Mammalian cyclic nucleotide phosphodiesterases: molecular mechanisms and physiological functions.

The superfamily of cyclic nucleotide (cN) phosphodiesterases (PDEs) is comprised of 11 families of enzymes. PDEs break down cAMP and/or cGMP and are major determinants of cellular cN levels and, consequently, the actions of cN-signaling pathways. PDEs exhibit a range of catalytic efficiencies for breakdown of cAMP and/or cGMP and are regulated by myriad processes including phosphorylation, cN binding to allosteric GAF domains, changes in expression levels, interaction with regulatory or anchoring proteins, and reversible translocation among subcellular compartments. Selective PDE inhibitors are currently in clinical use for treatment of erectile dysfunction, pulmonary hypertension, intermittent claudication, and chronic pulmonary obstructive disease; many new inhibitors are being developed for treatment of these and other maladies. Recently reported x-ray crystallographic structures have defined features that provide for specificity for cAMP or cGMP in PDE catalytic sites or their GAF domains, as well as mechanisms involved in catalysis, oligomerization, autoinhibition, and interactions with inhibitors. In addition, major advances have been made in understanding the physiological impact and the biochemical basis for selective localization and/or recruitment of specific PDE isoenzymes to particular subcellular compartments. The many recent advances in understanding PDE structures, functions, and physiological actions are discussed in this review.

cGMP-dependent protein kinases and cGMP phosphodiesterases in nitric oxide and cGMP action.

To date, studies suggest that biological signaling by nitric oxide (NO) is primarily mediated by cGMP, which is synthesized by NO-activated guanylyl cyclases and broken down by cyclic nucleotide phosphodiesterases (PDEs). Effects of cGMP occur through three main groups of cellular targets: cGMP-dependent protein kinases (PKGs), cGMP-gated cation channels, and PDEs. cGMP binding activates PKG, which phosphorylates serines and threonines on many cellular proteins, frequently resulting in changes in activity or function, subcellular localization, or regulatory features. The proteins that are so modified by PKG commonly regulate calcium homeostasis, calcium sensitivity of cellular proteins, platelet activation and adhesion, smooth muscle contraction, cardiac function, gene expression, feedback of the NO-signaling pathway, and other processes. Current therapies that have successfully targeted the NO-signaling pathway include nitrovasodilators (nitroglycerin), PDE5 inhibitors [sildenafil (Viagra and Revatio), vardenafil (Levitra), and tadalafil (Cialis and Adcirca)] for treatment of a number of vascular diseases including angina pectoris, erectile dysfunction, and pulmonary hypertension; the PDE3 inhibitors [cilostazol (Pletal) and milrinone (Primacor)] are used for treatment of intermittent claudication and acute heart failure, respectively. Potential for use of these medications in the treatment of other maladies continues to emerge.

Inhibition of cyclic nucleotide phosphodiesterases by methylxanthines and related compounds.

Naturally occurring methylxanthines were the first inhibitors of cyclic nucleotide (cN) phosphodiesterases (PDEs) to be discovered. To improve potency and specificity for inhibition of various PDEs in research and for treatment of diseases, thousands of compounds with related structures have now been synthesized. All known PDE inhibitors contain one or more rings that mimic the purine in the cN substrate and directly compete with cN for access to the catalytic site; this review focuses on inhibitors that contain a nucleus that is closely related to the xanthine ring of theophylline and caffeine and the purine ring of cNs. The specificity and potency of these compounds for blocking PDE action have been improved by appending groups at positions on the rings as well as by modification of the number and distribution of nitrogens and carbons in those rings. Several of these inhibitors are highly selective for particular PDEs; potent and largely selective PDE5 inhibitors are used clinically for treatment of erectile dysfunction [sildenafil (Viagra™), tadalafil (Cialis™) and vardenafil (Levitra™)] and pulmonary hypertension [sildenafil (Revatio™) and tadalafil (Adenocirca)]. Related compounds target other PDEs and show therapeutic promise for a number of maladies.

Probing the catalytic sites and activation mechanism of photoreceptor phosphodiesterase using radiolabeled phosphodiesterase inhibitors.

Retinal photoreceptor phosphodiesterase (PDE6) is unique among the phosphodiesterase enzyme family not only for its catalytic heterodimer but also for its regulatory gamma-subunits (Pgamma) whose inhibitory action is released upon binding to the G-protein transducin. It is generally assumed that during visual excitation both catalytic sites are relieved of Pgamma inhibition upon binding of two activated transducin molecules. Because PDE6 shares structural and pharmacological similarities with PDE5, we utilized radiolabeled PDE5 inhibitors to probe the catalytic sites of PDE6. The membrane filtration assay we used to quantify [(3)H]vardenafil binding to PDE6 required histone II-AS to stabilize drug binding to the active site. Under these conditions, [(3)H]vardenafil binds stoichiometrically to both the alpha- and beta-subunits of the activated PDE6 heterodimer. [(3)H]vardenafil fails to bind to either the PDE6 holoenzyme or the PDE6 catalytic dimer reconstituted with Pgamma, consistent with Pgamma blocking access to the drug-binding sites. Following transducin activation of membrane-associated PDE6 holoenzyme, [(3)H]vardenafil binding increases in proportion to the extent of PDE6 activation. Both [(3)H]vardenafil binding and hydrolytic activity of transducin-activated PDE6 fail to exceed 50% of the value for the PDE6 catalytic dimer. However, adding a 1000-fold excess of activated transducin can stimulate the hydrolytic activity of PDE6 to its maximum extent. These results demonstrate that both subunits of the PDE6 heterodimer are able to bind ligands to the enzyme active site. Furthermore, transducin relieves Pgamma inhibition of PDE6 in a biphasic manner, with only one-half of the maximum PDE6 activity efficiently attained during visual excitation.

Interactions between cyclic nucleotide phosphodiesterase 11 catalytic site and substrates or tadalafil and role of a critical Gln-869 hydrogen bond.

Poor understanding of the topography of cyclic nucleotide (CN) phosphodiesterase (PDE) catalytic sites compromises development of potent, selective inhibitors for therapeutic use. In the X-ray crystal structures of the catalytic domains of some PDEs, an invariant glutamine hydrogen bonds with groups at C6 and N1 or N7 on catalytic products or analogous positions of some inhibitors, inferring similar bonds with CNs (Nature 425:98-102, 2003; J Mol Biol 337:355-365, 2004; Mol Cell 15:279-286, 2004). A site-directed mutant (Q869A) lacking this invariant Gln in cGMP-/cAMP-hydrolyzing PDE11 had unaltered catalytic activity and affinity for sildenafil; but cGMP/cAMP or tadalafil affinity was reduced approximately 50- or 140-fold, respectively, and calculated free energy of binding suggested one hydrogen bond for each. A cGMP analog lacking the C6 oxygen had approximately 80-fold weakened affinity, modifications at N(2), N7, or 2'-OH diminished affinity approximately 16-fold, and analogs with groups appended at N1 had only 2- to 6-fold weakened affinity. Analogs with C8 substitutions were ineffective inhibitors, suggesting that cGMP binds in the anti conformation. Calculated decline in free energy of binding was consistent with that for one hydrogen bond only in the analog lacking binding potential at C6. In conclusion, Gln-869 interacts strongly with cGMP/cAMP and tadalafil, but not with sildenafil; interactions with CN analogs suggest a hydrogen bond only between Gln-869 and the C6 substituent. The results define interactions between the PDE11 catalytic site and substrates/inhibitors and advance potential for inhibitor design.

cGMP-hydrolytic activity and its inhibition by sildenafil in normal and failing human and mouse myocardium.

In mouse models of cardiac disease, the type 5 (PDE5)-selective cyclic nucleotide phosphodiesterase inhibitor sildenafil has antihypertrophic and cardioprotective effects attributable to the inhibition of cGMP hydrolysis. To investigate the relevance of these findings to humans, we quantified cGMP-hydrolytic activity and its inhibition by sildenafil in cytosolic and microsomal preparations from the left ventricular myocardium of normal and failing human hearts. The vast majority of cGMP-hydrolytic activity was attributable to PDE1 and PDE3. Sildenafil had no measurable effect on cGMP hydrolysis at 10 nM, at which it is selective for PDE5, but it had a marked effect on cGMP and cAMP hydrolysis at 1 microM, at which it inhibits PDE1. In contrast, in preparations from the left ventricles of normal mice and mice with heart failure resulting from coronary artery ligation, the effects of sildenafil on cGMP hydrolysis were attributable to inhibition of both PDE5 and PDE1; PDE5 comprised approximately 22 and approximately 43% of the cytosolic cGMP-hydrolytic activity in preparations from normal and failing mouse hearts, respectively. These differences in PDE5 activities in human and mouse hearts call into question the extent to which the effects of sildenafil in mouse models are likely to be applicable in humans and raise the possibility of PDE1 as an alternative therapeutic target.

Phosphorylation increases affinity of the phosphodiesterase-5 catalytic site for tadalafil.

Phosphodiesterase-5 (PDE5) is phosphorylated at a single serine residue by cyclic nucleotide-dependent protein kinases. To test for a direct effect of phosphorylation on the PDE5 catalytic site, independent of cGMP binding to the allosteric sites of the enzyme, binding of the catalytic site-specific substrate analog [(3)H]tadalafil to PDE5 was measured. Phosphorylation increased [(3)H]tadalafil binding 3-fold, whereas cGMP caused a 1.6-fold increase. Combination of both treatments caused more than 4-fold increase in [(3)H]tadalafil binding, and effects were additive only at submaximal stimulation. Consistent with the increase in affinity, phosphorylation slowed the [(3)H]tadalafil exchange-dissociation rate from PDE5 more than 6-fold. Finally, phosphorylation increased affinity for hydrolysis of a catalytic site-specific cGMP analog, 2'-O-anthraniloyl-cGMP, by approximately 3-fold. The combined results showed that phosphorylation activates PDE5 catalytic site independently of cGMP binding to the allosteric sites. The results suggested that phosphorylation acts in concert with allosteric cGMP binding to stimulate the PDE5 catalytic site, which should promote negative feedback regulation of the cGMP pathway in intact cells. By increasing the affinity of the catalytic site, phosphorylation should also consequently increase the potency and duration of PDE5 inhibitor action.

Phosphorylation of phosphodiesterase-5 is promoted by a conformational change induced by sildenafil, vardenafil, or tadalafil.

Phosphodiesterase-5 (PDE5) inhibitors (sildenafil, vardenafil, or tadalafil) or phosphorylation by cyclic nucleotide-dependent protein kinase causes an apparent conformational change in PDE5, as indicated by a shift in migration on non-denaturing PAGE gels and an altered pattern of tryptic digestion. Combination of cGMP and a PDE5 inhibitor or phosphorylation does not cause a further gel shift or change in tryptic digest. Phosphorylation of PDE5 is stimulated by inhibitors, and combination of cGMP and inhibitor does not cause further phosphorylation. Dephosphorylation of PDE5 by either purified phosphoprotein phosphatase-1 or -2A catalytic subunit or by a crude phosphatase mixture is not affected by cGMP or inhibitors, suggesting that phosphorylation itself maintains conformational exposure of the phosphorylation site. The combined results imply that cGMP binding to the catalytic site initiates negative feedback control of many cellular cGMP signaling pathways by directly stimulating phosphorylation and activation of PDE5; by exploiting this molecular mechanism, PDE5 inhibitors stimulate their own potencies.

Interrupted Glucagon Signaling Reveals Hepatic α Cell Axis and Role for L-Glutamine in α Cell Proliferation.

Decreasing glucagon action lowers the blood glucose and may be useful therapeutically for diabetes. However, interrupted glucagon signaling leads to α cell proliferation. To identify postulated hepatic-derived circulating factor(s) responsible for α cell proliferation, we used transcriptomics/proteomics/metabolomics in three models of interrupted glucagon signaling and found that proliferation of mouse, zebrafish, and human α cells was mTOR and FoxP transcription factor dependent. Changes in hepatic amino acid (AA) catabolism gene expression predicted the observed increase in circulating AAs. Mimicking these AA levels stimulated α cell proliferation in a newly developed in vitro assay with L-glutamine being a critical AA. α cell expression of the AA transporter Slc38a5 was markedly increased in mice with interrupted glucagon signaling and played a role in α cell proliferation. These results indicate a hepatic α islet cell axis where glucagon regulates serum AA availability and AAs, especially L-glutamine, regulate α cell proliferation and mass via mTOR-dependent nutrient sensing.

Molecular properties of mammalian proteins that interact with cGMP: protein kinases, cation channels, phosphodiesterases, and multi-drug anion transporters.

Cyclic GMP is a critical second messenger signaling molecule in many mammalian cell types. It is synthesized by a family of guanylyl cyclases that is activated in response to stimuli from hormones such as natriuretic peptides, members of the guanylin family, and chemical stimuli including nitric oxide and carbon monoxide. The resulting elevation of cGMP modulates myriad physiological processes. Three major groups of cellular proteins bind cGMP specifically at allosteric sites; interaction of cGMP with these sites modulates the activities and functions of other domains within these protein groups to bring about physiological effects. These proteins include the cyclic nucleotide (cN)-dependent protein kinases, cN-gated cation channels, and cGMP-binding phosphodiesterases (PDE). Cyclic GMP also interacts with the catalytic sites of many cN PDEs and with some members of the multi-drug anion transporter family (MRPs) which can extrude nucleotides from cells. The allosteric cN-binding sites in the kinases and the cN-gated channels are evolutionarily and biochemically related, whereas the allosteric cGMP-binding sites in PDEs (also known as GAF domains), the catalytic sites of PDEs , and the ligand-binding sites in the MRPs are evolutionarily and biochemically distinct from each other and from those in the kinase and channel families. The sites that interact with cGMP within each of these groups of proteins have unique properties that provide for cGMP binding. Within a given cell, cGMP can potentially interact with members of all these groups of proteins if they are present. The relative abundance and affinities of these various cGMP-binding sites in conjunction with their subcellular compartmentation, proximity to cyclases and PDEs, and post-translational modification contribute importantly in determining the impact of these respective proteins to cGMP signaling within a particular cell.

Phosphodiesterase-5 inhibition: the molecular biology of erectile function and dysfunction.

This article discusses the role of phosphodiesterase-5 (PDE-5) inhibition in the molecular biology of erectile function and dysfunction. Commercially marketed PDE-5 inhibitors are highly specific for PDE-5, and in the face of continuing cyclic GMP (cGMP) synthesis,elevate cellular cGMP. This elevation results from direct competitive inhibition of PDE-5 and from blocking the negative feedback regulation of the enzyme. Elevation of cGMP activates cGMP-dependent protein kinase, which mediates the effects of the cGMP-signaling pathway to decrease smooth muscle tone and dilate penile vascular smooth muscle. By exploiting features of PDE-5 regulatory mechanisms that modulate PDE-5 function, the inhibitors enhance their own potencies.

Radiolabeled ligand binding to the catalytic or allosteric sites of PDE5 and PDE11.

Cyclic nucleotide phosphodiesterases (PDEs) have been investigated for years as targets for therapeutic intervention in a number of pathophysiological processes. Phosphodiesterase-5 (PDE5), which is highly specific for guanosine 3'-5'-cyclic-monophosphate (cGMP) at both its catalytic site and its allosteric sites, has generated particular interest because it is potently and specifically inhibited by three drugs: sildenafil (Viagra, Pfizer), tadalafil (Cialis, Lilly-ICOS), and vardenafil (Levitra, Bayer GSK). Previously, we have used [(3)H]cGMP to directly study the interaction of cGMP with the allosteric sites of PDE5, but because cGMP binds with relatively low affinity to the catalytic site, it has been difficult to devise a binding assay for this particular binding reaction. This approach using measurement of radiolabeled ligand binding continues to allow us to more precisely define functional features of the enzyme. We now use a similar approach to study the characteristics of high-affinity [(3)H]inhibitor binding to the PDE5 catalytic domain. For these studies, we have prepared [(3)H]sildenafil and [(3)H]tadalafil, two structurally different competitive inhibitors of PDE5. The results demonstrate that radiolabeled ligands can be used as probes for both catalytic site and allosteric site functions of PDE5. We describe herein the methods that we have established for studying the binding of radiolabeled ligands to both types of sites on PDE5. These techniques have also been successfully applied to the study of binding of radiolabeled PDE5 inhibitors to PDE11, suggesting that these methods are applicable to the study of other PDEs, and perhaps other enzyme families.

Sildenafil: efficacy, safety, tolerability and mechanism of action in treating erectile dysfunction.

Sildenafil citrate is marketed under the trademark name Viagra and is widely used to treat male erectile dysfunction; therapeutic uses of this medication for other diseases related to vascular dysfunction are emerging. When used as recommended, the drug has a strong clinical efficacy and safety profile in a broad spectrum of the male population. Its widespread use and effects of long-term exposure to the drug due to particular treatment regimens or inappropriate use mandate an ongoing update of its molecular mechanism, pharmacological profile and associated safety issues. This review focuses on biochemical and pharmacological features of sildenafil, the active component in Viagra, interaction of sildenafil with phosphodiesterase 5, pharmacokinetic parameters, action in smooth muscle, side effects, safety profile and prospects for other uses.

Allosteric sites of phosphodiesterase-5 sequester cyclic GMP.

Phosphodiesterase-5 (PDE5) and cGMP-dependent protein kinase (PKG) play key roles in cGMP signaling. PDE5 has a catalytic domain (C domain) that hydrolyzes cGMP and a regulatory domain (R domain) that binds cGMP at allosteric sites. We recently demonstrated that in corpus cavernosum, PDE5 concentration exceeds basal cGMP by ~5-fold making it possible that its allosteric sites could bind a significant fraction of the total cellular cGMP. It is hypothesized that the allosteric sites regulate cGMP signaling by sequestering cGMP. At 60 nM cGMP in vitro, which approaches a stimulated concentration of cGMP in rabbit corpus cavernosum, isolated R domain inhibits both cGMP hydrolysis by C domain and activation of PKG (IC50 values of 388 and 100 nM, respectively). Prior phosphorylation of R domain by cyclic nucleotide-dependent protein kinases, which increases its cGMP-binding affinity, also increases its potency for inhibiting both cGMP hydrolysis by C domain and cGMP activation of PKG (IC50 values of 58 and 38 nM, respectively). In rabbit corpus cavernosum, PDE5 concentration (94 nM) exceeds these values. These findings support our hypothesis that physiological concentrations of R domain regulate cGMP signaling by sequestering this nucleotide and that phosphorylation of R domain modulates this effect. This could provide for negative feedback control of cGMP-signaling.

Mechanisms of autoinhibition in cyclic nucleotide-dependent protein kinases.

Cyclic AMP-dependent protein kinase (PKA) and cGMP-dependent protein kinase (PKG) are autoinhibited through multiple interactions between their respective regulatory and catalytic domains. A large portion of this autoinhibition occurs through interactions between residues within the catalytic domain and those within either a substrate-like sequence (-RRXSX-) or pseudosubstrate sequence (-RRXAX-) in the regulatory domains. These contacts effectively inhibit catalysis by blocking substrate binding. Particularly important contacts involve the P-2, P-3, and P+1 residues where either serine, which is potentially autophosphorylated, or alanine occupies the P0 position. The primary sequence is apparently less important for autoinhibition in PKGs than in PKAs, and a conserved serine at P+2 in PKGs is important for autoinhibitory contacts. Elements outside the substrate-related sequences also contribute to autoinhibition in both PKA and PKG. For example, synthetic peptides with relatively short pseudosubstrate sequences are weak inhibitors; while heat-denatured RII subunit does not inhibit catalytic subunit, it is still rapidly autophosphorylated; and truncated PKGs lacking the substrate-like sequence are still partially autoinhibited. Thus, capacity for autoinhibition of PKA or PKG is provided by contacts involving direct interactions with the catalytic site and by contacts that stabilize an inactive conformation.

Vardenafil: structural basis for higher potency over sildenafil in inhibiting cGMP-specific phosphodiesterase-5 (PDE5).

Phosphodiesterase-5 (PDE5) inhibitors act by competing with the substrate, cGMP, for the catalytic site of the enzyme. Two commercialized PDE5 inhibitors, sildenafil and vardenafil, are being used to treat erectile dysfunction. These two compounds differ in the heterocyclic ring system used to mimic the purine ring of cGMP. They also differ in the substituent (ethyl/methyl) of a piperazine side chain. Although these are the only two structural differences, vardenafil has more than 20-fold greater potency than sildenafil for inhibiting purified PDE5. The molecular structural basis for the difference in potency of the two compounds was investigated by synthesizing an analog of sildenafil ("methyl-sildenafil") that contained the sildenafil ring system but with the appended ethyl group found in vardenafil, and an analog of vardenafil ("demethyl-vardenafil") that contained the vardenafil ring system but with the appended methyl group found in sildenafil. The IC50 of methyl-sildenafil for inhibiting PDE5 indicated that it was 64 times less potent than demethyl-vardenafil, which was similar to the finding that, based on IC50, sildenafil was 40 times less potent than vardenafil. Similarly, the EC50 of methyl-sildenafil for inhibiting [3H]vardenafil binding to PDE5 indicated that it was 84 times less potent than demethyl-vardenafil, while the EC50 for sildenafil indicated that it was 31 times less potent than vardenafil. It is concluded that the methyl/ethyl appended group on the piperazine moiety plays very little role in the difference in potency between sildenafil and vardenafil for inhibiting PDE5, whereas the differences in the ring systems play a critical role in higher potency of vardenafil over sildenafil.

Stimulation of serotonin transport by the cyclic GMP phosphodiesterase-5 inhibitor sildenafil.

The serotonin (5-hydroxtryptamine, 5-HT) transporter (SERT) plays a critical role in the inactivation of synaptic 5-HT and has been implicated in multiple psychiatric and peripheral disorders. SERT regulation studies demonstrate that activation of cyclic guanosine monophosphate (cGMP)/protein kinase G (PKG)-linked pathways can increase SERT activity. As cGMP actions are limited by cGMP-specific phosphodiesterase (PDEs), we investigated whether the cGMP-specific PDE5 inhibitor sildenafil (Viagra) can stimulate 5-HT uptake and potentiate cGMP-mediated regulation. In RBL-2H3 cells, SERT activity was stimulated by sildenafil in a concentration- and time-dependent manner. Sildenafil also enhanced the stimulation of SERT triggered by the adenosine receptor agonist 5'-N-ethylcarboxamidoadenosine (NECA), effects blocked by the PKG inhibitor N-[2-(methylamino)ethy]-5-isoquinoline-sulfonamide (H8). Sildenafil stimulation of 5-HT uptake arises from an increase in 5-HT transport Vmax and is paralleled by elevated SERT surface antagonist binding, also H8-sensitive. These findings implicate cGMP-targeted PDEs in limiting the regulation of antidepressant-sensitive 5-HT transport.

Catalytic site amino acids of PKGI-alpha influence allosteric cGMP binding.

Ser-64, an autophosphorylation site in the autoinhibitory subdomain of cGMP-dependent protein kinase type I-alpha (PKGI-alpha), lowers affinity for cGMP and suppresses catalytic activity (1). Using the structure of homologous cAMP-dependent protein kinase as a model, three conserved residues (Gln-401, His-404, Cys-518) in the PKGI-alpha catalytic site are predicted to be juxtaposed to Ser-64 (2). Individual point mutants (Q401A, H404A and C518A) and a double mutant (S64A/H404A) have been generated. cGMP or cAMP affinities (K(a)) of each mutant protein for phosphotransferase activation and allosteric (3H)cGMP-binding affinity (K(D)) of each mutant protein are significantly improved over those of wild-type (WT) PKGI-alpha. However, affinities (K(m)) of the mutant PKGs for peptide substrates or ATP are unaltered. Kinase activity ratio (-GMP/+cGMP) of H404A is greater than that for WT, Q401A, or C518A, and similar to that for S64A and S64A/H404A. These results reveal a unique mechanism whereby catalytic domain residues predicted to be spatially close to Ser-64 of the regulatory domain weaken the intrinsically high affinity of PKGI-alpha for cGMP and provide for autoinhibition of catalytic activity.

Calcineurin regulates homologous desensitization of natriuretic peptide receptor-A and inhibits ANP-induced testosterone production in MA-10 cells.

Receptor desensitization is a ubiquitous regulatory mechanism that defines the activatable pool of receptors, and thus, the ability of cells to respond to environmental stimuli. In recent years, the molecular mechanisms controlling the desensitization of a variety of receptors have been established. However, little is known about the molecular mechanisms that underlie desensitization of natriuretic peptide receptors, including natriuretic peptide receptor-A (NPR-A). Here we report that calcineurin (protein phosphatase 2B, PP2B, PPP3C) regulates homologous desensitization of NPR-A in murine Leydig tumor (MA-10) cells. We demonstrate that both pharmacological inhibition of calcineurin activity and siRNA-mediated suppression of calcineurin expression potentiate atrial natriuretic peptide (ANP)-induced cGMP synthesis. Treatment of MA-10 cells with inhibitors of other phosphoprotein phosphatases had little or no effect on ANP-induced cGMP accumulation. In addition, overexpression of calcineurin blunts ANP-induced cGMP synthesis. We also present data indicating that the inhibition of calcineurin potentiates ANP-induced testosterone production. To better understand the contribution of calcineurin in the regulation of NPR-A activity, we examined the kinetics of ANP-induced cGMP signals. We observed transient ANP-induced cGMP signals, even in the presence of phosphodiesterase inhibitors. Inhibition of both calcineurin and phosphodiesterase dramatically slowed the decay in the response. These observations are consistent with a model in which calcineurin mediated dephosphorylation and desensitization of NPR-A is associated with significant inhibition of cGMP synthesis. PDE activity hydrolyzes cGMP, thus lowering intracellular cGMP toward the basal level. Taken together, these data suggest that calcineurin plays a previously unrecognized role in the desensitization of NPR-A and, thereby, inhibits ANP-mediated increases in testosterone production.