyme activity conforming to the predictions of a simple receptor model. No tonic GC activity attributable to long-term NO binding was detected after exposure to the full range of active NO concentrations (peaking at 2-500 nM). Comparable results were obtained by using neural cells isolated from the cerebellum. After exposure to high NO concentrations, persistent GC activity could be recorded, but this activity was caused artifactually by secondary NO sources being formed in the medium. The new scheme for regulation of GC activity by NO is of doubtful relevance to cells.