Involvement of natriuretic peptide system in C2C12 myocytes.

The natriuretic peptide system, a key regulator of cGMP signaling, comprises three types of natriuretic peptides, osteocrin/musclin (OSTN), and their natriuretic peptide receptors. Although this system plays important roles in many organs, its physiological roles in skeletal muscle have not been clearly described. In the present study, we investigated the role of the natriuretic peptide system in C2C12 myocytes. All three natriuretic peptide receptors were expressed by cells differentiating from myoblasts to myotubes, and natriuretic peptide receptor B (NPR-B) transcripts were detected at the highest levels. Further, higher levels of cGMP were generated in response to stimulation with C-type natriuretic peptide (CNP) versus atrial natriuretic peptide (ANP), which reflected receptor expression levels. A cGMP analog downregulated the expression of a few ER stress-related genes. Furthermore, OSTN gene expression was strongly upregulated after 20 days of differentiation. Augmented gene expression was found to correlate closely with endoplasmic reticulum (ER) stress, and C/EBP [CCAAT-enhancer-binding protein] homologous protein (CHOP), which is known to be activated by ER stress, affected the expression of OSTN. Together, these results suggest a role for natriuretic peptide signaling in the ER stress response of myocytes.

Morphology-Based Analysis of Myoblasts for Prediction of Myotube Formation.

The development of new drugs depends on the efficiency of drug screening. Phenotype-based screening has attracted interest due to its considerable potency for the discovery of first-in-class drugs. In general, fluorescently labeled imagery is the leading technique for phenotype-based screening; however, there are growing requirements to understand total culture profiles, which are unclear after end-point assays. In this study, we demonstrate that morphology-based cellular evaluation of unlabeled cells is an efficient approach to evaluate myotube formation assays. One of our aims was to study the myogenic differentiation process in C2C12 cells to discern the differences between cellular responses to different medium conditions (serum concentrations and insulin dosages). Our results show that predictive morphological profiles that strongly correlate with myogenic differentiation can be generated from myotube images, even in the confluent stage. The differentiation rate after 14 days can be quantitatively predicted with the highest accuracy by means of images taken on days 0-11.5. In addition, for the application of our morphology-based cellular evaluation, the effect of cyclic guanosine monophosphate (cGMP) on myogenic differentiation was analyzed. Our results show that the quantitated morphological profile from these images can be an effective descriptor for analysis of the myotube-recovering effect of cGMP.

Discovery of 2-[(E)-2-(7-Fluoro-3-methylquinoxalin-2-yl)vinyl]-6-pyrrolidin-1-yl-N-(tetrahydro-2H-pyran-4-yl)pyrimidin-4-amine Hydrochloride as a Highly Selective PDE10A Inhibitor.

Phosphodiesterase (PDE) 10A is a dual hydrolase of cAMP and cGMP and highly expressed in striatal medium spiny neurons. Inhibition of PDE10A modulates the activity of medium spiny neurons (MSN) via the regulation of cAMP and cGMP. Signal control of MSN is considered associated with psychotic symptoms. Therefore PDE10A inhibitor is expected as a therapeutic method for psychosis disease such as schizophrenia. Avanafil (1) is a PDE5 inhibitor (treatment for erectile dysfunction) discovered by our company. We paid attention to the homology of PDE10A and PDE5 and took advantage of PDE5 inhibitor library to discover PDE10A inhibitors, and found a series of compounds that exhibit higher potency for PDE10A than PDE5. We transformed the afforded derivatives, which had weak inhibitory activity against PDE10A, and discovered stilbene as a PDE10A inhibitor. Brain penetration of this compound was improved by further conversion of N-containing heterocycles and their substituents. The afforded dimethylaminopyrimidine was effective for rat conditioned avoidance response (CAR) test; however, it did not exhibit good brain penetration. We performed in-depth optimization focusing on substituents of the quinoxaline ring, and produced 3-methyl-7-fluoro quinoxaline. This compound was the most effective in rat CAR test due to its strong PDE10A inhibitory activity and good pharmacokinetics.

8-(3-chloro-4-methoxybenzyl)-8H-pyrido[2,3-d]pyrimidin-7-one derivatives as potent and selective phosphodiesterase 5 inhibitors.

A novel series of highly selective phosphodiesterase 5 (PDE5) inhibitors was found. 8H-Pyrido[2,3-d]pyrimidin-7-one derivatives bearing an (S)-2-(hydroxymethyl)pyrrolidin-1-yl group at the 2-position and a 3-chloro-4-methoxybenzyl group at the 8-position exhibited potent PDE5 inhibitory activities and high PDE5 selectivity over PDE6. Among the synthesized compounds, the 5-methyl analogue (5b) showed the most potent relaxant effect on isolated rabbit corpus cavernosum with an EC30 value of 0.85 nM.

Girdin is phosphorylated on tyrosine 1798 when associated with structures required for migration.

The mammalian protein Girdin interacts with several key molecules such as actin, and it functions as a regulator of the cytoskeleton. Silencing of Girdin mRNA results in defective migration in a variety of cultured cells. Moreover, knockout of Girdin causes phenotypes related to defective migration, including hypoplasia of olfactory bulbs and a widened rostral migratory stream (RMS) in mice. To elucidate the molecular basis underlying cellular migration, we generated site- and phosphorylation state-specific antibodies against human Girdin peptides carrying four putative phosphorylation sites (serine1386 [S1386], S1416, tyrosine1764 [Y1764] and Y1798) that had been identified by mutagenesis analyses or mass spectrometric studies. We found that these residues were phosphorylated in an epidermal growth factor (EGF)-dependent manner. Among the four antibodies we developed, the antibody that targeted Girdin when phosphorylated at Y1798 (pY1798) worked well for immunohistochemistry of paraffin-embedded tissues as well as for cultured cells. Immunocytochemistry of HEK293FT cells transfected with an EGF receptor expression plasmid exhibited punctate signals with pY1798. These signals colocalized with those of endocytosed EGF receptors after EGF stimulation. Signals from pY1798 were also observed on lamellipodia, filopodia, focal adhesion and stress fibers in NIH3T3 cells under conventional culture conditions. Immunohistochemistry of paraffin-embedded mouse brain at P14 using anti-pY1798 antibody displayed signals at the hilum-side (internal side) of the dentate gyrus of the hippocampus, the RMS, the accessory olfactory bulb and the olfactory bulb in which Girdin expression was detected. Primary culture of RMS neurons showed punctate signals of pY1798 at the tips of leading processes as well as in the cytoplasm, whereas no signals were observed when neurons were treated with Src inhibitor, PP2. Our data revealed the changes in the phosphorylation status of Y1798 in Girdin when it associated with migration-related structures in vitro and in vivo.

Design and synthesis of novel 5-(3,4,5-trimethoxybenzoyl)-4-aminopyrimidine derivatives as potent and selective phosphodiesterase 5 inhibitors: scaffold hopping using a pseudo-ring by intramolecular hydrogen bond formation.

5-(3,4,5-Trimethoxybenzoyl)-4-amimopyrimidine derivatives were found as a novel chemical class of potent and highly selective phosphodiesterase 5 inhibitors. A pseudo-ring formed by an intramolecular hydrogen bond constrained the conformation of 3-chloro-4-methoxybenzylamino and 3,4,5-trimethoxybenzoyl substituents and led to the discovery of T-6932 (19a) with a potent PDE5 inhibitory activity (IC50 = 0.13 nM) and a high selectivity over PDE6 (IC50 ratio: PDE6/PDE5 = 2400). Further modification at the 2-position of T-6932 resulted in the finding of 26, which exhibited potent relaxant effects on isolated rabbit corpus cavernosum (EC30 = 11 nM) with a high PDE5 selectivity over PDE6 (IC50 ratio: PDE6/PDE5 = 2800).

The discovery of avanafil for the treatment of erectile dysfunction: a novel pyrimidine-5-carboxamide derivative as a potent and highly selective phosphodiesterase 5 inhibitor.

Novel pyrimidine-5-carboxamide derivatives bearing a 3-chloro-4-methoxybenzylamino group at the 4-position were identified as potent and highly selective phosphodiesterase 5 inhibitors. Among them, we successfully found 10j (avanafil) which exhibited a potent relaxant effect on isolated rabbit cavernosum (EC30=2.1 nM) and a high isozyme selectivity.

Selectivity of avanafil, a PDE5 inhibitor for the treatment of erectile dysfunction: implications for clinical safety and improved tolerability.

INTRODUCTION: Phosphodiesterase type 5 (PDE5) inhibitors are indicated for the treatment of erectile dysfunction (ED); however, they can also inhibit other PDE isozymes, affecting their target tissues (e.g., PDE1: heart; PDE6: retina; and PDE11: skeletal muscle), which in some cases can cause unwanted side effects and therapy discontinuation. Data from in vitro studies showed that avanafil, a PDE5 inhibitor for the treatment of ED, exhibited strong selectivity toward PDE5 and against all other PDE isozymes. AIM: To review the inhibitory effects of avanafil for PDE isozymes compared with those of sildenafil, tadalafil, and vardenafil and to discuss these results within the context of clinical trial safety observations. METHODS: Review of in vitro selectivity data for avanafil (published primary data from a peer-reviewed journal and scientific congress abstracts); PubMed search for pertinent publications on PDE5 inhibitor safety data; and review of published articles and abstracts from avanafil phase 1, 2, and 3 clinical trials. MAIN OUTCOME MEASURES: A low incidence of some PDE-related adverse events may be reflected by the high selectivity of avanafil against non-PDE5 isozymes. RESULTS: Avanafil is highly selective toward PDE5 and against all other PDE isozymes tested. Lower selectivity against PDE1, PDE6, and PDE11 is consistent with results from randomized, placebo-controlled, phase 3 trials in which musculoskeletal and hemodynamic adverse events were reported in <2% of patients and no color vision-related abnormalities were reported with avanafil doses up to 200 mg once daily. CONCLUSIONS: Data suggest that avanafil may confer a safety benefit, in terms of a lower incidence of specific adverse events, by virtue of its high specificity to PDE5 and its overall selectivity against other PDE isozymes.

Avanafil, a potent and highly selective phosphodiesterase-5 inhibitor for erectile dysfunction.

PURPOSE: We investigated the in vitro inhibitory effects of avanafil, a novel, potent inhibitor of phosphodiesterase-5, on 11 phosphodiesterases. We also studied its potentiation of penile tumescence in dogs. MATERIALS AND METHODS: Phosphodiesterase assay was done with the 4 phosphodiesterase-5 inhibitors avanafil, sildenafil, vardenafil and tadalafil using 11 phosphodiesterase isozymes. In anesthetized dogs the pelvic nerve was repeatedly stimulated to evoke tumescence. Intracavernous pressure was measured after avanafil or sildenafil administration. RESULTS: Avanafil specifically inhibited phosphodiesterase-5 activity at a 50% inhibitory concentration of 5.2 nM. Avanafil showed higher selectivity (121-fold) against phosphodiesterase-6 than sildenafil and vardenafil (16 to 21-fold) and showed excellent selectivity (greater than 10,000-fold) against phosphodiesterase-1 compared with sildenafil (375-fold). Avanafil also had higher selectivity against phosphodiesterase-11 than tadalafil (greater than 19,000 vs 25-fold). Avanafil also showed excellent selectivity against all other phosphodiesterases. After intravenous administration in anesthetized dogs the 200% effective dose of avanafil and sildenafil on the penile tumescence was 37.5 and 34.6 µg/kg, respectively. After intraduodenal administration the 200% effective dose of avanafil and sildenafil on tumescence was 151.7 and 79.0 µg/kg at the peak time, respectively. Time to peak response with avanafil and sildenafil was 10 and 30 minutes, respectively, indicating a more rapid onset of avanafil. CONCLUSIONS: Avanafil has a favorable phosphodiesterase-5 selectivity profile compared to that of marketed phosphodiesterase-5 inhibitors. Avanafil shows excellent in vitro and in vivo potency, and fast onset of action for penile erection. Cumulative data suggest that avanafil has a promising pharmacological profile for erectile dysfunction.

Crystal structure of the GAF-B domain from human phosphodiesterase 10A complexed with its ligand, cAMP.

Cyclic nucleotide phosphodiesterases (PDEs) catalyze the degradation of the cyclic nucleotides cAMP and cGMP, which are important second messengers. Five of the 11 mammalian PDE families have tandem GAF domains at their N termini. PDE10A may be the only mammalian PDE for which cAMP is the GAF domain ligand, and it may be allosterically stimulated by cAMP. PDE10A is highly expressed in striatal medium spiny neurons. Here we report the crystal structure of the C-terminal GAF domain (GAF-B) of human PDE10A complexed with cAMP at 2.1-angstroms resolution. The conformation of the PDE10A GAF-B domain monomer closely resembles those of the GAF domains of PDE2A and the cyanobacterium Anabaena cyaB2 adenylyl cyclase, except for the helical bundle consisting of alpha1, alpha2, and alpha5. The PDE10A GAF-B domain forms a dimer in the crystal and in solution. The dimerization is mainly mediated by hydrophobic interactions between the helical bundles in a parallel arrangement, with a large buried surface area. In the PDE10A GAF-B domain, cAMP tightly binds to a cNMP-binding pocket. The residues in the alpha3 and alpha4 helices, the beta6 strand, the loop between 3(10) and alpha4, and the loop between alpha4 and beta5 are involved in the recognition of the phosphate and ribose moieties. This recognition mode is similar to those of the GAF domains of PDE2A and cyaB2. In contrast, the adenine base is specifically recognized by the PDE10A GAF-B domain in a unique manner, through residues in the beta1 and beta2 strands.

Overview of PDEs and their regulation.

Contraction and relaxation of vascular smooth muscle and cardiac myocytes are key physiological events in the cardiovascular system. These events are regulated by second messengers, cAMP and cGMP, in response to extracellular stimulants. The strength of signal transduction is controlled by intracellular cyclic nucleotide concentrations, which are determined by a balance in production and degradation of cAMP and cGMP. Degradation of cyclic nucleotides is catalyzed by 3',5'-cyclic nucleotide phosphodiesterases (PDEs), and therefore regulation of PDEs hydrolytic activity is important for modulation of cellular functions. Mammalian PDEs are composed of 21 genes and are categorized into 11 families based on sequence homology, enzymatic properties, and sensitivity to inhibitors. PDE families contain many splice variants that mostly are unique in tissue-expression patterns, gene regulation, enzymatic regulation by phosphorylation and regulatory proteins, subcellular localization, and interaction with association proteins. Each unique variant is closely related to the regulation of a specific cellular signaling. Thus, multiple PDEs function as a particular modulator of each cardiovascular function and regulate physiological homeostasis.

PfPDE1, a novel cGMP-specific phosphodiesterase from the human malaria parasite Plasmodium falciparum.

This is the first report of molecular characterization of a novel cyclic nucleotide PDE (phosphodiesterase), isolated from the human malaria parasite Plasmodium falciparum and designated PfPDE1. PfPDE1 cDNA encodes an 884-amino-acid protein, including six putative transmembrane domains in the N-terminus followed by a catalytic domain. The PfPDE1 gene is a single-copy gene consisting of two exons and a 170 bp intron. PfPDE1 transcripts were abundant in the ring form of the asexual blood stages of the parasite. The C-terminal catalytic domain of PfPDE1, produced in Escherichia coli, specifically hydrolysed cGMP with a K(m) value of 0.65 microM. Among the PDE inhibitors tested, a PDE5 inhibitor, zaprinast, was the most effective, having an IC50 value of 3.8 microM. The non-specific PDE inhibitors IBMX (3-isobutyl-1-methylxanthine), theophylline and the antimalarial chloroquine had IC50 values of over 100 microM. Membrane fractions prepared from P. falciparum at mixed asexual blood stages showed potent cGMP hydrolytic activity compared with cytosolic fractions. This hydrolytic activity was sensitive to zaprinast with an IC50 value of 4.1 microM, but insensitive to IBMX and theophylline. Furthermore, an in vitro antimalarial activity assay demonstrated that zaprinast inhibited the growth of the asexual blood parasites, with an ED50 value of 35 microM. The impact of cyclic nucleotide signalling on the cellular development of this parasite has previously been discussed. Thus this enzyme is suggested to be a novel potential target for the treatment of the disease malaria.

Transcriptional activation of phosphodiesterase 7B1 by dopamine D1 receptor stimulation through the cyclic AMP/cyclic AMP-dependent protein kinase/cyclic AMP-response element binding protein pathway in primary striatal neurons.

Phosphodiesterase (PDE) 7B, a cAMP-specific PDE which is dominantly expressed in striatum, is expected to be involved in dopaminergic signaling in striatal neurons. Here we show, for the first time, the involvement of the dopaminergic signaling pathway in transcriptional activation of rat PDE7B in primary striatal culture. RT-PCR analysis revealed that dopamine, D1 agonist, forskolin and 8-Br-cAMP stimulation potentiated PDE7B transcription in striatal neurons, while D2 agonist failed to activate the PDE7B transcription. Pre-treatment with D1 antagonist abolished the dopamine- or D1 agonist-induced transcriptional activation of PDE7B. The activation of PDE7B transcription by these stimulators was completely ablated by pre-treatment of the cells with a cAMP-dependent protein kinase inhibitor, H-89. RT-PCR using splice variant-specific primers revealed that transcription of PDE7B1, but not of other splice variants, was activated by D1 agonist. We determined the putative transcription start site of PDE7B1, a brain-specific splice variant of PDE7B, by 5'-RACE and identified a promoter region of PDE7B1. Sequence analysis of the PDE7B1 promoter revealed the presence of a canonical cAMP-response element at 166 bp upstream of the putative transcription start site. The cAMP-responsiveness of the PDE7B1 promoter was demonstrated by functional promoter analysis using the luciferase reporter system. Deletion and mutation of the cAMP-response element site in the PDE7B1 promoter abolished the forskolin-induced activation of the PDE7B1 promoter activity. Electrophoretic mobility shift assay showed the binding of cAMP-response element binding protein to the PDE7B1 promoter. These data demonstrate the dopamine D1 receptor-mediated transcriptional activation of PDE7B through the cAMP/cAMP-dependent protein kinase/cAMP-response element binding protein pathway in striatal neurons.

Subcellular localization of cyclic nucleotide phosphodiesterase type 10A variants, and alteration of the localization by cAMP-dependent protein kinase-dependent phosphorylation.

Our previous studies have suggested that two phosphodiesterase type 10A (PDE10A) variants, PDE10A1 and PDE10A2 transcripts, are mainly expressed in humans and that PDE10A2 and PDE10A3 transcripts are major variants in rats. In the present study, immunoblot analysis demonstrated that PDE10A proteins, especially PDE10A2, are more abundant in membrane fractions than in cytosolic fractions of rat striatum. Recombinant PDE10A1 and PDE10A3 were produced only in cytosolic fractions of transfected PC12h cells. By contrast, recombinant PDE10A2 was present mainly in membrane fractions. This finding agreed well with the result of subcellular fractionation of PDE10A in rat striatum. Immunocytochemical analysis showed that PDE10A2 was localized in the Golgi apparatus of transfected PC12h cells. PDE10A2 was phosphorylated by cAMP-dependent protein kinase (PKA) at Thr16. Interestingly, recombinant protein of wild-type PDE10A2, but not PDE10A2 mutant with an Ala replacement at Thr16, was distributed to cytosolic fractions by co-transfection with a plasmid encoding the catalytic subunit of PKA. A PDE10A2 mutant with Glu substitution at Thr16, which can be a mimic of phosphorylation, was localized in the cytosolic fractions of transfected PC12h cells. These observations implied that phosphorylation of PDE10A2 at Thr16 by PKA caused alteration of subcellular localization of PDE10A2 from the Golgi apparatus to cytosol. It is hypothesized that cAMP signaling in the Golgi area and the cytosol in neurons is controlled through alteration of subcellular localization of PDE10A brought by activation of PKA in response to intracellular elevations of cAMP.

Molecular comparison of rat cyclic nucleotide phosphodiesterase 8 family: unique expression of PDE8B in rat brain.

Cyclic nucleotide phosphodiestease (PDE) type 8 is categorized into a family of 3-isobutyl-1-methylxanthine-insensitive PDE hydrolyzing cAMP with high affinity. We have isolated cDNAs encoding rat PDE8A and PDE8B from brain and testis, respectively. The sequence analysis demonstrated that rat PDE8A was a protein of 823 amino acid residues. Rat PDE8B protein was predicted as an N-terminal truncated form of 760 amino acid residues. Both of rat PDE8 proteins include REC, PAS and catalytic PDE domains. Tissue-specific expression patterns of rat PDE8A and PDE8B transcripts were demonstrated by Northern blot analysis. Rat PDE8A transcripts were rich in the liver and testis, and those of rat PDE8B were particularly abundant in the brain and were not expressed in the thyroid gland, while human thyroid gland contains PDE8B transcripts at a high level. Rat PDE8B transcripts were found in all brain regions other than cerebellum and shown to exist in the neuronal cells in in situ hybridization. Mouse PDE8B1 sequence was also identified by a database search and sequence alignment, revealing a protein of 885 amino acid residues, which is 99% and 96% identical to rat and human PDE8B1, respectively. As well as rat PDE8B, expression of mouse PDE8B transcripts was not confined to the thyroid gland. Species-dependent tissue expression pattern was quite unique features of PDE8B.

mRNA expression patterns of the cGMP-hydrolyzing phosphodiesterases types 2, 5, and 9 during development of the rat brain.

Recent evidence indicates that cGMP plays an important role in neural development and neurotransmission. Since cGMP levels depend critically on the activities of phosphodiesterase (PDE) enzymes, mRNA expression patterns were examined for several key cGMP-hydrolyzing PDEs (type 2 [PDE2], 5 [PDE5], and 9 [PDE9]) in rat brain at defined developmental stages. Riboprobes were used for nonradioactive in situ hybridization on sections derived from embryonic animals at 15 days gestation (E15) and several postnatal stages (P0, P5, P10, P21) until adulthood (3 months). At all stages PDE9 mRNA was present throughout the whole central nervous system, with highest levels observed in cerebellar Purkinje cells, whereas PDE2 and PDE5 mRNA expression was more restricted. Like PDE9, PDE5 mRNA was abundant in cerebellar Purkinje cells, although it was observed only on and after postnatal day 10 in these cells. In other brain regions, PDE5 mRNA expression was minimal, detected in olfactory bulb, cortical layers, and in hippocampus. PDE2 mRNA was distributed more widely, with highest levels in medial habenula, and abundant expression in olfactory bulb, olfactory tubercle, cortex, amygdala, striatum, and hippocampus. Double immunostaining of PDE2, PDE5, or PDE9 mRNAs with the neuronal marker NeuN and the glial cell marker glial fibrillary acidic protein revealed that these mRNAs were predominantly expressed in neuronal cell bodies. Our data indicate that three cGMP-hydrolyzing PDE families have distinct expression patterns, although specific cell types coexpress mRNAs for all three enzymes. Thus, it appears that differential expression of PDE isoforms may provide a mechanism to match cGMP hydrolysis to the functional demands of individual brain regions.

Novel membrane protein containing glycerophosphodiester phosphodiesterase motif is transiently expressed during osteoblast differentiation.

Osteoblast maturation is a multistep series of events characterized by an integrated cascade of gene expression that are accompanied by specific phenotypic alterations. To find new osteoblast-related genes we cloned differentially expressed cDNAs characteristic of specific differentiation stages in the mouse osteoblast-like MC3T3-E1 cells by a differential display method. We identified a novel cDNA encoding a putative glycerophosphodiester phosphodiesterase, GDE3, which specifically was expressed at the stage of matrix maturation. Interestingly, the deduced amino acid sequence contains 539 amino acids including seven putative transmembrane domains and a glycerophosphodiester phosphodiesterase region in one of the extracellular loops. Northern blot analysis revealed that GDE3 was also expressed in spleen as well as primary calvarial osteoblasts and femur. We next transfected HEK293T cells with GDE3 with green fluorescent protein fused to the C terminus. The green fluorescent protein-fused protein accumulated at the cell periphery, and the transfected cells overexpressing the protein changed from a spread form to rounded form with disappearance of actin filaments. Immunofluorescence staining with GDE3 antibody and phalloidin in MC3T3-E1 cells indicated that endogenous GDE3 might be co-localized with the actin cytoskeleton. To identify a role for GDE3 in osteoblast differentiation, MC3T3-E1 cells stably expressing the full-length protein were constructed. Expression of GDE3 showed morphological changes, resulting in dramatic increases in alkaline phosphatase activity and calcium deposit. These results suggest that GDE3 might be a novel seven-transmembrane protein with a GP-PDE-like extracellular motif expressed during the osteoblast differentiation that dramatically accelerates the program of osteoblast differentiation and is involved in the morphological change of cells.

cGMP-phosphodiesterase activity is up-regulated in response to pressure overload of rat ventricles.

Although expression of natriuretic peptides in cardiac tissues is up-regulated in response to pressure overload, no significant change in cGMP level in hypertrophied ventricles was observed. Activities of two cyclic nucleotide phosphodiesterase (PDE) isoforms, Ca2+/calmodulin-stimulated PDE (PDE1) and cGMP-stimulated PDE (PDE2), were significantly higher in rat left ventricles 14 days after aortic banding. The absence of significant changes in PDE1A and PDE2A mRNA levels indicated that the two PDE activities were post-transcriptionally up-regulated. These results suggested that the increased cGMP-PDE activity in response to pressure overload plays an important role in neutralizing cGMP action in cardiac tissue.