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Introduction: Preclinical studies suggest a link between cAMP/PKA signalling, phosphodiesterase 4 (PDE4) expression and excessive daytime sleepiness (EDS). Here, we investigated in vivo the association between PDE4 expression and EDS in Parkinson's disease (PD) patients using [ 11 C]rolipram PET and MR imaging. Methods: Eighteen participants, 12 PD and 6 healthy controls, underwent one [ 11 C]rolipram PET and a multi-modal MRI scan. Probabilistic tractography was performed on subjects’ diffusion data to functionally parcellate the striatum according with projections to limbic cortical areas. The severity of EDS was assessed using the Epworth Sleepiness Scale (ESS). To assess PDE4 expression in PD patients with EDS, the PD cohort was divided according to the presence (n = 5) or absence (n = 7) of EDS, defined using validated cut-off of score ≥10 on the ESS as score ≥10 on the ESS. Results: PD patients with EDS showed significantly increased [ 11 C]rolipram volume of distribution (V T ) in the caudate (P = 0.029), hypothalamus (P = 0.013), hippocampus (P = 0.036) and limbic striatum (P = 0.030) compared to patients without EDS. Furthermore, higher ESS scores correlated with increased [ 11 C]rolipram V T in the caudate (r = 0.77; P = 0.003), hypothalamus (r = 0.84; P = 0.001), hippocampus (r = 0.81; P = 0.001) and limbic subdivisions of the striatum (r = 0.80; P = 0.003). Conclusion: Our findings translate into humans preclinical data indicating that EDS is associated with elevated PDE4 in regions regulating sleep. The severity of EDS in PD was associated with elevated PDE4 expression; thus, suggesting a role of PDE4 in the pathophysiology of EDS in PD.

Synaptic plasticity, such as long term potentiation (LTP) and long term depression (LTD), underlies the cellular mechanism of learning and memory. Chemical-induced LTP (cLTP), which facilitates biochemical analysis of molecular changes in brain slices or neuronal cultures, has been accepted as an in vitro model to explore synaptic plasticity. cLTP, by either forskolin and rolipram (F&R) or glycine, is thought to be dependent on NMDA receptor. However, subunit-specific dependence and regulation of the NMDA receptor in cLTP remain poorly understood. In the present study, we found that phosphorylation level of GluN2B at tyrosine 1472 was modulated by F&R-induced LTP but not by glycine-induced LTP in hippocampal slices. Furthermore, an increased phosphorylation level of GluA1 at serine 845 by F&R-induced LTP rather than glycine-induced LTP was dependent on the activation of GluN2B, which is supported by the results from GluN2B antagonists, small interfering peptide and CRISPR-Cas9-mediated knock out of GluN2B. Taken together, we reveal the significant role of GluN2B in F&R-induced LTP, uncovering the role of GluN2B subunit of NMDA receptor in a specified cLTP.

Development of drug addictive behaviors is modulated by both genetic and environmental risk factors. However, the molecular mechanisms remain unknown. To address the role of adolescent stress in the development of drug addiction, we combined a transgenic mouse model in which a putative dominant-negative form of DISC1 under expressional control of the prion protein promoter is used as a genetic risk factor and adolescent social isolation stress as a gene-environmental interaction (GXE). Repeated cocaine exposure induced greater locomotion in the GXE group than in the other groups. In a conditioned place preference (CPP) test, GXE mice exhibited a significant place preference to the cocaine-conditioned area compared with the other groups. In the nucleus accumbens (NAc) of GXE mice, we found increased enzyme activity of phosphodiesterase-4 (PDE4), predominantly located in NAc D2-receptor-expressing neurons, and enhanced effects of the PDE4 inhibitor rolipram, but not the D1 agonist SKF81297, on the phosphorylation of DARPP-32 and GluA1 at PKA sites. Rolipram injection before cocaine exposure completely inhibited cocaine-induced hyperlocomotion and CPP in the GXE group. These results indicate that GXE enhances sensitivity to repeated cocaine exposure via an increase in PDE4 activity in NAc D2-recptor-expressing neurons, leading to the development of cocaine addictive behaviors.

Antinociception caused by cannabinoids may have a partial peripheral origin in addition to its central site of action. In fact, we have observed that anandamide selectively and reversibly inhibits GABAA receptors of putative nociceptive neurons of mouse trigeminal sensory ganglia via CB1 receptor activation to inhibit adenylyl cyclase and decrease cAMP with downstream posttranslational alterations. Since cannabinoids are often used chronically, we studied changes in cAMP levels and GABA-mediated currents of trigeminal neurons following 24 h application of anandamide (0.5 μM) or the synthetic cannabinoid WIN 55,212-2 (5 μM). With this protocol GABA responses were similar to control despite persistent fall in cAMP levels. Inhibition by WIN 55,212-2 of GABA effects recovered after 30 min washout and was not associated with changes in CB1 receptor expression, indicating lack of CB1 receptor inactivation and transient loss of negative coupling between CB1 receptors and GABAA receptors. The phosphodiesterase inhibitor rolipram (100 μM; 24 h) enhanced cAMP levels and GABA-mediated currents, suggesting GABAA receptors were sensitive to persistent upregulation via cAMP. While the adenylyl cyclase activator forskolin (1–20 μM) facilitated cAMP levels and GABA currents following 30 min application, this action was lost after 24 h in line with the drug limited lifespan. The PKA inhibitor PKI 14–22 (10 μM) increased cAMP without changing GABA currents. These data indicate that modulation of GABAA receptors by intracellular cAMP could be lost following persistent application of cannabinoids. Thus, these observations provide an insight into the waning antinociceptive effects of these compounds.

Growing evidence confirms the potential of PDE4 inhibitors for the treatment of Parkinson's disease. Our reported PDE4 inhibitors FCPR16 and FCPR03 have displayed neuroprotective effects in SH-SY5Y cells, but have very low oral bioavailability. To access analogues with improved bioavailability, a new series of arylbenzylamine derivatives were designed and synthesized. Preliminary screening results of the series showed that arylbenzylamine derivatives bearing a pyridin-3-amine side chain displayed good inhibitory activities against human PDE4B1 and PDE4D7 isoforms. Moreover, kinetic studies revealed that the most potent compounds 11r and 11s with mid-nanomolar IC50 values partially bind to PDE4B1 (Imax = 93% and 90% respectively). Molecular docking results revealed the possible interactions of compounds 11r and 11s with upstream conserved region 2 (UCR2) of PDE4B1, which illuminate possible reasons for their partial inhibition against PDE4. Using a cell-based model of PD, compounds 11r and 11s were found to alleviate cellular apoptosis in SH-SY5Y cells induced by MPP+ (1-methyl-4-phenylpyridinium), with this neuroprotective effect being greater than PDE4 inhibitor rolipram. Furthermore, compound 11r displayed nearly sevenfold oral bioavailability (8.20%) than FCPR03 (1.23%).

In this paper, we report the assembling of libraries of β-arylated short/medium/long chain-based non-α-amino acid (aminoalkanoic acid) derivatives via the Pd(II)-catalyzed, bidentate directing group 8-aminoquinoline-aided sp3 β-C-H activation/arylation method. Short/medium chain-based unnatural amino acid derivatives containing an aryl group at the β-position are promising small molecules with therapeutic properties. Thus, it is necessary to enrich the libraries of short/medium/long chain-based unnatural amino acid derivatives containing an aryl group at the β-position. Considering the importance of β-arylated short/medium/long chain-based non-α-amino acid derivatives, an inclusive attention was paid to explore the Pd(II)-catalyzed sp3 β-C-H arylation of short/medium/long chain-based non-α-amino acids. Representative synthetic transformations including a short route for the assembling of rolipram and related compounds and 3-arylated GABA derivatives such as, baclofen, phenibut and tolibut were shown using selected β-C-H arylated non-α-amino acid derivatives.

Demirel-Yilmaz, Emine/0000-0002-2476-6225; Derici, Mehmet Kursat/0000-0002-8260-7492; Guray, Tulin/0000-0003-4572-1429 WOS: 000455010800015 PubMed: 30529467 Phosphodiesterase enzymes (PDEs) are responsible for the adjustment of cyclic nucleotide levels. Alterations in PDE expressions in different tissues cause conflicts between functional and clinical effects of PDE inhibitors. Therefore, the aim of this study was to investigate the gene and protein expressions and the functional role of PDEs in atrium and ventricle of rat heart The expressions of PDEs were examined in cardiac intact tissues and enzymatically isolated cells. The effects of PDE1-5 inhibitors (vinpocetine, EHNA, milrinone, rolipram, sildenafil, and IBMX) on basal and isoprenaline-stimulated contractions and sinus rate were recorded in the isolated spontaneously beating right atrium and electrically stimulated left papillary muscles. The mRNA and protein levels of PDEs were significantly different in atrial and ventricular intact tissues and isolated myocytes. Atrial contractions were increased with vinpocetine while suppressed by EHNA, milrinone, rolipram, sildenafil and IBMX. Milrinone, sildenafil and IBMX increased the heart rate whereas vinpocetine caused negative chronotropy. Papillary muscle contractions have been increased only with the vinpocetine and IBMX. Both the expression and the action of PDE-1-5 show atrial and ventricular differences. Therefore, these differences should be taken into account in the experimental or therapeutic approaches of the heart. Ankara University Research Foundation Ankara, TurkeyAnkara University [13B3330007] This study was supported by a research grants, 13B3330007 from the Ankara University Research Foundation Ankara, Turkey.

Recent studies have underscored the importance of the CA2 area in social memory formation. This area, a narrow transition zone between hippocampal CA3 and CA1 areas, is endowed with special connectivity and a distinctive molecular composition. In particular, adenosine A1 receptors (A1R) are enriched in CA2, and based on the prominent synaptic potentiation induced by A1R antagonists (e.g., caffeine) in this area, it has been proposed that CA2 is under the strong tonic control of A1R activation. It is unclear whether this special sensitivity of CA2 to A1R antagonists is due to an elevated extracellular concentration of adenosine or to a different A1R function. Here, using the recording of field potentials evoked simultaneously in CA2 and CA1 by Schaffer collateral stimulation, we confirm that the application of A1R antagonists, caffeine and DPCPX has a stronger effect on synaptic responses in CA2 than in those evoked in CA1. This difference was, at least partially, explained by the action of A1R antagonists on presynaptic A1Rs. We found that caffeine-induced potentiation in CA2 was restricted to Schaffer collateral synapses, but not to those formed by temporoammonic inputs. We also observed that the apparent affinity of an A1R agonist is similar for A1R in both CA2 and CA1 areas, which indicates that the tonic activation of A1R in both areas is comparable. Furthermore, we show that the direct activation of adenylyl cyclase with forskolin in the presence of rolipram, a phosphodiesterase inhibitor, greatly enhances the synaptic potentials in CA2 compared to CA1. The forskolin-induced potentiation was exacerbated in the presence of caffeine or DPCPX, accentuating the differences between the two areas. These results indicate that the tonic activation of A1Rs in area CA2 is not different to that of other hippocampal areas, but it is more efficiently coupled to the downstream effectors.

Neuropathic pain arises as a consequence of a lesion or disease affecting the somatosensory nervous system. It is accompanied by neuronal and non-neuronal alterations, including alterations in intracellular second messenger pathways. Cellular levels of 3',5'-cyclic adenosine monophosphate (cAMP) and 3',5'-cyclic guanosine monophosphate (cGMP) are regulated by phosphodiesterase (PDE) enzymes. Here, we studied the impact of PDE inhibitors (PDEi) in a mouse model of peripheral nerve injury induced by placing a cuff around the main branch of the sciatic nerve. Mechanical hypersensitivity, evaluated using von Frey filaments, was relieved by sustained treatment with the non-selective PDEi theophylline and ibudilast (AV-411), with PDE4i rolipram, etazolate and YM-976, and with PDE5i sildenafil, zaprinast and MY-5445, but not by treatments with PDE1i vinpocetine, PDE2i EHNA or PDE3i milrinone. Using pharmacological and knock-out approaches, we show a preferential implication of delta opioid receptors in the action of the PDE4i rolipram and of both mu and delta opioid receptors in the action of the PDE5i sildenafil. Calcium imaging highlighted a preferential action of rolipram on dorsal root ganglia non-neuronal cells, through PDE4B and PDE4D inhibition. Rolipram had anti-neuroimmune action, as shown by its impact on levels of the pro-inflammatory cytokine tumor necrosis factor-α (TNFα) in the dorsal root ganglia of mice with peripheral nerve injury, as well as in human peripheral blood mononuclear cells (PBMCs) stimulated with lipopolysaccharides. This study suggests that PDEs, especially PDE4 and 5, may be targets of interest in the treatment of neuropathic pain.

The purpose of this study was to evaluate in vitro the effect of the combination of BRL 37344 (s3-adrenoceptor agonist) with tadalafil (phosphodiesterase type 5 inhibitor) or rolipram (phosphodiesterase type 4 inhibitor) in an experimental model of detrusor overactivity. The experiments were carried out in two phases using bladder strips of mice. In the first phase, on the top of 40 mM potassium-induced contraction, strips isolated from control mice were exposed to increasing concentrations of each study drug. In another series of experiments, prior to contraction, strips were incubated with either tadalafil or rolipram, followed by the addition of increasing concentrations of BRL 37344. In the second phase, the same protocols were performed with animals previously treated with L-NAME for 30 days. Chronic L-NAME administration leads to detrusor overactivity due to nitric oxide synthase inhibition. In phase one, preincubation with tadalafil enhanced relaxation response to BRL 37344 at two concentrations. Pretreatment with rolipram had no effect on BRL 37344-induced relaxation. In L-NAME-treated mice, rolipram induced more relaxation than the other drugs, enhancing relaxation response to BRL 37344 at almost all concentrations, but no synergistic effect with tadalafil was observed. The relaxant effect of BRL 37344 was enhanced by rolipram but not by tadalafil, suggesting that PDE4 inhi bition, especially when associated with s3-adrenoceptor stimulation , could represent a potential treatment for overactive bladder.

Most pregnant women have symptoms of gastroesophageal reflux disease (GERD) during pregnancy. Postmenopausal hormone replacement therapy is associated with GERD. The effects of estradiol on lower esophageal sphincter (LES) motility and GERD are not clearly known. The purpose of this study is to investigate the effects of estradiol on the motility of the porcine LES. Relaxations of clasp and sling strips of porcine LES caused by estradiol were measured using isometric transducers. We investigated the mechanism of estradiol-induced relaxation of the porcine LES using tetraethylammonium, apamine, iberiotoxin, glibenclamide, KT5720, KT5823, NG-nitro-l-arginine, tetrodotoxin, and ω-conotoxin GVIA. Reverse transcription polymerase chain reaction (PCR) analysis and immunohistochemistry (IHC) were performed to determine the existence of the G protein-coupled estrogen receptor (GPER) in the porcine LES. In endothelin-1-precontracted porcine LES strips, estradiol caused marked relaxations in a concentration-dependent manner. The mechanism of estradiol-induced relaxation on the porcine LES was associated with the potassium channel. Reverse transcription PCR analysis and IHC revealed that GPER was expressed in the sling and clasp fibers of the porcine LES. This finding suggests that GPER mediates the relaxation of the porcine LES. Estradiol may play a role in LES motility.

Dexmedetomidine (DMED) is a potent and highly selective α2-adrenergic receptor agonist and is widely used for short-term sedation. However, the mechanism of DMED-induced sedation has not been deciphered. In the present study, we investigated the mechanism of Gαi and Gβγ subunits on DMED-induced sedation. An ED50 of DMED-induced loss of righting reflex (200.0nmol/kg) was increased to 375.0 or 433.3nmol/kg after pre-treatment with cAMP analog dbcAMP (50nmol/5 μl/mouse, i.c.v.) or the phosphodiesterase 4 inhibitor rolipram (100nmol/5 μl/mouse, i.c.v.). Conversely, the ED50 of DMED-induced LORR decreased to 113.6 or 136.5 nmol/kg after pre-treated with Gβγ subunit inhibitor M119 (100 mg/kg, i.p.) or gallein (100 mg/kg, i.p.) respectively. Administration of dbcAMP, rolipram, gallein or M119 alone had no effect on LORR. Gallein (10 μM) significantly inhibited forskolin-stimulated cAMP accumulation in α2A-AR -CHO cells. Compared with Gβγ subunit inhibitors or DMED alone, [Ca2+]i and pERK1/2 was significantly increased after co-administration with Gβγ subunit inhibitors and DMED. DbcAMP (5 μM) or rolipram (5 μM) alone had no effect on ERK1/2 phosphorylation, but decreased DMED-induced ERK1/2 phosphorylation after co-administration with DMED. Gβγ subunit inhibitor treatment increased DMED-induced phosphorylation of CREB, whereas dbcAMP or rolipram had no effect on pCREB induced by DMED. From our results we conclude that, Gβγ subunit may inhibit DMED-induced sedation through the cAMP and pERK1/2 pathway.

This paper describes our medicinal chemistry efforts on 7-(cyclopentyloxy)-6-methoxy1,2,3,4-tetrahydroisoquinoline scaffold: design, synthesis and biological evaluation using conformational restriction approach and bioisosteric replacement strategy. Biological data revealed that the majority of the synthesized compounds of this series displayed moderate to potent inhibitory activity against PDE4B and strong inhibition of LPS-induced TNFα release. Among them, compound 19 exhibited the strongest inhibition against PDE4B with an IC50 of 0.88 µM and 21 times more potent selectivity toward PDE4B over PDE4D when compared to rolipram. A primary structure-activity relationship study showed that the attachment of CH3O group or CF3O group to the phenyl ring at the para-position was helpful to enhance the inhibitory activity against PDE4B. Moreover, sulfonamide group played a key role in improving the inhibitory activity against PDE4B and subtype selectivity. In addition, the attachment of the additional rigid substituents at the C-3 position of 1,2,3,4-tetrahydroisoquinoline ring was favored to subtype selectivity, which was consistent well with the observed docking simulation.

Phosphodiesterase-4 (PDE4) plays a fundamental role in a range of central nervous system (CNS) insults, however, the role of PDE4 in early brain injury (EBI) after subarachnoid hemorrhage (SAH) remains unclear. The current study was designed to investigate the role of PDE4 in EBI after SAH and explore the potential mechanism. The SAH model in Sprague-Dawley rat was established by endovascular perforation process. Rats were randomly divided into: sham group, SAH + vehicle group, SAH + rolipram (PDE4 inhibitor) group, SAH + rolipram + sirtinol (SIRT1 inhibitor) group and SAH + rolipram+MK2206 (Akt inhibitor) group. Mortality, SAH grades, neurological function, brain edema, immunofluorescence staining and western blotting were performed. Double fluorescence labeling staining indicated that PDE4 was located predominately in neurons after SAH. Rolipram reduced brain edema, improved neurological function in the rat model of SAH. Moreover, rolipram increased the expression of Sirtuin1 (SIRT1) and up-regulated the phosphorylation of Akt, which was accompanied by the reduction of neuronal apoptosis. Administration of sirtinol inhibited the phosphorylation of Akt. Moreover, all the beneficial effects of rolipram against SAH were abolished by both sirtinol and MK2206. These data indicated that PDE4 inhibition by rolipram protected rats against EBI after SAH via suppressing neuronal apoptosis through the SIRT1/Akt pathway. Rolipram might be an important therapeutic drug for SAH.

© 2017 Elsevier B.V. Atrazine (ATR) alters female reproductive functions in different animal species. Here, we analyzed whether ATR disturbs steroidogenic and ovulatory processes in hormone-stimulated human cumulus granulosa cells and mechanism of its action. Results showed that treatment of human cumulus granulosa cells with 20 μM ATR for 48 h resulted in lower FSH-stimulated estradiol and progesterone production. ATR reduced mRNA levels of aromatase (CYP19A1), steroidogenic acute regulatory protein (STAR) and luteinizing hormone/choriogonadotropin receptor (LHCGR). Addition of hCG 48 h after FSH and ATR treatment did not trigger maximal expression of the ovulatory genes amphiregulin (AREG) and epiregulin (EREG). Mechanistic experiments showed that ATR activated cPDE and decreased cAMP level. Addition of total PDE and specific PDE4 inhibitors, IBMX and rolipram, prevented ATR's action on CYP19A1 and STAR mRNA expression in FSH-stimulated human cumulus granulosa cells. This study suggests that ATR alters steroidogenesis and ovulatory process in human cumulus granulosa cells jeopardizing female reproduction.

The bioassay-guided phytochemical study of a traditional Chinese medicine Morus alba led to the isolation of 18 prenylated flavonoids (1–18), of which (±)-cyclomorusin (1/2), a pair of enantiomers, and 14-methoxy-dihydromorusin (3) are the new ones. Subsequent structural modification of the selected components by methylation, esterification, hydrogenation, and oxidative cyclization led to 14 more derivatives (19–32). The small library was screened for its inhibition against phosphodiesterase-4 (PDE4), which is a drug target for the treatment of asthma and chronic obstructive pulmonary disease (COPD). Among them, nine compounds (1–5, 8, 10, 16, and 17) exhibited remarkable activities with IC50 values ranging from 0.0054 to 0.40 μM, being more active than the positive control rolipram (IC50 = 0.62 μM). (+)-Cyclomorusin (1), the most active natural PDE4 inhibitor reported so far, also showed a high selectivity across other PDE members with the selective fold greater than 55. The SAR study revealed that the presence of prenyls at C-3 and/or C-8, 2H-pyran ring D, and the phenolic hydroxyl groups were important to the activity, which was further supported by the recognition mechanism study of the inhibitors with PDE4 by using molecular modeling.

Quercetin and isoquercitrin were reported to have anti-inflammatory effects on typical asthma. Therefore, we investigated quercetin-3-O-glycosides to clarify the potential for treating atypical asthma. The inhibitions of quercetin-3-O-glycosides on PDE1–5 activities and on high-affinity rolipram binding sites were measured. The sensitized guinea-pig trachealis was challenged by ovalbumin (OVA) to induce contractions. Ten female BABL/c mice in each group were sensitized by OVA on days 0 and 14. On day 21, these mice were injected a mixture of 1% OVA and Freund’s complete adjuvant (1:1). Mice were challenged using 1% OVA in saline for 30 min on days 28, 29, and 30 by ultrasonic nebulization. Twenty-four hours after the last nebulization, the blood and bronchoalveolar lavage fluid (BALF) of these mice were collected. Total immunoglobulin (Ig)E or IgG2a level in the serum and cytokines in the both were determined. The number of inflammatory cells was counted using a hemocytometer. Rutin (30 and100 μmol/kg, p.o.) and quercitrin (100 μmol/kg, p.o.) significantly suppressed the increases in the inflammatory cells and cytokines in the BALF of mice. In contrast to rutin, quercitrin significantly increased the IFN-γ level. Both significantly increased the IgG2a level in the serum and suppressed total and OVA-specific IgE levels in the serum and BALF of mice. Rutin, quercitrin, and isoquercitrin did not affect xylazine/ketamine-induced anesthesia. In conclusion, the above results suggest that quercetin-3-O-glycosides have few adverse effects and that rutin and quercitrin, but not isoquercitrin, may have the potential for treating atypical asthma.

We previously constructed a collection of fission yeast strains that express various mammalian cyclic nucleotide phosphodiesterases (PDEs) and developed a cell-based high throughput screen (HTS) for small molecule PDE inhibitors. Here we describe a compound, BC54, that is a selective inhibitor of enzymes from the cAMP-specific PDE4 and PDE7 families. Consistent with the biological effect of other PDE4 and PDE7 inhibitors, BC54 displays potent anti-inflammatory properties and is superior to a combination of rolipram (a PDE4 inhibitor) and BRL50481 (a PDE7A inhibitor) for inducing apoptosis in chronic lymphocytic leukemia (CLL) cells. We further exploited PKA-regulated growth phenotypes in fission yeast to isolate two mutant alleles of the human PDE4B2 gene that encode enzymes possessing single amino acid changes that confer partial resistance to BC54. We confirm this resistance to both BC54 and rolipram via yeast-based assays and, for PDE4B2T407A, in vitro enzyme assays. Thus, we are able to use this system for both chemical screens to identify biologically-active PDE inhibitors and molecular genetic studies to characterize the interaction of these molecules with their target enzymes. Based on its potency, selectivity, and effectiveness in cell culture, BC54 should be a useful tool to study biological processes regulated by PDE4 and PDE7 enzymes.

Background There is ongoing interest in generating cardiomyocytes derived from human induced pluripotent stem cells (hiPSC) to study human cardiac physiology and pathophysiology. Recently we found that norepinephrine-stimulated calcium currents (ICa) in hiPSC-cardiomyocytes were smaller in conventional monolayers (ML) than in engineered heart tissue (EHT). In order to elucidate culture specific regulation of β1-adrenoceptor (β1-AR) responses we investigated whether action of phosphodiesterases (PDEs) may limit norepinephrine effects on ICa and on cytosolic cAMP in hiPSC-cardiomyocytes. Results were compared to adult human atrial cardiomyocytes. Methods Adult human atrial cardiomyocytes were isolated from tissue samples obtained during open heart surgery. All patients were in sinus rhythm. HiPSC-cardiomyocytes were dissociated from ML and EHT. Forster-resonance energy transfer (FRET) was used to monitor cytosolic cAMP (Epac1-camps sensor, transfected by adenovirus). ICa was recorded by whole-cell patch clamp technique. Cilostamide (300 nM) and rolipram (10 μM) were used to inhibit PDE3 and PDE4, respectively. β1-AR were stimulated with the physiological agonist norepinephrine (100 μM). Results In adult human atrial cardiomyocytes, norepinephrine increased cytosolic cAMP FRET ratio by +13.7 ± 1.5% (n = 10/9, mean ± SEM, number of cells/number patients) and ICa by +10.4 ± 1.5 pA/pF (n = 15/10). This effect was not further increased in the concomitant presence of rolipram, cilostamide and norepinephrine, indicating saturation by norepinephrine alone. In ML hiPSC-cardiomyocytes, norepinephrine exerted smaller increases in cytosolic cAMP and ICa (FRET +9.6 ± 0.5% n = 52/21, number of cells/number of ML or EHT, and ICa + 1.4 ± 0.2 pA/pF n = 34/7, p Conclusion Our results show culture-dependent differences in hiPSC-cardiomyocytes. In conventional ML but not in EHT, maximum norepinephrine effects on cytosolic cAMP depend on PDE3 and PDE4, suggesting immaturity when compared to the situation in adult human atrial cardiomyocytes. The smaller ICa responses to norepinephrine in ML and EHT vs. adult human atrial cardiomyocytes depend at least partially on a non-physiological large impact of PDE4 in hiPSC-cardiomyocytes.

Improvement of subtype selectivity of an inhibitor's binding activity using the conformational restriction approach has become an effective strategy in drug discovery. In this study, we applied this approach to PDE4 inhibitors and designed a series of novel oxazolidinone-fused 1,2,3,4-tetrahydroisoquinoline derivatives as conformationally restricted analogues of rolipram. The bioassay results demonstrated the oxazolidinone-fused tetrahydroisoquinoline derivatives exhibited moderate to good inhibitory activity against PDE4B and high selectivity for PDE4B/PDE4D. Among these derivatives, compound 12 showed both the strongest inhibition activity (IC50=0.60μM) as well as good selectivity against PDE4B and good in vivo activity in animal models of asthma/COPD and sepsis induced by LPS. The primary SAR study showed that restricting the conformation of the catechol moiety in rolipram with the scaffold of oxazolidinone-fused tetrahydroisoquinoline could lead to an increase in selectivity for PDE4B over PDE4D, which was consistent with the observed docking simulation.

The study investigates the potential of Rolipram a phosphodiesterase-4 inhibitor in cognitive deficits induced by streptozotocin (STZ, 3mg/kg intracerebroventricularly) and natural ageing in mice. Morris water maze (MWM) test was employed to evaluate learning and memory of the animals. Extent of oxidative stress was measured by estimating the levels of brain glutathione (GSH) and thiobarbituric acid reactive species (TBARS). Brain acetylcholinestrase (AChE) activity was also estimated. The brain activity of myeloperoxidase (MPO) was measured as a marker of inflammation. STZ and ageing results in marked decline in MWM performance of the animals, reflecting impairment of learning and memory. STZ treated mice and aged mice exhibited a marked accentuation of AChE activity, TBARS and MPO activity along with fall in GSH level. Further the stained micrographs of STZ treated mice and aged mice indicate pathological changes, severe neutrophilic infiltration and amyloid deposition. Rolipram treatment significantly attenuated STZ induced and age related memory deficits, biochemical and histopathological alterations. The findings demonstrate the potential of Rolipram in memory dysfunctions which may probably be attributed to its anti-cholinesterase, anti-amyloid, anti-oxidative and anti-inflammatory effects. The study concludes that PDE-4 can be explored as a potential therapeutic target in dementia.

G-protein coupled receptors may have a role in mediating the antiepileptogenic effect of low-frequency stimulation (LFS) on kindling acquisition. This effect is accompanied by changes at the intracellular level of cAMP. In the present study, the effect of rolipram as a phosphodiesterase inhibitor on the antiepileptogenic effect of LFS was investigated. Meanwhile, the expression of α s - and α i -subunit of G proteins and regulators of G-protein signaling (RGS) proteins following LFS application was measured. Male Wistar rats were kindled by perforant path stimulation in a semi-rapid kindling manner (12 stimulations per day) during a period of 6 days. Application of LFS (0.1 ms pulse duration at 1 Hz, 200 pulses, 50–150 μA, 5 min after termination of daily kindling stimulations) to the perforant path retarded the kindling development and prevented the kindling-induced potentiation and kindling-induced changes in paired pulse indices in the dentate gyrus. Intra-cerebroventricular microinjection of rolipram (0.25 μM) partially prevented these LFS effects. Twenty-four hours after the last kindling stimulation, the dentate gyrus was removed and changes in protein expression were measured by Western blotting. There was no significant difference in the expression of α-subunit of G s and G i/o proteins in different experimental groups. However, application of LFS during the kindling procedure decreased the expression RGS4 and RGS10 proteins (that reduce the activity of G i/o ) and prevented the kindling-induced decrease of RGS2 protein (which reduces the G s activity). Therefore, it can be postulated that the G i/o protein signaling pathways may be involved in antiepileptogenetic effect of LFS, and this is why decreasing the cAMP metabolism by rolipram attenuates this effect of LFS.

Thus far, phosphodiesterase-4 (PDE4) inhibitors have not been approved for application in Alzheimer's disease (AD) in a clinical setting due to severe side effects, such as nausea and vomiting. In this study, we investigated the effect of FFPM, a novel PDE4 inhibitor, on learning and memory abilities, as well as the underlying mechanism in the APP/PS1 mouse model of AD. Pharmacokinetic studies have revealed that FFPM efficiently permeates into the brain, and reached peak values in plasma 2 h after orally dosing. A 3-week treatment with FFPM, at doses of 0.25 mg/kg and 0.5 mg/kg, significantly improved the learning and memory abilities of APP/PS1 transgenic mice in the Morris water maze and the Step-down passive avoidance task. Interestingly, we found that while rolipram (0.5 mg/kg) reduced the duration of the α2 adrenergic receptor-mediated anesthesia induced by xylazine/ketamine, FFPM (0.5 mg/kg) or the vehicle did not have an evident effect. FFPM increased the cAMP, PKA and CREB phosphorylation and BDNF levels, and reduced the NF-κB p65, iNOS, TNF-α and IL-1β levels in the hippocampi of APP/PS1 trangenic mice, as observed by ELISA and Western blot analysis. Taken together, our data demonstrated that the reversal effect of FFPM on cognitive deficits in APP/PS1 transgenic mice might be related to stimulation of the cAMP/PKA/CREB/BDNF pathway and anti-inflammatory effects. Moreover, FFPM appears to have potential as an effective PDE4 inhibitor in AD treatment with little emetic potential.

Aims Glioblastoma cancer stem-like cells (GCSCs) promote themselves proliferation by secreting the vascular endothelial growth factor A (VEGF A ) in an autocrine manner, positively regulated by phosphodiesterase IV (PDE4). In the current study, we investigated the putative cytotoxic effect of bevacizumab, a VEGF A blocker, alone and in combination with a specific inhibitor of PDE4 called rolipram on GCSCs isolated from human surgical tumor specimen with a focus on PI3K/AKT pathway. Main methods CD133 +/CD15 + GCSCs were characterized by flow cytometry and expanded in a serum-free primary culture system. The cell survival, apoptosis, and protein expression values were measured using MTT assay, TUNEL staining and western blot, successively. Intracellular cAMP and free secreted VEGF A levels were assessed by cAMP enzyme immunoassay and ELISA, respectively. Key findings Bevacizumab suppressed GCSCs survival with IC 50 ~ 6.5 μg/ml and enhanced the levels of apoptosis, p53 and cleaved-caspase3 along with a decrease in free VEGF A levels and ERKs activation. However, there was no significant modulation of AKT phosphorylation on serine 473, the intracellular PDE4A, VEGF A and cAMP levels. More cytotoxicity in co-treated cells coupled with a more substantial decline in the free VEGF A levels and a greater increase in the quantities of p53 and cleaved-caspase3 compared to those treated with bevacizumab alone. Co-treatment reduced phospho-AKT, endogenous VEGF A and PDE4A values but elevated cAMP levels. Significance This study highlighted a booster cytotoxic effect of combined rolipram and bevacizumab treatment on the GCSCs primary culture, suggesting that this approach is warranted in treatment of GBMs overexpressing VEGF A and PDE4A.

A novel, efficient and protecting group free enantioselective synthetic approach of (−)-( R )- 1 and (+)-( S )-rolipram 2 is described employing the organocatalyzed asymmetric Michael addition, Henry condensation, Wittig olefination and reductive lactamization reactions as key steps.

Lysosomal integral membrane protein-2 (LIMP-2) is an important protein in lysosomal biogenesis and function and also plays a role in the tissue inflammatory response. It is known that lysosomes play a central role in acute pancreatitis, with inflammatory cell infiltration triggering the disease early on. In this study we report increases in pancreatic LIMP-2 protein and mRNA levels as early events that occur during the development of cerulein (Cer)-induced acute pancreatitis (AP) in rats. GdCl3, a macrophage inhibitor, but not FK506, a T lymphocyte inhibitor, was able to reverse the increase in LIMP-2 expression after Cer treatment, although such reversion was abolished if the animals were depleted of neutrophils due to a vinblastine sulfate pre-treatment. Immunostaining revealed that the cellular source of LIMP-2 was mainly acinar cells. Additionally, pre-treatments with the MAPKs inhibitors SP600125 and PD98059, inhibitors of JNK and ERK½ activation, respectively, but not of rolipram, a type IV phosphodiesterase inhibitor, suppressed the increase in the expression of LIMP-2 after Cer administration. Together, these results indicate that neutrophils are able to drive a macrophage activation that would regulate the increase in LIMP-2 expression during the early phase of Cer-induced AP, with the stress kinases JNK and ERK½ also playing a coordinated role in the increase of LIMP-2 expression due to Cer.

Cumulus cells sustain the development and fertilization of the mammalian oocyte. These cells are retained around the oocyte by a hyaluronan-rich extracellular matrix synthesized before ovulation, a process called cumulus cell-oocyte complex (COC) expansion. Hyaluronan release and dispersion of the cumulus cells progressively occur after ovulation, paralleling the decline of oocyte fertilization. We show here that, in mice, postovulatory changes of matrix are temporally correlated to cumulus cell death. Cumulus cell apoptosis and matrix disassembly also occurred in ovulated COCs cultured in vitro. COCs expanded in vitro with FSH or EGF underwent the same changes, whereas those expanded with 8-bromo-adenosine-3′,5′-cyclic monophosphate (8-Br-cAMP) maintained integrity for a longer time. It is noteworthy that 8-Br-cAMP treatment was also effective on ovulated COCs cultured in vitro, prolonging the vitality of the cumulus cells and the stability of the matrix from a few hours to >2 days. Stimulation of endogenous adenylate cyclase with forskolin or inhibition of phosphodiesterase with rolipram produced similar effects. The treatment with selective cAMP analogues suggests that the effects of cAMP elevation are exerted through an EPAC-independent, PKA type II-dependent signaling pathway, probably acting at the post-transcriptional level. Finally, overnight culture of ovulated COCs with 8-Br-cAMP significantly counteracted the decrease of fertilization rate, doubling the number of fertilized oocytes compared with control conditions. In conclusion, these studies suggest that cAMP-elevating agents prevent cumulus cell senescence and allow them to continue to exert beneficial effects on oocyte and sperm, thereby extending in vitro the time frame of oocyte fertilizability.

Diabetics suffer from a higher risk of cognitive decline. cAMP response element-binding protein (CREB) is a transcription factor associated with memory and synaptic plasticity. Here, we investigated the molecular changes in the hippocampus correlated with diabetes associated cognitive decline (DACD) from a CREB-centered perspective in a rat model of type 2 diabetes. Furthermore, we tested the therapeutic effect of rolipram on DACD. High-fat diet and low-dose streptozocin were adopted to induce diabetes in SD rats. Results show that supplementation with rolipram for 23 days (0.5mg/kg, once a day) improved the performance of diabetic rats in Morris water navigation task with increased level of CREB, brain-derived neurotrophic factor (BDNF), and Arc protein in the hippocampus. Rolipram, acting as an inhibitor of PDE4, was found to repair the imbalance in the CREB/BDNF/Arc pathway. This study may provide important insights into the mechanisms underlying DACD and provide new therapeutic targets for clinical treatment.

Ethnopharmacological relevance The endemic succulent South African plant, Sceletium tortuosum (L.) N.E. Br. (synonym Mesembryanthemum tortuosum L.), of the family Mesembryathemaceae, has an ancient oral tradition history of use by San and Khoikhoi people as an integral part of the indigenous culture and materia medica . A special standardized extract of Sceletium tortuosum (Zembrin®) has been developed and tested pre-clinically in rats, and clinically in healthy subjects. Aim of the study The present investigation aimed at the construction of electropharmacograms of Zembrin® in the presence of three dosages (2.5, 5.0 and 10.0 mg/kg), and comparative electropharmacograms and discriminatory analyses for other herbal extracts, citicoline and rolipram. Material and methods Seventeen adult Fischer rats were each implanted with a set consisting of four bipolar concentric steel electrodes fixed by dental cement and three screws driven into the scalp. After two weeks of recovery from surgery the animals were adapted to oral administration by gavage and to experimental conditions (45 min pre-drug period and 5 h of recording after a rest of 5 min for calming down). Data were transmitted wirelessly and processed using a Fast Fourier Transformation (FFT). Spectral power was evaluated for 8 frequency ranges, namely delta, theta, alpha1, alpha2, beta1a, beta1b, beta2 and gamma power. Results Zembrin® dose dependently attenuated all frequency ranges, to varying degrees. The most prominent was the statistically significant reduction in alpha2 and beta1a waves, correlated with activation of the dopaminergic and glutamatergic transmitter systems respectively. This feature is common to all synthetic and herbal stimulants tested to date. The second strongest effects were reduction in both the delta and the theta frequency ranges, correlated with changes in the cholinergic and norepinephrine systems respectively, a pattern seen in preparations prescribed for neurodegenerative diseases. Theta wave reduction in common with the delta, alpha2 and beta1 attenuation has been noted for analgesic drugs. Attenuation of alpha1 waves emerged during the highest dosage in all brain areas, a feature seen in all antidepressants. Discussion The electropharmacogram of Zembrin® was compared to the electropharmacograms of herbal extracts archived in our database. Extracts of Oenothera biennis and Cimicifuga racemosa gave a very similar electropharmacograms to that of Zembrin®, and extracts of Ginkgo biloba and Rhodiola rosea gave rather similar electropharmacograms to Zembrin®. Linear discriminant analysis confirmed these similarities and demonstrated that all three dosages of Zembrin® plotted in close neighbourhood to each other. Citocoline, a synthetic compound originally developed for cognitive enhancement, had a similar electropharmacogram to Zembrin®. Similarity to the electropharmacograms of the synthetic phosphodiesterase-4 inhibitor, rolipram, suggests Zembrin® has antidepressant and cognitive function enhancing potential. Conclusion The combined results from the electropharmacograms and comparative discriminatory analyses suggest that Zembrin® has dose dependent activity, with potential applications as a cognitive function enhancer, as an antidepressant, and as an analgesic.

The wnt protein family has important members involved in cell differentiation, proliferation and plasticity expression; however, little is known about its biosynthesis processes. On the other hand, an increase in the intracerebral cyclic adenosine 3', 5'-monophosphate (cAMP) level leads to synaptic plasticity via the de novo synthesis of any protein. Here, the effect of dibutyryl cAMP (dbcAMP), a membrane permeability cAMP analog, on the wnt family was investigated in rat primary-cultured glial cells containing astrocytes and microglia. Among wnt3a, 4, 5a, 7a and 11 mRNA, only wnt4 expression was increased by longer treatment (24 h), compared with short treatment (2 h), with dbcAMP in a concentration-dependent manner, and its effect reached statistical significance at 1 mM. In cultures of isolated astrocytes or microglia, wnt4 expression was not affected by 1 mM dbcAMP for 24 h, and microglial wnt4 protein was undetectable even when cells were treated with the drug. Mixed glial cells treated for 24 h with 1 mM dbcAMP showed significantly increased wnt4 protein, as well as mRNA. Immunofluorescence manifested that cells that expressed wnt4 protein were astrocytes, but not microglia. Intraperitoneal injection of 1.25 mg/kg rolipram, a phosphodiesterase (PDE) IV inhibitor that can pass through the blood brain barrier and inhibits cAMP degradation specifically, showed a tendency to increase wnt4 expression in the adult rat brain after 24 h, and the increases in wnt4 mRNA and protein levels reached statistical significance in the hippocampus and striatum, respectively. This is the first finding to help elucidate the selective biosynthesis of central wnt4 through cAMP-stimulated microglia and astrocytes interaction.

In this study, a series of catechol-based amides (8a-n) with different amide linkers linking the catecholic moiety to the terminal phenyl ring was designed and synthesized as potent phosphodiesterase (PDE) 4D inhibitors. The inhibitory activities of these compounds were evaluated against the core catalytic domains of human PDE4 (PDE4CAT), full-length PDE4B1 and PDE4D7 enzymes, and other PDE family members. The results indicated the majority of compounds 8a-n displayed moderate to good inhibitory activities against PDE4CAT. Among these compounds, compound 8 j with a short amide linker (-CONHCH2-) displayed comparable PDE4CAT inhibitory activity (IC50=410 nM) with rolipram. More interestingly, compound 8 g, a potent and selective PDE4D inhibitor (IC50=94 nM), exhibited a 10-fold selectivity over the PDE4B subtypes and an over 1000-fold selectivity against other PDE family members. Docking simulations suggested that 8 g forms three extra H-bonds with the N-H of residue Asn487 and two water molecules.

Objective In chronic inflammation, prevention of cAMP degradation by phosphodiesterase-4 (PDE4) inhibition can be anti-inflammatory therapy. However, PDE4 inhibition was uneffective in rheumatoid arthritis (RA). Recent studies demonstrated that PDE4/β-arrestin interaction at β-adrenoceptors resulted in switching from Gαs to Gαi signaling and ERK1/2 activation. Such a switch in signaling might elicit proinflammatory effects. We aimed to investigate this possible Gαs to Gαi signaling switch in RA and osteoarthritis (OA) mixed synoviocytes. Methods Synoviocytes were treated alone or with combinations of adrenergic, dopaminergic, and adenosinergic drugs, rolipram (PDE4 inhibitor), inhibitors of Gαi signaling (pertussis toxin), and blockers of protein kinase A (PKA). Under normoxic or hypoxic conditions, proinflammatory TNF was the readout-parameter. We investigated co-expression and interaction of PDE4 and β-arrestin by imaging techniques. Expression of pERK1/2 was analyzed by western blotting. Results Mixed synoviocytes in RA and OA possessed all major Gαs-coupled neurotransmitter receptors. Under hypoxia, particularly in RA cells, Gαs-coupled receptor agonists unexpectedly increased TNF and respective antagonists decreased TNF. Under hypoxia, rolipram alone or rolipram plus Gαs agonists increased TNF, which was reversed by pertussis toxin or PKA inhibition. Co-localization and interaction of PDE4 and β-arrestin in synovial tissue and cells was demonstrated. Gαs agonists or rolipram plus Gαs agonists increased pERK1/2 expression. Conclusions This study in human arthritic synovial tissue presents an unexpected proinflammatory switch from Gαs to Gαi signaling, which depends on PDE4/β-arrestin interaction. This phenomenon is most probably responsible for reduced efficacy of PDE4 inhibitors and Gαs agonists in RA.

Inhibitors of Type 4 cAMP-phosphodiesterases (PDE4s) exert a number of promising therapeutic benefits, including potent anti-inflammatory, memory- and cognition-enhancing, metabolic, and antineoplastic effects. We report here that treatment with a number of distinct PDE4 inhibitors, including Rolipram, Piclamilast, Roflumilast and RS25344, but not treatment with the PDE3-selective inhibitor Cilostamide, induces a rapid (10–30 min), substantial (−5 °C) and long-lasting (up to 5 h) decrease in core body temperature of C57BL/6 mice; thus, identifying a critical role of PDE4 also in the regulation of body temperature. As little as 0.04 mg/kg of the archetypal PDE4 inhibitor Rolipram induces hypothermia. As similar or higher doses of Rolipram were used in a majority of published animal studies, most of the reported findings are likely paralleled by, or potentially impacted by hypothermia induced by these drugs. We further show that PDE4 inhibition affects central body temperature regulation and acts by lowering the cold-defense balance point of behavioral (including posture and locomotion) and autonomous (including cutaneous tail vasodilation) cold-defense mechanisms. In line with the idea of an effect on central body temperature regulation, hypothermia is induced by moderate doses of various brain-penetrant PDE4 inhibitors, but not by similar doses of YM976, a PDE4 inhibitor that does not efficiently cross the blood–brain barrier. Finally, to begin delineating the mechanism of drug-induced hypothermia, we show that blockade of D2/3-type dopaminergic, but not β-adrenergic, H1-histaminergic or opiate receptors, can alleviate PDE4 inhibitor-induced hypothermia. We thus propose that increased D2/3-type dopaminergic signaling, triggered by PDE4 inhibitor-induced and cAMP-mediated dopamine release in the thermoregulatory centers of the hypothalamus, is a significant contributor to PDE4 inhibitor-induced hypothermia.

The involvement of specific phosphodiesterases (PDEs) in the modulation of cAMP and thereby spontaneous meiotic resumption remains poorly understood. This work aims to evaluate the effects of cilostamide and rolipram (PDE 3A and PDE 4D inhibitors) on spontaneous meiotic resumption from diplotene arrest in rat oocytes cultured in vitro. For this purpose, diplotene-arrested cumulus oocyte complexes (COCs) were collected from rat ovary. The COCs and denuded oocytes were exposed to various concentrations of cilostamide (0.0, 2.5, 5.0 and 10 μM) and rolipram (0, 10, 50 and 100 μM) for various times (0, 3, 5, 7, 14, 16, 18, 20, 22 and 24 h). Cilostamide inhibited spontaneous meiotic resumption in a concentration- and time-dependent manner in COCs and denuded oocytes. Although rolipram showed inhibition of spontaneous meiotic resumption up to some extent, cilostamide was more potent to prevent spontaneous meiotic resumption in both COCs and denuded oocytes. Cilostamide significantly reduced PDE 3A expression, increased cAMP level and prevented spontaneous meiotic resumption in COCs and denuded oocytes. Although rolipram inhibited PDE 4D expression in cumulus cells, increased cAMP level but was not sufficient to prevent spontaneous meiotic resumption. We conclude that both drugs prevent spontaneous resumption from diplotene-arrest through PDE 3A/PDE 4D-cAMP mediated pathway. However, as compare to rolipram, cilostamide was more potent in preventing spontaneous resumption from diplotene-arrest in rat oocytes cultured in vitro. Thus, cilostamide could be used as a potential candidate for the development of female contraceptive drug in future.

Phosphodiesterases (PDEs) consisted of 11 subtypes (PDE1 to PDE11) and over 40 isoforms that regulate levels of cyclic guanosine monophosphate (cGMP) and cyclic adenosine monophosphate (cAMP), the second messengers in cell functions. PDE inhibitors (PDEIs) have been attractive therapeutic targets due to their involvement in diverse medical conditions, e.g. cardiovascular diseases, autoimmune diseases, Alzheimer's disease (AD), etc. Among them; AD with a complex pathology is a progressive neurodegenerative disorder which affect mostly senile people in the world and only symptomatic treatment particularly using cholinesterase inhibitors in clinic is available at the moment for AD. Consequently, novel treatment strategies towards AD are still searched extensively. Since PDEs are broadly expressed in the brain, PDEIs are considered to modulate neurodegenerative conditions through regulating cAMP and cGMP in the brain. In this sense, several synthetic or natural molecules inhibiting various PDE subtypes such as rolipram and roflumilast (PDE4 inhibitors), vinpocetine (PDE1 inhibitor), cilostazol and milrinone (PDE3 inhibitors), sildenafil and tadalafil (PDE5 inhibitors), etc have been reported showing encouraging results for the treatment of AD. In this review, PDE superfamily will be scrutinized from the view point of structural features, isoforms, functions and pharmacology particularly attributed to PDEs as target for AD therapy.

Cyclic nucleotide phosphodiesterase (PDE)3 and PDE4 provide the major PDE activity in cardiac myocytes and shape β1-adrenoceptor-dependent cardiac cAMP signaling but their role in regulating β2-adrenoceptor-mediated responses is less well known. We investigated potential differences in PDE3 and PDE4 activities between right (RV) and left (LV) ventricular myocardium, and their role in regulating β2-adrenoceptor effects. PDE3 activity in the microsomal fraction was lower in RV than in LV but was the same in the cytosolic fraction. However, no significant difference between RV and LV was found when the PDE4 activity was studied. β2-adrenoceptor activation increased inotropism and lusitropism in LV when measured in the presence of either the PDE3 inhibitor cilostamide, the PDE4 inhibitor rolipram or a non-selective PDE inhibitor IBMX. However, the joint inhibition of both PDE3 and PDE4 was necessary in RV to uncover β2-adrenoceptor-induced inotropic and lusitropic effects. Our results indicate different regulation of β2-adrenoceptor-mediated contractility by PDE3 and PDE4 in RV and LV of the rat heart. In the case of PDE3 due to a different contribution of the enzyme in the microsomal fraction whereas in the case of PDE4 it can be attributed to differences in the intracellular distribution and coupling to β2-adrenoceptors.

cAMP response element binding protein (CREB) signaling is involved in the heroin reward, but whether the CREB signaling is involved in the incubation of heroin-seeking remains unknown. Here we aim to explore the expression of p-CREB and the p-ERK, an upstream molecular of CREB, in the nucleus accumbens (NAc) in the incubation of heroin-seeking induced by cue after withdrawal. First, rats were trained to self-administer heroin for 14 days, and then assessed heroin-seeking induced by context cue (CC)or by conditioned cues (CS)after 1 or 14 d withdrawal. We found that the active responses induced by CC or CS was higher after 14 d withdrawal than that after 1 d withdrawal, and the extent increased was more significant by CS than that by CC. Meanwhile, the expression of p-ERK decreased significantly when rats exposed to the CS, and decreased more after 14 d withdrawal. In contrast, reduction of the expression of p-CREB was more obvious with exposure to CS after 14 d withdrawal. Furthermore, microinjection of rolipram into the NAc decreased the heroin-seeking behavior induced by CS after 14 d withdrawal, which was correlated to an enhancement in the expression of p-CREB in the NAc. These findings suggest that the inactivation of CREB and ERK may be involved in the incubation of heroin-seeking induced by cues after prolonged withdrawal.

Adenosine A1 receptor is highly expressed in hippocampus where it inhibits neurotransmitter release and has neuroprotective activity. Similar actions are obtained by increasing cGMP concentration, but a clear link between adenosine A1 receptor and cGMP levels remains to be established. The present work aims to investigate if cGMP formation is modulated by adenosine A1 receptors at the hippocampus and if this effect is gender dependent. cGMP accumulation, induced by phosphodiesterases inhibitors Zaprinast (100 μM) and Bay 60-7550 (10 μM), and cAMP accumulation, induced by Forskolin (20 μM) and Rolipram (50 μM), were quantified in rat hippocampal slices using specific enzymatic immunoassays. N6-cyclopentyladenosine (CPA, 100 nM) alone failed to modify basal cGMP accumulation. However, the presence of adenosine deaminase (ADA, 2 U/ml) unmasked a CPA (0.03-300 nM) stimulatory effect on basal cGMP accumulation (EC50: 4.2±1.4 nM; Emax: 17±0.9%). ADA influence on CPA activity was specific for cGMP, since inhibition of cAMP accumulation by CPA was not affected by the presence of ADA, though ADA inhibited cAMP accumulation in the absence of CPA. Increasing cGMP accumulation, by about four-fold, with sodium nitroprusside (SNP, 100 μM) abolished the CPA (100 nM) effect on cGMP accumulation in males but did not modify the effect of CPA in female rats. This effect was reversed by 8-Cyclopentyl-1,3-dipropylxanthine (DPCPX, 100 nM), indicating an adenosine A1 receptor mediated effect on cGMP accumulation. In conclusion, adenosine A1 receptors increase intracellular cGMP formation at hippocampus both in males and females under basal conditions, but only in females when cGMP levels are increased by SNP.

Peripheral nerve injury is common especially among young individuals. Although injured neurons have the ability to regenerate, the rate is slow and functional outcomes are often poor. Several potential therapeutic agents have shown considerable promise for improving the survival and regenerative capacity of injured neurons. These agents are reviewed within the context of their molecular mechanisms. The PI3K/Akt and Ras/ERK signaling cascades play a key role in neuronal survival. A number of agents that target these pathways, including erythropoietin, tacrolimus, acetyl-l-carnitine, n-acetylcysteine and geldanamycin have been shown to be effective. Trk receptor signaling events that up-regulate cAMP play an important role in enhancing the rate of axonal outgrowth. Agents that target this pathway including rolipram, testosterone, fasudil, ibuprofen and chondroitinase ABC hold considerable promise for human application. A tantalizing prospect is to combine different molecular targeting strategies in complementary pathways to optimize their therapeutic effects. Although further study is needed prior to human trials, these modalities could open a new horizon in the clinical arena that has so far been elusive.

Cyclic nucleotide phosphodiesterases (PDEs) regulate the intracellular concentrations and effects of adenosine 3′,5′-cyclic monophosphate (cAMP) and guanosine 3′,5′-cyclic monophosphate (cGMP). The role of PDEs in malignant tumor cells is still uncertain. The role of PDEs, especially PDE2, in human malignant melanoma PMP cell line was examined in this study. In PMP cells, 8-bromo-cAMP, a cAMP analog, inhibited cell growth and invasion. However, 8-bromo-cGMP, a cGMP analog, had little or no effect. PDE2 and PDE4, but not PDE3, were expressed in PMP cells. Growth and invasion of PMP cells were inhibited by erythro-9-(2-hydroxy-3-nonyl) adenine (EHNA), a specific PDE2 inhibitor, but not by rolipram, a specific PDE4 inhibitor. Moreover, cell growth and invasion were inhibited by transfection of small interfering RNAs (siRNAs) specific for PDE2A and a catalytically-dead mutant of PDE2A. After treating cells with EHNA or rolipram, intracellular cAMP concentrations were increased. Growth and invasion were stimulated by PKA14-22, a PKA inhibitor, and inhibited by N6-benzoyl-c AMP, a PKA specific cAMP analog, whereas 8-(4-chlorophenylthio)-2′-O-methyl-cAMP, an Epac specific cAMP analog, did not. Invasion, but not growth, was stimulated by A-kinase anchor protein (AKAP) St-Ht31 inhibitory peptide. Based on these results, PDE2 appears to play an important role in growth and invasion of the human malignant melanoma PMP cell line. Selectively suppressing PDE2 might possibly inhibit growth and invasion of other malignant tumor cell lines.

Background Progression to a chronic state after an initial, mostly asymptomatic, acute phase is the normal fate of Toxoplasma infection in immunocompetent hosts. The inter-conversion between tachyzoite and bradyzoite life stages is a key to Toxoplasma survival and pathogenesis. Chronic toxoplasmosis is resistant to all commercially available medications. Reactivation of chronic stage has serious complications on immunocompromised hosts including encephalitis and death. Cyclic AMP, TNF-α and Th1 immune response have pivotal roles in establishing chronic disease. Rolipram, a PDE4 inhibitor, has regulatory roles on these factors. This is the first study to investigate a possible modulatory effect of rolipram on Toxoplasma infection in an experimental murine model. The ability, of this novel regimen to prevent or reverse Toxoplasma-induced pathology was evaluated. Materials and methods Toxoplasma-infected mice received rolipram (10 mg/kg) daily for three weeks. Life expectancy, serum ALT and histopathology of liver and brain were the parameters used to evaluate therapeutic and/or toxic impact of rolipram in comparison with infection and healthy control groups of mice. Results Toxoplasma-induced pathology was never completely cleared by rolipram. However, a significant impact in aborting chronic state and in mitigating pathology was detected. The drug almost normalized Toxoplasma-induced high ALT levels reversing the hepatotoxic effect of the parasite. Conclusion This study is the first to demonstrate relative merits of rolipram therapy to toxoplasmosis with illustration of its efficacy and toxicity. It reports a novel approach with potential future benefits using PDE inhibitors to abort resistant latent phases of biologically related infectious agents.

Small-molecule, ligand-activated G protein-coupled receptors are generally thought to be rapidly desensitized within a period of minutes through receptor phosphorylation and internalization after repeated or prolonged stimulation. This transient G protein-coupled receptor activation remains at odds with many observed long-lasting cellular and physiological responses. Here, using live cell imaging of cAMP with a FRET-based biosensor and myocyte contraction assay, we show that the catecholamine-activated β1 adrenergic receptor (β1AR) continuously stimulates second messenger cAMP synthesis in primary cardiac myocytes and neurons, which lasts for more than 8 h (a decay t½ of 3.9 h) in cardiac myocytes. However, the β1AR-induced cAMP signal is counterbalanced and masked by the receptor-bound phosphodiesterase (PDE) 4D8-dependent cAMP hydrolysis. Inhibition of PDE4 activity recovers the receptor-induced cAMP signal and promotes contractile response in mouse hearts during extended periods of agonist stimulation. β1AR associates with PDE4D8 through the receptor C-terminal PDZ motif-dependent binding to synaptic-associated protein 97 (SAP97). Knockdown of SAP97 or mutation of the β1AR PDZ motif disrupts the complex and promotes sustained agonist-induced cAMP activity, PKA phosphorylation, and cardiac myocyte contraction response. Together, these findings unveil a long lasting adrenergic signal in neurons and myocytes under prolonged stimulation and an underappreciated role of PDE that is essential in classic receptor signaling desensitization and in maintaining a long lasting cAMP equilibrium for ligand-induced physiological response.

Complete control of emesis caused by cyclophosphamide (CPA) is of immense interest to both patients and physicians. Serotonin 5-HT3- and tachykinin NK1-receptor antagonists are widely used antiemetics in clinic, but they fail to completely control CPA-induced emesis. New antiemetic targets for the full control of CPA-induced vomiting are lacking. We therefore examined the effects of CPA on emetic targets downstream of 5-HT3- and NK1- receptors in an attempt to better understand the molecular bases of CPA-induced emesis. Acute CPA (200 mg/kg, i.p.) administration in the least shrew caused a biphasic pattern of emesis over a 40 h observation period, with maximal peak vomit frequency during the 1st hour of treatment (acute phase), followed by a delayed-phase which peaks at 27th hour. The NK1 receptor mRNA levels increased significantly at 8 h post-CPA treatment in the brainstem, and at 28 h in the whole intestine. Substance P mRNA levels tended to increase both in the brainstem and intestine at most time-points post-CPA injection, however due to large variability, they failed to attain significance. Likewise, protein expression profiles of both NK1- and 5-HT3 -receptors in the brainstem were unchanged at any time-point. However, phosphorylation levels of protein kinase A (PKA), but not of extracellular signal-regulated protein kinase 1/2 (ERK1/2), were increased at 2, 8, 22, 28, and 33 h time-points after the treatment with CPA. Moreover, brainstem but not frontal cortex cAMP tissue levels tended to be elevated at most time-points, but significant increases occurred only at 1 and 2 h post-CPA treatment. The phosphodiesterase inhibitor, rolipram, caused significant increases in shrew brainstem cAMP levels which were associated with its capacity to produce vomiting, while pretreatment with SQ22536, an inhibitor of adenylyl cyclase, prevented rolipram-induced emesis. The results demonstrate that accumulation of cAMP and subsequent activation of PKA in the brainstem may help to initiate and sustain emesis induced by CPA in the least shrew. Our findings suggest that suppression of the cAMP/PKA cascade may have antiemetic potential in the management of CPA-induced emesis.

In simple systems, lifespan can be extended by various methods including dietary restriction, mutations in the insulin/insulin-like growth factor (IGF) pathway or mitochondria among other processes. It is widely held that the mechanisms that extend lifespan may be adapted for diminishing age-associated pathologies. We tested whether a number of compounds reported to extend lifespan in C. elegans could reduce age-dependent toxicity caused by mutant TAR DNA-binding protein-43 in C. elegans motor neurons. Only half of the compounds tested show protective properties against neurodegeneration, suggesting that extended lifespan is not a strong predictor for neuroprotective properties. We report here that resveratrol, rolipram, reserpine, trolox, propyl gallate, and ethosuximide protect against mutant TAR DNA-binding protein-43 neuronal toxicity. Finally, of all the compounds tested, only resveratrol required daf-16 and sir-2.1 for protection, and ethosuximide showed dependence on daf-16 for its activity.

Blood–brain-barrier (BBB) disruption, inflammation and thrombosis are important steps in the pathophysiology of acute ischemic stroke but are still inaccessible to therapeutic interventions. Rolipram specifically inhibits the enzyme phosphodiesterase (PDE) 4 thereby preventing the inactivation of the intracellular second messenger cyclic adenosine monophosphate (cAMP). Rolipram has been shown to relief inflammation and BBB damage in a variety of neurological disorders. We investigated the therapeutic potential of rolipram in a model of brain ischemia/reperfusion injury in mice. Treatment with 10 mg/kg rolipram, but not 2 mg/kg rolipram, 2 h after 60 min of transient middle cerebral artery occlusion (tMCAO) reduced infarct volumes by 50% and significantly improved clinical scores on day 1 compared with vehicle-treated controls. Rolipram maintained BBB function upon stroke as indicated by preserved expression of the tight junction proteins occludin and claudin-5. Accordingly, the formation of vascular brain edema was strongly attenuated in mice receiving rolipram. Moreover, rolipram reduced the invasion of neutrophils as well as the expression of the proinflammatory cytokines IL-1β and TNFα but increased the levels of TGFβ-1. Finally, rolipram exerted antithrombotic effects upon stroke and fewer neurons in the rolipram group underwent apoptosis. Rolipram is a multifaceted antiinflammatory and antithrombotic compound that protects from ischemic neurodegeneration in clinically meaningful settings.

The phosphodiesterase (PDE)4 molecule exists as two distinct conformers, PDE4H and PDE4L, which have high and low affinities, respectively, for the selective PDE4 inhibitor, rolipram. The inhibition of PDE4H and PDE4L is associated with adverse responses, such as nausea, vomiting, and gastric hypersecretion, and with anti-inflammatory and bronchodilator effects, respectively. We determined the therapeutic (PDE4H/PDE4L) ratios of hesperetin-7-O-methylether, hesperetin-5,7,3′-O-trimethylether (HTME), hesperetin-7-O-acetate, hesperetin-7,3′-O-diacetate, hesperetin-5,7,3′-O-triacetate (HTA), hesperetin-5,7,3′-O-tripropionate, hesperetin-5,7,3′-O-tributyrate, hesperetin-5,7,3′-O-triisobutyrate, and hesperetin-5,7,3′-O-tripivatate, and compared these ratios to those of hesperetin, hesperetin-7,3′-O-dimethylether, hesperidin, and hesperidin-3′-O-methylether to identify derivatives with therapeutic ratios and to characterize the structure–activity relationships among these compounds. The activities of PDE isozymes 1 through 5 were measured using a two-step procedure using [3H]adenosine 3′,5′-cyclic monophosphate or [3H]guanosine 3′,5′-cyclic monophosphate as substrates. The inhibitory concentration (IC50) for 50% of PDE4 inhibition and effective concentration (EC50) for replacing 50% of [3H]rolipram binding on high-affinity rolipram-binding sites was taken as the PDE4L and PDE4H value, respectively. The HTME and the HTA dually inhibited PDE3 and PDE4, and displayed PDE4H/PDE4L ratios of 18.3 and 20.8, respectively, suggesting that they may be candidate drugs for treating asthma and chronic obstructive pulmonary disease (COPD) because the combined inhibition of PDE3 and PDE4 has synergistically anti-inflammatory and bronchodilator effects in COPD patients. © 2013 Wiley Periodicals, Inc. and the American Pharmacists Association J Pharm Sci 102:2120–2127, 2013

Methylprednisolone (MP) has been widely used as a standard therapeutic agent for the treatment of spinal cord injury (SCI). Because of its controversial beneficial effects, the combination of MP and other pharmacological agents aimed at enhancing functional recovery is desirable. The phosphodiesterase 4 (PDE4) inhibitor rolipram has been implicated in promotion of regeneration due to elevating cAMP. In the present study, we sought to determine the effects of MP and rolipram, administered in combination, after spinal cord injury (SCI) in adult rats. Here we show that in vitro administration of rolipram and MP significantly increased neuron survival and promoted neurite outgrowth of neurons on the inhibitory substrate CSPGs by upregulation of MMP-2 expression; in vivo administration of rolipram and MP inhibited CSPG expression and increase CSPG digestion after rat SCI. Rolipram and MP combining treatment promoted significant neuroprotection through reduced motoneuron death, minimized lesion cavity, and increased regeneration of lesioned corticospinal tract (CST) axons beyond the lesion site after SCI. Enhanced functional recovery was also observed. Overall, our study strongly suggested that the combination treatment of MP and rolipram may represent a promising strategy for clinically applicable pharmacological therapy for rapid initiation of neuroprotection after SCI.

Cigarette smoke, a major causative agent of chronic obstructive pulmonary disease (COPD), induces lung cell death by incompletely understood mechanisms. The induction of apoptosis in lung structural cells by cigarette smoke may contribute to the pathogenesis of emphysema. Phosphodiesterase-4 (PDE4) inhibitors are anti-inflammatory agents used in COPD therapy that can prevent cigarette smoke-induced emphysema in mice. We investigated the effect of rolipram, a first generation PDE4 inhibitor, on the regulation of cigarette smoke-induced apoptosis. Human lung fibroblast (MRC-5) cells were exposed to cigarette smoke extract (CSE). Cell viability and apoptosis were determined by MTT assay and Annexin-V staining, respectively. Caspase activation was determined by Western immunoblot analysis. Rolipram protected against cell death and increased viability in MRC-5 fibroblasts after CSE exposure. Furthermore, rolipram protected against apoptosis, decreased caspase-3 and -8 cleavage in MRC-5 cells exposed to CSE. Pre-treatment with rolipram enhanced Akt phosphorylation and associated cytoprotection in CSE-treated cells, which could be reversed by the PI3K inhibitor LY294002 partly. In conclusion, rolipram protects against apoptosis of MRC-5 cells through inhibition of caspase-3 and caspase-8. Rolipram may represent an effective therapeutic agent to reduce cigarette smoke-induced apoptosis of lung fibroblasts.

A mixed micellar liquid chromatographic (LC) method, the mobile phase consisting of anionic and non-ionic surfactants, has been developed for the high-speed direct radiometabolite analysis of positron emission tomography (PET) radioligands in plasma. The addition of Triton X-100 on an anionic surfactant sodium dodecyl sulphate (SDS) mobile phase improved elution strength and peak efficiency for many PET radioligands. Several radioligands could be easily separated from their radioactive metabolites with short run time of only 4 min using a “pure” (without organic solvent) mixed micellar mobile phase and semi-preparative monolithic C 18 -bonded silica column by simple isocratic elution without any treatment of plasma. Moreover, the use of “hybrid” mixed micellar mobile phase containing anionic, non-ionic surfactants and organic solvent was effective to further enhance peak efficiency and elute highly retained hydrophobic PET radioligands. These characteristics enabled significant shorting the radiometabolite analysis procedure of PET radioligands and simplifying the experimental setup.