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,...

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