In vivo reconstitution of the negative feedback in nitric oxide/cGMP signaling: role of phosphodiesterase type 5 phosphorylation

Abstract

Most effects of the messenger molecule nitric oxide (NO) are mediated by cGMP, which is formed by NO-sensitive guanylyl cyclase (GC) and degraded by phosphodiesterases (PDEs). In platelets, NO elicits a spike-like cGMP response and causes a sustained desensitization. Both characteristics have been attributed to PDE5 activation caused by cGMP binding to its regulatory GAF domain. Activation is paralleled by phosphorylation whose precise function remains unknown. Here, we report reconstitution of all features of the NO-induced cGMP response in human embryonic kidney cells by coexpressing NO-sensitive GC and PDE5. The spike-like cGMP response was blunted when PDE5 phosphorylation was enhanced by additional overexpression of cGMP-dependent protein kinase. Analysis of PDE5 activation in vitro revealed a discrepancy between the cGMP concentrations required for activation (micromolar) and reversal of activation (nanomolar), indicating the conversion of a low-affinity state to a high-affinity state upon binding of cGMP. Phosphorylation even increased the high apparent affinity enabling PDE5 activation to persist at extremely low cGMP concentrations. Our data suggest that the spike-like shape and the desensitization of the cGMP response are potentially inherent to every GC- and PDE5-expressing cell. Phosphorylation of PDE5 seems to act as memory switch for activation leading to long-term desensitization of the signaling pathway.

Figure 1.

Western blot detection of stably expressed proteins. The α₁ and β₁ subunits of NO-sensitive GC and human PDE5 were detected in HEK 293 cells after stable transfection of the respective cDNAs. Untransfected control cells (HEK) are shown in the first lane; lanes 2-6 contain the cell lines used in this study. The β₁ subunit of HEK-GC_low cells is below the detection limit but becomes visible upon longer exposure.

Figure 2.

NO-induced cGMP accumulation in HEK cells stably expressing NO-sensitive GC. (A) cGMP response in untransfected control cells (HEK) and in cells with high (HEK-GC) and low (HEK-GC_low) expression level of NO-sensitive GC after stimulation with GSNO (100 μM) in the absence or presence of YC-1 (100 μM). (B) Relative GTP content of HEK-GC cells 5 min after the incubation with or without GSNO (left). GTP was quantified by determination of area under the curve after chromatographic separation of nucleotides using a Mono-Q column. Right, original trace data of a representative elution profile.

Figure 3.

NO-induced cGMP response in HEK cells stably expressing NO-sensitive GC and PDE5. (A) HEK-GC cells with low (HEK-GC/PDE5) or high (HEK-GC/PDE5_high) expression level of PDE5 were stimulated with GSNO (100 μM), and cGMP levels were measured at the indicated time points. (B) For comparison, the NO-induced cGMP responses in human platelets (open squares) and rat aortic strips (open triangles) are shown in the same graph as the response in HEK-GC/PDE5; note that these data are expressed as percentage of maximum cGMP. (C) HEK-GC/PDE5 and HEK-GC/PDE5_high cells were incubated with sildenafil (100 μM) for 10 min and then stimulated with GSNO (100 μM); cGMP levels were determined at the indicated time points.

Figure 4.

NO-induced desensitization of the cGMP response. (A) HEK-GC/PDE5 cells were incubated with either 0 μM (control) or 3 μM GSNO for 5 min, washed with buffer to remove NO, and further kept at 37°C. After 60 min, cells were restimulated with GSNO (100 μM), and cGMP levels were measured at the indicated time points; the resulting cGMP response was reduced compared with control. In each experiment, data were normalized to the maximal cGMP level measured under control conditions. (B) HEK-GC/PDE5 cells were incubated with or without 3 μM GSNO for 5 min, washed with buffer to remove NO, and further kept at 37°C for 60 min. PDE5 activity was determined in supernatant fractions before incubation (control), directly after incubation with 3 μM GSNO (5 min), and 60 min after removal of NO (60 min).

Figure 6.

NO-induced cGMP response in HEK-GC/PDE5 cells overexpressing PKG. (A) cGMP was determined after stimulation of the cells with GSNO (100 μM) at the indicated time points. In each experiment, data were normalized to the maximal cGMP value measured in the mock-transfected cells. The cGMP response was clearly blunted in cells expressing PKG. (B) Western blot detection of PKG and phosphorylated PDE5 after NO stimulation (100 μM GSNO) of mock-transfected (left) and PKG-transfected HEK-GC/PDE5 cells (right). Expression of PDE5, α₁ and β₁ subunits of NO-sensitive GC also is depicted; GAPDH is shown as control for equal loading of the lanes.

Figure 5.

(A) NO-induced cGMP response in HEK-GC cells stably expressing the phosphorylation site mutant PDE5(S102A). HEK-GC cells stably expressing PDE5 mutated at the phosphorylation site [HEK-GC/PDE5(S102A)] were stimulated with GSNO (100 μM), and cGMP was determined at the indicated time points. (B) NO/cGMP-mediated activation of wild-type (WT) or mutant PDE5. Intact HEK-GC cells expressing WT (filled columns) or mutant PDE5 (S102A) (open columns) were stimulated with GSNO (100 μM; 60 s) and lysed. PDE5 activity was determined in the supernatant fractions.

Figure 7.

Effect of phosphorylation on activation and deactivation of PDE5. (A) Western blot detection of phosphorylated PDE5 (top) after incubation of cytosols with increasing cGMP concentrations in the absence of presence of PKG/ATP; bottom shows loading of PDE5. (B) Activation of PDE5 by preincubation of cytosols with increasing cGMP concentrations. (C) Deactivation of PDE5. PDE5 was activated by preincubation with 30 μM cGMP in the presence or absence of PKG/ATP to obtain phospho- or a nonphospho-PDE5. Then samples were diluted to yield a cGMP concentration of 10 nM (solid lines) and further incubated. At the indicated time points, PDE5 activity was determined. The dashed line represents deactivation of nonphospho-PDE5 at a cGMP concentration of 50 nM.