In the molecular scheme of living organisms, adenosine 3',5'-monophosphate (cyclic AMP or cAMP) has been a universal second messenger. In eukaryotic cells, the primary receptors for cAMP are the regulatory subunits of cAMP-dependent protein kinase. The crystal structure of a 1-91 deletion mutant of the type I alpha regulatory subunit was refined to 2.8 A resolution. Each of the two tandem cAMP binding domains provides an extensive network of hydrogen bonds that buries the cyclic phosphate and the ribose between two beta strands that are linked by a short alpha helix. Each adenine base stacks against an aromatic ring that lies outside the beta barrel. This structure provides a molecular basis for understanding how cAMP binds cooperatively to its receptor protein, thus mediating activation of the kinase.

Cyclic guanosine monophosphate (cGMP) is a second messenger molecule that transduces nitric oxide (NO) and natriuretic peptide (NP) coupled signaling, stimulating phosphorylation changes by protein kinase G (PKG). Enhancing cGMP synthesis or blocking its degradation by phosphodiesterase type 5A (PDE5A) protects against cardiovascular disease1,2. However, cGMP stimulation alone is limited by counter-adaptions including PDE upregulation3. Furthermore, though PDE5A regulates NO-generated cGMP4,5, NO-signaling is often depressed by heart disease6. PDEs controlling NP-coupled cGMP remain uncertain. Here we show that cGMP-selective PDE9A7,8 is expressed in mammalian heart including humans, and is upregulated by hypertrophy and cardiac failure. PDE9A regulates NP rather than NO-stimulated cGMP in heart myocytes and muscle, and its genetic or selective pharmacological inhibition protects against pathological responses to neuro-hormones, and sustained pressure-overload stress. PDE9A inhibition reverses pre-established heart disease independent of NO-synthase (NOS) activity, whereas PDE5A inhibition requires active NOS. Transcription factor activation and phospho-proteome analyses of myocytes with each PDE selectively inhibited reveals substantial differential targeting, with phosphorylation changes from PDE5A inhibition being more sensitive to NOS activation. Thus, unlike PDE5A, PDE9A can regulate cGMP signaling independent of the NO-pathway, and its role in stress-induced heart disease suggests potential as a therapeutic target.

Abstract-␤-Adrenergic agonists stimulate cardiac contractility and simultaneously blunt this response by coactivating NO synthase (NOS3) to enhance cGMP synthesis and activate protein kinase G (PKG-1). cGMP is also catabolically regulated by phosphodiesterase 5A (PDE5A). PDE5A inhibition by sildenafil (Viagra) increases cGMP and is used widely to treat erectile dysfunction; however, its role in the heart and its interaction with ␤-adrenergic and NOS3/cGMP stimulation is largely unknown. In nontransgenic (control) murine in vivo hearts and isolated myocytes, PDE5A inhibition (sildenafil) minimally altered rest function. However, when the hearts or isolated myocytes were stimulated with isoproterenol, PDE5A inhibition was associated with a suppression of contractility that was coupled to elevated cGMP and increased PKG-1 activity. In contrast, NOS3-null hearts or controls with NOS inhibited by N G -nitro-Larginine methyl ester, or soluble guanylate cyclase (sGC) inhibited by 1H-[1,2,4]oxadiazolo[4,3-a]quinoxaline-1-one, showed no effect of PDE5A inhibition on ␤-stimulated contractility or PKG-1 activation. This lack of response was not attributable to altered PDE5A gene or protein expression or in vitro PDE5A activity, but rather to an absence of sGC-generated cGMP specifically targeted to PDE5A catabolism and to a loss of PDE5A localization to z-bands. Re-expression of active NOS3 in NOS3-null hearts by adenoviral gene transfer restored PDE5A z-band localization and the antiadrenergic efficacy of PDE5A inhibition. These data support a novel regulatory role of PDE5A in hearts under adrenergic stimulation and highlight specific coupling of PDE5A catabolic regulation with NOS3-derived cGMP attributable to protein subcellular localization and targeted synthetic/catabolic coupling. Key Words: PDE5 Ⅲ phosphodiesterase Ⅲ sildenafil Ⅲ nitric oxide synthase Ⅲ contractility Ⅲ z-band B eta-adrenergic regulation of cardiac contraction is coupled to elevations in adenosine (cAMP) and guanosine (cGMP) cyclic nucleotides. Increased cAMP enhances contractility 1,2 by activating protein kinase A (PKA), whereas concomitant stimulation of cGMP opposes this in part by activating protein kinase G (PKG-1). 3,4 The latter response is thought to be attributable to stimulation of soluble guanylate cyclase (sGC) by NO. 3-9 Cyclic GMP is also synthesized by receptor GC (rGC) coupled to natriuretic peptide stimulation, and both sources can modulate cardiac function and structure, particularly in hearts stimulated by neurohormones or mechanical stress. 3-5,10 -13 cGMP is also regulated by catabolic phosphodiesterases such as phosphodiesterase 5A (PDE5A), and PDE5A inhibition by sildenafil (Viagra; SIL) and similar compounds augments cGMP in vascular tissue and is the primary therapy for erectile dysfunction. 14,15 However, the role for PDE5A in regulating cardiac function has remained unclear. 16 -18 Such clarification has become increasingly important because PDE5A inhibitors are poised to become chronic treatments for diseases such as pul...

Abstract-␤-Adrenergic signaling via cAMP generation and PKA activation mediates the positive inotropic effect of catecholamines on heart cells. Given the large diversity of protein kinase A targets within cardiac cells, a precisely regulated and confined activity of such signaling pathway is essential for specificity of response. Phosphodiesterases (PDEs) are the only route for degrading cAMP and are thus poised to regulate intracellular cAMP gradients. Their spatial confinement to discrete compartments and functional coupling to individual receptors provides an efficient way to control local [cAMP] i in a stimulus-specific manner. By performing real-time imaging of cyclic nucleotides in living ventriculocytes we identify a prominent role of PDE2 in selectively shaping the cAMP response to catecholamines via a pathway involving ␤ 3 -adrenergic receptors, NO generation and cGMP production. In cardiac myocytes, PDE2, being tightly coupled to the pool of adenylyl cyclases activated by ␤-adrenergic receptor stimulation, coordinates cGMP and cAMP signaling in a novel feedback control loop of the ␤-adrenergic pathway. In this, activation of ␤ 3 -adrenergic receptors counteracts cAMP generation obtained via stimulation of ␤ 1 /␤ 2 -adrenoceptors. Our study illustrates the key role of compartmentalized PDE2 in the control of catecholamine-generated cAMP and furthers our understanding of localized cAMP signaling.

The indoleamine 5-hydroxytryptamine (serotonin, 5-HT) 1 plays a pivotal, modulatory role in a variety of centrally controlled physiological processes, including respiration, arousal, aggression, and mood, and in the periphery supports gastrointestinal, platelet, and placental function (1). 5-HT is inactivated following vesicular release by a presynaptic, antidepressant-sensitive 5-HT transporter (SERT, 5-HTT), a member of the Na ϩ /Cl Ϫ -dependent solute transporter family (SLC6A4) (2-4). SERT knock-out mice display altered presynaptic 5-HT homeostasis, modified 5-HT receptor sensitivities, and stressdependent behavioral modulation as well as altered responses to psychostimulants (5). In humans, altered SERT gene expression and/or transport function have been linked to multiple disorders including autism, obsessive-compulsive disorder, depression, and suicide (6 -11).Previous studies have demonstrated that both genetic and posttranscriptional processes regulate SERT activity (12, 13). SERT activity in native cells and transfected models can be rapidly (in minutes) modulated by multiple signaling pathways (14, 15). Observations with transfected HEK cells expressing human SERT (hSERT) demonstrated that protein kinase C activators or protein phosphatase 1/2A inhibitors trigger hSERT phosphorylation and a parallel decrease in hSERT cell surface density, effects that can be attenuated by . Protein kinase A and protein kinase G (PKG) activation can also trigger SERT phosphorylation (17), although the functional consequences of these stimuli are only beginning to be appreciated. SERTs appear to form homomultimers at the plasma membrane (19) and also interact with a growing list of associated proteins, including syntaxin 1A (20 -22)

To investigate the dynamics of guanosine 3,5-cyclic monophosphate (cGMP) in single living cells, we constructed genetically encoded, fluorescent cGMP indicators by bracketing cGMP-dependent protein kinase (cGPK), minus residues 1-77, between cyan and yellow mutants of green fluorescent protein. cGMP decreased fluorescence resonance energy transfer (FRET) and increased the ratio of cyan to yellow emissions by up to 1.5-fold with apparent dissociation constants of Ϸ2 M and >100:1 selectivity for cGMP over cAMP. To eliminate constitutive kinase activity, Thr 516 of cGPK was mutated to Ala. Emission ratio imaging of the indicators transfected into rat fetal lung fibroblast (RFL)-6 showed cGMP transients resulting from activation of soluble and particulate guanylyl cyclase, respectively, by nitric oxide (NO) and C-type natriuretic peptide (CNP). Whereas all naive cells tested responded to CNP, only 68% responded to NO. Both sets of signals showed large and variable (0.5-4 min) latencies. The phosphodiesterase (PDE) inhibitor 3-isobutyl-1-methylxanthine (IBMX) did not elevate cGMP on its own but consistently amplified responses to NO or CNP, suggesting that basal activity of guanylate cyclase is very low and emphasizing the importance of PDEs in cGMP recycling. A fraction of RFL cells showed slowly propagating tides of cGMP spreading across the cell in response to delocalized application of NO. Biolistically transfected Purkinje neurons showed cGMP responses to parallel fiber activity and NO donors, confirming that single-cell increases in cGMP occur under conditions appropriate to cause synaptic plasticity.cGPK ͉ FRET ͉ green fluorescent protein T he importance of the guanosine 3Ј,5Ј-cyclic monophosphate (cGMP) second messenger cascade has been steadily gaining recognition because of the fact that it is different from the cAMP system and thus, points us to novel answers of the biological problem of receptor-effector coupling in intracellular signal transduction. cGMP is a key player in the regulation of various physiological processes, including smooth muscle tone, neuronal excitability, epithelial electrolyte transport, phototransduction in the retina, and cell adhesion (for reviews see refs. 1-5). However, cGMP is still an unruly member of the cyclic nucleotide family because of several peculiarities of the cGMP signal transduction system. (i) The pathways that control cGMP levels are complex, with receptors coupled not only to two different forms of guanylyl cyclases (6, 7), but also to a number of cGMP-specific phosphodiesterases (PDEs) (1). (ii) The intracellular actions of cGMP are primarily mediated by cGMPdependent protein kinases (cGPKs) (2), but several types of cyclic nucleotide-activated ion channels also appear to be involved (4). (iii) Many of the enzymes that participate in the cGMP cascade are restricted to a limited subset of tissues. The necessity of working out multiple mechanisms, and the difficulty of studying cGMP in broken cell preparations, have been experimental and conceptual stumbling block...

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cAMP-dependent protein kinase (cAPK) is a heterotetramer containing two regulatory (R) and two catalytic (C) subunits. Each R-subunit contains two tandem cAMP-binding domains, and activation of cAPK is mediated by the cooperative, high affinity binding of cAMP to these two domains. Mutant R-subunits containing one intact high affinity cAMP-binding site and one defective site were used to define the pathway for activation and to delineate the unique roles that each cAMP-binding domain plays. Two mutations were introduced by replacing the essential Arg in each cAMP-binding site with Lys (R209K in Site A and R333K in Site B). Also, the double mutant (R209/333K) was constructed. Analysis of cAMP binding and dissociation and the apparent constants for holoenzyme activation and R-and C-subunit interaction, measured by analytical gel filtration and surface plasmon resonance, established the following: (1) For rR(R209K), occupancy of Site B is not sufficient to activate the holoenzyme; the low affinity Site A must also be occupied. In rR(R333K), Site A retains its high affinity for cAMP, but Site A cannot bind until the low affinity Site B is occupied. Thus, both mutants, for different reasons, have similar K a 's for activation that are approximately 20-fold higher than that of the wild-type holoenzyme. The double mutant with two defective sites is no worse than either single mutant. (2) Kinetic analysis of cAMP binding showed that the mutation in Site A or B abolishes high affinity cAMP binding to that site and slightly weakens the affinity of the adjacent site for cAMP. (3) In the presence of MgATP, both mutants rapidly form a stable holoenzyme even in the presence of cAMP in contrast to the wild-type R where holoenzyme forms slowly in Vitro and requires dialysis. Regarding the mechanism of activation based on these and other mutants and from kinetic data, the following conclusions are reached: Site A provides the major contact site with the C-subunit; Site B is not essential for holoenzyme formation. Occupancy of Site A by cAMP mediates dissociation of the C-subunit. Site A is inaccessible to cAMP in the full length holoenzyme, while Site B is fully accessible. Access of cAMP to Site A is mediated by Site B. Thus Site B not only helps to shield Site A, it also provides the specific signal that "opens up" Site A. Finally, a nonfunctional Site A in the holoenzyme prevents stable binding of cAMP to Site B in the absence of subunit dissociation.The regulatory subunits of cAMP-dependent protein kinase (cAPK) 1 serve as negative regulators of cAPK and maintain an inactive holoenzyme complex in the absence of cAMP. All R-subunits contain an inhibitory site that resembles either a substrate or an inhibitor followed by two contiguous cAMP-binding domains in each protomer (Takio et al., 1984;Taylor et al., 1990;Titani et al., 1984). The cooperative activation of the holoenzyme is mediated by the sequential binding of cAMP to these two sites, which then leads to the dissociation of the active C-subunit (Øgreid & Døsk...