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Background and purpose Asthenozoospermia is a leading cause of male infertility, but development of pharmacological agents to improve sperm motility is hindered by the lack of effective screening platforms and knowledge of suitable molecular targets. We have demonstrated that a high-throughput screening (HTS) strategy and established in vitro tests can identify and characterise compounds that improve sperm motility. Here, we applied HTS to identify new compounds from a novel small molecule library that increase intracellular calcium ([Ca2+ ]i ), promote human sperm cell motility, and systematically determine the mechanism of action. Experimental approach A validated HTS fluorometric [Ca2+ ]i assay was used to screen an in-house library of compounds. Trequinsin hydrochloride (a PDE3 inhibitor) was selected for detailed molecular (plate reader assays, electrophysiology, and cyclic nucleotide measurement) and functional (motility and acrosome reaction) testing in sperm from healthy volunteer donors and, where possible, patients. Key results Fluorometric assays identified trequinsin as an efficacious agonist of [Ca2+ ]i , although less potent than progesterone. Functionally, trequinsin significantly increased cell hyperactivation and penetration into viscous medium in all donor sperm samples and cell hyperactivation in 22/25 (88%) patient sperm samples. Trequinsin-induced [Ca2+ ]i responses were cross-desensitised consistently by PGE1 but not progesterone. Whole-cell patch clamp electrophysiology confirmed that trequinsin activated CatSper and partly inhibited potassium channel activity. Trequinsin also increased intracellular cGMP. Conclusion and implications Trequinsin exhibits a novel pharmacological profile in human sperm and may be a suitable lead compound for the development of new agents to improve patient sperm function and fertilisation potential.

The paucity of currently available therapies for glioblastoma multiforme requires novel approaches to the treatment of this brain tumour. Disrupting cyclic nucleotide-signalling through phosphodiesterase (PDE) inhibition may be a promising way of suppressing glioblastoma growth. Here, we examined the effects of 28 PDE inhibitors, covering all the major PDE classes, on the proliferation of the human U87MG, A172 and T98G glioblastoma cells. The PDE10A inhibitors PF-2545920, PQ10 and papaverine, the PDE3/4 inhibitor trequinsin and the putative PDE5 inhibitor MY-5445 potently decreased glioblastoma cell proliferation. The synergistic suppression of glioblastoma cell proliferation was achieved by combining PF-2545920 and MY-5445. Furthermore, a co-incubation with drugs that block the activity of the multidrug resistance-associated protein 1 (MRP1) augmented these effects. In particular, a combination comprising the MRP1 inhibitor reversan, PF-2545920 and MY-5445, all at low micromolar concentrations, afforded nearly complete inhibition of glioblastoma cell growth. Thus, the potent suppression of glioblastoma cell viability may be achieved by combining MRP1 inhibitors with PDE inhibitors at a lower toxicity than that of the standard chemotherapeutic agents, thereby providing a new combination therapy for this challenging malignancy.

With the identification of novel cAMP binding effecter molecules in Trypanosoma, role of cAMP in kinetopalstida parasites gained an intriguing break through. Despite earlier demonstrations of role of cAMP in survival of Leishmania during macrophage infection, there is essential need to specifically clarify involvement of cAMP in various cellular processes in the parasite. In this context, we sought to gain a comprehensive understanding of the effect of cAMPanalogs and cAMP-phosphodiesterase (PDE) inhibitors on proliferation of log phase parasites. Administration of both hydrolysable (8-pCPT-cAMP) and non-hydrolysable analogs (Sp-8-pCPT-cAMPS) of cAMP resulted in significant decrease of Leishmania proliferation. Amongst the various PDE inhibitors, etazolate was found to be potently anti-proliferative. BrdU cell proliferation and K/N/F-enumeration microscopic study revealed that both cAMP analogues and selective PDE inhibitors resulted in significant cell cycle arrest at G1 phase with reduced S-phase population. Furthermore, careful examination of the flagellar motility patterns revealed significantly reduced coordinated forward flagellar movement of the promastigotes with a concomitant decrease in cellular ATP levels. Alongside, 8-pCPT-cAMP and PDE inhibitors etazolate and trequinsin showed marked reduction in mitochondrial membrane potential. Treatment of etazolate at subcytotoxic concentration to infected macrophages significantly reduced parasite burden and administration of etazolate to Leishmania-infected BALB/c mice showed reduced liver and spleen parasite burden. Collectively, these results imply involvement of cAMP in various crucial processes paving the avenue for developing potent anti-leishmanial agent.Author SummaryLeishmania donovani is the causative agent of fatal Visceral Leishmaniasis. The current available medications are toxic, expensive and require long term daily administrations. With an aim to develop improved therapeutic, components of cAMP homeostasis, particularly cAMP-phosphodiesteares, has been targeted for Leishmania and other kinetoplastid pathogens. cAMP plays diverse roles in functional processes involved in cell division, transition into different stages of the life cycle of Leishmania and motility. In this study, the authors found administration of both hydrolysable and non-hydrolysable analogs of cAMP and certain PDE inhibitors resulted in remarkable decrease proliferation with considerable cytopathic impact on promastigotes. The mammalian phosphodiestearse inhibitor etazolate caused significant reduction in parasite load in L. donovani infected macrophages and demonstrated considerable reduction of liver and spleen parasite burden in in vivo mouse infection model. The study suggested that etazolate, with its slightest impact on mammalian host, can be repurposed for developing effective anti-leishmanials.

SUMMARYSupressed levels of intracellular cAMP have been associated with malignancy. Thus, elevating cAMP through activation of adenylyl cyclase (AC) or by inhibition of phosphodiesterase (PDE) may be therapeutically beneficial. Here, we demonstrate that elevated cAMP levels suppress growth in C6 cells (a model of glioma) through treatment with forskolin, an AC activator, or a range of small molecule PDE inhibitors with differing selectivity profiles. Forskolin suppressed cell growth in a protein kinase A (PKA)-dependent manner by inducing a G2/M phase cell cycle arrest. In contrast, trequinsin (a non-selective PDE2/3/7 inhibitor), not only inhibited cell growth via PKA, but also stimulated (independent of PKA) caspase-3/-7 and induced an aneuploidy phenotype. Interestingly, a cocktail of individual PDE 2,3,7 inhibitors suppressed cell growth in a manner analogous to forskolin but not trequinsin. Finally, we demonstrate that concomitant targeting of both AC and PDEs synergistically elevated intracellular cAMP levels thereby potentiating their antiproliferative actions.

Recent studies demonstrate that mitochondrial dysfunction is a mediator of acute kidney injury (AKI). Consequently, restoration of mitochondrial function after AKI may be key to the recovery of renal function. Mitochondrial function can be restored through the generation of new, functional mitochondria in a process called mitochondrial biogenesis (MB). Despite its potential therapeutic significance, very few pharmacological agents have been identified to induce MB. To examine the efficacy of phosphodiesterase (PDE) inhibitors (PDE3: cAMP and cGMP activity; and PDE4: cAMP activity) in stimulating MB, primary cultures of renal proximal tubular cells (RPTCs) were treated with a panel of inhibitors for 24 hours. PDE3, but not PDE4, inhibitors increased the FCCP-uncoupled oxygen consumption rate (OCR), a marker of MB. Exposure of RPTCs to the PDE3 inhibitors, cilostamide and trequinsin, for 24 hours increased peroxisome proliferator–activated receptor γ coactivator-1α, and multiple mitochondrial electron transport chain genes. Cilostamide and trequinsin also increased mRNA expression of mitochondrial genes and mitochondrial DNA copy number in mice renal cortex. Consistent with these experiments, 8-Br-cGMP increased FCCP-uncoupled OCR and mitochondrial gene expression, whereas 8-Br-cAMP had no effect. The cGMP-specific PDE5 inhibitor sildenafil also induced MB in RPTCs and in vivo in mouse renal cortex. Treatment of mice with sildenafil after folic acid–induced AKI promoted restoration of MB and renal recovery. These data provide strong evidence that specific PDE inhibitors that increase cGMP are inducers of MB in vitro and in vivo, and suggest their potential efficacy in AKI and other diseases characterized by mitochondrial dysfunction and suppressed MB.

We report here the generation and pharmacological characterization of a phosphodiesterase 2A (PDE2A) reporter cell line. Human PDE2A was stably transfected in a parental cell line expressing the atrial natriuretic peptide (ANP) receptor and the cyclic nucleotide-gated (CNG) cation channel CNGA2, acting as the biosensor for intracellular cGMP. In this reporter cell line, cGMP levels can be monitored in real-time via aequorin luminescence stimulated by calcium influx through the CNG channel. By using different PDE inhibitors, we could show that our PDE2A reporter assay specifically monitors PDE2A inhibition with high sensitivity. In the absence of ANP stimulation, the PDE2A selective inhibitors EHNA, BAY 60-7550 and PDP did not increase basal luminescence levels in this experimental setting. However, in combination with ANP, these inhibitors stimulated luminescence signals and induced leftward shifts of ANP concentration-response curves. Similar results were obtained when using IBMX, trequinsin and dipyridamole, which inhibit PDE2A nonselectively with lower potency. PDP, the most potent PDE2A inhibitor known to date, was found to exhibit much lower cellular activity as anticipated from its biochemical PDE2A inhibitory activity. By cellular uptake and transport studies we could show that PDP's cell permeability is low and that the compound is a substrate for an efflux transporter. Other PDE inhibitors including vinpocetine, milrinone, rolipram, sildenafil, zaprinast, BRL 50481 and BAY 73-6691 did not stimulate luminescence signals on our PDE2A reporter cell line. The results imply that this novel PDE2A reporter assay provides an efficient, high throughput means for the identification and characterization of PDE2A inhibitors.

cAMP is a key modulator for glucose-dependent insulin secretion (GDIS). Members of the phosphodiesterase (PDEs) gene family regulate intracellular levels of cAMP by hydrolyzing cAMP to the corresponding inactive 5'AMP derivative. These studies examined the expression and function of all 18 cAMP-specific PDEs in the rat insulinoma derived INS-1 (832/13) cell and isolated rat islets using quantitative PCR and siRNA-mediated gene-specific knockdown. PDE1C, PDE3B, PDE4C, PDE8B, PDE10A, and PDE11A were significantly expressed in rat islets and INS-1 (832/13) cells at the mRNA level. PDE1C, PDE10A and PDE11A were also expressed in brain, along with PDE3B, PDE4C and PDE8B which were also highly expressed in liver, and PDE3B was present in adipose tissue and PDE4C in skeletal muscle. siRNA mediated knockdown of PDE1C, PDE3B, PDE8B and PDE4C, but not PDE10A and PDE11A, significantly enhanced GDIS in rat INS-1 (832/13) cells. Also, selective inhibitors of PDE3 (trequinsin) and PDE4 (roflumilast and L-826,141) significantly augmented GDIS in both INS-1 (832/13) cells and rat islets. The combination of PDE3 and PDE4 selective inhibitors demonstrate that these enzymes comprise a significant proportion of the cAMP metabolizing activity in INS-1 cells and rat islets.

Efferent dorsal unpaired median neurons are pacemaker neurosecretory cells. A Ca2+ background current contributing to the pacemaker activity of cockroach dorsal unpaired median neurons is up-regulated by neurohormone D (NHD), an octapeptide belonging to the adipokinetic hormone family. This modulation accelerates spiking and increases [Ca2+]i. Using patch clamp, calcium imaging, and immunocytochemistry, we investigated the signaling pathway of NHD-induced current modulation. The membrane depolarization produced by NHD was related to the increase in membrane conductance for Ca2+, Ba2+, or Sr2+. This increase was abolished by LOE 908, an inhibitor of noncapacitive Ca2+ entry (NCCE), and it was strongly attenuated by the phospholipase C inhibitor U37122 and the diacylglycerol lipase inhibitor RHC80267. Arachidonic acid and ETYA mimicked the NHD effect on background current. This was abolished by l-NAME and ODQ, inhibitors of NO synthase and NO-sensitive guanylyl cyclase, respectively, but mimicked by the NO donor sodium nitroprusside and 8-bromo-cGMP. Immunocytochemistry using cGMP antibodies indicated that NHD and ETYA increase cGMP. Inhibition of protein kinase G with KT5823 and Rp-8-pCPT-cGMPS had no effect, whereas zaprinast, a cGMP-specific phosphodiesterase 5,6,9 inhibitor, mimicked the NHD effect. Furthermore, inhibition of the cGMP-activated phosphodiesterase 2 by EHNA and trequinsin abolished the effect of NHD. We conclude that the final step of the NHD signal transduction is the phosphodiesterase 2-induced down-regulation of the cAMP level. This removes a depression of NCCE directly attributed to cAMP because inhibition of protein kinase A with KT5720, Rp-cAMPS, and PKI14-22 amide did not mimic the NHD effect. We also demonstrate that any mechanism increasing the cGMP level can induce NCCE.

It was previously demonstrated that inhibition of cAMP degradation with phosphodiesterase type 3 (PDE3) inhibitors resulted in the maintenance of bovine cumulus-oocyte complexes (COC) and denuded oocytes (DO) in meiotic arrest, while a PDE4 inhibitor was without effect. In this study, different inhibitors of PDE3 and PDE4 were tested for their effects on bovine oocyte nuclear maturation. Bovine COC and DO were cultured in TCM-199+10% fetal bovine serum (FBS) with or without different concentrations of the PDE inhibitors. The PDE3 inhibitor trequinsin significantly increased the percentage of COC remaining at the germinal vesicle (GV) stage after 7h of culture (19.3, 60.3, and 67.8% GV for control and trequinsin 10 and 50 nM, respectively) while Ro 20-1724 (a PDE4 inhibitor) was without effect. In DO, only trequinsin at 10 nM had a significant effect after 7h of culture (51.3 and 86.1% GV for control and trequinsin 10 nM, respectively). Trequinsin reduced the percentage of COC reaching the mature phase after 22h, but was without effect on DO. The protein kinase A (PKA) inhibitor H-89 reversed the inhibitory effect of trequinsin in COC and DO, indicating that inhibition of nuclear maturation by trequinsin involves activation of PKA. Trequinsin increased cAMP concentrations in COC but not in DO, suggesting that cumulus cells may also contain a PDE3 isoenzyme.

Compounds that activate adenylate cyclase, increasing intracellular cyclic adenosine monophosphate (cAMP), inhibit equine platelet aggregation. Cyclic AMP is broken down by phosphodiesterase (PDE) and, in the present study, the effects of theophylline, a nonselective PDE inhibitor, and selective inhibitors of PDE isoenzymes PDE3, PDE4 and PDE5, on equine platelet aggregation in response to platelet activating factor (PAF) and adenosine diphosphate (ADP) have been examined. Theophylline and the PDE3 inhibitors, trequinsin and quazinone, inhibited both PAF and ADP-induced aggregation in a concentration dependent manner. The inhibition of PAF-induced aggregation was, however, significantly greater than that of the response to ADP. The inhibitory effects of theophylline and the PDE3 inhibitors on ADP- but not PAF-, induced aggregation were reversed by addition of the calcium ionophore, A23187. Rolipram and zaprinast, inhibitors of PDE4 and PDE5, respectively, had no effect on either PAF- or ADP-induced aggregation. These results demonstrate that inhibition of aggregation caused by PAF or ADP can be achieved by selective inhibition of PDE3 but suggest that there may be agonist-specific differences in the intracellular signalling pathways that regulate equine platelet aggregation.

We tested the hypothesis that cGMP stimulates renin release through inhibition of the cAMP-specific phosphodiesterase 3 (PDE3) in isolated rat juxtaglomerular (JG) cells. In addition, we assessed the involvement of PDE4 in JG-cell function. JG cells expressed PDE3A and PDE3B, and the PDE3 inhibitor trequinsin increased cellular cAMP content, enhanced forskolin-induced cAMP formation, and stimulated renin release from incubated and superfused JG cells. Trequinsin-mediated stimulation of renin release was inhibited by the permeable protein kinase A antagonist Rp-8-CPT-cAMPS. PDE4C was also expressed, and the PDE4 inhibitor rolipram enhanced cellular cAMP content. Dialysis of single JG cells with cAMP in whole-cell patch-clamp experiments led to concentration-dependent, biphasic changes in cell membrane capacitance (Cm) with a marked increase in Cmat 1 μmol/L, no net change at 10 μmol/L, and a decrease at 100 μmol/L cAMP. cGMP also had a dual effect on Cmat 10-fold higher concentration compared with cAMP. Trequinsin, milrinone, and rolipram mimicked the effect of cAMP on Cm. Trequinsin, cAMP, and cGMP enhanced outward current 2- to 3-fold at positive membrane potentials. The effects of cAMP, cGMP, and trequinsin on Cmand cell currents were abolished by inhibition of protein kinase A with Rp-cAMPs. We conclude that degradation of cAMP by PDE3 and PDE4 contributes to regulation of renin release from JG cells. Our data provide evidence at the cellular level that stimulation of renin release by cGMP involves inhibition of PDE3 resulting in enhanced cAMP formation and activation of the cAMP sensitive protein kinase.

The intracellular messenger cyclic guanosine monophosphate (cGMP) has an important role in regulating smooth muscle tone. An increase in intracellular cGMP levels is a prerequisite for penile erection. Inhibition of cGMP degradation by cGMP specific phosphodiesterase 5 has been used for treating erectile dysfunction. In addition to degradation by phosphodiesterase, cGMP is exported from cells by multidrug resistance protein 5 (MRP5), also called ABCC5, which we recently identified as an adenosine triphosphate dependent export pump for cGMP. MRP5 is potently inhibited by substances known as phosphodiesterase inhibitors, including sildenafil and trequinsin. Therefore, we analyzed whether MRP5 is expressed in tissues of the human genitourinary system and whether MRP5 and phosphodiesterase 5 proteins are localized in the same cell types.Localization of MRP5 and phosphodiesterase 5 was analyzed by immunofluorescence microscopy in cryosections of various tissues of the human genitourinary system.MRP5 and phosphodiesterase 5 were co-expressed in smooth muscle cells of the corpus cavernosum, ureter, urethra and bladder. In addition, MRP5 and phosphodiesterase 5 were localized in epithelial cells of the mucosa in the ureter and urethra, and in blood vessels of the lamina propria.The co-expression of MRP5 and phosphodiesterase 5 in smooth muscle cells of the genitourinary system indicates 2 distinct pathways for cGMP removal. Thus, MRP5 inhibition represents a new approach for enhancing cGMP levels in smooth muscle cells and developing drugs for erectile dysfunction.

This study reports the identification and characterization of a cAMP-specific phosphodiesterase from the parasitic hemoflagellate Trypanosoma brucei. TbPDE2A is a class I phosphodiesterase. Its catalytic domain exhibits 30-40% sequence identity with those of all 11 mammalian phosphodiesterase (PDE) families, as well as with PDE2 from Saccharomyces cerevisiae, dunce from Drosophila melanogaster, and regA from Dictyostelium discoideum. The overall structure of TbPDE2A resembles that of human PDE11A in that its N-terminal region contains a single GAF domain. This domain is very similar to those of the mammalian PDE2, -5, -6, -10, and -11, where it constitutes a potential cGMP binding site. TbPDE2A can be expressed in S. cerevisiae, and it complements an S. cerevisiae PDE deletion strain. Recombinant TbPDE2A is specific for cAMP, with a K(m) of approximately 2 micrometer. It is entirely resistant to the nonselective PDE inhibitor 3-isobutyl-1-methylxanthine, but it is sensitive to trequinsin, dipyridamole, sildenafil, and ethaverine with IC(50) values of 5.4, 5.9, 9.4, and 14.2 micrometer, respectively. All four compounds inhibit proliferation of bloodstream form trypanosomes in culture, indicating that TbPDE2A is an essential enzyme.

Nitric oxide (NO) donors increase heart rate (HR) through a guanylyl cyclase-dependent stimulation of the pacemaker current I(f), without affecting basal I(Ca-L). The activity of I(f)is known to be enhanced by cyclic nucleotides and by an increase in cytosolic Ca(2+). We examined the role of cGMP-dependent signaling pathways and intracellular Ca(2+)stores in mediating the positive chronotropic effect of NO donors. In isolated guinea pig atria, the increase in HR in response to 1-100 micromol/l 3-morpholino-sydnonimine (SIN-1; with superoxide dismutase, n=6) or diethylamine-NO (DEA-NO, n=8) was significantly attenuated by blockers of the cGMP-inhibited phosphodiesterase (PDE3; trequinsin, milrinone or Ro-13-6438, n=22). In addition, the rate response to DEA-NO or sodium nitroprusside (SNP) was significantly reduced following inhibition of PKA (KT5720 or H-89, n=15) but not PKG (KT5728 or Rp-8-pCPT-cGMPs, n=16). Suppression of sarcoplasmic (SR) Ca(2+)release by pretreatment of isolated atria with ryanodine or cyclopiazonic acid (2 micromol/l and 60 micromol/l, n=16) significantly reduced the chronotropic response to 1-100 micromol/l SIN-1 or DEA-NO. Moreover, in isolated guinea pig sinoatrial node cells 5 micromol/l SNP significantly increased diastolic and peak Ca(2+)fluorescence (+13+/-1% and +28+/-1%, n=6, P

Cellular export of cyclic nucleotides has been observed in various tissues and may represent an elimination pathway for these signaling molecules, in addition to degradation by phosphodiesterases. In the present study we provide evidence that this export is mediated by the multidrug resistance protein isoform MRP5 (gene symbol ABCC5). The transport function of MRP5 was studied in V79 hamster lung fibroblasts transfected with a human MRP5 cDNA. An MRP5-specific antibody detected an overexpression of the glycoprotein of 185 +/- 15 kDa in membranes from MRP5-transfected cells and a low basal expression of hamster Mrp5 in control membranes. ATP-dependent transport of 3',5'-cyclic GMP at a substrate concentration of 1 micrometer was 4-fold higher in membrane vesicles from MRP5-transfected cells than in control membranes. This transport was saturable with a K(m) value of 2.1 micrometer. MRP5-mediated transport was also detected for 3',5'-cyclic AMP at a lower affinity, with a K(m) value of 379 micrometer. A potent inhibition of MRP5-mediated transport was observed by several compounds, known as phosphodiesterase modulators, including trequinsin, with a K(i) of 240 nm, and sildenafil, with a K(i) value of 267 nm. Thus, cyclic nucleotides are physiological substrates for MRP5; moreover, MRP5 may represent a novel pharmacological target for the enhancement of tissue levels of cGMP.

Natural killer (NK) cells are lymphocytes that are capable of destroying tumor cells and virally infected cells (cytolysis) without prior sensitization. When cyclic AMP (cAMP) is elevated artificially in NK cells, it is a potent inhibitor of their cytolytic function. Recently, we have shown that when NK cells are exposed to a range of lysis-sensitive (LS) tumor target cells, there is an increase in intracellular cAMP levels in the NK cells over a 60-min period. There is no increase in NK-cell cAMP in response to lysis-resistant (LR) tumor target cells. We determined that this cAMP elevation is due, in part, to an LS target-induced activation of adenylyl cyclase (AC), and that the AC-activation component appears to require a protein tyrosine kinase (PTK) activity. In the present study, we demonstrated that an LS target-induced inhibition of phosphodiesterase (PDE) is also contributing to the overall elevation of cAMP. Direct measurement of PDE activity showed an inhibition in lymphocytes that were exposed to LS targets but not in those exposed to LR targets. The inhibition of PDE activity was maximal by 30 min. Lymphocytes were exposed to targets and then lysed, so that PDE activity could be measured. Addition of class-selective inhibitors of PDE (at levels sufficient to completely block that class of PDE) to the lysate focused the measurement of PDE activity on those classes of PDE that were unaffected by the selective inhibitor. Using the PDE IV selective inhibitor rolipram and the PDE III selective inhibitors trequinsin and milrinone, we showed that a PDE III is being inhibited in lymphocytes by exposure to LS targets. As PDE III is known to be inhibited by elevated cyclic GMP (cGMP) levels, increased cGMP in NK cells following exposure to LS targets was a possible mechanism by which a PDE III in NK cells might be inhibited. However, when we measured cGMP levels in control and LS target-stimulated lymphocytes, we saw no change.

Proliferation of vascular smooth muscle cells (SMC) in response to platelet-derived growth factor (PDGF) and other mitogens plays an important role in restenosis following coronary angioplasty. Elevation of adenosine 3',5'-cyclic monophosphate (cAMP) concentration in SMC has been shown to inhibit SMC mitogenesis and could be obtained either directly by stimulation of adenylyl cyclase-coupled receptors or indirectly by inhibition of cAMP-specific phosphodiesterase (PDE4) or the cyclic guanosine 3', 5'-monophosphate-inhibitable phosphodiesterase (PDE3). This study compared the effects of the selective PDE3 inhibitors trequinsin and quazinone with the selective PDE4 inhibitors Ro 20-1724 and rolipram on PDGF-induced DNA synthesis, mitogen-activated protein (MAP) kinase activation, cAMP levels, and protein kinase A (PKA) activation in SMC. Both PDE3 and PDE4 inhibitors stimulated intracellular PKA activation as seen from phosphorylation of vasodilator-stimulated phosphoprotein (VASP). However, only PDE3 inhibitors, and not inhibitors of PDE4, reduced PDGF-induced DNA synthesis and inhibited p42/p44 MAP kinase phosphorylation. At antimitogenic concentrations, the PDE3 inhibitors had only minor effects on cAMP levels. In contrast, PDE4 inhibitors increased the forskolin-induced cellular cAMP concentration 13- to 17-fold above control. These data demonstrate that inhibitors of PDE3 are potent antimitogenic agents and that a general increase in cellular cAMP levels and PKA activation per se are not sufficient to inhibit PDGF-induced SMC mitogenesis.

Four cyclic-nucleotide phosphodiesterase (PDE) genes belonging to the PDE4 family (PDE4A, 4B, 4C and 4D) have been identified. All four isogenes, including several deletions and alterations of the amino, carboxyl and central catalytic domains, were expressed in insect cells. Lysates were characterised for enzyme activity by using the Km for substrate and the EC50 for activation by the cofactor Mg2+. The catalytic domain alone appears to be sufficient for the normal enzymatic function of PDE4 proteins. Substrate affinity varied by less than 2-fold between catalytic-domain forms of the PDE4A, 4B and 4D isogenes and the long forms (PDE4A5, PDE4B1 and PDE4D3). The affinity for Mg2+ varied by less than 4-fold between long and catalytic-domain forms of PDE4A and 4B. The catalytic-domain form of PDE4D, however, had a 12-fold lower affinity for Mg2+ that was restored by including a portion of the amino-terminal domain, upstream conserved region-2 (UCR2). This result suggests that the Mg2+-binding site of PDE4D involves the UCR2 region. Inhibition of the PDE4 proteins by synthetic compounds is apparently affected differently by the domains. For PDE4B, the catalytic domain is sufficient for interactions with the inhibitors studied: IBMX, trequinsin, rolipram, TVX 2706, RP 73401 and RS-25344. For PDE4D the catalytic-domain form is less sensitive than the long form to inhibition by RS-25344, rolipram and TVX 2706, by 1463-, 11-and 12-fold, respectively. Addition of UCR2 to the catalytic-domain form of PDE4D restored all the lost sensitivities. The catalytic-domain form of PDE4A showed a reduced inhibitor affinity with RS-25344 and TVX 2706 by 77- and 90-fold, respectively. Both catalytic-domain and long forms of PDE4 isogenes interacted with equal affinity with the non-specific inhibitors IBMX and trequinsin, as well as the very potent PDE4-specific inhibitor RP 73401. Other potent and specific PDE4 inhibitors, such as rolipram, RS-25344 or TVX 2706, appear to utilize non-catalytic domain interactions with PDE4D and 4A to supplement those within the catalytic domains. These observations suggest a different relation between amino and catalytic domains in PDE4D relative to PDE4B. We therefore propose a model to illustrate these isogene-specific PDE4 domain interactions with substrate, inhibitors and the co-factor Mg2+. The model for PDE4D is also discussed in relation to changes in the activation curve for Mg2+ and sensitivity to RS-25344 that accompany phosphorylation of the long form by protein kinase A.

This study aimed to characterize the cellular pathways along which nitric oxide (NO) stimulates renin secretion from the kidney. Using the isolated perfused rat kidney model we found that renin secretion stimulated 4- to 8-fold by low perfusion pressure (40 mmHg), by macula densa inhibition (100 μmol/liter of bumetanide), and by adenylate cyclase activation (3 nmol/liter of isoproterenol) was markedly attenuated by the NO synthase inhibitor nitro- l -arginine methyl ester ( l -Name) (1 mM) and that the inhibition by l -Name was compensated by the NO-donor sodium nitroprusside (SNP) (10 μmol/liter). Similarly, inhibition of cAMP degradation by blockade of phosphodiesterase 1 (PDE-1) (20 μmol/liter of 8-methoxymethyl-1-methyl-3-(2-methylpropyl)xanthine) or of PDE-4 (20 μmol/liter of rolipram) caused a 3- to 4-fold stimulation of renin secretion that was attenuated by l -Name and that was even overcompensated by sodium nitroprusside. Inhibition of PDE-3 by 20 μmol/liter of milrinone or by 200 nmol/liter of trequinsin caused a 5- to 6-fold stimulation of renin secretion that was slightly enhanced by NO synthase inhibition and moderately attenuated by NO donation. Because PDE-3 is a cGMP-inhibited cAMP-PDE the role of endogenous cGMP for the effects of NO was examined by the use of the specific guanylate cyclase inhibitor 1-H-(1,2,4)oxodiazolo(4,3a)quinoxalin-1-one (20 μmol). In the presence of 1 H -[1,2,4]oxodiazolo[4,3- a ]quinoxalin-1-one the effect of NO on renin secretion was abolished, whereas PDE-3 inhibitors exerted their normal effects. These findings suggest that PDE-3 plays a major role for the cAMP control of renin secretion. Our findings are compatible with the idea that the stimulatory effects of endogenous and exogenous NO on renin secretion are mediated by a cGMP-induced inhibition of cAMP degradation.

The beta-adrenoceptor agonists, isoprenaline, salbutamol and salmeterol, the non-selective phosphodiesterase (PDE) isoenzyme inhibitors, theophylline, trequinsin; the PDE3 isoenzyme inhibitor, milrinone; the PDE3/4 isoenzyme inhibitor, benzafentrine; and the PDE4 isoenzyme inhibitors, denbufylline, nitraquazone, RP 73401, Ro-20-1724, rolipram and tibenelast all induced concentration-dependent reversal of prostaglandin F2α-induced contraction of guinea-pig supervised trachea in vitro . The relaxant response of the non-selective PDE isoenzyme inhibitor trequinsin was slow in onset and demonstrated very slow recovery, similar to that observed with the long-acting beta 2 -adrenoceptor agonist, salmeterol and the PDE4 inhibitor, RP 73401. The relaxant agonists also significantly reversed bombesin-induced bronchospasm in anaesthetised guinea-pigs and there was a highly significant correlation between the ability of drugs to reverse PGF2α-induced contraction of guinea-pig isolated trachea in vitro and bombesin-induced bronchoconstriction in vivo . Furthermore, both salmeterol and trequinsin demonstrated long lasting bronchodilator responses consistent with the in vitro data. These results show that PDE isoenzyme inhibitors demonstrate different pharmacodynamic profiles that is not determined by PDE4 inhibitory potency and indicate that other factors may be important in this regard.

1. In the present study we examined whether interleukin-1 beta (IL-1 beta) increases the activity of adenylyl cyclase in vascular smooth muscle cells and determined its role in the cytokine-induced expression of the inducible nitric oxide synthase (iNOS) and activation of nuclear transcription factor-kappa B (NF-kappa B). In addition the interaction between cyclic AMP- and cyclic GMP-elevating agonists on the IL-1 beta-stimulated expression of iNOS was examined. 2. Exposure of vascular smooth muscle cells to IL-1 beta stimulated the formation of cyclic AMP but not of cyclic GMP. The intracellular level of cyclic AMP reached a maximum within 1 h and then gradually declined over the next 5 h. This IL-1 beta (60 u ml-1)-stimulated formation of cyclic AMP was modest (about 3 fold at 60 u ml-1 for 1 h) compared to that evoked by isoprenaline (about 9 fold at 3 x 10(-6) M for 2 min). 3. The IL-1 beta (60 u ml-1 for 24 h)-stimulated accumulation of nitrite, which was taken as an index of NO production, was concentration-dependently increased by preferential inhibitors of cyclic AMP-dependent phosphodiesterases (rolipram and trequinsin). This effect was reproduced by a specific activator of the cyclic AMP-dependent protein kinase(s) A, Sp-8-CPT-cAMPS (10(-4) M) but was prevented by a specific inhibitor of cyclic AMP-dependent protein kinase(s) A, Rp-8-CPT-cAMPS (10(-4) M). These compounds alone [rolipram (10(-6) M), trequinsin (3 x 10(-6) M) and Sp-8-CPT-cAMPS (10(-4) M)] slightly but significantly increased the release of nitric oxide while Rp-8-CPT-cAMPS elicited no such effect. 4. Inducible NOS protein was expressed in IL-1 beta (30 u ml-1, 24 h)-stimulated smooth muscle cells as assessed by Western blot analysis. The level of iNOS protein was markedly increased in smooth muscle cells which had been exposed to IL-1 beta in combination with either rolipram (3 x 10(-6) M) or Sp-8-CPT-cAMPS (10(-4) M) but was reduced in those exposed to IL-1 beta and Rp-8-CPT-cAMPS (10(-4) M). A weak expression of iNOS protein was found in smooth muscle cells which had been exposed to either Sp-8-CPT-cAMPS or rolipram alone for 24 h while Rp-8-CPT-cAMPS elicited no such effect. 5. Exposure of smooth muscle cells to IL-1 beta (30 u ml-1) for 30 min increased the level of NF-kappa B-DNA complexes in nuclear extracts as detected by electrophoretic mobility shift assay. Similar levels of NF-kappa B-DNA complexes were found in cells which had been exposed to IL-1 beta in combination with either Sp-8-CPT-cAMPS (10(-4) M), trequinsin (10(-6) M) or rolipram (10(-6) M). None of the modulators alone affected the basal level of NF-kappa B binding activity. 6. NO-donors [sodium nitroprusside (SNP) 10(-4) M; dinitrosyl-iron-di-L-cysteine-complex (DNIC), 10(-4) M; 3-morpholino-sydnonimine (SIN-1), 10(-4) M] and atrial natriuretic factor (10(-6) M) significantly increased the IL-1 beta (30 or 60 u ml-1, 24 h)-stimulated expression of iNOS protein and activity as assessed indirectly by the conversion of oxyhaemoglobin to methaemoglobin. In the absence of IL-1 beta, SNP (10(-4) M, 24 h) but not the other cyclic GMP-dependent vasodilators caused a modest expression of iNOS protein. No such effect was found in smooth muscle cells exposed to SNP in combination with Rp-8-CPT-cAMPS (10(-4) M) while an increased level of iNOS protein was found in those exposed to SNP in combination with either Sp-8-CPT-cAMPS (10(-4) M) or rolipram (3 x 10(-6) M). 7. Exposure of vascular smooth muscle cells to either S-nitroso-L-cysteine (Cys-SNO, 10(-4) M), SNP (10(-4) M) or SIN-1 (10(-4) M) for 35 min affected minimally the basal activation of NF-kappa B but abolished that evoked by IL-1 beta (30 u ml-1 added during the last 30 min). However, addition of Cys-SNO following the stimulation with IL-1 beta (during the last 5 min of the 30 min exposure period) reduced the level of NF-kappa B-DNA complexes only slightly. 8. These data indicate that the cyclic AMP-dependent pathway plays a decisi

The effects of isoenzyme selective phosphodiesterase (PDE) inhibitors and other anti-asthma drugs on antigen-induced eosinophil recruitment and activation in guinea-pig airways was studied. Guinea-pigs were sensitized and subsequently challenged with aerosolized ovalbumin (OVA). Bronchoalveolar lavage (BAL) was performed 24 h later. A significant increase in eosinophils and eosinophil peroxidase (EPO) was detected in BAL fluid and BAL fluid supernatant respectively from OVA immunized guinea-pigs compared with sham treated animals. Guinea-pigs were treated for 7 days prior to antigen challenge with either the following drugs or the appropriate vehicle (i.p.). The selective beta 2 agonist, salbutamol (0.3 mg/kg), the PDE III inhibitor, milrinone (15 mg/kg) and the non-selective PDE inhibitor, trequinsin (1 mg/kg) had no effect on eosinophil number or EPO levels. The PDE IV inhibitor, rolipram (15 mg/kg), the mixed type III/IV PDE inhibitor, benzafentrine (15 mg/kg) and the non-selective PDE inhibitor, aminophylline (31.5 mg/kg) had no effect on eosinophil number but reduced the amount of EPO detected. The anti-inflammatory glucocorticosteroids, beclomethasone (10 mg/kg) and betamethasone (4 mg/kg) and the type IV PDE inhibitor, RP 73401 (5 mg/kg) reduced both eosinophil numbers and EPO levels. These results suggest a role for the type IV PDE isoenzyme in the control of eosinophil recruitment and possibly activation in the airways.

In most cells, the steady-state level of cAMP ultimately depends on the rate of cAMP synthesis by adenylyl cyclase and the rate of cAMP hydrolysis by cyclic nucleotide phosphodiesterases (PDEs). PDEs exist in multiple forms that have been grouped into seven families based on their substrate specificity, mode of regulation and kinetic properties. Selective inhibitors of many PDE families are now available. Examples are milrinone and trequinsin (PDE3); rolipram and Ro 20-1724 (PDE4); and zaprinast, sildenafil and didyridamole (PDE5). These inhibitors have proven to be valuable tools to investigate the role of PDEs in cell function. Representatives of most PDE families are present in the kidneys, and recent studies in this and other laboratories have provided evidence that some of them participate in the regulation of renin secretion. In particular, administration of selective PDE inhibitors has marked effects on renin secretion. For example, the PDE3 inhibitors milrinone and trequinsin increase resting renin in conscious rabbits and enhance the renin secretory response to beta-adrenergic stimulation. Milrinone also increases renin secretion in human subjects. The PDE4 inhibitors rolipram and Ro 20-1724 both increase renin secretion in rabbits and also enhance the renin response to beta-adrenergic stimulation. Studies in other laboratories have implicated other PDE families in the control of renin secretion. The aim of this review is to present current concepts concerning the PDEs and to discuss their role in the control of renin secretion by the kidneys.

Treatment of membranes from guinea-pig peritoneal eosinophils with deoxycholate and NaCl solubilized greater than 95% of the particulate cyclic AMP-specific phosphodiesterase (PDE IV). Solubilized PDE IV was at least 10 times more potently inhibited by selective PDE IV inhibitors (e.g. rolipram, denbufylline) than bound enzyme. Vanadate/glutathione complex (V/GSH) activated membrane-bound PDE IV and also increased potencies of these same inhibitors by at least 10-fold. Neither solubilization nor V/GSH markedly influenced the inhibitory activities of non-selective inhibitors (e.g. trequinsin, dipyridamole). Inhibitor effects on solubilized PDE IV and cyclic AMP accumulation in intact cells were strongly correlated. These results suggest a biologically important site on eosinophil PDE IV which is concealed or partially concealed in freshly prepared membranes and is exposed by solubilization or V/GSH.

Sulindac displays promising antineoplastic activity, but toxicities from cyclooxygenase inhibition limit its use for chemoprevention. Previous reports suggest that its anticancer properties may be attributed to a cyclooxygenase-independent mechanism, although alternative targets have not been well defined. Here, we show that sulindac sulfide (SS) induces apoptosis and inhibits the growth of human breast tumor cells with IC50 values of 60 to 85 μmol/L. Within the same concentration range, SS inhibited cyclic GMP (cGMP) hydrolysis in tumor cell lysates but did not affect cyclic AMP hydrolysis. SS did not induce apoptosis of normal human mammary epithelial cells (HMEC) nor did it inhibit phosphodiesterase (PDE) activity in HMEC lysates. SS increased intracellular cGMP levels and activated protein kinase G in breast tumor cells but not HMEC. The guanylyl cyclase (GC) activator, NOR-3, and cGMP PDE inhibitors, trequinsin and MY5445, displayed similar growth-inhibitory activity as SS, but the adenylyl cyclase activator, forskolin, and other PDE inhibitors had no effect. Moreover, GC activation increased the sensitivity of tumor cells to SS, whereas GC inhibition reduced sensitivity. By comparing PDE isozyme profiles in breast tumor cells with HMEC and determining the sensitivity of recombinant PDE isozymes to SS, PDE5 was found to be overexpressed in breast tumor cells and selectively inhibited by SS. The mechanism of SS binding to the catalytic domain of PDE5 was revealed by molecular modeling. These data suggest that PDE5 inhibition is responsible for the breast tumor cell growth–inhibitory and apoptosis-inducing activity of SS and may contribute to the chemopreventive properties of sulindac. [Mol Cancer Ther 2009;8(12):3331–40]

Experiments have been performed to characterize guinea-pig peritoneal eosinophil cyclic nucleotide phosphodiesterase (PDE) activity and establish whether it is involved in regulating superoxide (.O − 2 ) generation. Eosinophils were found to contain a predominantly membrane-bound cAMP PDE(s) (92.5 ± 2.4% of total activity) which was resistant to solubilization with Triton X-100 (1%). This particulate PDE exhibited complex kinetics ( K m = 1.3 and 31.4 μ M) and was unaffected by cGMP ( ic 50 > 100 μ M ) or CaCl 2 (2 mM) + calmodulin (10 units/mL). Little cGMP PDE activity was detected in either the soluble or particulate fractions. Inhibitors of the Ro-20-1724-inhibited (Type IV) cAMP PDE, namely Ro-20-1724 ( ic 50 = 0.92 ± 0.43 μ M ) , rolipram ( ic 50 = 0.20 ± 0.04 μ M ) and denbufylline ( ic 50 = 0.20 ± 0.01 μ M ) , potently inhibited the particulate cAMP PDE, as did the non-selective inhibitors trequinsin ( ic 50 = 0.11 ± 0.02 μ M ) and AH-21-132 ( ic 50 = 2.57 ± 0.02 μ M ) . Eosinophil cAMP PDE was resistant to SK&F 94120 ( ic 50 > 1000 μM), the cGMP-inhibited (Type III) cAMP PDE inhibitor, and the cGMP PDE (Type I) inhibitor, zaprinast, was only weakly active ( ic 50 = 35.33 ± 10.74 μ M ) . O − 2 release from resting cells was potently inhibited by rolipram ( ic 50 = 0.05 ± 0.03 μ M ) and denbufylline ( ic 50 = 0.06 ± 0.04 μ M ) but surprisingly, in view of its potent cAMP PDE inhibitory activity, was only weakly decreased by trequinsin ( ic 50 = 8.0 ± 2.7 μ M ) . AH-21-132 ( ic 50 > 10 μ M ) , SK&F 94120 ( ic 50 > 10 μ M ) and zaprinast ( ic 50 > 10 μ M ) were without effect. Rolipram and denbufylline alone exerted little effect on cAMP in intact cells but, in the presence of 10 μM isoprenaline, potently increased intracellular accumulation ( ec 50 = 0.45 ± 0.16 and 0.28 ± 0.08 μ M , respectively). Trequinsin and AH21–132 only weakly enhanced isoprenaline-stimulated cAMP accumulation. Although it induced a marked rise in cAMP only in the presence of isoprenaline, rolipram (50 μM) alone was able to increase the activity ratio of cAMP-dependent protein kinase from 0.24 to 0.84. The results suggest that Ro-201724-inhibited cAMP PDE plays a role in regulating eosinophil. O − 2 generation. The poor correlation between the PDE inhibitory actions of certain compounds and their effectiveness in elevating cAMP and inhibiting. O − 2 suggests the existence of a barrier impeding access to the enzyme.

Experiments were carried out in order to isolate and characterize the cyclic nucleotide phosphodiesterase activities in primary and low passages of cultured bovine aortic endothelial cells. The subcellular characterization of the cyclic nucleotide hydrolytic activity showed that both cAMP and cGMP hydrolytic activities were predominant in the cytosolic rather than the particulate fraction of the endothelial cell homogenate. At a low substrate concentration (0.25 μM), the major hydrolytic activity was for cAMP while at a high concentration (20 μM) it was for both cAMP and cGMP. Both cAMP and cGMP hydrolytic activities were insensitive to calmodulin. Cytosolic cyclic nucleotide phosphodiesterase activity was resolved into two distinct phosphodiesterase forms using HPLC. The first eluted form was designated cGS-PDE: it hydrolysed both cAMP and cGMP and its cAMP hydrolytic activity was markedly enhanced by the presence of cGMP. The second form was designated cAMP-PDE: it selectively hydrolysed cAMP. The cytosolic cAMP-PDE was inhibited by micromolar concentrations of cAMP-PDE inhibitors such as trequinsin, rolipram, dipyridamole or papaverine. The cGS-PDE was inhibited by micromolar concentrations of trequinsin, dipyridamole and papaverine and was insensitive to rolipram, except for the hydrolysis of cAMP which was inhibited in the micromolar range. Both the cAMP-PDE and the cGS-PDE were relatively insensitive to the selective cGMP-PDE inhibitor, zaprinast which was about 750-fold less potent on endothelial PDEs than on smooth muscle cGMP-PDE. The identification of selective and specific PDE inhibitors of the different PDE forms may allow a better understanding of the regulation and the role of cyclic nucleotides in endothelial cells.

Zur näheren Charakterisierung der nicht-adrenerg nicht-cholinerg vermittelten Relaxation am Magenfundus der Ratte wurde die Wirkung inhibitorischer Stimuli (elektrische Feldstimulation (EFS), vasoaktives intestinales Polypeptid (VIP), atriales natriuretisches Peptid (ANP) und Diethylamin-NO (DEA-NO)) auf das Motilitätsmuster isolierter Muskelstreifen untersucht. Dabei zeigte sich, daß die NO induzierte Relaxation cGMP abhängig über eine Aktivierung der löslichen Guanylatzyklase vermittelt und durch eine cGMP spezifische Phosphodiesterase (PDE V) limitiert wird. Die Proteinkinase A bzw. cAMP abhängige Mechanismen sind dabei nicht von Bedeutung. Die VIP und ANP vermittelte Relaxation wird über eine Aktivierung der Proteinkinase A vermittelt und durch eine cGMP inhibierte, cAMP spezifische Phosphodiesterase (PDE III) limitiert. Eine Beteiligung der Guanylatzyklase bzw. cGMP abhängiger Mechanismen liegt nicht vor. Bei der EFS induzierten Relaxation scheinen die Purine (z.B. ATP), die ihre Wirkung unter anderem über einen Ca2+ abhängigen K+ Kanal mit niedriger Leitfähigkeit entfalten, nicht wesentlich beteiligt zu sein. The aim of this study was to further characterize the mechanisms of non-adrenergic non-cholinergic inhibitory neurotransmission in the rat gastric fundus, using isolated gastric fundus muscle strips. Electrical field stimulation (EFS), vasoactive intestinal polypeptide (VIP), atrial natriuretic peptide (ANP) and the NO donor diethylamine-NO (DEA NO) were used as inhibitory stimuli. NO induced relaxation follows cGMP dependent pathways, involving activation of the soluble guanylate cyclase and is limited by a cGMP specific phosphodiesterase (PDE V). Proteinkinase A and cAMP dependent mechanisms are not involved. Relaxation induced by VIP and ANP includes activation of proteinkinase A, without involvement of guanylate cyclase and cGMP, respectively, and it´s extent is limited by a cGMP inhibited cAMP specific phosphodiesterase (PDE III). Concerning EFS induced relaxation, there is no evidence for significant involvement of the purins (e.g. ATP), that act via acivation of small conductance Ca2+ dependent K+ channels.

Cellular injury can involve the aberrant stimulation of cell cycle proteins in part through activation of phosphodiesterases (PDEs) and downstream expression of cell-cycle components such as cyclin D1. In mature non-proliferating cells activation of the cell cycle can lead to the induction of programmed cell death. In the present study, we investigated the in vitro neuroprotective efficacy and mechanism of action of vinpocetine (PDE1 inhibitor), trequinsin (PDE3 inhibitor), and rolipram (PDE4 inhibitor) in four mechanistically-distinct models of injury to primary rat cortical neurons as related to cell cycle regulation and apoptosis. Cellular injury was induced by hypoxia/hypoglycemia, veratridine (10 microM), staurosporine (1 microM), or glutamate (100 microM), resulting in average neuronal cell death rates of 43-48% as determined by MTT assay. Treatment with each PDE inhibitor (PDEI) resulted in a similar concentration-dependent neuroprotection profile with maximal effective concentrations of 5-10 microM (55-77% neuroprotection) in all four neurotoxicity models. Direct cytotoxicity due to PDE inhibition alone was not observed at concentrations below 100 microM. Further studies indicated that PDEIs can suppress the excitotoxic upregulation of cyclin D1 similar to the effects of flavopiridol, a cyclin-dependent kinase inhibitor, including suppression of pro-apoptotic caspase-3 activity. Overall, these data indicate that PDEIs are broad-spectrum neuroprotective agents acting through modulation of cell cycle elements and may offer a novel mode of therapy against acute injury to the brain.

The diseases of cystic fibrosis, chronic obstructive pulmonary disease (COPD), and chronic bronchitis are characterized by mucus-congested and inflamed airways. Anti-inflammatory agents that can simultaneously restore or enhance mucociliary clearance through cystic fibrosis transmembrane conductance regulator (CFTR) activation may represent new therapeutics in their treatment. Herein, we report the activation of CFTR-mediated chloride secretion by phosphodiesterase (PDE) 4 inhibitors in T84 monolayer using (125)I anion as tracer. In the absence of forskolin, the iodide secretion was insensitive to PDE4 inhibitor L-826,141 [4-[2-(3,4-bis-difluoromethoxyphenyl)-2-[4-(1,1,1,3,3,3-hexafluoro-2-hydroxypropan-2-yl)phenyl]-ethyl]-3-methylpyridine-1-oxide], roflumilast, or to PDE3 inhibitor trequinsin. However, these inhibitors potently augmented iodide secretion after forskolin stimulation, with efficacy coupled to the activation states of adenylyl cyclase. The iodide secretion from PDE3 or PDE4 inhibition was characterized at first by a prolonged efflux duration, followed by progressively elevated peak efflux rates at higher inhibitor concentrations. Paralleled with an increased phosphor-cAMP response element-binding protein formation, the CFTR activation dissociated from a global cAMP elevation and was blocked by H89 [N-[2-((p-bromocinnamyl)amino)ethyl]-5-isoquinolinesulfonamide]. 2-(4-Fluorophenoxy)-N-[(1S)-1-(4-methoxyphenyl)ethyl]nicotinamide, a stereoselective PDE4D inhibitor, augmented iodide efflux more efficiently than its less potent (R)-isomer. The peak efflux from maximal PDE4 and PDE3 inhibition matched that from full adenylyl cyclase activation. These data suggest that PDE3 and PDE4 (mainly PDE4D) form the major cAMP diffusion barrier in T84 cells to ensure a compartmentalized CFTR signaling. Together with their potent anti-inflammatory properties, the potentially enhanced airway mucociliary clearance from CFTR activation may have contributed to the efficacy of PDE4 inhibitors in COPD and asthmatic patients. PDE4 inhibitors may represent new opportunities to combat cystic fibrosis and other respiratory diseases in future.

Gravity alteration (micro- and hypergravity) is known to influence cell functions. As guanosine 3',5'-cyclic monophosphate (cGMP) plays an important role in human melanocyte functions and different guanylyl cyclase isoforms are responsible for cGMP synthesis in human non-metastatic and metastatic melanoma cells, we investigated the effects of hypergravity on the regulation of cGMP levels in cultured human melanocytes and in melanoma cell lines with different metastatic potentials. Hypergravity was produced by horizontal centrifugal acceleration. Here we report that long-term application of hypergravity (up to 5 g for 24 h) stimulated cGMP efflux in cultured melanocytes and in non-metastatic melanoma cells in the presence of 0.1 mM 3-isobutyl-1-methylxanthine (IBMX), a non-selective phosphodiesterase (PDE) inhibitor. Under these conditions, cAMP synthesis and melanin production were up-regulated in pigmented melanocytes and non-metastatic melanoma cells. Hypergravity also stimulated cGMP transport in the presence of 1 microM trequinsin, an inhibitor of cGMP-binding PDE (PDE5) and of transport by multidrug resistance proteins MRP4/5, whereas 50 microM trequinsin partially inhibited cGMP transport. Transport was further inhibited by probenecid, an inhibitor of endogenous non-selective transporters as well as of MRP4/5 and by cycloheximide as an inhibitor of de novo protein synthesis. In contrast, hypergravity did not affect cGMP efflux in metastatic melanoma cells, which might be related to an up-regulated cGMP efflux at 1 g. The results of the present study indicate that hypergravity may stimulate cGMP efflux in melanocytes and in non-metastatic melanoma cells most probably by an enhanced expression of endogenous transporters and/or MRP4/5. Thus, an altered acceleration vector may induce signaling events in melanocytic cells.

PURPOSE: Pharmacologic treatments are gaining widespread acceptance as first-line therapy for anal fissure. However, existing treatments have limited clinical usefulness because of side effects and incomplete healing rates. METHODS: Fresh human surgical resection specimens containing internal anal sphincter and rectal circular muscle were collected. Strips of smooth muscle were cut from each muscle group and mounted in a superfusion organ bath. The effects of increasing concentrations of phosphodiesterase inhibitors were evaluated. RESULTS: All phosphodiesterase inhibitors tested caused a dose-dependent reduction in the tone of the internal anal sphincter, with potencies as follows: vinpocentine (phosphodiesterase-1 inhibitor; 50 percent maximum inhibition concentration = 0.87 ± 0.10 μM), erythro-9-(2-hydroxy-3-nonyl) adenine hydrochloride (phosphodiesterase-2 inhibitor; 32 ± 4.8 μM), trequinsin (phosphodiesterase-3 inhibitor; 0.28 ± 0.041 μM), rolipram (phosphodiesterase-4 inhibitor; 63 ± 9 μM), zaprinast (phosphodiesterase-1,5,6,9,11 inhibitor; 3 ± 0.69 μM), and dipyridamole (phosphodiesterase-5,6,8,10,11 inhibitor; 5.5 ± 2 μM). Although all inhibitors were also effective on rectal circular muscle strips, erythro-9-(2-hydroxy-3-nonyl) adenine hydrochloride, trequinsin, and rolipram were at least an order of magnitude more potent in this tissue than in the internal anal sphincter. CONCLUSIONS: There are several functionally important phosphodiesterases in the internal anal sphincter and rectal circular muscle. Both adenosine 3′, 5′-cyclic monophosphate and guanosine 3′,5′-cyclic monophosphate appear to be important in the myogenic tone of the internal anal sphincter, and this study provides further evidence of the sphincteric specialization of this tissue. Phosphodiesterase inhibitors might represent a new therapy for the treatment of anal fissure.

The effects of family selective inhibitors of phosphodiesterase (PDEI, PDE2, PDE3, PDE4, and PDE5) on the behavior of rats under either a differential-reinforcement-of-low-rate (DRL) 72-s schedule or a variable-interval (VI) 30-s schedule were determined; previous work has shown that antidepressant drugs increase reinforcement rate under long DRL schedules. The PDE4-selective inhibitor rolipram (0.03-0.1 mg/kg) reduced response rate and increased reinforcement rate under the DRL schedule in a dose-dependent manner; similar effects were observed with the tricyclic antidepressant drug desipramine (3-10 mg/kg). Both of these drugs produced biphasic effects on behavior maintained under the VI schedule, increasing response rate at the lower doses tested (rolipram: 0.003 mg/kg; desipramine: 0.03 mg/kg) and decreasing response rate at higher doses (rolipram: 0.1 mg/kg; desipramine: 0.3-18 mg/kg). Of the other PDE inhibitors tested, only the PDE5-selective inhibitor zaprinast (10 mg/kg) produced an antidepressant-like effect on DRL behavior. However, in contrast to the biphasic effects of rolipram and desipramine on VI behavior, zaprinast produced monotonic decreases in response rate (10-30 mg/kg). The PDE2-selective inhibitor trequinsin produced biphasic effects on response rate under the VI schedule, increasing rates at low doses (3-5.6 mg/kg) and decreasing rates at higher doses (18-30 mg/kg). Trequinsin also reduced response rate under the DRL schedule (30 mg/kg); however, the reduction in response rate was not accompanied by increased reinforcement rate. The PDE3-selective inhibitor milrinone (1-10 mg/kg) tended to increase response rates under both schedules while the PDE1-selective inhibitor vinpocetine did not affect behavior at the dose range tested (1-30 mg/kg). These findings suggest that inhibition of PDE4 results in a rather unique pattern of behavioral effects, most notably an antidepressant-like effect on DRL behavior. It remains to be determined if a similar effect produced by zaprinast also implicates PDE5 in the mediation of antidepressant activity or represents an effect of this drug on PDE4 activity at high doses.

Rationale and objectives This study investigated the involvement of cyclic adenosine monophosphate (cAMP) in contrast medium-induced renal vasomotor effects and the efficacy of selective phosphodiesterase (PDE) inhibitors influencing cAMP in preventing contrast medium-induced renal vasospasm. Methods Isometric contractions of rabbit renal artery rings were subjected to increasing concentrations of the ionic contrast medium sodium/meglumine diatrizoate (DIA) and the nonionic contrast media iopamidol (IOP) and iodixanol (IOD) and compared with a potassium chloride control. Subsequently increasing concentrations of the nonselective phosphodiesterase inhibitors theophylline and papaverine and the following selective phosphodiesterase inhibitors were applied: vinpocetine, trequinsin, zardaverine, rolipram, and dipyridamole (subtypes I-V) before restimulation of the arterial tissue with contrast medium. Results Diatrizoate, iopamidol, and iodixanol induced contractions up to 30%, 15%, and 3.5% of the potassium chloride control, respectively. All phosphodiesterase inhibitors markedly inhibited the contrast medium-induced contractions in a dose-dependent manner. The selective phosphodiesterase inhibitors rolipram and trequinsin attenuated these contractions significantly more (92% and 94%) than did zardaverine, dipyridamole, and vinpocetine, with an inhibitory potency of 37%, 41%, and 62%, respectively. Conclusions Nonionic contrast media induced renal vasoconstriction less potently than ionic contrast media. Significant differences in the ability to prevent contrast medium-induced vasoconstriction were observed among the various phosphodiesterase subtypes studied. selective phosphodiesterase inhibition with inhibitor subtypes II and IV showed the most promising results in specifically preventing contrast medium-induced renal vasospasm.

Phosphodiesterases (PDEs) are responsible for the hydrolysis of cAMP and cGMP which act as intracellular second messengers in a variety of cellular functions. In this paper we report that PDE3 and PDE4 were two dominant classes of PDEs expressed in HL60 cells. The influence of specific PDE inhibitors on apoptosis in HL60 cells was studied. The non-specific inhibitor IBMX and PDE3 specific inhibitors (milrinone and trequinsin) did not promote apoptosis. They inhibited apoptosis induced by paclitaxel or thapsigargin. However, PDE4 specific inhibitors (rolipram and RO-20-1724) promoted apoptosis within 5 h. In HL60 cells, other cAMP-eliciting reagents (8-bromo-cAMP, Sp-cAMP and forskolin) also inhibited apoptosis, while cell-permeable cGMP analogs did not affect apoptosis. Therefore, IBMX and PDE3 specific inhibitors may prevent HL60 cells from apoptosis by increasing intracellular cAMP. However, apoptosis induced by PDE4 specific inhibitors is not likely due to increased cAMP level. These results suggest that rolipram and RO-20-1724 promoted apoptosis in HL60 cells through cAMP-independent mechanism.

Hepatocellular carcinoma (HCC) is the fifth common malignancy worldwide and the third leading cause of cancer-related death. Targeted therapies for HCC are being extensively developed with the limited success of sorafinib. In the present study, we investigated the potential antitumor activity of zardaverine, a dual-selective phosphodiesterase (PDE) 3/4 inhibitor in HCC cells both in vitro and in vivo. Although all zardaverine, PDE3 inhibitor trequinsin and PDE4 inhibitor rolipram increased intracellular cAMP levels through inhibiting PDE activity, only zardaverine significantly and selectively inhibited the proliferation of certain HCC cells, indicating that the antitumor activity of zardaverine is independent of PDE3/4 inhibition and intracellular cAMP levels. Further studies demonstrated that zardaverine induced G0/G1 phase cell cycle arrest of sensitive HCC cells through dysregulating cell cycle-associated proteins, including Cdk4, Cdk6, Cdk2, Cyclin A, Cyclin E, p21 and Rb. Notably, Rb expression was reversely related to the cell sensitivity to zardaverine. The present findings indicate that zardaverine may have potential as targeted therapies for some HCC, and the likely mechanism of action underlying its selective antitumor activity may be related to its regulation of Rb or Rb-associated signaling in cell cycles.

cAMP-specific phosphodiesterase inhibitors display a range of activities in vitro and in vivo which suggest they may be useful in the treatment of inflammatory diseases. However, these compounds elicit a number of side-effects which may limit their therapeutic potential. Certain side-effects of PDE4 inhibitors such as emesis and gastric acid secretion are associated with their actions at a high affinity rolipram binding site (HARBS). In contrast, a number of anti-inflammatory actions of PDE4 inhibitors are better correlated with inhibition of PDE4 catalytic activity than with displacement of [3H] rolipram from HARBS. This suggests that native PDE4s in different cell-types can be discriminated pharmacologically. Although known to be associated with PDE4, the nature of HARBS is uncertain. The majority of evidence suggests it represents particular conformational states of PDE subtypes with which rolipram interacts with high potency (KD approximately 2 nM) (High-affinity PDE4, HPDE4). Rolipram is generally moderately or weakly active (IC50-200 nM-2000 nM) in inhibiting catalytic activity of the majority of crude, partially-purified or recombinant PDE4-preparations (Low-affinity PDE4, LPDE4). Solubilization or V/GSH treatment of particulate eosinophil PDE4, cAMP-dependent kinase activation of RNPDE4D3 and membrane association of HSPDE4A4 increase the potencies of some (e.g., rolipram) but not other (e.g., trequinsin) inhibitors. In eosinophils, the changes in enzyme properties brought about by solubilization result in a close correlation between the potency order of compounds in inhibiting cAMP hydrolysis and displacing [3H] rolipram from HARBS. The identification of distinct pharmacological PDE4 forms may have therapeutic consequences since it may be possible to synthesize potent inhibitors of LPDE4 with low affinity for HARBS which should, theoretically, be less emetic. Most inhibitors synthesized to date (rolipram, denbufylline nitraquazone, etc.) display high-affinity for HARBS but are much weaker in inhibiting cAMP hydrolysis. Other compounds (RP 73401, trequinsin, CDP 840) display slightly higher potency against LPDE4 or do not discriminate between the two putative PDE4 forms. Recently, inhibitors have been synthesized which are considerably more active against LPDE4 than HPDE4. Such compounds with appropriate pharmacokinetic properties may retain anti-inflammatory activity but have a reduced capacity to cause nausea and emesis and, consequently, have a wider therapeutic window than compounds currently undergoing clincial evaluation.

Phosphodiesterase (PDE) inhibitors were shown to inhibit proliferation of various cell types. The present investigation was designed to study the activity of selective PDE inhibitors (8-MeoMIX, milrinone, trequinsin, rolipram, RO-201724, zaprinast, and MY-5445) on the proliferation of the Dami cell line in relation to their effects on cAMP levels and PDE isoenzymes isolated from Dami cells. All compounds, except 8-MeoMIX, elicited antiproliferative effects. Trequinsin, RO-201724, and MY-5445 (100 microM) were found to inhibit cell growth up to 60%, 83%, and 85%, respectively; milrinone, rolipram and zaprinast elicited only weak effects (19-21% at 100 microM). Their growth-inhibitory effects could not be related to their effects on cAMP levels. In addition, although PDE type III and IV inhibitors potentiated cAMP formation due to adenylycyclase activation, no potentiation could be observed when considering their antiproliferative effect. Separation and characterization of PDE of Dami cells revealed the existence of types III, IV, and V isoenzymes. The PDE inhibition found for the PDE inhibitors could not explain their antiproliferative effects. The lack of correlation with cAMP concentrations or PDE inhibition and the high concentrations needed to elicit antiproliferative effects suggest the implication of other parameters, such as cytotoxicity or lipophilicity, or other targets in addition to PDE for the PDE inhibitors tested. Lipophilicity did not seem to be of importance in antiproliferative effects. In contrast, cytotoxic effects, in particular those of trequinsin and MY-5445, could partially explain their negative action on cell growth.

The relaxing effects of several specific and nonspecific inhibitors of phosphodiesterases (PDE) on rabbit isolated corpus cavernosum (CC) and spongiosum (CS) were investigated. Preparations were mounted in organ baths, and isometric tension was recorded. The results were compared with the effects of direct administration of analogs of the second messenger cyclic nucleotides and the effects of forskolin, a direct stimulator of adenylate cyclase, and the nitric oxide donor 3-morpholinosydnonimine (SIN 1). All drugs relaxed the phenylephrine-induced contractions in CC and CS in a dose-dependent fashion. In CC and CS, type III (SKF 95654) and type V (zaprinast and dipyramidole) PDE inhibitors, as well as the nonspecific inhibitors papaverine and trequinsin, showed no differences in IC50. The type IV inhibitor rolipram relaxed CC and CS at significantly lower concentrations (p0.005) than any other PDE inhibitor, and in CC the type III and IV inhibitor zardaverine was more potent (p0.05) than SKF 95654. SIN 1 stimulates guanylate cyclase and effectively inhibits contractions in CC and CS. Activation of adenylate cyclase by forskolin also was highly effective (p0.005). It is concluded that PDE inhibition constitutes an effective relaxing mechanism in rabbit CC and CS. The marked effects of the different types of PDE inhibitors support the importance of cyclic guanosine 3',5'-monophosphate and cyclic adenosine 3',5'-monophosphate in smooth muscle relaxation in erectile tissue.

Ultraviolet irradiation of human platelet cytosol in the presence of 32P-labelled cyclic GMP (cGMP) can specifically label 110, 80, 55, 49 and 38 kDa proteins; the 110 kDa species is the subunit of cGMP-inhibited phosphodiesterase (PDE III) and the 80 kDa species that of cGMP-dependent protein kinase (Tang et al., 1993, Biochem. J. 294, 329). We have now shown that although photolabelling of platelet PDE III was inhibited by unlabelled cGMP, 8-bromo-cGMP and cyclic AMP (cAMP), it was not affected by phosphorothioate analogues of these cyclic nucleotides. Specific concentration-dependent inhibitions of the photolabelling of PDE III were observed with the following PDE inhibitors: trequinsin (IC50 = 13 +/- 2 nM), lixazinone (IC50 = 22 +/- 4 nM), milrinone (IC50 = 56 +/- 12 nM), cilostamide (IC50 = 70 +/- 9 nM), siguazodan (IC50 = 117 +/- 29 nM) and 3-isobutyl 1-methylxanthine (IBMX) (IC50 = 3950 +/- 22 nM). Thus, measurements of the inhibitory effects of compounds on the photolabelling of platelet PDE III provide a simple quantitative means of investigating their actions at a molecular level that avoids the need to purify the enzyme. Photolabelling of rat platelet lysate or rat heart homogenate by [32P]cGMP showed that the 110 kDa PDE III present in human material was replaced by a 115 kDa protein, labelling of which was also blocked by PDE III inhibitors. Heart and other rat tissues contained much less of this putative 115 kDa PDE III than rat platelets. In contrast, the 80 kDa protein was labelled much less in platelets than in many other rat tissue homogenates (e.g., heart, aorta, uterus and lung). Thus, comparison of the relative amounts of specific photolabelled proteins in different cells may provide an indication of different patterns of cyclic nucleotide action. We compared the abilities of phosphodiesterase inhibitors to block the photolabelling of PDE III in human platelet cytosol and to increase the iloprost-stimulated accumulation of cAMP in intact platelets. Whereas trequinsin (EC50 = 19 +/- 3 nM), lixazinone (EC50 = 122 +/- 8 nM), milrinone (EC50 = 5320 +/- 970 nM) and siguazodan (EC50 = 18880 +/- 3110 nM) all increased platelet cAMP to the same maximum extent, cilostamide and IBMX increased cAMP further, indicating that they inhibited a PDE isozyme in addition to PDE III.

Nonsteroidal anti-inflammatory drugs (NSAIDs) and cyclooxygenase (COX)-2 selective inhibitors display promising antineoplastic activity, but the gastrointestinal, renal, and cardiovascular toxicities that result from COX inhibition limit their clinical utility for cancer chemoprevention. Previous studies suggest that the pro-apoptotic activity of these drugs may be due to a COX-independent mechanism involving cyclic guanosine monophosphate (cGMP) phosphodiesterase (PDE) inhibition. However, the specific PDE isozyme(s) responsible has not yet been identified. Here we used the NSAID sulindac sulfide (SS) to further study cGMP PDE as a potential molecular target of the NSAIDs for breast cancer chemoprevention. SS inhibited growth and induced apoptosis of human SK-BR-3 and MDA-MB-231 breast tumor cells, while normal human mammary epithelial cells (HMEC) were less sensitive. SS also inhibited cGMP PDE activity from tumor cell lysates at concentrations comparable to those required for apoptosis, while appreciably higher concentrations were necessary to inhibit activity from HMEC lysates or cAMP PDE activity from any of the cell lysates. Additionally, SS increased intracellular cGMP concentrations and induced protein kinase G (PKG) activity in MDA-MB-231 cells as determined by ELISA and increased levels of phosphorylated VASP, a known PKG substrate, respectively. Moreover, pretreatment with the guanylyl cyclase inhibitor LY83583 desensitized the breast tumor cells to the growth-inhibitory activity of SS, likely by suppressing intracellular cGMP levels. The guanylyl cyclase activator NOR-3 and the cGMP PDE inhibitors MY5445 and trequinsin inhibited tumor cell growth, while the adenylyl cyclase activator forskolin and other PDE inhibitors had no effect. HMEC were found to express several cGMP hydrolyzing PDE isozymes (PDE1, 5, and 9), while the breast tumor cells expressed predominantly PDE5, which was confirmed to be sensitive to inhibition by SS using a recombinant enzyme. This work demonstrates that inhibition of PDE5A, elevation of cGMP, and activation of PKG are responsible for the pro-apoptotic effects of SS in human breast tumor cells and that differential expression of PDE isozymes between normal and tumor cells may explain the tumor selectivity of NSAIDs. These data also suggest that it may be feasible to develop NSAID derivatives with increased selectivity for inhibiting PDE5 and decreased COX-inhibitory activity, which would potentially improve safety and efficacy for breast cancer chemoprevention. Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 1041.

This article is a review of cyclic nucleotide phosphodiesterase(s) (CN PDE) from the point of view of the relationships between the newer aspects of the complex enzymology of CN PDE and recent major advances in CN PDE pharmacology. A consolidation of isozyme nomenclature to the proposed family designations is recommended. Emphasis is placed on the importance of defining the subcellular localization of isozymes expressed in a given tissue and cyclic GMP substrate and regulatory roles in CN PDE isozyme function. CN PDe inhibitors that may be useful for experimental and clinical purposes are discussed. Examples of these inhibitors include CGS 9343B, TCV-3B, KW-6, MIMAX, Dihydroisoquinolines, Trequinsin, bipyridine and dihydropyridazinone cardiotonics, Rolipram, SQ 65442, Zaprinast and Dipyridamole.

Primary sequence information has been reported for more than 15 different mammalian cyclic nucleotide phosphodiesterases. Moreover, recent observations suggest that many of these isozymes are selectively expressed in a limited number of cell types. The fact that nearly all these different phosphodiesterases have unique primary sequences in their catalytic or regulatory domains and that they are often selectively expressed implies that it may be possible to modulate individual isozymes using specific drugs. Joe Beavo and David Reifsnyder summarize much of the evidence that has led to our current understanding of multiple isozymes of phosphodiesterase, with emphasis on aspects that may be relevant to drug design. They also discuss why many previous attempts to isolate isozyme-selective inhibitors may have failed.

Two cyclic nucleotide phosphodiesterase (PDE) activities were identified in pig aortic endothelial cells, a cyclic GMP-stimulated PDE and a cyclic AMP PDE. Cyclic GMP-stimulated PDE had Km values of 367 microM for cyclic AMP and 24 microM for cyclic GMP, and low concentrations (1 microM) of cyclic GMP increased the affinity of the enzyme for cyclic AMP (Km = 13 microM) without changing the Vmax. This isoenzyme was inhibited by trequinsin [IC50 (concn. giving 50% inhibition of substrate hydrolysis) = 0.6 microM for cyclic AMP hydrolysis in the presence of cyclic GMP; IC50 = 0.6 microM for cyclic GMP hydrolysis] and dipyridamole (IC50 = 5 microM for cyclic AMP hydrolysis in the presence of cyclic GMP; IC50 = 3 microM for cyclic GMP hydrolysis). Cyclic AMP PDE exhibited a Km of 2 microM for cyclic AMP and did not hydrolyse cyclic GMP. This activity was inhibited by trequinsin (IC50 = 0.2 microM), dipyridamole (IC50 = 6 microM) and, selectively, by rolipram (IC50 = 3 microM). Inhibitors of cyclic GMP PDE (M&B 22948) and of low Km (Type III) cyclic AMP PDE (SK&F 94120) only weakly inhibited the two endothelial PDEs. Incubation of intact cells with trequinsin and dipyridamole induced large increases in cyclic GMP, which were completely blocked by LY-83583. Rolipram, SK&F 94120 and M&B 22948 did not significantly influence cyclic GMP accumulation. Dipyridamole enhanced the increase in cyclic GMP induced by sodium nitroprusside. Cyclic AMP accumulation was stimulated by dipyridamole and trequinsin with and without forskolin. Rolipram, although without effect alone, increased cyclic AMP in the presence of forskolin, whereas M&B 22948 and SK&F 94120 had no effects on resting or forskolin-stimulated levels. These results suggest that cyclic GMP-stimulated PDE regulates cyclic GMP levels and that both endothelial PDE isoenzymes contribute to the control of cyclic AMP.

A cGMP-specific phosphodiesterase (PDE6) preferentially hydrolyzes cGMP in the outer segments of photoreceptors. The level of cGMP controls the polarization state of photoreceptors through cGMP-gated channels in the disc membranes. Preliminary experiments suggest that PDE3B, a cAMP hydrolyzing, cGMP-inhibited PDE, may be found in photoreceptors PDE3B, a physiologically important mediator of insulin, IGF, and leptin, mediates an effect of cGMP on cAMP function in the smooth muscle. High expression of cGMP-inhibited PDE3B may mediate a similar process in photoreceptors by lowering the levels of intracellular cAMP, thus decreasing protein kinase A (PKA) activity. PKA phosphorylates phosducin, which when phosphorylated, does not bind to the beta-gamma subunit of transducin. Immunocytochemistry localize PDE3B to bovine retina with highest signal found over the cone outer segments, as well as to both the inner and outer segments of gecko rods. This was confirmed with western blots using specific antisera to the C-terminal 1/3 region of PDE3B, which was blocked by preincubation with PDE3B-GST fusion protein. There were no significant changes in the photoreceptor light response measured by single cell recordings of individual salamander rod and cone cells in the presence of 10μM milrinone (a PDE3 specific inhibitor). Intraperitoneal injections of 3mg/kg milrinone and 0.3mg/kg trequinsin (another more potent PDE3 inhibitor) did not significantly influence the A or B wave of mouse electroretinograms in response to flashes of light. These findings report a cAMP hydrolyzing, cGMP-inhibited PDE highly expressed in bovine and gecko photoreceptor cells. Under the conditions tested, PDE3B may exist in an inactive state in photoreceptors.

Three isoforms of cyclic nucleotide phosphodiesterase (PDE) have been recently isolated from aortic tissue and two of them specifically hydrolyzed adenosine 3',5'-cyclic monophosphate (cAMP) and guanosine 3':5'-cyclic monophosphate (cGMP), respectively (Lugnier et al., Biochem. Pharmac. 35, 1743, 1986). The role of these forms in controlling cyclic nucleotide levels and smooth muscle tone was investigated by the use of PDE inhibitors. The effects of selective inhibitors of the two forms specifically hydrolyzing cAMP or cGMP (cAMP-PDE and cGMP-PDE, respectively) were compared to those of non-selective inhibitors of the three aortic PDE forms, including the calmodulin-sensitive one (CaM-PDE). Relaxation responses and accumulation of tissue cAMP and cGMP induced by these drugs were studied in precontracted rat isolated aorta, and compared to the effects of isoprenaline and forskolin (stimulants of adenylate cyclase) or sodium nitroprusside (SNP) and sodium azide (stimulants of guanylate cyclase). The eight PDE inhibitors tested all relaxed aorta with potencies that correlated with their potencies as inhibitors of cAMP-PDE, but not of cGMP-PDE. At a concentration producing half-maximal relaxation, all PDE inhibitors induced a moderate but significant accumulation of cAMP, which was comparable to the accumulation of cAMP elicited by half-maximally relaxing concentrations of adenylate cyclase stimulating agents. At this concentration, some PDE inhibitors (M&B 22,948, dipyridamole and to a lesser extent, trequinsin) also induced a significant increase in cGMP levels, of the same order of magnitude as that caused by agents stimulating guanylate cyclase. However, the cGMP-increasing effect of these inhibitors was dissociated from their relaxing effect. In particular, the relaxing concentrations of M&B 22,948 (a selective inhibitor of cGMP-PDE) were clearly higher than the cGMP-increasing concentrations of the compound. At a concentration at which they elicited 10% relaxation by themselves, the selective cAMP-PDE inhibitor, rolipram, as well as the mixed inhibitor of cAMP- and cGMP-PDE, AAL 05 (a cilostamide analogue) enhanced both the cAMP-increasing and the relaxing effect of isoprenaline. Under the same conditions, no clear enhancement of the relaxation induced by SNP was observed. Only M&B 22,948 showed a slight potentiating effect on SNP-induced relaxation, but this effect was limited to low concentrations of SNP (less than 10 nM).(ABSTRACT TRUNCATED AT 400 WORDS)

Series of 3-substituted-9,10-dimethoxy-3,4,6,7-tetrahydro-2H-pyrimido [6,1-a]isoquinoline-2,4-diones and 2-substituted-9,10-dimethoxy-6,7-dihydro-4H-pyrimido [6,1-a]isoquinolin-4-ones were synthesized and tested for blood pressure lowering properties in anesthetized normotensive cats and conscious spontaneously hypertensive rats. Several compounds in the 2-(arylamino)-9,10-dimethoxy-6,7-dihydro-4H-pyrimido [6,1-a]isoquinolin-4-one series display a high order of activity. The most active compounds are the alkyl derivatives of the 2-mesitylamino/2-mesitylimino tautomeric forms. The 2-(mesitylimino)-3-methyl analogue trequinsin is a potent antihypertensive agent and displays a hemodynamic profile characteristic of an arteriolar dilator. It is also a potent inhibitor of both cAMP phosphodiesterase and platelet aggregation.