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 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.

Fig. 1.

Behavior of GC_lowPDE_HEK cells in response to perfusion (1 min) of different clamped NO concentrations. (A) Responses of a single cell, illustrating how baselining was applied (orange broken line); the mean population responses (seven cells) are shown in B. The red line fits the data to the GC/PDE_HEK model (Fig. 2 and SI Materials and Methods) with GC_max = 0.235 μM/s and PDE_max = 0.036 μM/s; for the final response, PDE_max was raised to 0.08 μM/s to fit the faster decay (blue line). The changes in the principal NO receptor species during the course of the experiment, according to the model, are shown in C; with reference to Fig. 2A, Unliganded, GC; Active, NOGC*; and Desensitized, GC*NO. (D) Predicted changes in cGMP concentration giving rise to the fluorescent changes depicted in B, together with the profiles of applied NO concentration (calibrated from the kinetics of fluorescein wash-in and wash-out; see SI Materials and Methods).

Fig. 2.

Model for NO-activated GC (A) and PDE5 (B). The parameters in A had the following values (15): k₁ = 3 × 10⁸ M^-1 s^-1, k_-1 = 6 s^-1, k₂ = k_-2 = 28 s^-1, k₃ = 10⁷ M^-1 s^-1, k_-3 = 1000 s^-1, k₄ = 2000 s^-1, k_-4 = 1.8 × 10⁶ M^-1 s^-1, k₅ = 7.34 × 10^-4 s^-1, k_-5 = 4 × 10⁸ M^-1 s^-1, k₆ = 1 s^-1, k_-6 = 10^-3 s^-1. The PDE5 parameters are given in Table 2 and the text. Data analysis (see SI Materials and Methods) generated indistinguishable K_p and PDE_max values for the two active PDE5 species (tPDE5* and pPDE5*), and so they were designated equal.

Fig. 3.

Behavior of GC_highPDE_HEK cells (A, 5 cells), GC_highPDE5_low cells (C, 17 cells), and GC_midPDE5_high cells (D, 22 cells) on perfusion of clamped NO concentrations. A also shows the effect of removal of the NO scavenger, CPTIO (100 μM), followed by perfusion of the NO antagonist, ODQ (3 μM), and restoration of the fluorescence with 8-bromo-cGMP (8-Br-cGMP, 1 mM) at the end. The red lines are fits to the GC/PDE_HEK model (A, GC_max = 20 μM/s, PDE_max = 0.035 μM/s; C, GC_max = 5 μM/s, PDE_max = 2.5 μM/s; D, GC_max = 3 μM/s, PDE_max = 18 μM/s). (B) Changes in NO and cGMP concentrations for the simulation in A. The Inset in C shows the response of a cell in a different experiment to a 95-s exposure to 30 pM NO (points, Left) simulated with the model (red line), and the predicted changes in cGMP concentration, GC, and PDE activities (Right; scale bar for GC and PDE activities, 0.05 μM/s; GC_max = 21.5 μM/s, PDE_max = 3.2 μM/s). The Inset in D is an expansion of the response to 10 nM NO (Left) together with the predicted corresponding change in cGMP and the NO concentration profile (right). Note in C and D the small rapid undershoots and slow rebound increases in fluorescence following exposure to the high NO concentrations. Their origin has not been investigated because of their small size and poor reproducibility between experiments.

Fig. 4.

Puff applications of NO to GC_highPDE5_low cells. (A) Sequence of images showing the transient δ-FlincG fluorescence increase in a field of cells subjected to a 2-s puff of NO from a neighboring pipette. The final frame shows the δ-FlincG response (red line) in the cell outlined in red in the first frame in relation to the delivered NO concentration (black broken line). (B) Four NO puffs were applied 12 s apart; data are means of five runs (six cells) and are fitted by the GC/PDE5 model (GC_max = 15.2 μM/s, PDE_max = 2.34 μM/s). The Inset shows the responses aligned, numbered according to their order. (C) Four NO puffs were given 30 s apart, followed by a test puff 4 min after the last one, showing only partial recovery (three cells). The red line is a fit to the model (GC_max = 35.5 μM/s, PDE_max = 4.17 μM/s). (D) Predicted changes in cGMP concentration from the model in relation to the NO concentration profiles in the experiment in B. (E) The cGMP concentration changes predicted for the experiment in C. (F) Rates of recovery in four experiments (each in a different color) quantified from measurement of the width of the responses at half-height (using the “Peak Analyzer” in OriginPro 8) and expressed relative to the width of the last of the induction puffs (6.94 ± 0.69 s). (G) Changes in the amount of transiently (tPDE5*) and persistently (pPDE5*) active PDE5 species during the experiment shown in C, according to the model.

Fig. 5.

Effect of varying the NO puff duration. (A) NO puffs lasting 0.1–30 s were delivered to a GC_highPDE5_low cell, with repeat 1-s puffs at the end. The Inset plots the peak NO concentration and peak δ-FlincG response for the different puff durations. The red line is a fit to the GC/PDE5 model (GC_max = 17.5 μM/s, PDE_max = 3.23 μM/s). (B) Predicted changes in cGMP concentration in relation to the NO concentration profiles. The Inset illustrates the effect of diffusion through unstirred layers on the access of NO to the intracellular compartment at two sample NO puff durations (see SI Materials and Methods). (C) Changes in the amount of transiently (tPDE5*) and persistently (pPDE5*) active PDE5 species during the experiment shown in A, according to the model.