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

Abstract

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 cGMPhydrolyzing 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.is an evolutionarily ancient transmitter of fundamental importance to the physiology of the mammalian cardiovascular, nervous, and other systems (1, 2). NO signal transduction takes place through the simplest one-component type of receptor, comprising an NO binding site (a prosthetic heme) coupled to a guanylyl cyclase (GC) transduction domain (3, 4). The elevation in cellular cGMP that follows NO binding is curtailed by one or more phosphodiesterase (PDE) enzymes that convert it to GMP. There have been countless descriptions of changes in the levels of cGMP in association with NOmediated transmission in different cells and tissues. It has become increasingly evident, however, that the levels of cGMP measurable by traditional methods, such as radioimmunoassay, may not be relevant to physiological NO signaling. For example, smooth muscle relaxation (5) and cGMP-dependent phosphorylation events in platelets (6) are seen at NO concentrations below those giving increases in cGMP measurable with such methods; NO can still relax vascular smooth muscle despite deletion of 94% of the NO-activated GC (7); and engagement of 2% or less of the available GC activity is sufficient to stimulate cGMPdependent phosphorylation in platelets (8). These findings indicate that cells possess a very large receptor excess and that new approaches are required to understand the elementary properties of NO capture and signal amplification by cells under physiological conditions. A related gap in knowledge is in...

Antiinflammatory actions of inorganic nitrate stabilize the atherosclerotic plaque

“…Nevertheless, treatment with KNO 3 resulted in elevations of both nitrate and nitrite across all compartments, with no evidence of any particular organ selectivity, indicating that at least in ApoE KO mice the pathways for uptake of the two anions is universally present and that dietary nitrate effectively restores, and can elevate beyond, normal physiological levels. In support of the suggestion that dietary nitrate ultimately results in NO delivery in vivo are our observations demonstrating that nitrate feeding also results in elevations of cGMP, perhaps the most sensitive indicator of NO bioactivity (28). Whereas a HFD regimen increased plaque burden compared with NCD in ApoE KO mice, dietary nitrate treatment for 12 wk with KNO 3 (15 mmol/L)-enriched water did not alter the total plaque burden.…”

Dynamic Ligand Exchange in Soluble Guanylyl Cyclase (sGC)

“…Thus, under the premises of the "tonic" activation hypothesis, under physiological conditions, NO/cGMP signaling should be very inefficient. This conclusion is inconsistent with experimental observations of an effective cGMP synthesis in response even to picomolar puffs of NO (20).…”

“…At the same time, it has been estimated that intracellular concentrations of sGC, at least in platelets and cerebellar astrocytes, are in the micromolar range (20). These assessments indicate that, under normal conditions, the interaction between NO and sGC occurs at substoichiometric ratios.…”

“…Previous observations that 1 eq of NO does not fully activate sGC (21,22) gave rise to the hypothesis that, after its formation, the cellular sGC-NO complex is in a "tonically" activated state, which requires additional NO for full activation and efficient NO/cGMP signaling (21,22,27). However, under normal non-inflammatory conditions, the sGC concentration may be several orders of magnitude higher than the NO concentration (20), and only a fraction of sGC may convert into the sGC-NO complex. Thus, under the premises of the "tonic" activation hypothesis, under physiological conditions, NO/cGMP signaling should be very inefficient.…”

“…5, B and C). (17)(18)(19) and estimation of intracellular concentrations of sGC (20) indicate that, under normal conditions, cellular sGC encounters and is stimulated by substoichiometric NO. The goal of this study was to investigate the dynamics of NO interaction with sGC and the functional outcome of such interaction.…”