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cGMP inhibits hypertrophy, decreases fibrosis, and protects against cardiac ischemia-reperfusion (I/R) injury. Gene-targeting studies have not defined a clear role for its major downstream effector, cGMP-dependent protein kinase I (cGKI), in cardiac hypertrophy, but do implicate cGMP-cGKI signaling in fibrosis and I/R injury. No direct cGKI activators have advanced to clinical trials, whereas cardiac trials of agents that modulate cGMP via particulate or soluble guanylyl cyclases (GCs) and phosphodiesterase 5 (PDE5) are ongoing. Here we review concerns arising from preclinical and clinical studies that question whether targeting the cGMP pathway remains an encouraging concept for management of heart dysfunction. So far, trial results for GC modulators are inconclusive, and sildenafil, a PDE5 inhibitor, although cardioprotective in mouse models, has not shown positive clinical results. Preclinical cardioprotection observed for sildenafil may result from inhibition of PDE5 in non-cardiomyocytes or off-target effects, possibly on PDE1C. On the basis of such mechanistic considerations, re-evaluation of the cellular localization of drug target(s) and intervention protocols for cGMP-elevating agents may be needed.

Microglial phagocytosis plays a key role in neuroprotective and neurodegenerative responses of the innate immune system in the brain. Here we investigated the regulatory function of phosphoinositide 3-kinase γ (PI3Kγ) in phagocytosis of bacteria and Zymosan particles by mouse brain microglia in vitro and in vivo. Using genetic and pharmacological approaches our data revealed PI3Kγ as an essential mediator of microglial phagocytosis. Unexpectedly, microglia expressing lipid kinase deficient mutant PI3Kγ exhibited similar phagocytosis as wild-type cells. These data suggest kinase-independent stimulation of cAMP phosphodiesterase activity by PI3Kγ as a crucial mediator of phagocytosis. In sum our findings indicate PI3Kγ-dependent suppression of cAMP signaling as a critical regulatory element of microglial phagocytosis.

The meiotic cell cycle of mammalian oocytes in preovulatory follicles is held in prophase arrest by diffusion of cGMP from the surrounding granulosa cells into the oocyte. Luteinizing hormone (LH) then releases meiotic arrest by lowering cGMP in the granulosa cells. The LH-induced reduction of cGMP is caused in part by a decrease in guanylyl cyclase activity, but the observation that the cGMP phosphodiesterase PDE5 is phosphorylated during LH signaling suggests that an increase in PDE5 activity could also contribute. To investigate this idea, we measured cGMP-hydrolytic activity in rat ovarian follicles. Basal activity was due primarily to PDE1A and PDE5, and LH increased PDE5 activity. The increase in PDE5 activity was accompanied by phosphorylation of PDE5 at serine 92, a protein kinase A/G consensus site. Both the phosphorylation and the increase in activity were promoted by elevating cAMP and opposed by inhibiting protein kinase A, supporting the hypothesis that LH activates PDE5 by stimulating its phosphorylation by protein kinase A. Inhibition of PDE5 activity partially suppressed LH-induced meiotic resumption as indicated by nuclear envelope breakdown, but inhibition of both PDE5 and PDE1 activities was needed to completely inhibit this response. These results show that activities of both PDE5 and PDE1 contribute to the LH-induced resumption of meiosis in rat oocytes, and that phosphorylation and activation of PDE5 is a regulatory mechanism.

It has been suggested that cGMP kinase I (cGKI) dampens cardiac hypertrophy. We have compared the effect of isoproterenol (ISO) and transverse aortic constriction (TAC) on hypertrophy in WT [control (CTR)] mice, total cGKI-KO mice, and cGKIβ rescue mice (βRM) lacking cGKI specifically in cardiomyocytes (CMs). Infusion of ISO did not change the expression of cGKI in the hearts of CTR mice or βRM but raised the heart weight by ∼20% in both. An identical hypertrophic growth response was measured in CMs from CTR mice and βRM and in isolated adult CMs cultured with or without 1 μM ISO. In both genotypes, ISO infusion induced similar changes in the expression of hypertrophy-associated cardiac genes and significant elevation of serum atrial natriuretic peptide and total cardiac cGMP. No differences in cardiac hypertrophy were obtained by 7-day ISO infusion in 4- to 6-week-old conventional cGKI-KO and CTR mice. Furthermore, TAC-induced hypertrophy of CTR mice and βRM was not different and did not result in changes of the cGMP-hydrolyzing phosphodiesterase activities in hypertropic hearts or CMs. These results strongly suggest that cardiac myocyte cGKI does not affect the development of heart hypertrophy induced by pressure overload or chronic ISO infusion.

cGMP-specific phosphodiesterase (PDE5) has become a target for drug development for the treatment of a number of physiological dysfunctions, affected by changes in the cGMP/cGMP-dependent protein kinase (PKG) signaling pathway. PDE5 has two highly homologous regulatory domains, GAF-A and GAF-B. We showed previously that PDE5 could be converted from a low-activity (nonactivated) state to a high-activity state upon cGMP binding to the GAF-A domain with higher sensitivities toward sildenafil (EMBO J 22:469-478, 2003). Here we investigated whether sildenafil sensitivity of PDE5 could be modified by cGMP-independent mechanisms. Individually expressed recombinant GAF-A and GAF-B proteins were tested for their ability to modulate full-length recombinant PDE5 affinity to sildenafil. The GAF-A domain protein had the most dramatic effect on the affinity of the nonactivated recombinant PDE5 for sildenafil, revealing much higher sensitivity to sildenafil inhibition. The apparent affinity for sildenafil increased from the nanomolar range to the picomolar range, providing evidence for the presence of a "super-high" sensitivity state of PDE5 for sildenafil inhibition. In human platelet, higher sensitivity of PDE5 for sildenafil inhibition has been detected after blocking cGMP-binding sites of the GAF-A domain. Thus, our data demonstrate that high sensitivity of PDE5 for sildenafil can be obtained not only through cGMP-induced activation of PDE, but also through cGMP-independent modulation of PDE5 in the nonactivated state, possibly through protein-protein interaction. Furthermore, data suggest that nonactivated PDE5 with "super-high" affinities for sildenafil inhibition may be responsible for therapeutic effects of long-term treatments with low doses of PDE5 inhibitors.

The meiotic cell cycle of mammalian oocytes in preovulatory follicles is held in prophase arrest by diffusion of cGMP from the surrounding granulosa cells into the oocyte. Luteinizing hormone (LH) then releases meiotic arrest by lowering cGMP in the granulosa cells. The LH-induced reduction of cGMP is caused in part by a decrease in guanylyl cyclase activity, but the observation that the cGMP phosphodiesterase PDE5 is phosphorylated during LH signaling suggests that an increase in PDE5 activity could also contribute. To investigate this idea, we measured cGMP-hydrolytic activity in rat ovarian follicles. Basal activity was due primarily to PDE1A and PDE5, and LH increased PDE5 activity. The increase in PDE5 activity was accompanied by phosphorylation of PDE5 at serine 92, a protein kinase A/G consensus site. Both the phosphorylation and the increase in activity were promoted by elevating cAMP and opposed by inhibiting protein kinase A, supporting the hypothesis that LH activates PDE5 by stimulating its phosphorylation by protein kinase A. Inhibition of PDE5 activity partially suppressed LH-induced meiotic resumption as indicated by nuclear envelope breakdown, but inhibition of both PDE5 and PDE1 activities was needed to completely inhibit this response. These results show that activities of both PDE5 and PDE1 contribute to the LH-induced resumption of meiosis in rat oocytes, and that phosphorylation and activation of PDE5 is a regulatory mechanism.

The G protein-coupled, receptor-activated phosphoinositide 3-kinase gamma (PI3Kgamma) mediates inflammatory responses and negatively controls cardiac contractility by reducing cAMP concentration. Here, we report that mice carrying a targeted mutation in the PI3Kgamma gene causing loss of kinase activity (PI3KgammaKD/KD) display reduced inflammatory reactions but no alterations in cardiac contractility. We show that, in PI3KgammaKD/KD hearts, cAMP levels are normal and that PI3Kgamma-deficient mice but not PI3KgammaKD/KD mice develop dramatic myocardial damage after chronic pressure overload induced by transverse aortic constriction (TAC). Finally, our data indicate that PI3Kgamma is an essential component of a complex controlling PDE3B phosphodiesterase-mediated cAMP destruction. Thus, cardiac PI3Kgamma participates in two distinct signaling pathways: a kinase-dependent activity that controls PKB/Akt as well as MAPK phosphorylation and contributes to TAC-induced cardiac remodeling, and a kinase-independent activity that relies on protein interactions to regulate PDE3B activity and negatively modulates cardiac contractility.

The nitric oxide (NO)-cGMP pathway has been implicated as playing a crucial role in the induction of cerebellar long-term depression (LTD). The amplitude and duration of the cGMP signal is controlled by cyclic nucleotide phosphodiesterases (PDEs). Here we identify PDE5 and PDE1B as the two major cGMP-hydrolyzing PDEs specifically and differentially expressed in the Purkinje neurons of mouse cerebellum. PDE5 was found in all Purkinje neurons, whereas PDE1B was detected only in a subset of these cells, suggesting that individual Purkinje cells may differentially regulate cGMP, depending on the PDE isozymes expressed.Although expression of guanylate cyclase and/or cGMP-dependent protein kinase (PKG) in Purkinje cells have been reported, neither cGMP accumulation nor PKG activation in these cellsin vivohas been demonstrated. To determine if changes in PKG activation and PDE5 regulation occurin vivowe have examined the phosphorylation of PDE5 in mouse cerebellar Purkinje cells by immunocytochemistry and Western blot analyses using a phosphospecific PDE5 antibody. Injection of sodium nitroprusside or selective PKG activators into the lateral ventricle of mouse brain induced PDE5 phosphorylationin vivo, but was completely missing in Purkinje cell-specific PKG I knock-out mice. In cerebellar slices, treatment with sildenafil or IBMX led to different levels of phospho-PDE5 accumulation and activation of PDE5. These results suggest that phosphorylation of PDE5 in Purkinje neurons after cGMP-PKG activation performs a critical role in the termination of the cGMP signal during LTD progression; moreover, PDE5 phosphorylation may be used as anin vivoindicator for PKG activation.

cGMP-specific, cGMP-binding phosphodiesterase (PDE5) regulates such physiological processes as smooth muscle relaxation and neuronal survival. PDE5 contains two N-terminal domains (GAF A and GAF B), but the functional roles of these domains have not been determined. Here we show that recombinant PDE5 is activated directly upon cGMP binding to the GAF A domain, and this effect does not require PDE5 phosphorylation. PDE5 exhibited time- and concentration-dependent reversible activation in response to cGMP, with the highest activation (9- to 11-fold) observed at low substrate concentrations (0.1 micro M cGMP). A monoclonal antibody directed against GAF A blocked cGMP binding, prevented PDE5 activation and decreased basal activity, revealing that PDE5 in its non-activated state has low intrinsic catalytic activity. Activated PDE5 showed higher sensitivity towards sildenafil than non-activated PDE5. The stimulatory effect of cGMP binding on the catalytic activity of PDE5 suggests that this mechanism of enzyme activation may be common among other GAF domain-containing proteins. The data also suggest that development of agonists and antagonists of PDE5 activity based on binding to this site might be possible.

IntroductionSmooth muscle cells (SMCs) in the arterial wall are normally found in a contractile, nonproliferative state. These SMCs are continuously exposed to agents that elevate cyclic AMP (cAMP) and cyclic GMP (cGMP), such as prostacyclin and nitric oxide (NO) released from the endothelium. Both cAMP and cGMP potently inhibit SMC proliferation by antagonizing major signaling pathways induced by growth factors, such as platelet-derived growth factor. Different forms of injury to the endothelium result in proliferation of the SMCs and can develop into atherosclerosis, restenosis, or arterial thickening associated with neonatal pulmonary hypertension. In human adult aorta, cAMP and cGMP levels are tightly regulated by synthesis and degradation. The latter is mediated by at least two different cAMP phosphodiesterases (PDEs), PDE3 and PDE4, and three different PDEs that degrade mainly cGMP, PDE1A, PDE1B, and PDE5. Strikingly, expression of a new PDE that degrades both cAMP and cGMP, PDE1C, is markedly induced in proliferating SMCs from the same aorta in culture, whereas its expression is nondetectable in contractile aorta. We propose that, for a human arterial SMC to be able to efficiently proliferate, and perhaps even to undergo a phenotypic change from a quiescent to a proliferative phenotype, it has to efficiently degrade inhibitory cAMP and cGMP. In human SMCs, PDE1C is induced to degrade these cyclic nucleotides. Understanding the role of PDE1C in SMC proliferation may provide the basic information necessary for development of highly specific PDE1C inhibitors that target proliferating SMCs in cardiovascular disease.

The diversity among cyclic nucleotide phosphodiesterases provides multiple mechanisms for regulation of cAMP and cGMP in the cardiovascular system. Here we report that a calmodulin-stimulated phosphodiesterase (PDE1C) is highly expressed in proliferating human arterial smooth muscle cells (SMCs) in primary culture, but not in the quiescent SMCs of intact human aorta. High levels of PDE1C were found in primary cultures of SMCs derived from explants of human newborn and adult aortas, and in SMCs cultured from severe atherosclerotic lesions. PDE1C was the major cAMP hydrolytic activity in these SMCs. PDE expression patterns in primary SMC cultures from monkey and rat aortas were different from those from human cells. In monkey, high expression of PDE1B was found, whereas PDE1C was not detected. In rat SMCs, PDE1A was the only detectable calmodulin-stimulated PDE. These findings suggest that many of the commonly used animal species may not provide good models for studying the roles of PDEs in proliferation of human SMCs. More importantly, the observation that PDE1C is induced only in proliferating SMCs suggests that it may be both an indicator of proliferation and a possible target for treatment of atherosclerosis or restenosis after angioplasty, conditions in which proliferation of arterial SMCs is negatively modulated by cyclic nucleotides.

Cyclic nucleotides (cAMP and cGMP) as second messengers regulate a wide variety of biological processes such as cellular growth, secretary signaling and neuroplasticity (1). These processes can be regulated by increasing the synthesis of cyclic nucleotides (cyclases), by regulation of cAMP and cGMP effector proteins such as cAMP- and cGMP-dependent protein kinases, or by regulation of cyclic nucleotide degradation via cyclic nucleotide phosphodiestases (PDEs) (2). At present PDEs are classified into 11 gene families, each containing several different isoforms and splice variants. All PDEs share considerable homology in their catalytic domains but substantially differ in their N-terminal regions, that contain different types of regulatory domains (3). The different PDEs show complex substrate specificity. PDE5, PDE6 and PDE9 are considered to be cGMP specific, while PDE1, PDE2, PDE3, PDE10 and PDE11 can hydrolyze both cGMP and cAMP. PDE4, PDE7 and PDE8 use mainly cAMP as their substrates at physiological substrate levels. Here we described two methods designed for measuring cGMP (cAMP) hydrolytic activities. The first one is a tradiotional method using radioactive substrates and the second one is a recently developed non-radioactive method based on Isothermal Titration Calorimetry.

Recent results show that the classic PDE5 inhibitor, Viagra® (sildenafil), can ameliorate much of the cardiac pathology resulting from several different forms of cardiac damage. These results have elicited great interest in the use of this drug as a therapeutic agent, particularly for treatment of diastolic cardiac dysfunction. In fact several clinical trials are now enrolling and one published trial shows promising results. Unfortunately, it is not clear how this drug works at a molecular level to improve cardiac function and not even clear what the initial molecular target(s) are for Viagra® in any of these studies. Recent data from our group and others strongly suggest that sildenafil, a “classical” PDE5 inhibitor, ameliorates much of the diastolic dysfunction seen in older mdx mice, animals having a disruption in the dystrophin gene. However, our preliminary data suggests that tadalafil does not cause this effect. Moreover, the usual accepted target for sildenafil, PDE5, is not even expressed in adult mouse cardiomyocyes; but an alternate or additional target, PDE1C is highly expressed in this cell type. Since PDE1C, also can be substantially inhibited by sildenafil (but not by tadalafil) we feel that the most likely molecular target for Viagra® is a direct effect on PDE1C in the cardiomyocyte itself. There also may be additional effects on PDE5, but probably in another cell type. These topics will be discussed in this talk.

Concerning the letter by Kass and Takimoto (1) in PNAS regarding our recent publication (2), we would like to make the following comments. First, we agree that our results showing a “lack of importance” for cGMP-dependent protein kinase I (cGKI) as a modulator of cardiac hypertrophy only refers to the “basal” condition as we clearly stated in the manuscript (1, 2). We purposely did not use guanylyl cyclase stimulators or phosphodiesterase (PDE) inhibitors in these studies. However, we do note that, particularly under the stress conditions of isoproterenol infusion or aortic constriction, atrial natriuretic peptide is substantially increased. This provides substantial drive on cGMP synthesis. It is possible that cGKI needs to be induced by very high cGMP to have protective effects, although induction is not a requirement for the importance of this kinase in other tissues. The point here is that ablation of cGKI from cardiomyocytes and most other tissues (except vasculature) does not potentiate the ability of transverse aortic constriction (TAC) or isoproterenol (ISO) to cause hypertrophy. To us, this is an observation that needs to be reconciled with current dogma. The degrees of hypertrophy obtained in our studies were equal to or even higher than those obtained in many other studies.

Rationale: Cyclic nucleotide phosphodiesterases (PDEs) through the degradation of cGMP play critical roles in maintaining cardiomyocyte homeostasis. Ca 2+ /calmodulin (CaM)-activated cGMP-hydrolyzing PDE1 family may play a pivotal role in balancing intracellular Ca 2+ /CaM and cGMP signaling; however, its function in cardiomyocytes is unknown. Objective: Herein, we investigate the role of Ca 2+ /CaM-stimulated PDE1 in regulating pathological cardiomyocyte hypertrophy in neonatal and adult rat ventricular myocytes and in the heart in vivo. Methods and Results: Inhibition of PDE1 activity using a PDE1-selective inhibitor, IC86340, or downregulation of PDE1A using siRNA prevented phenylephrine induced pathological myocyte hypertrophy and hypertrophic marker expression in neonatal and adult rat ventricular myocytes. Importantly, administration of the PDE1 inhibitor IC86340 attenuated cardiac hypertrophy induced by chronic isoproterenol infusion in vivo. Both PDE1A and PDE1C mRNA and protein were detected in human hearts; however, PDE1A expression was conserved in rodent hearts. Moreover, PDE1A expression was significantly upregulated in vivo in the heart and myocytes from various pathological hypertrophy animal models and in vitro in isolated neonatal and adult rat ventricular myocytes treated with neurohumoral stimuli such as angiotensin II (Ang II) and isoproterenol. Furthermore, PDE1A plays a critical role in phenylephrine-induced reduction of intracellular cGMP- and cGMP-dependent protein kinase (PKG) activity and thereby cardiomyocyte hypertrophy in vitro. Conclusions: These results elucidate a novel role for Ca 2+ /CaM-stimulated PDE1, particularly PDE1A, in regulating pathological cardiomyocyte hypertrophy via a cGMP/PKG-dependent mechanism, thereby demonstrating Ca 2+ and cGMP signaling cross-talk during cardiac hypertrophy.

Oral presentation from 3rd International Conference on cGMP Generators, Effectors and Therapeutic Implications Dresden, Germany. 15–17 June 2007

Meeting abstracts - A single PDF containing all abstracts in this Supplement is available here .

Sildenafil (Viagra®), a selective inhibitor of phosphodiesterase 5 (PDE5), induces headache and migraine. Although previously supposed to be a “vascular” headache, no significant cerebral artery dilatation was found in vivo. Thus, we hypothesised that PDE5 may not be present or that sildenafil is less effective on the cGMP hydrolysis in cerebral arteries, and that sildenafil may not be an effective dilator of cerebral arteries under baseline conditions. We evaluated the presence of PDE5 mRNA and protein in human arteries. Furthermore, the effects of two selective PDE5 inhibitors, sildenafil and UK-114,542, and a PDE1 inhibitor UK-90,234 on cGMP hydrolysis were investigated in human and guinea pig cerebral arteries. The vasoactive responses of the compounds were evaluated in guinea pig basilar arteries in vitro, with concomitant measurements of cAMP and cGMP. PDE5 was found in human middle cerebral arteries. Sildenafil and UK-114,542 inhibited cGMP hydrolysis concentration-dependently in both species. In guinea pig arteries, sildenafil induced an endothelium-dependent vasodilatation only at concentrations above 10 nM, which was augmented by sodium nitroprusside and attenuated by reduction of cGMP, but was cGMP independent at high concentrations. UK-114,542 was more and UK-90,234 was less potent than sildenafil. In conclusion, PDE5 is present in human and guinea pig cerebral arteries, and is inhibited by sildenafil at micromolar levels. Sildenafil in vitro is a poor dilator of guinea pig cerebral arteries unless a nitric oxide donor is co-administered, corresponding to the previous findings in vivo.

Angiotensin II (Ang II) and nitric oxide (NO)/natriuretic peptide (NP) signaling pathways mutually regulate each other. Imbalance of Ang II and NO/NP has been implicated in the pathophysiology of many vascular diseases. cGMP functions as a key mediator in the interaction between Ang II and NO/NP. Cyclic nucleotide phosphodiesterase 5A (PDE5A) is important in modulating cGMP signaling by hydrolyzing cGMP in vascular smooth muscle cells (VSMC). Therefore, we examined whether Ang II negatively modulates intracellular cGMP signaling in VSMC by regulating PDE5A. Ang II rapidly and transiently increased PDE5A mRNA levels in rat aortic VSMC. Upregulation of PDE5A mRNA was associated with a time-dependent increase of both PDE5 protein expression and activity. Increased PDE5A mRNA level was transcription-dependent and mediated by the Ang II type 1 receptor. Ang II-mediated activation of extracellular signal-regulated kinases 1/2 (ERK1/2) was essential for Ang II-induced PDE5A upregulation. Pretreatment of VSMC with Ang II inhibited C-type NP (CNP) stimulated cGMP signaling, such as cGMP dependent protein kinase (PKG)-mediated phosphorylation of vasodilator-stimulated-phosphoprotein (VASP). Ang II-mediated inhibition of PKG was blocked when PDE5 activity was decreased by selective PDE5 inhibitors, suggesting that upregulation of PDE5A expression is an important mechanism for Ang II to attenuate cGMP signaling. PDE5A may also play a critical role in the growth promoting effects of Ang II because inhibition of PDE5A activity significantly decreased Ang II-stimulated VSMC growth. These observations establish a new mechanism by which Ang II antagonizes cGMP signaling and stimulates VSMC growth.

Cyclic GMP (cGMP) made in response to atrial natriuretic peptide (ANP) or nitric oxide (NO) is an important regulator of short-term changes in smooth muscle tone and longer-term responses to chronic drug treatment or proliferative signals. The ability of smooth muscle cells (SMCs) to utilize different combinations of phosphodiesterase (PDE) isozymes allows cGMP to mediate these multiple processes. For example, PDE5 as a major cGMP-hydrolyzing PDE effectively controls the development of smooth muscle relaxation. In order for contraction to occur, PDE5 is activated and cGMP falls. Conversely, blockade of PDE5 activity allows the relaxation cycle to be prolonged and enhanced. A recently shown direct activation of PDE5 by cGMP binding to the GAF A domain suggests that this regulatory site might be a target for new drug development. The calcium surge associated with vasoconstrictor initiated contraction also activates a calcium/calmodulin-dependent PDE (PDE1A). Together, PDE5 and PDE1A lower cGMP sufficiently to allow contraction. Longer term, both PDE5 and PDE1A mRNA are induced by chronic stimulation of guanylyl cyclase. This induction is a major cause of the tolerance that develops to NO-releasing drugs. Finally, high levels of cGMP or cAMP also act as a brake to attenuate the proliferative response of SMCs to many mitogens. After vessel damage, in order for SMC proliferation to occur, the levels of cGMP and cAMP must be decreased. In humans, this decrease is caused in large part by induction of another Ca 2+ /calmodulin-dependent PDE (PDE1C) that allows the brake to be released and proliferation to start.

Proliferation of arterial smooth muscle cells (SMCs) is a key event in the formation of advanced atherosclerotic lesions and restenosis after angioplasty. Cyclic nucleotides (cAMP and cGMP) inhibit arterial SMC proliferation, and elevation of cyclic nucleotides reduces neointimal formation after angioplasty in animal models. Degradation of cAMP and cGMP is catalyzed by cyclic nucleotide phosphodiesterases (PDEs). One of these, PDE1C, hydrolyzes cAMP and cGMP and is expressed in proliferating human SMCs but is absent in quiescent human aorta. Thus, PDE1C expression is low in cultured human SMCs made quiescent by attaching to fibrillar collagen type I. After release from the fibrillar collagen, PDE1C expression is induced and associated with traverse through S-phase of the cell cycle. Further, PDE1C is expressed in vivo in human fetal aorta containing proliferating SMCs, but not in newborn aorta in which SMC proliferation has ceased. Inhibition of PDE1C in SMCs isolated from normal aorta or from lesions of atherosclerosis using antisense oligonucleotides or a PDE1 inhibitor results in suppression of SMC proliferation. In conclusion, PDE1C expression is a marker of human SMC proliferation ex vivo and in vivo. Inhibition of PDE1C leads to inhibition of human SMC proliferation. Because PDE1C is absent in quiescent SMCs, PDE1C inhibitors may target proliferating SMCs in lesions of atherosclerosis or restenosis.

Background The efficacy of nitroglycerin (NTG) as a vasodilator is limited by tolerance, which develops shortly after treatment begins. In vascular smooth muscle cells (VSMCs), NTG is denitrated to form nitric oxide (NO), which activates guanylyl cyclase and generates cGMP. cGMP plays a key role in nitrate-induced vasodilation by reducing intracellular Ca 2+ concentration. Therefore, one possible mechanism for development of nitrate tolerance would be increased activity of the cGMP phosphodiesterase (PDE), which decreases cGMP levels. Methods and Results To test this hypothesis, rats were made tolerant by continuous infusion of NTG for 3 days (10 μg · kg −1 · min −1 SC) with an osmotic pump. Analysis of PDE activities showed an increased function of Ca 2+ /calmodulin (CaM)–stimulated PDE (PDE1A1), which preferentially hydrolyzes cGMP after NTG treatment. Western blot analysis for the Ca 2+ /CaM-stimulated PDE revealed that PDE1A1 was increased 2.3-fold in NTG-tolerant rat aortas. Increased PDE1A1 was due to mRNA upregulation as measured by relative quantitative reverse transcription–polymerase chain reaction. The PDE1-specific inhibitor vinpocetine partially restored the sensitivity of the tolerant vasculature to subsequent NTG exposure. In cultured rat aortic VSMCs, angiotensin II (Ang II) increased PDE1A1 activity, and vinpocetine blocked the effect of Ang II on decrease in cGMP accumulation. Conclusions Induction of PDE1A1 in nitrate-tolerant vessels may be one mechanism by which NO/cGMP-mediated vasodilation is desensitized and Ca 2+ -mediated vasoconstriction is supersensitized. Inhibiting PDE1A1 expression and/or activity could be a novel therapeutic approach to limit nitrate tolerance.

The aim was to investigate the presence and activity of cGMP hydrolysing phosphodiesterases in guinea pig basilar arteries and the effect of selective and non-selective phosphodiesterase inhibitors on cerebral artery dilatation involving the nitric oxide (NO)-guanosine cyclic 3'5-monophosphate (cGMP) pathway. Immunoreactivity to phosphodiesterases 1A, 1B and 5, but not phosphodiesterase 1C was found in fractions of homogenised cerebral arteries eluted by high-pressure liquid chromatography (HPLC). Both the phosphodiesterase 1 inhibitor 8-methoxymethyl-1-methyl-3-(2methylpropyl)-xanthine (8-MM-IBMX) and the phosphodiesterase 5 inhibitors zaprinast and dipyridamole induced dilatation of cerebral arteries. The dilatory response to 8-MM-IBMX was reduced by 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ) (10 microM) and endothelial removal and restored by sodium nitroprusside (0.1 microM) pretreatment, indicating a close relation to the nitric oxide-cGMP pathway. The responses to zaprinast and dipyridamole, however, were not only moderately affected, but also restored by sodium nitroprusside (0.1 microM) pretreatment. At high concentrations, the dilatory effects of zaprinast and dipyridamole were partly caused by cGMP-independent mechanisms. Targeting the phosphodiesterases present in cerebral arteries, with selective inhibitors or activators of phosphodiesterase, may be a possible new way of treating cerebrovascular disease.

An experimental ovine fetal model for perinatal pulmonary hypertension of the neonate (PPHN) was characterized by altered pulmonary vasoreactivity and structure. Because past studies had suggested impaired nitric oxide-cGMP cascade in this experimental model, we hypothesized that elevated phosphodiesterase (PDE) activity may contribute to altered vascular reactivity and structure in experimental PPHN. Therefore, we studied the effects of the PDE inhibitors zaprinast and dipyridamole on fetal pulmonary vascular resistance and PDE5 activity, protein, mRNA, and localization in normal and pulmonary hypertensive fetal lambs. Infusion of dipyridamole and zaprinast lowered pulmonary vascular resistance by 55 and 35%, respectively, in hypertensive animals. In comparison with control animals, lung cGMP PDE activity was elevated in hypertensive fetal lambs (150%). Increased PDE5 activity was not associated with either an increased PDE5 protein or mRNA level. Immunocytochemistry demonstrated that PDE5 was localized to vascular smooth muscle. We concluded that PDE5 activity was increased in experimental PPHN, possibly by posttranslational phosphorylation. We speculated that these increases in cGMP PDE activity contributed to altered pulmonary vasoreactivity in experimental perinatal pulmonary hypertension.

During transitional circulation, the pulmonary vascular bed undergoes a rapid and profound reduction in both tone and vascular smooth-muscle (VSM) content. 3',5'-Guanylate cyclic monophosphate (cGMP) is a crucial mediator in the regulation of pulmonary vascular resistance (PVR) and VSM proliferation. Hydrolysis of cGMP is achieved predominately by cGMP-specific phosphodiesterases (PDEs). Among the cGMP-specific PDEs, PDE5 is quantitatively prevalent in lung tissue. We have investigated the levels of pulmonary PDE5 enzymatic activity, protein, and messenger RNA (mRNA) in ovine and mouse lung during perinatal development. We report that within 1 h following birth, PDE5 activity, protein, and mRNA levels decrease in both species, in a manner that correlates with known decreases in PVR in early transition. However, from 4 to 7 d following birth, a secondary increase in PDE5 activity, protein, and mRNA occurs in both ovine and mouse lung, suggesting a complex regulation of PVR and VSM proliferation in late perinatal development. Our data imply that PDE5 may be an important mediator in the regulation of PVR in normal and possibly in pathologic states, and may ultimately provide a basis for PDE5 inhibitors as a treatment for pulmonary hypertension.

Background The heart responds to maladaptive pro-hypertrophic stimuli by stimulating intrinsic signals that contrast and dampen the onset and development of hypertrophy. Cyclic guanosine monophosphate (cGMP) and its downstream effector cGMP kinase I (cGKI) have been suggested to be an important anti-hypertrophic signalling pathway (1). Intracellular levels of cGMP can be raised by the action of nitric oxide (NO) and natriuretic peptides (ANP, BNP), or by inhibiting cGMP-degrading phosphodiesterases (PDE).

Background Calpain, intracellular non-lysosomal calcium-dependent proteinase, is activated by a number of signaling pathways that lead to the elevation of intraplatelet calcium concentrations. Physiological roles of calpain, highly expressed in platelets, include regulation of platelet aggregation and adhesion, while excessive activation of calpain in such pathological disorders as diabetes could cause changes in platelet functions, resulting in platelet hyperactivation. Platelets from diabetic patients are also characterized by resistance to nitric oxide inhibition. Here we examined if calpain could regulate major cAMP and cGMP hydrolyzing phosphodiesterases (PDEs), PDE3A and PDE5, in human platelets.

Background Duchenne muscular dystrophy (DMD) is the most common inherited form of muscular dystrophy, which results in skeletal muscle weakness by age 6. In its later stages DMD leads to dilated cardiomyopathy and heart failure with high level of mortality. At present there are no effective treatments for most of the cardiac pathology in DMD patients. Results