Kinetics of a Cellular Nitric Oxide/cGMP/Phosphodiesterase-5 Pathway

Abstract

Rat platelets served as a model to evaluate quantitatively how guanylate cyclase (GC)-coupled nitric oxide (NO) receptors and phosphodiesterases (here phosphodiesterase-5) interact to transduce NO signals in cells. The platelets expressed mRNA only for the ␣ 1 and ␤ 1 GC-coupled receptor subunits. In intact platelets, the potency of NO for elevating cGMP (EC 50 ‫؍‬ 10 nM) was lower than in lysed platelets (EC 50 ‫؍‬ 1.7 nM). The limiting activities of GC and phosphodiesterase in intact platelets were both very high, being equivalent to about 100 M/s. With low phosphodiesterase activity (imposed by 100 M sildenafil), the cGMP response over time was hyperbolic in shape for a range of NO concentrations or GC activities due to GC desensitization. Without a phosphodiesterase inhibitor, NO generated only brief cGMP transients, peaking after 2-5 s but amounting maximally to about 150 M cGMP. The transients were caused partly by GC desensitization, which varied in rate (halftime up to 3 s) and extent (up to 80%) depending on the NO concentration, and partly by an enhancement of the phosphodiesterase catalytic activity with time, which was deduced to be up to 30-fold and to occur with a half-time of up to 5 s. The results were simulated by a quantitative model, which also explains the varied shapes of cGMP responses to NO found in other cells. Downstream phosphorylation in platelets was detectable within 2 s, and, with continuous exposure (1 min), this pathway could be engaged by subnanomolar NO concentrations (EC 50 ‫؍‬ 0.5 nM).Whereas the spatio-temporal dynamics of some of the primary steps of cellular signal transduction, such as synaptic transmission and changes in intracellular Ca 2ϩ , have become well understood in quantitative terms (1, 2), knowledge of downstream signaling cascades remains largely qualitative. Nevertheless, the kinetic properties of the second messenger cascades are likely to be similarly instrumental in determining how cells respond.A case in point is nitric oxide (NO) 1 signaling. NO acts as a diffusible chemical messenger throughout the body, where it subserves diverse functions, including smooth muscle relaxation, inhibition of platelet aggregation, and the induction of synaptic plasticity (3,4). NO elicits these and other physiological effects by binding to its guanylyl cyclase (GC)-coupled receptors, thereby evoking the accumulation of cGMP in target cells. The receptors are ␣␤-heterodimers, of which two main isoforms, ␣ 1 ␤ 1 and ␣ 2 ␤ 1 , exist at differing levels in different tissues (5). Studies on purified receptors (6) and intact cells (7) suggest that activation of GC occurs very rapidly, within a few ms or less of adding NO, and complete deactivation in cells (upon removal of NO) requires only about 500 ms (7). Furthermore, when studied in isolation, the receptors are highly sensitive NO detectors, with only about 1 nM being needed to evoke half-maximal GC activity (8). The shape of the resulting cGMP response in cells, however, varies greatly from one cell to another. For example,...

Modeling Cooperative Volume Signaling in a Plexus of Nitric Oxide Synthase-Expressing Neurons

“…It is clear from results presented here and previously (Philippides et al, 2000(Philippides et al, , 2003 that this is the case regardless of what constitutes an effective NO concentration [a subject of ongoing debate (Artz et al, 2001;Mo et al, 2004)]. The limiting effect of source size has been confirmed experimentally in clusters of macrophages comprising variable numbers of cells (Porterfield et al, 2001).…”

“…In interpreting the data, it is important to note that there is continuing uncertainty in the literature about the half-maximally effective concentration (EC 50 ) of NO on its primary receptor, soluble guanylyl cyclase (sGC). The range reported is from ϳ1 M (Artz et al, 2001) to ϳ1 nM (Mo et al, 2004). The reported sensitivity of another NO target, the respiratory enzyme cytochrome c oxidase, whose inhibition by NO causes synaptic depression, falls in the middle of this range (IC 50 values of Ϸ120 nM at 20 -30 M O 2 ) Garthwaite 2001, 2002;Bellamy et al, 2002).…”

Differential patterning of cGMP in vascular smooth muscle cells revealed by single GFP-linked biosensors

“…An important criterion for intracellular cGMP sensors is that they exhibit appropriately high sensitivity to NO, as previously reported for sGC (half-maximal NO: 1.7 nM for the purified enzyme, 11 nM for intact platelets, 10 nM for cerebellar cells) (17,18). DEA-NO and PROLI-NO were used as NO-donors due to their overall favorable fast chemistry of NO-release (see Materials and Methods).…”

“…Furthermore, at low, physiological NO-concentrations (Ͻ10 nM), FRET-based cGMP indicators are limited in their use to detect fluctuations in [cGMP] i (12). However, recent studies using purified cyclase, intact platelets and cerebellar cells have shown that sGC is activated at low-nanomolar NO concentrations (1-10 nM) (17,18). FRETbased cGMP indicators are also limited in resolving possible compartmentalized intracellular cGMP signaling events using confocal microscopy.…”

Immunohistochemical assessment of cyclic guanosine monophosphate (cGMP) and soluble guanylate cyclase (sGC) within the rostral ventrolateral medulla

“…Basal cGMP levels in neurons are the result of a dynamic equilibrium between synthesis, driven by NOsGC, and the rate of degradation by PDEs, [61]. The rate of synthesis can change with time due to desensitisation of sGC, and cGMP kinetics can vary between not only different cell types but also different regions of the brain [62]. The expression of IBMX-insensitive PDEs, such as PDE9, which is specific for cGMP [46,63], may be another important factor limiting detection of cGMP accumulation in regions such as the RVLM.…”