PDE4 Inhibitor Rolipram Research Articles

The regulation of TRPA1 expression and function by Th1 and Th2-type inflammation in human A549 lung epithelial cells

BackgroundTransient Receptor Potential Ankyrin 1 (TRPA1) is a cation channel that mediates pain, itch, cough, and neurogenic inflammation in response to pungent compounds such as acrolein in cigarette smoke. TRPA1 is also activated by endogenous factors and promotes inflammation in asthma models. We have recently shown that TRPA1 is upregulated by inflammatory cytokines in A549 human lung epithelial cells. Here, we explored the effects of Th1 and Th2-type inflammation on TRPA1.Methods and resultsTRPA1 expression and function was studied in A549 human lung epithelial cells. To induce inflammation, the cells were exposed to a combination of cytokines TNF-α and IL-1β; and to model Th1 or Th2-type responses, IFN-γ or IL-4/IL-13 was added, respectively. TRPA1 expression (measured by RT-PCR and Western blot) and function (assessed by Fluo-3AM intracellular calcium measurement) was enhanced under the influence of TNF-α + IL-1β. IFN-γ further enhanced TRPA1 expression and function, whereas IL-4 and IL-13 suppressed them. The effects of IFN-γ and IL-4 on TRPA1 expression were reversed by the Janus kinase (JAK) inhibitors baricitinib and tofacitinib, and those of IL-4 also by the STAT6 inhibitor AS1517499. The glucocorticoid dexamethasone downregulated TRPA1 expression, whereas the PDE4 inhibitor rolipram had no effect. Under all conditions, TRPA1 blockade was found to reduce the production of LCN2 and CXCL6.ConclusionsTRPA1 expression and function in lung epithelial cells was upregulated under inflammatory conditions. IFN-γ further increased TRPA1 expression while IL-4 and IL-13 suppressed that in a JAK-STAT6 dependent manner which is novel. TRPA1 also modulated the expression of genes relevant to innate immunity and lung disease. We propose that the paradigm of Th1 and Th2 inflammation is a major determinant of TRPA1 expression and function, which should be considered when targeting TRPA1 for pharmacotherapy in inflammatory (lung) disease.

Scouting Different Phosphodiesterase 4 Inhibitor Chemotypes in Silico To Guide the Design of Anti-inflammatory/Antioxidant Agents.

During the last years, we developed a large library of new selective phosphodiesterase 4D inhibitors, maintaining the catechol portion of the well-known PDE4 inhibitor Rolipram, featuring different substitutions in place of the lactam group of this reference compound. Based on the X-ray analysis of PDE4 inhibitors (PDE4Is) previously synthesized by us and of naphthyridine- and naphthyridinone-containing derivatives exhibiting PDE4 inhibitory ability described in the literature, we designed and synthesized new compounds 1-3. All of them were screened in silico as putative PDE4Is, via molecular docking studies to exploit structural variation at the catechol group to gain further contacts especially with the flat aromatic residues (Phe506 and Phe538) of enzyme. Subsequent in silico prediction of ADMET properties and in vitro biological assays on platelets and endothelial cells are in good agreement with our previous data concerning the antioxidant/anti-inflammatory activity exhibited by our previous PDE4Is and similarly to other well-known PDE4Is.

Phosphodiesterase 4D contributes to angiotensin II-induced abdominal aortic aneurysm through smooth muscle cell apoptosis

Abdominal aortic aneurysm (AAA) is a permanent expansion of the abdominal aorta that has a high mortality but limited treatment options. Phosphodiesterase (PDE) 4 family members are cAMP-specific hydrolyzing enzymes and have four isoforms (PDE4A-PDE4D). Several pan-PDE4 inhibitors are used clinically. However, the regulation and function of PDE4 in AAA remain largely unknown. Herein, we showed that PDE4D expression is upregulated in human and angiotensin II-induced mouse AAA tissues using RT-PCR, western blotting, and immunohistochemical staining. Furthermore, smooth muscle cell (SMC)-specific Pde4d knockout mice showed significantly reduced vascular destabilization and AAA development in an experimental AAA model. The PDE4 inhibitor rolipram also suppressed vascular pathogenesis and AAA formation in mice. In addition, PDE4D deficiency inhibited caspase 3 cleavage and SMC apoptosis in vivo and in vitro, as shown by bulk RNA-seq, western blotting, flow cytometry and TUNEL staining. Mechanistic studies revealed that PDE4D promotes apoptosis by suppressing the activation of cAMP-activated protein kinase A (PKA) instead of the exchange protein directly activated by cAMP (Epac). Additionally, the phosphorylation of BCL2-antagonist of cell death (Bad) was reversed by PDE4D siRNA in vitro, which indicates that PDE4D regulates SMC apoptosis via the cAMP-PKA-pBad axis. Overall, these findings indicate that PDE4D upregulation in SMCs plays a causative role in AAA development and suggest that pharmacological inhibition of PDE4 may represent a potential therapeutic strategy.

Gs signaling pathway distinguishes hallucinogenic and nonhallucinogenic 5-HT2AR agonists induced head twitch response in mice

5- HT2A receptor is a member of the family A G-protein-coupled receptor. It is involved in many psychiatric disorders, such as depression, addiction and Parkinson's disease. 5-HT2AR targeted drugs play an important role in regulating cognition, memory, emotion and other physiological function by coupling G proteins, and their most notable function is stimulating the serotonergic hallucination. However, not all 5-HT2AR agonists exhibit hallucinogenic activity, such as lisuride. Molecular mechanisms of these different effects are not well illustrated. This study suggested that 5-HT2AR coupled both Gs and Gq protein under hallucinogenic agonists DOM and 25CN–NBOH stimulation, but nonhallucinogenic agonist lisuride and TBG only activates Gq signaling. Moreover, in head twitch response (HTR) model, we found that cAMP analogs 8-Bromo-cAMP and PDE4 inhibitor Rolipram could increase HTR, while Gs protein inhibitor Melittin could reduce HTR. Collectively, these results revealed that Gs signaling is a key signaling pathway that may distinguish hallucinogenic agonists and nonhallucinogenic agonists.

The protective effect of the PDE-4 inhibitor rolipram on intracerebral haemorrhage is associated with the cAMP/AMPK/SIRT1 pathway

Rolipram specifically inhibits phosphodiesterase (PDE) 4, thereby preventing inactivation of the intracellular second messenger cyclic adenosine monophosphate (cAMP). Rolipram has been shown to play a neuroprotective role in some central nervous system (CNS) diseases. However, the role of PDE4 and the potential protective effect of rolipram on the pathophysiological process of intracerebral haemorrhage (ICH) are still not entirely clear. In this study, a mouse model of ICH was established by the collagenase method. Rolipram reduced brain oedema, blood–brain barrier (BBB) leakage, neuronal apoptosis and inflammatory cytokine release and improved neurological function in our mouse model of ICH. Moreover, rolipram increased the levels of cAMP and silent information regulator 1 (SIRT1) and upregulated the phosphorylation of AMP-activated protein kinase (AMPK). Furthermore, these effects of rolipram could be reversed by the SIRT1 inhibitor sirtinol. In conclusion, rolipram can play a neuroprotective role in the pathological process of ICH by activating the cAMP/AMPK/SIRT1 pathway.

B-PO02-020 PDE4D MEDIATES DESENSITIZATION OF Β-ADRENERGIC RECEPTORS IN THE SINOATRIAL NODE IN HYPERTENSIVE HEART DISEASE

Chronotropic incompetence is common in hypertensive heart disease due to impaired β-adrenergic receptor (β-AR) responsiveness of the sinoatrial node (SAN). To determine the mechanism for impaired β-AR signaling in the SAN of angiotensin II (AngII) induced hypertensive heart disease. C57BL/6 mice were infused with saline or AngII (2.5mg/kg/day) for 21 days to induce hypertensive heart disease. Heart rate (HR) and SAN function in response to the β-AR agonist isoproterenol (ISO) were studied by in vivo electrocardiography, in isolated atrial preparations by optical mapping, in isolated SAN myocytes by patch-clamping, and by molecular techniques. AngII infused mice had impaired HR increases in response to ISO (100mg/kg) in vivo (171.1±7.1 vs. 134±9.8 bpm; n=5-6 mice; P<0.05) and in isolated atrial preparations (10μM ISO; 160.4±9.7 vs. 96.6±7.8, n=5-6 hearts; P<0.05). ISO (10μM) induced increases in SAN conduction velocity were also smaller in AngII infused hearts (1.6±0.1 vs. 1.1±0.1 cm/s, n=5-6 hearts; P<0.05). The effects of ISO (10μM) in isolated SAN myocytes after AngII infusion were impaired as indicated by smaller increases in spontaneous action potential (AP) firing (54.5±4.6 vs. 28.3±2.7 AP/min; P<0.001), diastolic depolarization (DD) slope (33.2±3.8 vs. 19.0±3.5 mV/s; P<0.05), peak “funny” current (-3.7±0.2 vs. -1.8±0.1 pA/pF; P<0.05), and peak L-type Ca2+ current (-6.9±1.3 vs. -3.5±0.7 pA/pF; P<0.05; n=7-15 cells). SAN myocytes from AngII infused mice had diminished cAMP generation in response to ISO (126.5±4.7 vs. 103.7±5.3 nM/1000 cells; P<0.05). No differences were seen in the protein expression of β1- or β2-ARs (both n=6; P>0.05); however, AngII infused mice had increased SAN PDE4 activity (1.4±0.3 vs. 3.9±0.9 pmol/mg/min, n=5; P<0.05). Total PDE4 expression was unaltered in the SAN of AngII infused mice but localization of PDE4D surrounding both β1- and β2-ARs was increased (n=6; P<0.05). Acute application of the PDE4 inhibitor rolipram (10μM) normalized ISO (10nM) induced increases in AP firing and DD slope in SAN myocytes (n=8-10) from AngII infused mice. AngII infusion results in blunted HR responses to β-AR stimulation due to upregulation of PDE4D specifically around β-ARs in the SAN.

Regulation of basal and norepinephrine-induced cAMP and ICa in hiPSC-cardiomyocytes: Effects of culture conditions and comparison to adult human atrial cardiomyocytes

BackgroundThere 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. MethodsAdult 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. Förster-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). ResultsIn 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 < 0.05 each) and both were augmented in the presence of the PDE4 inhibitor rolipram (FRET +16.7 ± 0.8% n = 94/26 and ICa + 5.6 ± 1.4 pA/pF n = 11/5, p < 0.05 each). Cilostamide increased the response to norepinephrine on FRET (+12.7 ± 0.5% n = 91/19, p < 0.05), but not on ICa. In EHT hiPSC-cardiomyocytes, norepinephrine responses on both, FRET and ICa, were larger than in ML (FRET +12.1 ± 0.3% n = 87/32 and ICa + 3.3 ± 0.2 pA/pF n = 13/5, p < 0.05 each). Rolipram augmented the norepinephrine effect on ICa (+6.2 ± 1.6 pA/pF; p < 0.05 vs. norepinephrine alone, n = 10/4), but not on FRET. ConclusionOur 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.

Impact of phosphodiesterases PDE3 and PDE4 on 5-hydroxytryptamine receptor4-mediated increase of cAMP in human atrial fibrillation

Atrial fibrillation (AF)–associated remodeling includes contractile dysfunction whose reasons are only partially resolved. Serotonin (5-HT) increases contractile force and causes arrhythmias in atrial trabeculae from patients in sinus rhythm (SR). In persistent atrial fibrillation (peAF), the force responses to 5-HT are blunted and arrhythmic effects are abolished. Since force but not arrhythmic responses to 5-HT in peAF could be restored by PDE3 + PDE4 inhibition, we sought to perform real-time measurements of cAMP to understand whether peAF alters PDE3 + PDE4-mediated compartmentation of 5-HT4 receptor-cAMP responses. Isolated human atrial myocytes from patients in SR, with paroxysmal AF (paAF) or peAF, were adenovirally transduced to express the FRET-based cAMP sensor Epac1-camps. Forty-eight hours later, cAMP responses to 5-HT (100 μM) were measured in the absence or concomitant presence of the PDE3 inhibitor cilostamide (0.3 μM) and the PDE4 inhibitor rolipram (1 μM). We successfully established real-time cAMP imaging in AF myocytes. 5-HT increased cAMP in SR, paAF, and peAF, but in line with previous findings on contractility, this increase was considerably smaller in peAF than in SR or paAF. The maximal cAMP response to forskolin (10 μM) was preserved in all groups. The diminished cAMP response to 5-HT in peAF was recovered by preincubation with cilostamide + rolipram. We uncovered a significantly diminished cAMP response to 5-HT4 receptor stimulation which may explain the blunted 5-HT inotropic responses observed in peAF. Since both cAMP and force responses but not arrhythmic responses were recovered after concomitant inhibition of PDE3 + PDE4, they might be regulated in different subcellular microdomains.

Inhibition of type 4 cAMP-phosphodiesterases (PDE4s) in mice induces hypothermia via effects on behavioral and central autonomous thermoregulation

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.

Rutaecarpine derivative Cpd-6c alleviates acute kidney injury by targeting PDE4B, a key enzyme mediating inflammation in cisplatin nephropathy

Acute kidney injury (AKI), characterized by a rapid decline in renal function, is triggered by an acute inflammatory response that leads to kidney damage. An effective treatment for AKI is lacking. Using in vitro and in vivo AKI models, our laboratory has identified a series of anti-inflammatory molecules and their derivatives. In the current study, we identified the protective role of rutaecarpine (Ru) on renal tubules. We obtained a series of 3-aromatic sulphonamide-substituted Ru derivatives exhibiting enhanced renoprotective and anti-inflammatory function. We identified Compound-6c(Cpd-6c) as having the best activity and examined its protective effect against cisplatin nephropathy both in vivo and in vitro in cisplatin-stimulated tubular epithelial cells (TECs). Our results showed that Cpd-6c restored renal function more effectively than Ru, as evidenced by reduced blood urea nitrogen and serum creatinine levels in mice. Cpd-6c alleviated tubular injury, as shown by PAS staining and molecular analysis of kidney injury molecule-1 (KIM-1), with both prevention and treatment protocols in cisplatin-treated mice. Moreover, Cpd-6c decreased kidney inflammation, oxidative stress and programmed cell death. These results have also been confirmed in cisplatin-treated TECs. Using web-prediction algorithms, molecular docking, and cellular thermal shift assay (CETSA), we identified phosphodiesterase 4B (PDE4B) as a Cpd-6c target. In addition, we firstly found that PDE4B was up-regulated significantly in the serum of AKI patients. After identifying the function of PDE4B in cisplatin-treated tubular epithelial cells by siRNA transfection or PDE4 inhibitor rolipram, we showed that Cpd-6c treatment did not protect against cisplatin-induced injury in PDE4B knockdown TECs, thus indicating that Cpd-6c exerts its renoprotective and anti-oxidative effects via the PDE4B-dependent pathway. Collectively, Cpd-6c might serve as a potential therapeutic agent for AKI and PDE4B may be highly involved in the initiation and progression of AKI.

The phosphodiesterase-4 and glycine transporter-1 inhibitors enhance in vivo hippocampal theta network connectivity and synaptic plasticity, whereas D-serine does not

Dysfunctional N-methyl-D-aspartate receptors (NMDARs) and cyclic adenosine monophosphate (cAMP) have been associated with deficits in synaptic plasticity and cognition found in neurodegenerative and neuropsychiatric disorders such as Alzheimer’s disease (AD) and schizophrenia. Therapeutic approaches that indirectly enhance NMDAR function through increases in glycine and/or D-serine levels as well as inhibition of phosphodiesterases that reduces degradation of cAMP, are expected to enhance synaptic strength, connectivity and to potentially impact cognition processes. The present in vivo study investigated effects of subcutaneous administration of D-serine, the glycine transporter 1 (GlyT1) inhibitor SSR504734 and the PDE4 inhibitor rolipram, on network oscillations, connectivity and long-term potentiation (LTP) at the hippocampi circuits in Sprague-Dawley rats. In conscious animals, multichannel EEG recordings assessed network oscillations and connectivity at frontal and hippocampal CA1–CA3 circuits. Under urethane anaesthesia, field excitatory postsynaptic potentials (fEPSPs) were measured in the CA1 subfield of the hippocampus after high-frequency stimulation (HFS) of the Schaffer collateral-CA1 (SC) pathway. SSR504734 and rolipram significantly increased slow theta oscillations (4–6.5 Hz) at the CA1–CA3, slow gamma oscillations (30–50 Hz) in the frontal areas and enhanced coherence in the CA1–CA3 network, which were dissociated from motor behaviour. SSR504734 enhanced short-term potentiation (STP) and fEPSP responses were extended into LTP response, whereas the potentiation of EPSP slope was short-lived to STP with rolipram. Unlike glycine, increased levels of D-serine had no effect on network oscillations and limits the LTP induction and expression. The present data support a facilitating role of glycine and cAMP on network oscillations and synaptic efficacy at the CA3–CA1 circuit in rats, whereas raising endogenous D-serine levels had no such beneficial effects.

Inhibition of PDE4 Induces Hypothermia in Mice

Type 4 cyclic nucleotide phosphodiesterases (PDE4s), a group of isoenzymes that hydrolyze and inactivate the second messenger cAMP, are established targets for development of enzyme inhibitors with anti‐inflammatory properties. While testing the anti‐inflammatory effects of a nonselective PDE4 inhibitor in a murine model of bacterial lung infection, we noticed that PDE4 inhibition per se induced significant hypothermia in the animals in the absence of bacterial infection. Because drug‐induced hypothermia may critically impact our animal model, and the role cAMP‐phosphodiesterases play in the regulation of body temperature is poorly understood, we further explored this observation. We found that treatment with a number of structurally distinct PAN‐PDE4 inhibitors, including Rolipram, Roflumilast and RS25344 (all 1 mg/kg, i.p.), but not the PDE3‐selective inhibitor Cilostamide, induced a rapid decrease in core body temperature of C57Bl6 mice as measured with a rectal thermometer at 30 min after drug application, suggesting that hypothermia in mice is a class‐effect of PDE4 inhibitors. PDE4 inhibitor‐induced hypothermia was considerable (~4°C drop), rapid in onset (max effect is reached within 20–30 min), and long lasting (core body temperature remains below controls for up to 5 h). As little as 40 μg/kg of the archetypal PDE4 inhibitor Rolipram induced a hypothermic effect. Similar or higher doses of Rolipram were used in most published animal studies. Thus, the reported findings are likely associated with, or at least paralleled by, drug‐induced hypothermia. The initial decrease in body temperature after PDE4 inhibitor injection is so rapid that it is no different from heat dissipation after euthanasia, suggesting that PDE4 inhibition affects central body temperature regulation so that both heat‐generating (e.g. shivering/non‐shivering thermogenesis) as well as heat‐preserving (e.g. skin vasoconstriction) mechanisms are simultaneously suspended, either by lowering the cold defense set point or by impairing its enforcement. Indeed, PDE4 inhibitor treatment induced a temporary heat‐loss via tail vasodilation as assessed by thermography. Consistent with the idea of an effect on central body temperature regulation, hypothermia was induced by moderate doses (≤1 mg/kg) of various brain‐penetrant PDE4 inhibitors. Conversely, YM976, a PDE4 inhibitor that does not cross the blood‐brain barrier, had no effect. Finally, to begin delineating the mechanism of drug‐induced hypothermia, we show that a blockade of serotonergic‐ and dopaminergic‐, but not β‐adrenergic‐, histaminergic‐ or opiate receptors, can alleviate PDE4 inhibitor‐induced hypothermia.Support or Funding InformationSupported by grants from the Cystic Fibrosis Foundation (SALEH18H0,RICHTE16GO) and the NIH (HL76125, HL141473, HL066299).

Cold-Inducible RNA-Binding Protein Prevents an Excessive Heart Rate Response to Stress by Targeting Phosphodiesterase.

The stress response of heart rate, which is determined by the plasticity of the sinoatrial node (SAN), is essential for cardiac function and survival in mammals. As an RNA-binding protein, CIRP (cold-inducible RNA-binding protein) can act as a stress regulator. Previously, we have documented that CIRP regulates cardiac electrophysiology at posttranscriptional level, suggesting its role in SAN plasticity, especially upon stress conditions. Our aim was to clarify the role of CIRP in SAN plasticity and heart rate regulation under stress conditions. Telemetric ECG monitoring demonstrated an excessive acceleration of heart rate under isoprenaline stimulation in conscious CIRP-KO (knockout) rats. Patch-clamp analysis and confocal microscopic Ca2+ imaging of isolated SAN cells demonstrated that isoprenaline stimulation induced a faster spontaneous firing rate in CIRP-KO SAN cells than that in WT (wild type) SAN cells. A higher concentration of cAMP-the key mediator of pacemaker activity-was detected in CIRP-KO SAN tissues than in WT SAN tissues. RNA sequencing and quantitative real-time polymerase chain reaction analyses of single cells revealed that the 4B and 4D subtypes of PDE (phosphodiesterase), which controls cAMP degradation, were significantly decreased in CIRP-KO SAN cells. A PDE4 inhibitor (rolipram) abolished the difference in beating rate resulting from CIRP deficiency. The mechanistic study showed that CIRP stabilized the mRNA of Pde4b and Pde4d by direct mRNA binding, thereby regulating the protein expression of PDE4B and PDE4D at posttranscriptional level. CIRP acts as an mRNA stabilizer of specific PDEs to control the cAMP concentration in SAN, maintaining the appropriate heart rate stress response.

Discovery and Optimization of α-Mangostin Derivatives as Novel PDE4 Inhibitors for the Treatment of Vascular Dementia.

To validate PDE4 inhibitors as novel therapeutic agents against vascular dementia (VaD), 25 derivatives were discovered from the natural inhibitor α-mangostin (IC50 = 1.31 μM). Hit-to-lead optimization identified a novel and selective PDE4 inhibitor 4e (IC50 = 17 nM), which adopted a different binding pattern from PDE4 inhibitors roflumilast and rolipram. Oral administration of 4e at a dose of 10 mg/kg exhibited remarkable therapeutic effects in a VaD model and did not cause emesis to beagle dogs, indicating its potential as a novel anti-VaD agent.

Rolipram treatment during consolidation ameliorates long-term object location memory in aged male mice

Normal aging is accompanied by cognitive and memory impairments that negatively impact quality of life for the growing elderly population. Hippocampal function is most vulnerable to the deleterious effects of aging, and deficits in hippocampus-dependent memories are common amongst aged individuals. Moreover, signaling networks such as the cAMP/PKA/CREB pathway, which are critical for memory consolidation, are dampened in healthy aged subjects. Phosphodiesterase (PDE) enzymes that break down cAMP are also affected by aging, and increased break down of cAMP by PDEs may contribute to reduced activity of the cAMP/PKA/CREB signaling network in the brain of aged individuals. Here, we report that the PDE4 inhibitor rolipram administered during consolidation of hippocampus-dependent object location memory improves aged-related spatial memory deficits in aged mice.

Влияние ролипрама на осморегулирующую функцию почек у крыс с различным уровнем вазопрессина в крови

The effect of the selective PDE-4 inhibitor rolipram, which eliminates cAMP degradation, on osmoregulatory function in WAG and Brattleboro rats with different blood vasopressin (AVP) level was studied. It was found that in WAG rats with high endogenous AVP level treatment of rilipram induced the significant acceleration of the urine flow rate as a result of the increase in sodium excretion and also the decrease in solute‐free water reabsorption. The changes in the functional parameters were followed by an increase in the content of glycosaminoglycans, which determine the permeability of the interstitial matrix in the AVP-sensitive renal medulla. At the same time in AVP-deficient Brattleboro rats with low adenylate cyclase system activity in the kidney rolipram treatment induced the development of the antidiuretic response whithout changes in the parameters of natriuretic function and the state of the interstitial renal matrix. These results showed a significant influence of rolipram, which eliminates the degradation of cAMP by the inhibition of PDE4, on the osmoregulatory renal function, changes parameters of which are mainly determined by the AVP level and the integration of effects mediated by V2- and V1a-like vasopressin receptors.

Agonist-specific desensitization of PGE2-stimulated cAMP signaling due to upregulated phosphodiesterase expression in human lung fibroblasts

Pulmonary fibrosis is characterized by fibroblasts persisting in an activated form, producing excessive fibrous material that destroys alveolar structure. The second messenger molecule cyclic 3′,5′-adenosine monophosphate (cAMP) has antifibrotic properties, and prostaglandin E2 (PGE2) can stimulate cAMP production through prostaglandin E (EP)2 and EP4 receptors. Although EP receptors are attractive therapeutic targets, the effects of long-term exposure to PGE2 have not been characterized. To determine the effects of long-term exposure of lung fibroblasts to PGE2, human fetal lung (HFL)-1 cells were treated for 24 h with 100 nM PGE2 or other cAMP-elevating agents. cAMP levels stimulated by acute exposure to PGE2 were measured using a fluorescent biosensor. Pretreatment for 24 h with PGE2 shifted the concentration-response curve to PGE2 rightward by approximately 22-fold but did not affect responses to the beta-adrenoceptor agonist isoproterenol. Neither isoproterenol nor forskolin pretreatment altered PGE2 responses, implying that other cAMP-elevating agents do not induce desensitization. Use of EP2- and EP4-selective agonists and antagonists suggested that PGE2-stimulated cAMP responses in HFL-1 cells are mediated by EP2 receptors. EP2 receptors are resistant to classical mechanisms of agonist-specific receptor desensitization, so we hypothesized that increased PDE activity mediates the loss of signaling after PGE2 pretreatment. PGE2 treatment upregulated messenger RNA for PDE3A, PDE3B, PDE4B, and PDE4D and increased overall PDE activity. The PDE4 inhibitor rolipram partially reversed PGE2-mediated desensitization and PDE4 activity was increased, but rolipram did not alter responses to isoproterenol. The PDE3 inhibitor cilostazol had minimal effect. These results show that long-term exposure to PGE2 causes agonist-specific desensitization of EP2 receptor-stimulated cAMP signaling through the increased expression of PDE isozymes, most likely of the PDE4 family.

Pro-cognitive effect of upregulating cyclic guanosine monophosphate signalling during memory acquisition or early consolidation is mediated by increased AMPA receptor trafficking.

Background:Episodic memory consists of different mnemonic phases, including acquisition and early and late consolidation. Each of these phases is characterised by distinct molecular processes. Although both cyclic adenosine monophosphate (cAMP) and cyclic guanosine monophosphate (cGMP) are implicated in the acquisition phase, early consolidation only depends on cGMP, whereas late consolidation is mediated by cAMP. Accordingly, the cGMP-selective phosphodiesterase 5 (PDE5) inhibitor vardenafil or the cAMP-selective PDE4 inhibitor rolipram can improve memory acquisition or consolidation when applied during their respective time windows.Aims:Considering the important role of glutamatergic α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors (AMPAR) during normal memory function, we aimed to investigate whether the differential actions of these PDE inhibitors are mediated through AMPAR dynamics.Methods:For biochemical analysis, mice were treated with either vardenafil or rolipram and sacrificed shortly after injection. For the behavioural studies, mice received either of the inhibitors during the different mnemonic phases, while their spatial memory was tested using the object location task, and they were sacrificed 24 hours later.Results:Administration of either vardenafil or rolipram causes rapid changes in AMPARs. Moreover, treatment with vardenafil during the acquisition or early consolidation of spatial memory resulted in increased surface levels of AMPARs which were still augmented 24 hours after learning. Membrane levels of AMPARs were not affected anymore 24 hours after learning when rolipram was administrated at either the acquisition or late consolidation phase.Conclusions:These results suggest that dissociative molecular mechanisms could mediate the pro-cognitive function of different classes of PDE inhibitors, and in the case of vardenafil, this phenomenon could be explained by changes in AMPAR dynamics.

Regulation of cAMP accumulation and activity by distinct phosphodiesterase subtypes in INS-1 cells and human pancreatic β-cells.

Pancreatic β-cells express multiple phosphodiesterase (PDE) subtypes, but the specific roles for each in β-cell function, particularly in humans, is not clear. We evaluated the cellular role of PDE1, PDE3, and PDE4 activity in the rat insulinoma cell line INS-1 and in primary human β-cells using subtype-selective PDE inhibitors. Using a genetically encoded, FRET-based cAMP sensor, we found that the PDE1 inhibitor 8MM-IBMX, elevated cAMP levels in the absence of glucose to a greater extent than either the PDE3 inhibitor cilostamide or the PDE4 inhibitor rolipram. In 18 mM glucose, PDE1 inhibition elevated cAMP levels to a greater extent than PDE3 inhibition in INS-1 cells, while PDE4 inhibition was without effect. Inhibition of PDE1 or PDE4, but not PDE3, potentiated glucose-stimulated insulin secretion in INS-1 cells. PDE1 inhibition, but not PDE3 or PDE4 inhibition, reduced palmitate-induced caspase-3/7 activation, and enhanced CREB phosphorylation in INS-1 cells. In human β-cells, only PDE3 or PDE4 inhibition increased cAMP levels in 1.7 mM glucose, but PDE1, PDE3, or PDE4 inhibition potentiated cAMP levels in 16.7 mM glucose. Inhibition of PDE1 or PDE4 increased cAMP levels to a greater extent in 16.7 mM glucose than in 1.7 mM glucose in human β-cells. In contrast, elevation of cAMP levels by PDE3 inhibition was not different at these glucose concentrations. PDE1 inhibition also potentiated insulin secretion from human islets, suggesting that the role of PDE1 may be conserved between INS-1 cells and human pancreatic β-cells. Our results suggest that inhibition of PDE1 may be a useful strategy to potentiate glucose-stimulated insulin secretion, and to protect β-cells from the toxic effects of excess fatty acids.

Divergent off-target effects of RSK N-terminal and C-terminal kinase inhibitors in cardiac myocytes

P90 ribosomal S6 kinases (RSK) are ubiquitously expressed and regulate responses to neurohumoral stimulation. To study the role of RSK signalling on cardiac myocyte function and protein phosphorylation, pharmacological RSK inhibitors were tested. Here, the ATP competitive N-terminal kinase domain-targeting compounds D1870 and SL0101 and the allosteric C-terminal kinase domain-targeting FMK were evaluated regarding their ability to modulate cardiac myocyte protein phosphorylation. Exposure to D1870 and SL0101 significantly enhanced phospholamban (PLN) Ser16 and cardiac troponin I (cTnI) Ser22/23 phosphorylation in response to D1870 and SL0101 upon exposure to phenylephrine (PE) that activates RSK. In contrast, FMK pretreatment significantly reduced phosphorylation of both proteins in response to PE. D1870-mediated enhancement of PLN Ser16 phosphorylation was also observed after exposure to isoprenaline or noradrenaline (NA) stimuli that do not activate RSK. Inhibition of β-adrenoceptors by atenolol or cAMP-dependent protein kinase (PKA) by H89 prevented the D1870-mediated increase in PLN phosphorylation, suggesting that PKA is the kinase responsible for the observed phosphorylation. Assessment of changes in cAMP formation by FRET measurements revealed increased cAMP formation in vicinity to PLN after exposure to D1870 and SL0101. D1870 inhibited phosphodiesterase activity similarly as established PDE inhibitors rolipram or 3-isobutyl-1-methylxanthine. Assessment of catecholamine-mediated force development in rat ventricular muscle strips revealed significantly reduced EC50 for NA after D1870 pretreatment (DMSO/NA: 2.33 μmol/L vs. D1870/NA: 1.30 μmol/L). The data reveal enhanced cardiac protein phosphorylation by D1870 and SL0101 that was not detectable in response to FMK. This disparate effect might be attributed to off-target inhibition of PDEs with impact on muscle function as demonstrated for D1870.