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To date, relative cellular levels of cGMP and cGMP-binding proteins have not been considered important in the regulation of smooth muscle or any other tissue. In rabbit penile corpus cavernosum, intracellular cGMP was determined to be 18 ± 4 nm, whereas the cGMP-binding sites of types Iα and Iβ cGMP-dependent protein kinase (PKG) and cGMP-binding cGMP-specific phosphodiesterase (PDE5) were 58 ± 14 nm and 188 ± 6 nm, respectively, as estimated by two different methods for each protein. Thus, total cGMP-binding sites (246 nm) greatly exceed total cGMP. Given this excess of cGMP-binding sites and the high affinities of PKG and PDE5 for cGMP, it is likely that a large portion of intracellular cGMP is associated with these proteins, which could provide a dynamic reservoir for cGMP. Phosphorylation of PDE5 by PKG is known to increase the affinity of PDE5 allosteric sites for cGMP, suggesting the potential for regulation of a reservoir of cGMP bound to this protein. Enhanced binding of cGMP by phosphorylated PDE5 could reduce the amount of cGMP available for activation of PKG, contributing to feedback inhibition of smooth muscle relaxation or other processes. This introduces a new concept for cyclic nucleotide signaling.
Cyclic GMP plays a key role in the regulation of the contractile state of smooth muscle tissues such as that in the corpus cavernosum of the penis [1–4]. Release of nitric oxide from nerves causes elevation of cGMP in this tissue, which produces relaxation of smooth muscle and accumulation of blood, resulting in penile erection. Inadequate blood flow or nerve function can compromise this process, resulting in male erectile dysfunction. New drugs such as sildenafil (Viagra™) maintain higher levels of cGMP by inhibiting phosphodiesterase-5 (PDE5), an enzyme that degrades cGMP . In addition to its catalytic site that is specific for degradation of cGMP, PDE5 contains allosteric (noncatalytic) binding sites that are highly specific for cGMP .
Practically all mammalian signaling pathways contain mechanisms for negative feedback control. This counterbalances the initial signal by dampening the response to this signal and facilitating termination of the signal. Most cAMPsignaling pathways are regulated in this manner. When extracellular signals increase cAMP levels, several negative feedback mechanisms are activated. Some of these involve PDEs. First, cAMP degradation by PDEs is stimulated by mass action, i.e. by increased substrate availability . Second, cellular PDE3 and PDE4 activities are increased acutely by cAMP activation of cAMP-dependent protein kinase (PKA), which phosphorylates these enzymes [7–10]. Third, PDE4 activity is increased by chronic elevation of cAMP, resulting in increased PDE4 gene transcription [11,12].
It is likely that cGMP-signaling pathways as well as cAMP-signaling pathways are regulated by negative feedback. The subject of the present paper is the possibility that overall availability of cellular cGMP could be reduced by being sequestered in the allosteric cGMP-binding sites of PDE5 and it would therefore be unavailable to target proteins such as cGMP-dependent protein kinase (PKG). Intracellular receptors for cGMP in body tissues include PKG, cGMP-binding PDEs, and cGMP-gated channels , although the latter may be present at relatively low levels. PKA may act under some conditions as a cGMP receptor, a process known as cross-activation . PKG and PDE5 are known to be present in penile corpus cavernosum. Each contains allosteric cGMP-binding sites. If smooth muscle cells of corpus cavernosum are rich in PDE5, allosteric sites of this enzyme could compete with those of PKG, thereby effectively sequestering a large fraction of cellular cGMP, which would reduce the extent of PKG activation. We have shown that under basal conditions the concentration of PKG in pig coronary arteries exceeds the concentration of cGMP , but levels of PDE2 and PDE5 were not considered in that study. Our recent evidence indicates that phosphorylation of PDE5 substantially increases the cGMP-binding affinity of its allosteric sites . After phosphorylation, affinity of PDE5 for cGMP is approximately that of PKG. Increased sequestration of cGMP by PDE5 when PKG becomes activated by this nucleotide in cells would provide negative feedback control of cGMP-stimulated processes. This effect would act in concert with the increase in PDE5 catalytic activity caused by phosphorylation of this enzyme  to produce dampening of the signaling pathway. In addition, unknown modulators may further modulate dissociation of cGMP from PDE5, thereby controlling its availability to activate PKG or other cyclic nucleotide receptors.
It has been determined that a major portion of photoreceptor cGMP is tightly associated with the allosteric sites of PDE6, an enzyme present at high concentrations in retinal rod outer segment . Thus, the allosteric cGMP-binding sites of PDE6 comprise a major reservoir for cGMP in that tissue by providing a sequestration site for cGMP which effectively lowers the concentration of free cGMP in the photoreceptor. If PDE5 or any other protein in corpus cavernosum functions effectively as a sequestration site for cGMP, it would be expected that the molar amount of this protein would approach that of cGMP. The present study establishes the quantitative cGMP-binding potential of PKG and PDE5 in corpus cavernosum. Results suggest that PDE5 could bind a significant portion of cGMP in this tissue. While bound, this portion of the cellular cGMP pool would be a relatively inactive fraction, or sequestered form, of total cellular cGMP. Such a reservoir of bound cGMP may be modulated, particularly after cGMP elevation and enzyme phosphorylation, and could represent an important cellular regulatory mechanism not previously recognized. Consideration of relative concentrations of cyclic nucleotides and their intracellular receptors and the impact of these relationships on signaling pathways is a novel concept that has not been addressed experimentally. This report represents an initial step in that direction.
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The present finding of significant levels of cGMP, PKG, and PDE5 in rabbit corpus cavernosum are consistent with the important role of cGMP signaling in smooth muscle tissues. Levels of cAMP and PKA also suggest important roles for these factors, although cAMP-PDE activity is low compared with cGMP-PDE activity. Whether or not cAMP could ‘cross-activate’ PKG in this tissue should be explored, but this nucleotide binds to PDE5 allosteric sites very weakly [28,29]
Results herein demonstrate that cGMP-binding sites of PKG are 3.2 times higher than cGMP molecules in unstimulated rabbit corpus cavernosum, which is close to the value of five times higher that we measured earlier in pig coronary artery smooth muscle , but considerably lower than the value of 37 times for these factors in platelets . Given the high affinity of cGMP for PKG, and the high level of PKG in smooth muscle cells, the intracellular relationship between cGMP and PKG would be expected to be nearly stoichiometric. Thus, PKG could be one-fifth to one-third saturated with cGMP in the unstimulated state. This does not provide a large window for activation of PKG for smooth muscle relaxation or penile erection as threefold to fivefold elevation in cGMP should produce maximum effect. This is in good agreement with our finding that the concentration of sodium nitroprusside which produces maximum relaxation of pig coronary artery smooth muscle raises cGMP by only threefold . A narrow window for cGMP regulation of penile erection or other processes should necessitate tight cellular control of cGMP levels. Over-stimulation of cGMP levels would be deleterious to many physiological processes. A narrow window for cAMP/PKA regulation of heart rate and force has already been demonstrated .
This report is the first to address the stoichiometric relationships among cGMP, PKG, and PDE5 in any tissue. These three factors play fundamental roles in the regulation of smooth muscle relaxation in corpus cavernosum and other tissues. The impetus for the present study was our recent finding that cGMP binding to PDE5 allosteric sites is regulated by phosphorylation . This binding activity may have more than a single physiological role, but one suggested here is that it could sequester cGMP away from its targets, such as PKG, for stimulation of smooth muscle relaxation in corpus cavernosum and other tissues. This sequestration would increase after phosphorylation. In order for sequestration of cGMP by PDE5 to be a significant mode of cell regulation, the stoichiometric amount of this enzyme should be relatively close to that of cGMP. Results herein demonstrate that molar ratio of PDE5 allosteric cGMP-binding sites to cGMP is about ten, which is clearly sufficient for PDE5 to bind a significant portion of cGMP, even after elevation of this nucleotide by nitric oxide or other agents. A similar level of PDE5 was obtained when quantified by either catalytic or cGMP-binding activity, verifying this stoichiometry.
As the specific enzyme activity of PDE5 is 5 µmol·min−1·mg−1, the kcat would be 8 mol·mol−1·s−1. According to calculations above, catalytic sites (half the number of binding sites) would exceed cGMP by a molar ratio of 5 : 1. Thus, assuming equal intracellular distributions of cGMP and PDE5, cellular cGMP would turn over every 1/40 s [(1/8 s·mol cGMP−1)/(1/5 mol cGMP·mol PDE5 catalytic site−1)] if cGMP were saturating for PDE5 catalysis. However, cGMP is only 18 nm in corpus cavernosum smooth muscle cells. As the Km of PDE5 for cGMP is 5.6 µm, the rate of cGMP hydrolysis would be about 0.018 µm/(5.6 µm × 2) = 0.0016 of the maximum rate. It could therefore be estimated that the time required for turnover of total cellular cGMP by PDE5 catalytic activity would be 1/40 s/0.0016 = 16 s. This is probably a minimum value as it assumes that binding of cGMP to PDE5 and PKG would not influence turnover rate. The value for basal cellular cGMP reported here (18 nm) is somewhat less than that reported by Bush et al. (50 nm) for rabbit corpus cavernosum .
It should be emphasized again that most cGMP molecules under intracellular conditions would be bound to PDE5 or PKG given the high affinities of these two proteins for cGMP. Using the KD value of 30 nm that we determined recently for phospho-PDE5 , and the intracellular values of 188 nm PDE5 and 18 nm for cGMP (Table 3), we used the simple equation for ligand (L) binding to receptor (R) Eqn (1) to calculate KD Eqn (2):
Inserting values for each entity, a concentration of 3.1 nm free cGMP (L) is calculated. As total intracellular cGMP is 18 nm, the PDE5-bound concentration is 83% of the total. This assumes that PKG does not bind cGMP at all. Because of the relatively low molar amount of PDE2 in corpus cavernosum it is doubtful that there is sufficient cGMP-binding capacity of this enzyme to play a significant role in sequestration of cGMP. This is probably also the case for cyclic nucleotide-gated channels [32,33], which are believed to be present at low molar levels in tissues. No peak of cGMP-binding activity other than those associated with PDE5 and PKG was obtained using the assay adopted for the present investigation. The model in Fig. 8 depicts our proposal for cellular negative feedback regulation of cGMP based on stoichiometric relationships of cGMP, PKG, and PDE5 determined in this study, as well as effects of phosphorylation of PDE5. The increased binding affinity of PDE5 for cGMP following phosphorylation of this enzyme would favor sequestration of cGMP in these sites, resulting in decreased activation of PKG. Increases in cellular cGMP level stimulate phosphorylation of PDE5 both by activation of PKG and by a substrate-directed effect, i.e. by cGMP binding to the allosteric sites of PDE5. Therefore, cGMP elevation would cause increased sequestration, resulting in dampening of the cGMP signal and facilitating termination of this signal. This process is apparently part of a concert of negative feedback processes for cGMP that have evolved for tight regulation of penile erection and other physiological events. These have all been described in this laboratory and include: (a) increased PDE5 catalytic activity due to mass action of elevated cGMP; (b) increased cGMP binding to PDE5 allosteric sites due to mass action of elevated cGMP; (c) increased PDE5 catalytic activity due to phosphorylation and activation of PDE5 by activated PKG , and (d) increased cGMP binding to PDE5 allosteric sites due to this phosphorylation. A fifth possible process is direct stimulation of the PDE5 catalytic site by allosteric cGMP binding to the enzyme, which would be predicted by the principle of reciprocity as discussed earlier . The presence of such an array of mechanisms for negative feedback of the cGMP pathway suggests that cells cannot readily tolerate excessive activation of PKG or other target proteins.
Figure 8. Model for physiological negative feedback regulation of cGMP by increased catalytic and cGMP-binding activities of PDE5. (A) unstimulated cells (low cGMP). (B) stimulated cells with elevated cGMP. Stoichiometric relationships are estimated from measurements in rabbit corpus cavernosum in this report. Elevated cGMP may be exaggerated in order to illustrate cGMP-binding proteins in different states of bound cGMP.
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Negative feedback control of cyclic nucleotide-stimulated pathways is not unique to cGMP pathways. We described a negative feedback pathway for cAMP  that involves stimulation of PDE3 by phosphorylation catalyzed by activated PKA . PDE4 is also activated by a similar mechanism in some tissues , and this enzyme is induced by chronic elevation of cAMP .
The fact that sildenafil, when taken as Viagra™ tablets, is rather selective for penile erection and does not have strong effects on other tissues, may have several possible explanations [4,36]. One that is particularly viable is that sexual arousal causes relatively specific nerve stimulation directed to the penis. Elevation of cGMP due to guanylyl cyclase activation by selective nitric oxide release in this organ would be potentiated by sildenafil. Another possibility presented here is that PDE5 is by far the predominant PDE that hydrolyzes nearly physiological concentrations of cGMP in crude extract of rabbit corpus cavernosum. Also, PDE5 is relatively abundant in this tissue, being at a level (94 nm PDE5 catalytic sites in rabbit) that is nearly stoichiometric with the free blood level of sildenafil, which is believed to be ≈ 50 nm in humans after a 100-mg tablet [37,38]. Under this condition, the high affinity of sildenafil for PDE5 would dictate that a high proportion of total cellular PDE5 would contain bound sildenafil. This would cause substantial inhibition of this enzyme and result in significant cGMP elevation and enhanced penile erection.