Abstract
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.
Highlights
Synthesis of cGMP from GTP that follows receptor activation can engage a number of downstream targets, including cGMPdependent protein kinase, to bring about alterations in cell function, such as smooth muscle relaxation and neural transmission [1,2,3]
Activity-dependent Plasticity in Cellular nitric oxide (NO)-cGMP Signaling persist beyond the period of NO application to influence subsequent cellular responsiveness and, if so, for how long, and what are their relative contributions to the plasticity? Central to the feasibility of obtaining answers was our recent development of a method for delivering repeated NO pulses of known amplitude and duration [14], allowing controlled conditioning NO exposures to be followed by test exposures after selected time intervals in NO-free solution
The outcome (Fig. 1C) showed a very slow rate of loss of cGMP that was quantitatively the same as predicted from the measured kinetics of inhibition by sildenafil under steady-state conditions in rat platelets. This result shows that there was no unsuspected PDE activity and attests to the expected very rapid penetration of sildenafil into the platelets. These findings demonstrate that brief periods of NO exposure induce a progressive reduction of subsequent responsiveness and that the tachyphylaxis is caused by a mixture of receptor desensitization and PDE5 activation, with the former dominating under the conditions of this experiment
Summary
Aliquots of platelets were inactivated in boiling buffer, and the amounts of cGMP and protein were measured as before [14]. In each experiment there were 3– 4 independent runs of each condition; results are presented as the means Ϯ S.E. Except where impractical, extracellular/bound cGMP sustained through prior NO exposure was measured by inactivating an aliquot of platelets immediately before re-exposure. PDE5 Phosphorylation—After incubation and treatment, aliquots of platelets (0.1 ml) were inactivated with 25 l of a Laemmli-type buffer, giving a final concentration of 62 mM TrisHCl, pH 6.8, 2% (w/v) SDS, 0.01% (w/v) bromphenol blue, 10% (w/v) glycerol, and 5% (v/v) -mercaptoethanol. Anti-phospho-PDE5 antibody was diluted 1/1000 into blocking buffer for overnight incubation at 4 °C with agitation. The results of the calculations were in molar units (concentrations or rates), which were converted into the experimental units (pmol/mg protein) by assuming 1 mg of protein is equivalent to 2.5 l [23]
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
