磷酸二酯酶5在小鼠卵巢内的表达

目的探讨cGMP特异性结合的磷酸二酯酶5（PDE5）在小鼠卵巢内的定位和表达情况。方法应用免疫组织化学对小鼠卵巢切片进行染色,检测PDE5在卵巢内不同部位的表达情况。利用Western blot检测PDE5在小鼠不同组织和细胞内的表达情况。结果PDE5在小鼠卵巢的黄体细胞（CL）和卵泡膜细胞（TC）上有强表达,卵母细胞（Oos）上以及大有腔卵泡的卵丘细胞（CCs）也有表达,而在小卵泡的颗粒细胞上没有表达。结论揭示了PDE5在小鼠卵巢内的表达情况,为进一步研究PDE5对卵巢功能的调节提供了生理学基础。     

Cyclic GMP-specific phosphodiesterase 5 regulates growth and apoptosis in pulmonary endothelial cells

Sustained increases in intracellular cGMP concentrations ([cGMP]i) inhibit cell growth and induce apoptosis. We now report that a cGMP-specific phosphodiesterase, PDE5, plays a dominant role in regulating [cGMP]i transitions that inhibit cell growth and control susceptibility to apoptosis in pulmonary endothelium. Atrial natriuretic peptide (ANP) activates guanylyl cyclase A/B and induces a rapid [cGMP]i rise 2-5 min after its application, in both pulmonary arterial endothelial cells (PAECs) and pulmonary microvascular endothelial cells (PMVECs). However, increased [cGMP]i in PAECs is transient and decays within 10 min due to cytosolic PDE5 hydrolytic activity. Increased [cGMP]i in PMVECs is sustained for >3 h due to the absence of PDE5. Indeed, at any ANP concentration, the sustained (30 min) [cGMP]i rise is greater in PMVECs than in PAECs, unless PAECs are also treated with the PDE5 inhibitor zaprinast. Using RT-PCR, Western blot analysis, immunoprecipitation, and DEAE chromatography, we resolved the expression and activity of PDE 5A1/A2 only in PAECs. Similarly, PDE5 expression was restricted to extra-alveolar endothelium in vivo. ANP induced growth inhibition and apoptosis in PMVECs, but similar effects were not seen in PAECs unless ANP treatment was combined with zaprinast. ANP blocked the VEGF-induced proliferation and migration in PMVECs. Collectively, these data suggest that PDE5-regulated [cGMP]i controls endothelial cell growth and apoptosis, representing a mechanism of heterogeneity between two endothelial phenotypes.     

Expression of PDE11A in normal and malignant human tissues.

Cyclic nucleotide phosphodiesterase 11A (PDE11A) is the newest member in the PDE family. Although the tissue distribution of PDE11A mRNA has been shown, its protein expression pattern has not been well studied. The goal of this report is to investigate the distribution of PDE11A proteins in a wide range of normal and malignant human tissues. We utilized a polyclonal antibody that recognized all four PDE11A isoforms. Its specificity was demonstrated by Western blot analysis on a recombinant human PDE11A protein and native PDE11A proteins in various human tissues. Immunohistochemistry showed that PDE11A is widely expressed. Various degrees of immunoreactivity were observed in the epithelial cells, endothelial cells, and smooth muscle cells of all tissues examined. The highest expression was in the epithelial, endothelial, and smooth muscle cells of the prostate, Leydig, and spermatogenic cells of the testis, the tubule epithelial cells in the kidney, the epithelial and endothelial cells in the adrenal, the epithelial cells and macrophages in the colon, and the epidermis in the skin. Furthermore, PDE11A expression was also detected in several human carcinomas. Our results suggest that PDE11A might be involved in multiple physiological processes in various organs via its ability to modulate intracellular cAMP and cGMP levels.     

Thrombin Has Biphasic Effects on the Nitric Oxide-cGMP Pathway in Endothelial Cells and Contributes to Experimental Pulmonary Hypertension

Background

A potential role for coagulation factors in pulmonary arterial hypertension has been recently described, but the mechanism of action is currently not known. Here, we investigated the interactions between thrombin and the nitric oxide-cGMP pathway in pulmonary endothelial cells and experimental pulmonary hypertension.

Principal Findings

Chronic treatment with the selective thrombin inhibitor melagatran (0.9 mg/kg daily via implanted minipumps) reduced right ventricular hypertrophy in the rat monocrotaline model of experimental pulmonary hypertension. In vitro, thrombin was found to have biphasic effects on key regulators of the nitric oxide-cGMP pathway in endothelial cells (HUVECs). Acute thrombin stimulation led to increased expression of the cGMP-elevating factors endothelial nitric oxide synthase (eNOS) and soluble guanylate cyclase (sGC) subunits, leading to increased cGMP levels. By contrast, prolonged exposition of pulmonary endothelial cells to thrombin revealed a characteristic pattern of differential expression of the key regulators of the nitric oxide-cGMP pathway, in which specifically the factors contributing to cGMP elevation (eNOS and sGC) were reduced and the cGMP-hydrolyzing PDE5 was elevated (qPCR and Western blot). In line with the differential expression of key regulators of the nitric oxide-cGMP pathway, a reduction of cGMP by prolonged thrombin stimulation was found. The effects of prolonged thrombin exposure were confirmed in endothelial cells of pulmonary origin (HPAECs and HPMECs). Similar effects could be induced by activation of protease-activated receptor-1 (PAR-1).

Conclusion

These findings suggest a link between thrombin generation and cGMP depletion in lung endothelial cells through negative regulation of the nitric oxide-cGMP pathway, possibly mediated via PAR-1, which could be of relevance in pulmonary arterial hypertension.     

Expression and activity of cAMP phosphodiesterase isoforms in pulmonary artery smooth muscle cells from patients with pulmonary hypertension: role for PDE1

Pulmonary hypertension (PHT) is associated with increased vascular resistance due to sustained contraction and enhanced proliferation of pulmonary arterial smooth muscle cells (PASMC); the abnormal tone and remodeling in the pulmonary vasculature may relate, at least in part, to decreased cyclic nucleotide levels. Cyclic nucleotide phosphodiesterases (PDEs), of which 11 families have been identified, catalyze the hydrolysis of cAMP and cGMP. We tested the hypothesis that PASMC isolated from patients with PHT, either idiopathic pulmonary arterial hypertension (IPAH) or secondary pulmonary hypertension (SPH), have increased expression and activity of PDE isoforms that reduce the responsiveness of agents that raise cellular cAMP. Real-time PCR and immunoblotting demonstrated that the expression of PDE1A, PDE1C, PDE3B, and PDE5A was enhanced in PASMC from both IPAH and SPH patients compared with control PASMC. Consistent with this enhanced expression of PDEs, agonist-stimulated cAMP levels were significantly reduced in IPAH and SPH PASMC unless a PDE inhibitor was present. The use of specific PDE inhibitors revealed that an increase in PDE1 and PDE3 activity largely accounted for reduced agonist-induced cAMP levels and increased proliferation in IPAH and SPH PASMC. Treatment with PDE1C-targeted small interference RNA enhanced cAMP accumulation and inhibited cellular proliferation to a greater extent in PHT PASMC than controls. The results imply that an increase in PDE isoforms, in particular PDE1C, contributes to decreased cAMP and increased proliferation of PASMC in patients with PHT. PDE1 isoforms may provide novel targets for the treatment of both primary and secondary forms of the disease.     

Cyclic nucleotide phosphodiesterase PDE1C1 in human cardiac myocytes

Isoforms in the PDE1 family of cyclic nucleotide phosphodiesterases were recently found to comprise a significant portion of the cGMP-inhibited cAMP hydrolytic activity in human hearts. We examined the expression of PDE1 isoforms in human myocardium, characterized their catalytic activity, and quantified their contribution to cAMP hydrolytic and cGMP hydrolytic activity in subcellular fractions of this tissue. Western blotting with isoform-selective anti-PDE1 monoclonal antibodies showed PDE1C1 to be the principal isoform expressed in human myocardium. Immunohistochemical analysis showed that PDE1C1 is distributed along the Z-lines and M-lines of cardiac myocytes in a striated pattern that differs from that of the other major dual-specificity cyclic nucleotide phosphodiesterase in human myocardium, PDE3A. Most of the PDE1C1 activity was recovered in soluble fractions of human myocardium. It binds both cAMP and cGMP with K(m) values of approximately 1 microm and hydrolyzes both substrates with similar catalytic rates. PDE1C1 activity in subcellular fractions was quantified using a new PDE1-selective inhibitor, IC295. At substrate concentrations of 0.1 microm, PDE1C1 constitutes the great majority of cAMP hydrolytic and cGMP hydrolytic activity in soluble fractions and the majority of cGMP hydrolytic activity in microsomal fractions, whereas PDE3 constitutes the majority of cAMP hydrolytic activity in microsomal fractions. These results indicate that PDE1C1 is expressed at high levels in human cardiac myocytes with an intracellular distribution distinct from that of PDE3A and that it may have a role in the integration of cGMP-, cAMP- and Ca(2+)-mediated signaling in these cells.     

Cyclic nucleotide phosphodiesterase (PDE) inhibitors: novel therapeutic agents for progressive renal disease

Cyclic nucleotides are recognized as critical mediators of many renal functions, including solute transport, regulation of vascular tone, proliferation of parenchymal cells, and inflammation. Although most studies have linked elevated cAMP levels to activation of protein kinase A, cAMP can also directly activate cyclic nucleotide gated ion channels and can signal through activation of GTP exchange factors. Cyclic AMP signaling is highly compartmentalized through plasma membrane localization of adenylyl cyclase and expression of scaffolding proteins that anchor protein kinase A to specific intracellular locations. Cyclic nucleotide levels are largely regulated through catabolic processes directed by phosphodiesterases (PDEs). The PDE superfamily is large and complex, with over 60 distinct isoforms that preferentially hydrolyze cAMP, cGMP, or both. PDEs contribute to compartmentalized cyclic nucleotide signaling. The unique cell- and tissue-specific distribution of PDEs has prompted the development of highly specific PDE inhibitors to treat a variety of inflammatory conditions. In experimental systems, PDE inhibitors have been employed to demonstrate functional compartmentalization of cyclic nucleotide signaling in the kidney. For example, mitogenesis in glomerular mesangial cells and normal tubular epithelial cells is negatively regulated by an intracellular pool of cAMP that is metabolized by PDE3, but not by other PDEs. In Madin-Darby canine kidney cells, an in vitro model of polycystic kidney disease, an intracellular pool of cAMP directed by PDE3 stimulates mitogenesis. In mesangial cells, an intracellular pool of cAMP directed by PDE4 inhibits reactive oxygen species and expression of the potent proin-flammatory cytokine monocyte chemoattractant protein 1. An intracellular pool of cGMP directed by PDE5 regulates solute transport. PDE5 inhibitors ameliorate renal injury in a chronic renal disease model. In this overview, we highlight recent studies to define relationships between PDE expression and renal function and to provide evidence that PDE inhibitors may be effective agents in treating chronic renal disease.     

The Emperor's New Clothes: PDE5 and the Heart

Phosphodiesterase-5 (PDE5) is highly expressed in the pulmonary vasculature, but its expression in the myocardium is controversial. Cyclic guanosine monophosphate (cGMP) activates protein kinase G (PKG), which has been hypothesized to blunt cardiac hypertrophy and negative remodeling in heart failure. Although PDE5 has been suggested to play a significant role in the breakdown of cGMP in cardiomyocytes and hence PKG regulation in the myocardium, the RELAX trial, which tested effect of PDE5 inhibition on exercise capacity in patients with heart failure with preserved ejection fraction (HFpEF) failed to show a beneficial effect. These results highlight the controversy regarding the role and expression of PDE5 in the healthy and failing heart. This study used one- and two-dimensional electrophoresis and Western blotting to examine PDE5 expression in mouse (before and after trans-aortic constriction), dog (control and HFpEF) as well as human (healthy and failing) heart. We were unable to detect PDE5 in any cardiac tissue lysate, whereas PDE5 was present in the murine and bovine lung samples used as positive controls. These results indicate that if PDE5 is expressed in cardiac tissue, it is present in very low quantities, as PDE5 was not detected in either humans or any model of heart failure examined. Therefore in cardiac muscle, it is unlikely that PDE5 is involved the regulation of cGMP-PKG signaling, and hence PDE5 does not represent a suitable drug target for the treatment of cardiac hypertrophy. These results highlight the importance of rigorous investigation prior to clinical trial design.     

Calmodulin-dependent cyclic nucleotide phosphodiesterase (PDE1) splice variants from bovine cardiac muscle

Calmodulin-dependent cyclic nucleotide phopshodiesterase (PDE1) has been extensively characterized and is a key enzyme involved in the complex interaction between cyclic nucleotide and Ca(2+) second-messenger systems. It is well established that PDE1 exists in different isozymes. For example, bovine brain tissue has two PDE1 isozymes (PDE1A2 and PDE1B1) whereas only one form (PDE1A1) is reported in bovine cardiac tissue. In this study, we report the cloning of two cDNA splice variants of PDE1: PDE1-small and PDE1-large, from bovine cardiac tissue. Their amino acid sequence similarity to PDE1 sequences from other mammalian species showed that all are very conserved, suggesting their importance in cellular functions. Interestingly, compared to other mammalian species, bovine PDE1A, PDE-small and PDE-large show a deletion at the C-terminal end of the catalytic domain of the gene. Although the significance of this deletion at this crucial location of the gene is not known, we have successfully over-expressed both PDE1-small and PDE1-large splice variants in E. coli and these splice variants are characterized in terms of Western blot, biotinylated calmodulin overlay and peptide mass fingerprinting. Results from these studies suggested that these two splice variants belong to the PDE1 superfamily. To our knowledge, this is the first report on cloning and characterization of these cDNA variants from bovine cardiac tissue. Since there are at least two isoforms of PDE1 in bovine heart tissue, this merits further in-depth study.     

Expression of C-type natriuretic peptide and its receptor NPR-B in cardiomyocytes

C-type natriuretic peptide (CNP) was recently found in myocardium at the mRNA and protein levels, but it is not known whether cardiomyocytes are able to produce CNP. The aim of this study was to determine the expression of CNP and its specific receptor NPR-B in cardiac cells, both in vitro and ex vivo. CNP, brain natriuretic peptide (BNP) and natriuretic peptide receptor (NPR)-B mRNA expression were examined by RT-PCR in the H9c2 rat cardiac myoblast cell line, in neonatal rat primary cardiomyocytes and in human umbilical vein endothelial cells (HUVECs) as control. CNP protein expression was probed in cardiac tissue sections obtained from adult male minipigs by immunohistochemistry, and in H9c2 cells both by immunocytochemistry and by specific radioimmunoassay. The results showed that cardiac cells as well as endothelial cells were able to produce CNP. Unlike cardiomyocytes, as expected, in endothelial cells expression of BNP was not detected. NPR-B mRNA expression was found in both cell types. Production of CNP in the heart muscle cells at protein level was confirmed by radioimmunological determination (H9c2: CNP = 0.86 ± 0.083 pg/mg) and by immunocytochemistry studies. By immunostaining of tissue sections, CNP was detected in both endothelium and cardiomyocytes. Expression of CNP in cardiac cells at gene and protein levels suggests that the heart is actively involved in the production of CNP.     

Immunocytochemical localization of the endogenous vasoactive peptide apelin to human vascular and endocardial endothelial cells

Apelin, the proposed endogenous peptide ligand of the novel G-protein-coupled receptor APJ, has been shown to possess potent vasodilator and positive inotropic effects in rats and humans in vivo. However, in humans, no endogenous source of apelin has been reported. Therefore, based on the presence of APJ and mRNA encoding apelin in human tissues, we investigated the expression of apelin in fresh-frozen human tissue from right atrium, left ventricle, lung, kidney, adrenal and large conduit vessels using immunocytochemistry. Apelin-like immunoreactivity (apelin-LI) was detected in vascular endothelial cells lining blood vessels in the human heart, kidney, adrenal gland and lung and in endothelial cells of large conduit vessels. Apelin-LI was also present in endocardial endothelial cells lining recesses of the right atrium. Apelin-LI was not present or below the level of detection in cardiomyocytes, Purkinje's cells, pulmonary or renal epithelial cells, secretory cells of the adrenal gland, vascular smooth muscle cells, adipocytes, nerves and connective tissue. The restricted presence of apelin-LI in endothelial cells suggests that endothelial apelin may play a role as a locally secreted cardiovascular mediator acting on APJ receptors present on the vascular smooth muscle and on cardiac myocytes to regulate vascular tone and cardiac contractility.     

Phosphodiesterase 3B (PDE3B) antagonizes the anti-angiogenic actions of PKA in human and murine endothelial cells

Recent reports show that protein kinase A (PKA), but not exchange protein activated by cAMP (EPAC), acts in a cell autonomous manner to constitutively reduce the angiogenic sprouting capacity of murine and human endothelial cells. Specificity in the cellular actions of individual cAMP-effectors can be achieved when a cyclic nucleotide phosphodiesterase (PDE) enzyme acts locally to control the “pool” of cAMP that activates the cAMP-effector. Here, we examined whether PDEs coordinate the actions of PKA during endothelial cell sprouting. Inhibiting each of the cAMP-hydrolyzing PDEs expressed in human endothelial cells revealed that phosphodiesterase 3 (PDE3) inhibition with cilostamide reduced angiogenic sprouting in vitro, while inhibitors of PDE2 and PDE4 family enzymes had no such effect. Identifying a critical role for PDE3B in the anti-angiogenic effects of cilostamide, silencing this PDE3 variant, but not PDE3A, markedly impaired sprouting. Importantly, using both in vitro and ex vivo models of angiogenesis, we show the hypo-sprouting phenotype induced by PDE3 inhibition or PDE3B silencing was reversed by PKA inhibition. Examination of the individual cellular events required for sprouting revealed that PDE3B and PKA each regulated angiogenic sprouting by controlling the invasive capacity of endothelial cells, more specifically, by regulating podosome rosette biogenesis and matrix degradation. In support of the idea that PDE3B acts to inhibit angiogenic sprouting by limiting PKA-mediated reductions in active cdc42, the effects of PDE3B and/or PKA on angiogenic sprouting were negated in cells with reduced cdc42 expression or activity. Since PDE3B and PKA were co-localized in a perinuclear region in human ECs, could be co-immunoprecipitated from lysates of these cells, and silencing PDE3B activated the perinuclear pool of PKA in these cells, we conclude that PDE3B-mediated hydrolysis of cAMP acts to limit the anti-angiogenic potential of PKA in ECs.     

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. (J Histochem Cytochem 57:933–949, 2009)     

Concerted regulation of cGMP and cAMP phosphodiesterases in early cardiac hypertrophy induced by angiotensin II

Left ventricular hypertrophy leads to heart failure and represents a high risk leading to premature death. Cyclic nucleotides (cAMP and cGMP) play a major role in heart contractility and cyclic nucleotide phosphodiesterases (PDEs) are involved in different stages of advanced cardiac diseases. We have investigated their contributions in the very initial stages of left ventricular hypertrophy development. Wistar male rats were treated over two weeks by chronic infusion of angiotensin II using osmotic mini-pumps. Left cardiac ventricles were used as total homogenates for analysis. PDE1 to PDE5 specific activities and protein and mRNA expressions were explored.Rats developed arterial hypertension associated with a slight cardiac hypertrophy (+24%). cAMP-PDE4 activity was specifically increased while cGMP-PDE activities were broadly increased (+130% for PDE1; +76% for PDE2; +113% for PDE5) and associated with increased expressions for PDE1A, PDE1C and PDE5A. The cGMP-PDE1 activation by Ca(2+)/CaM was reduced. BNP expression was increased by 3.5-fold, while NOX2 expression was reduced by 66% and AMP kinase activation was increased by 64%. In early cardiac hypertrophy induced by angiotensin II, all specific PDE activities in left cardiac ventricles were increased, favoring an increase in cGMP hydrolysis by PDE1, PDE2 and PDE5. Increased cAMP hydrolysis was related to PDE4. We observed the establishment of two cardioprotective mechanisms and we suggest that these mechanisms could lead to increase intracellular cGMP: i) increased expression of BNP could increase "particulate" cGMP pool; ii) increased activation of AMPK, subsequent to increase in PDE4 activity and 5'AMP generation, could elevate "soluble" cGMP pool by enhancing NO bioavailability through NOX2 down-regulation. More studies are needed to support these assumptions. Nevertheless, our results suggest a potential link between PDE4 and AMPK/NOX2 and they point out that cGMP-PDEs, especially PDE1 and PDE2, may be interesting therapeutic targets in preventing cardiac hypertrophy.     

Cyclic nucleotide phosphodiesterases (PDEs) in human osteoblastic cells; the effect of PDE inhibition on cAMP accumulation

The regulation of the secondary messengers, cyclic adenosine monophosphate (cAMP) and cyclic guanosine monophosphate (cGMP), is crucial in the hormonal regulation of bone metabolism. Both cAMP and cGMP are inactivated by cyclic nucleotide phosphodiesterases (PDEs), a superfamily of enzymes divided into 11 families (PDE1-11). We compared the PDEs of cultured human osteoblasts (NHOst) and SaOS-2 osteosarcoma cells. The PDE activity of NHOst cells consisted of PDE1, PDE3 and PDE7, whereas PDE1, PDE7 and PDE4, but no PDE3 activity was detected in SaOS-2 cells. In line with the difference in the PDE profiles, rolipram, a PDE4 inhibitor, increased the accumulation of cAMP in SaOS-2, but not in NHOst cells. Expression of PDE subtypes PDE1C, PDE3A, PDE4A, PDE4B, PDE7A and PDE7B was detected in both cell types. NHOst cells additionally expressed PDE1A.     

Comparative study of cellular and extracellular matrix composition of native and tissue engineered heart valves.

Tissue engineering of heart valves utilizes biodegradable or metabolizable scaffolds for remodeling by seeded autologous cells. The aim of this study was to determine and compare extracellular matrix (ECM) formations, cellular phenotypes and cell location of native and tissue engineered (TE) valve leaflets. Ovine carotid arteries, ovine and porcine hearts were obtained from slaughterhouses. Cells were isolated from carotid arteries and dissected ovine, porcine and TE leaflets. TE constructs were fabricated from decellularized porcine pulmonary valves, seeded ovine arterial cells and subsequent 16 days dynamic in vitro culture using a pulsatile bioreactor. Native and TE valves were studied by histology (hematoxylin-eosin, resorcin-fuchsin, Movat pentachrome), NIR femtosecond multiphoton laser scanning microscopy and scanning electron microscopy (SEM). Cells of native and TE tissues were identified and localized by immunohistochemistry. Arterial, valvular and re-isolated TE-construct cells were processed for immunocytochemistry and Western blotting. ECM analysis and SEM revealed characteristical and comparable structures in native and TE leaflets. Most cells in native leaflets stained strongly positive for vimentin. Cells positive to alpha-smooth muscle actin (alpha-SMA), myosin and calponin were only found at the ventricular (inflow) side of ovine aortic and porcine pulmonary valve leaflets. Cells from TE constructs had a strong expression of vimentin, alpha-SMA, myosin, calponin and h-caldesmon throughout the entire leaflet. Comparable ECM formation and endothelial cell lining of native and TE leaflets could be demonstrated. However, immunostaining revealed significant differences between valvular cell phenotypes of native and TE leaflets. These results may be essential for further cardiovascular tissue engineering efforts.     

Cloning and localization of the cGMP-specific phosphodiesterase type 9 in the rat brain

In this study, we report the cloning of the rat cGMP-specific phosphodiesterase type 9 (PDE9A) and its localization in rat and mouse brain by non-radioactive in situ hybridization. Rat PDE9A was 97.6% identical to mouse PDE9A1 and showed 92.1% similarity on the amino acid level to the human homologue. PDE9A mRNA was widely distributed throughout the rat and mouse brain, with the highest expression observed in cerebellar Purkinje cells. Furthermore, strong staining was detected in areas such as cortical layer V, olfactory tubercle, caudate putamen and hippocampal pyramidal and granule cells. Comparison of PDE9A mRNA expression by double staining with the cellular markers NeuN and glial fibrillary acidic protein demonstrated that PDE9A expression was mainly detected in neurons and in a subpopulation of astrocytes. Using cGMP-immunocytochemistry, the localization of cGMP was investigated in the cerebellum in which the highest PDE9 expression was demonstrated. Strong cGMP immunoreactivity was detected in the molecular layer in the presence of the non-selective PDE inhibitor 3-isobutyl-1-methylxanthine (IBMX). After treatment with soluble guanylyl cyclase activators the granular layer also showed cGMP staining, whereas no clear immunostaining was detected in Purkinje cells under all conditions investigated, which might be due to the presence of the IBMX-insensitive PDE9A in these cells. The present findings indicate that PDE9A is highly conserved between species and is widely distributed throughout the rodent brain. PDE9A is probably involved in maintenance of low cGMP levels in cells and might play an important role in a variety of brain functions involving cGMP-mediated signal transduction.