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 alpha(1) and beta(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 microm/s. With low phosphodiesterase activity (imposed by 100 microm 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 microm cGMP. The transients were caused partly by GC desensitization, which varied in rate (half-time 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).