Expression and localization of phosphodiesterase 2A in the submandibular gland of mice.

OBJECTIVES: Phosphodiesterases comprise a superfamily of enzymes that hydrolyze and inactivate cyclic AMP (cAMP) and/or cyclic GMP (cGMP), thereby regulating cellular signaling mechanisms. We herein investigated the production of phosphodiesterase 2A (PDE2A) in the mouse submandibular gland. DESIGN: The expression and localization of the mRNA and protein of PDE2A were examined in the submandibular gland of male and female mice using the reverse transcription-polymerase chain reaction, in situ hybridization, Western blotting, and immunohistochemistry. RESULTS: Among the different species of phosphodiesterases examined in the mouse submandibular gland, PDE2A, which hydrolyzes cAMP and cGMP, exhibited a marked sexual difference; it was more abundantly expressed in females. The mRNA and protein signals for PDE2A were intense in all acinar and duct portions, including the striated duct, in females, whereas in males, these signals were markedly weaker in the granular convoluted duct, the counterpart of the female striated duct, than in acini and other duct portions. Furthermore, the signals for protein kinases A and G1, which are intracellular effectors of cAMP and cGMP, respectively, were markedly weaker in the male granular convoluted duct. CONCLUSIONS: These results suggest that cyclic nucleotide-dependent signaling mechanisms function poorly in granular convoluted duct cells in the mouse submandibular gland.

Phosphodiesterase 2 Protects Against Catecholamine-Induced Arrhythmia and Preserves Contractile Function After Myocardial Infarction.

RATIONALE: Phosphodiesterase 2 is a dual substrate esterase, which has the unique property to be stimulated by cGMP, but primarily hydrolyzes cAMP. Myocardial phosphodiesterase 2 is upregulated in human heart failure, but its role in the heart is unknown. OBJECTIVE: To explore the role of phosphodiesterase 2 in cardiac function, propensity to arrhythmia, and myocardial infarction. METHODS AND RESULTS: Pharmacological inhibition of phosphodiesterase 2 (BAY 60-7550, BAY) led to a significant positive chronotropic effect on top of maximal ß-adrenoceptor activation in healthy mice. Under pathological conditions induced by chronic catecholamine infusions, BAY reversed both the attenuated ß-adrenoceptor-mediated inotropy and chronotropy. Conversely, ECG telemetry in heart-specific phosphodiesterase 2-transgenic (TG) mice showed a marked reduction in resting and in maximal heart rate, whereas cardiac output was completely preserved because of greater cardiac contraction. This well-tolerated phenotype persisted in elderly TG with no indications of cardiac pathology or premature death. During arrhythmia provocation induced by catecholamine injections, TG animals were resistant to triggered ventricular arrhythmias. Accordingly, Ca2+-spark analysis in isolated TG cardiomyocytes revealed remarkably reduced Ca2+ leakage and lower basal phosphorylation levels of Ca2+-cycling proteins including ryanodine receptor type 2. Moreover, TG demonstrated improved cardiac function after myocardial infarction. CONCLUSIONS: Endogenous phosphodiesterase 2 contributes to heart rate regulation. Greater phosphodiesterase 2 abundance protects against arrhythmias and improves contraction force after severe ischemic insult. Activating myocardial phosphodiesterase 2 may, thus, represent a novel intracellular antiadrenergic therapeutic strategy protecting the heart from arrhythmia and contractile dysfunction.

Identification of dually acylated proteins from complementary DNA resources by cell-free and cellular metabolic labeling.

To establish a strategy to identify dually fatty acylated proteins from cDNA resources, seven N-myristoylated proteins with cysteine (Cys) residues within the 10 N-terminal residues were selected as potential candidates among 27 N-myristoylated proteins identified from a model human cDNA resource. Seven proteins C-terminally tagged with FLAG tag or EGFP were generated and their susceptibility to protein N-myristoylation and S-palmitoylation were evaluated by metabolic labeling with [(3)H]myristic acid or [(3)H]palmitic acid either in an insect cell-free protein synthesis system or in transfected mammalian cells. As a result, EEPD1, one of five proteins (RFTN1, EEPD1, GNAI1, PDE2A, RNF11) found to be dually acylated, was shown to be a novel dually fatty acylated protein. Metabolic labeling experiments using G2A and C7S mutants of EEPD1-EGFP revealed that the palmitoylation site of EEPD1 is Cys at position 7. Analysis of the intracellular localization of EEPD1 C-terminally tagged with FLAG tag or EGFP and its G2A and C7S mutants revealed that the dual acylation directs EEPD1 to localize to the plasma membrane. Thus, dually fatty acylated proteins can be identified from cDNA resources by cell-free and cellular metabolic labeling of N-myristoylated proteins with Cys residue(s) close to the N-myristoylated N-terminus.

Phosphodiesterase 2A localized in the spinal cord contributes to inflammatory pain processing.

BACKGROUND: Phosphodiesterase 2A (PDE2A) is an evolutionarily conserved enzyme that catalyzes the degradation of the cyclic nucleotides, cyclic adenosine monophosphate, and/or cyclic guanosine monophosphate. Recent studies reported the expression of PDE2A in the dorsal horn of the spinal cord, pointing to a potential contribution to the processing of pain. However, the functions of PDE2A in spinal pain processing in vivo remained elusive. METHODS: Immunohistochemistry, laser microdissection, and quantitative real-time reverse transcription polymerase chain reaction experiments were performed to characterize the localization and regulation of PDE2A protein and messenger RNA in the mouse spinal cord. Effects of the selective PDE2A inhibitor, BAY 60-7550 (Cayman Chemical, Ann Arbor, MI), in animal models of inflammatory pain (n = 6 to 10), neuropathic pain (n = 5 to 6), and after intrathecal injection of cyclic nucleotides (n = 6 to 8) were examined. Also, cyclic adenosine monophosphate and cyclic guanosine monophosphate levels in spinal cord tissues were measured by liquid chromatography tandem mass spectrometry. RESULTS: The authors here demonstrate that PDE2A is distinctly expressed in neurons of the superficial dorsal horn of the spinal cord, and that its spinal expression is upregulated in response to hind paw inflammation. Administration of the selective PDE2A inhibitor, BAY 60-7550, increased the nociceptive behavior of mice in animal models of inflammatory pain. Moreover, BAY 60-7550 increased the pain hypersensitivity induced by intrathecal delivery of cyclic adenosine monophosphate, but not of cyclic guanosine monophosphate, and it increased the cyclic adenosine monophosphate levels in spinal cord tissues. CONCLUSION: Our findings indicate that PDE2A contributes to the processing of inflammatory pain in the spinal cord.

Phosphodiesterase-2 is up-regulated in human failing hearts and blunts ß-adrenergic responses in cardiomyocytes.

OBJECTIVES: This study investigated whether myocardial phosphodiesterase-2 (PDE2) is altered in heart failure (HF) and determined PDE2-mediated effects on beta-adrenergic receptor (ß-AR) signaling in healthy and diseased cardiomyocytes. BACKGROUND: Diminished cyclic adenosine monophosphate (cAMP) and augmented cyclic guanosine monophosphate (cGMP) signaling is characteristic for failing hearts. Among the PDE superfamily, PDE2 has the unique property of being able to be stimulated by cGMP, thus leading to a remarkable increase in cAMP hydrolysis mediating a negative cross talk between cGMP and cAMP signaling. However, the role of PDE2 in HF is poorly understood. METHODS: Immunoblotting, radioenzymatic- and fluorescence resonance energy transfer-based assays, video edge detection, epifluorescence microscopy, and L-type Ca2(+) current measurements were performed in myocardial tissues and/or isolated cardiomyocytes from human and/or experimental HF, respectively. RESULTS: Myocardial PDE2 expression and activity were ~2-fold higher in advanced human HF. Chronic ß-AR stimulation via catecholamine infusions in rats enhanced PDE2 expression ~2-fold and cAMP hydrolytic activity ~4-fold, which correlated with blunted cardiac ß-AR responsiveness. In diseased cardiomyocytes, higher PDE2 activity could be further enhanced by stimulation of cGMP synthesis via nitric oxide donors, whereas specific PDE2 inhibition partially restored ß-AR responsiveness. Accordingly, PDE2 overexpression in healthy cardiomyocytes reduced the rise in cAMP levels and L-type Ca2(+) current amplitude, and abolished the inotropic effect following acute ß-AR stimulation, without affecting basal contractility. Importantly, PDE2-overexpressing cardiomyocytes showed marked protection from norepinephrine-induced hypertrophic responses. CONCLUSIONS: PDE2 is markedly up-regulated in failing hearts and desensitizes against acute ß-AR stimulation. This may constitute an important defense mechanism during cardiac stress, for example, by antagonizing excessive ß-AR drive. Thus, activating myocardial PDE2 may represent a novel intracellular antiadrenergic therapeutic strategy in HF.

Ras protein/cAMP-dependent protein kinase signaling is negatively regulated by a deubiquitinating enzyme, Ubp3, in yeast.

Ras proteins and cAMP-dependent protein kinase (protein kinase A, PKA) are important components of a nutrient signaling pathway that mediates cellular responses to glucose in yeast. The molecular mechanisms that regulate Ras/PKA-mediated signaling remain to be fully understood. Here, we provide evidence that Ras/PKA signaling is negatively regulated by a deubiquitinating enzyme, Ubp3. Disrupting the activity of Ubp3 leads to hyperactivation of PKA, as evidenced by much enhanced phosphorylation of PKA substrates, decreased accumulation of glycogen, larger cell size, and increased sensitivity to heat shock. Levels of intracellular cAMP and the active forms of Ras proteins are also elevated in the ubp3Δ mutant. Consistent with a possibility that the increased cAMP is responsible for the abnormal signaling behavior of the ubp3Δ mutant, overexpressing PDE2, which encodes a phosphodiesterase that hydrolyzes cAMP, significantly relieves the cell size increase and heat shock sensitivity of the mutant. Further analysis reveals that Ubp3 interacts with a Ras GTPase-accelerating protein, Ira2, and regulates its level of ubiquitination. Together, our data indicate that Ubp3 is a new regulator of the Ras/PKA signaling pathway and suggest that Ubp3 regulates this pathway by controlling the ubiquitination of Ras GTPase-accelerating protein Ira2.

RAS/cyclic AMP and transcription factor Msn2 regulate mating and mating-type switching in the yeast Kluyveromyces lactis.

In response to harsh environmental conditions, ascomycetes produce stress-resistant spores to promote survival. As sporulation requires a diploid DNA content, species with a haploid lifestyle, such as Kluyveromyces lactis, first induce mating in response to stress. In K. lactis, mating and mating-type switching are induced by the DNA-binding protein Mts1. Mts1 expression is known to be upregulated by nutrient limitation, but the mechanism is unknown. We show that a ras2 mutation results in a hyperswitching phenotype. In contrast, strains lacking the phosphodiesterase Pde2 had lower switching rates compared to that of the wild type (WT). As Ras2 promotes cyclic AMP (cAMP) production and Pde2 degrades cAMP, these data suggest that low cAMP levels induce switching. Because the MTS1 regulatory region contains several Msn2 binding sites and Msn2 is a transcription factor that is activated by low cAMP levels, we investigated if Msn2 regulates MTS1 transcription. Consistently with this idea, an msn2 mutant strain displayed lower switching rates than the WT strain. The transcription of MTS1 is highly induced in the ras2 mutant strain. In contrast, an msn2 ras2 double mutant strain displays WT levels of the MTS1 transcript, showing that Msn2 is a critical inducer of MTS1 transcription. Strains lacking Msn2 and Pde2 also exhibit mating defects that can be complemented by the ectopic expression of Mts1. Finally, we show that MTS1 is subjected to negative autoregulation, presumably adding robustness to the mating and switching responses. We suggest a model in which Ras2/cAMP/Msn2 mediates the stress-induced mating and mating-type switching responses in K. lactis.

A phosphodiesterase 2A isoform localized to mitochondria regulates respiration.

Mitochondria are central organelles in cellular energy metabolism, apoptosis, and aging processes. A signaling network regulating these functions was recently shown to include soluble adenylyl cyclase as a local source of the second messenger cAMP in the mitochondrial matrix. However, a mitochondrial cAMP-degrading phosphodiesterase (PDE) necessary for switching off this cAMP signal has not yet been identified. Here, we describe the identification and characterization of a PDE2A isoform in mitochondria from rodent liver and brain. We find that mitochondrial PDE2A is located in the matrix and that the unique N terminus of PDE2A isoform 2 specifically leads to mitochondrial localization of this isoform. Functional assays show that mitochondrial PDE2A forms a local signaling system with soluble adenylyl cyclase in the matrix, which regulates the activity of the respiratory chain. Our findings complete a cAMP signaling cascade in mitochondria and have implications for understanding the regulation of mitochondrial processes and for their pharmacological modulation.

cGMP signals modulate cAMP levels in a compartment-specific manner to regulate catecholamine-dependent signaling in cardiac myocytes.

RATIONALE: cAMP and cGMP are intracellular second messengers involved in heart pathophysiology. cGMP can potentially affect cAMP signals via cGMP-regulated phosphodiesterases (PDEs). OBJECTIVE: To study the effect of cGMP signals on the local cAMP response to catecholamines in specific subcellular compartments. METHODS AND RESULTS: We used real-time FRET imaging of living rat ventriculocytes expressing targeted cAMP and cGMP biosensors to detect cyclic nucleotides levels in specific locales. We found that the compartmentalized, but not the global, cAMP response to isoproterenol is profoundly affected by cGMP signals. The effect of cGMP is to increase cAMP levels in the compartment where the protein kinase (PK)A-RI isoforms reside but to decrease cAMP in the compartment where the PKA-RII isoforms reside. These opposing effects are determined by the cGMP-regulated PDEs, namely PDE2 and PDE3, with the local activity of these PDEs being critically important. The cGMP-mediated modulation of cAMP also affects the phosphorylation of PKA targets and myocyte contractility. CONCLUSIONS: cGMP signals exert opposing effects on local cAMP levels via different PDEs the activity of which is exerted in spatially distinct subcellular domains. Inhibition of PDE2 selectively abolishes the negative effects of cGMP on cAMP and may have therapeutic potential.

Immunohistochemical localization of phosphodiesterase 2A in multiple mammalian species.

Phosphodiesterases (PDEs) comprise a family of enzymes that regulate the levels of cyclic nucleotides, key second messengers that mediate a diverse array of functions. PDE2A is an evolutionarily conserved cGMP-stimulated cAMP and cGMP PDE. In the present study, the regional and cellular distribution of PDE2A in tissues of rats, mice, cynomolgus monkeys, dogs, and humans was evaluated by immunohistochemistry. A polyclonal antibody directed to the C-terminal portion of PDE2A specifically detected PDE2A by Western blotting and by immunohistochemistry. The pattern of PDE2A immunoreactivity (ir) was consistent across all species. Western blot analysis demonstrated that PDE2A was most abundant in the brain relative to peripheral tissues. PDE2A ir was heterogeneously distributed within brain and was selectively expressed in particular peripheral tissues. In the brain, prominent immunoreactivity was apparent in components of the limbic system, including the isocortex, hippocampus, amygdala, habenula, basal ganglia, and interpeduncular nucleus. Cytoplasmic PDE2A staining was prominent in several peripheral tissues, including the adrenal zona glomerulosa, neurons of enteric ganglia, endothelial cells in all organs, lymphocytes of spleen and lymph nodes, and pituitary. These studies suggest that PDE2A is evolutionarily conserved across mammalian species and support the hypothesis that the enzyme plays a fundamental role in signal transduction.

Soluble guanylyl cyclase contributes to ventilator-induced lung injury in mice.

High tidal volume (HV(T)) ventilation causes pulmonary endothelial barrier dysfunction. HV(T) ventilation also increases lung nitric oxide (NO) and cGMP. NO contributes to HV(T) lung injury, but the role of cGMP is unknown. In the current study, ventilation of isolated C57BL/6 mouse lungs increased perfusate cGMP as a function of V(T). Ventilation with 20 ml/kg V(T) for 80 min increased the filtration coefficient (K(f)), an index of vascular permeability. The increased cGMP and K(f) caused by HV(T) were attenuated by nitric oxide synthase (NOS) inhibition and in lungs from endothelial NOS knockout mice. Inhibition of soluble guanylyl cyclase (sGC) in wild-type lungs (10 muM ODQ) also blocked cGMP generation and inhibited the increase in K(f), suggesting an injurious role for sGC-derived cGMP. sGC inhibition also attenuated lung Evans blue dye albumin extravasation and wet-to-dry weight ratio in an anesthetized mouse model of HV(T) injury. Additional activation of sGC (1.5 muM BAY 41-2272) in isolated lungs at 40 min increased cGMP production and K(f) in lungs ventilated with 15 ml/kg V(T). HV(T) endothelial barrier dysfunction was attenuated with a nonspecific phosphodiesterase (PDE) inhibitor (100 muM IBMX) as well as an inhibitor (10 muM BAY 60-7550) specific for the cGMP-stimulated PDE2A. Concordantly, we found a V(T)-dependent increase in lung cAMP hydrolytic activity and PDE2A protein expression with a decrease in lung cAMP concentration that was blocked by BAY 60-7550. We conclude that HV(T)-induced endothelial barrier dysfunction resulted from a simultaneous increase in NO/sGC-derived cGMP and PDE2A expression causing decreased cAMP.