Exquisite sensitivity to subsecond, picomolar nitric oxide transients conferred on cells by guanylyl cyclase-coupled receptors.

Nitric oxide (NO) functions as a diffusible transmitter in most tissues of the body and exerts its effects by binding to receptors harboring a guanylyl cyclase transduction domain, resulting in cGMP accumulation in target cells. Despite its widespread importance, very little is known about how this signaling pathway operates at physiological NO concentrations and in real time. To address these deficiencies, we have exploited the properties of a novel cGMP biosensor, named δ-FlincG, expressed in cells containing varying mixtures of NO-activated guanylyl cyclase and cGMP-hydrolyzing phosphodiesterase activity. Responsiveness to NO, signifying a physiologically relevant rise in cGMP to 30 nM or more, was seen at concentrations as low as 1 pM, making cells by far the most sensitive NO detectors yet encountered. Even cells coexpressing phosphodiesterase-5, a cGMP-activated isoform found in many NO target cells, responded to NO in concentrations as low as 10 pM. The dynamics of NO capture and signal transduction was revealed by administering timed puffs of NO from a local pipette. A puff lasting only 100 ms, giving a calculated peak intracellular NO concentration of 23 pM, was detectable. The results could be encapsulated in a quantitative model of cellular NO-cGMP signaling, which recapitulates the NO responsiveness reported previously from crude cGMP measurements on native cells, and which explains how NO is able to exert physiological effects at extremely low concentrations, when only a tiny proportion of its receptors would be occupied.

Mechanisms of activity-dependent plasticity in cellular nitric oxide-cGMP signaling.

Cellular responsiveness to nitric oxide (NO) is shaped by past history of NO exposure. The mechanisms behind this plasticity were explored using rat platelets in vitro, specifically to determine the relative contributions made by desensitization of NO receptors, which couple to cGMP formation, and by phosphodiesterase-5 (PDE5), which is activated by cGMP and also hydrolyzes it. Repeated delivery of brief NO pulses (50 nM peak) at 1-min intervals resulted in a progressive loss of the associated cGMP responses, which was the combined consequence of receptor desensitization and PDE5 activation, with the former dominating. Delivery of pulses of differing amplitude showed that NO stimulated and desensitized receptors with similar potency (EC50 = 10-20 nM). PDE5 activation was highly sensitive to NO, with a single pulse peaking at 2 nM being sufficient to evoke a 50% loss of response to a subsequent near-maximal NO pulse. However, the activated state of the PDE subsided quickly after removal of NO, the half-time for recovery being 25 s. In contrast, receptor desensitization reverted much more slowly, the half-time being 16 min. Accordingly, with long (20-min) exposures, NO concentrations as low as 600 pM provoked significant desensitization. The results indicate that PDE5 activation and receptor desensitization subserve distinct short term and longer term roles as mediators of plasticity in NO-cGMP signaling. A kinetic model explicitly describing the complex interplay between NO concentration, cGMP synthesis, PDE5 activation, and the resulting cGMP accumulation successfully simulated the present and previous data.

An enzyme-linked receptor mechanism for nitric oxide-activated guanylyl cyclase.

Nitric oxide (NO) exerts physiological effects by activating specialized receptors that are coupled to guanylyl cyclase activity, resulting in cGMP synthesis from GTP. Despite its widespread importance as a signal transduction pathway, the way it operates is still understood only in descriptive terms. The present work aimed to elucidate a formal mechanism for NO receptor activation and its modulation by GTP, ATP, and allosteric agents, such as YC-1 and BAY 41-2272. The model comprised a module in which NO, the nucleotides, and allosteric agents bind and the protein undergoes a conformational change, dovetailing with a catalytic module where GTP is converted to cGMP and pyrophosphate. Experiments on NO-activated guanylyl cyclase purified from bovine lung allowed values for all of the binding and isomerization constants to be derived. The catalytic module was a modified version of one describing the kinetics of adenylyl cyclase. The resulting enzyme-linked receptor mechanism faithfully reproduces all of the main functional properties of NO-activated guanylyl cyclase reported to date and provides a thermodynamically sound interpretation of those properties. With appropriate modification, it also replicates activation by carbon monoxide and the remarkable enhancement of that activity brought about by the allosteric agents. In addition, the mechanism enhances understanding of the behavior of the receptor in a cellular setting.