Specific Phosphodiesterase Inhibitors Research Articles

Cyclic nucleotide phosphodiesterases: therapeutic targets incardiac hypertrophy and failure

Cyclic nucleotide phosphodiesterases (PDEs) modulate neurohormonal regulation of cardiac function by degrading cAMP and cGMP. In cardiomyocytes, multiple isoforms of PDEs with different enzymatic properties and subcellular locally regulate cyclic nucleotide levels and associated cellular functions. This organisation is severely disrupted during hypertrophy and heart failure (HF), which may contribute to disease progression. Clinically, PDE inhibition has been seen as a promising approach to compensate for the catecholamine desensitisation that accompanies heart failure. Although PDE3 inhibitors such as milrinone or enoximone can be used clinically to improve systolic function and relieve the symptoms of acute CHF, their chronic use has proved detrimental. Other PDEs, such as PDE1, PDE2, PDE4, PDE5, PDE9 and PDE10, have emerged as potential new targets for the treatment of HF, each with a unique role in local cyclic nucleotide signalling pathways. In this review, we describe cAMP and cGMP signalling in cardiomyocytes and present the different families of PDEs expressed in the heart and their modifications in pathological cardiac hypertrophy and HF. We also review results from preclinical models and clinical data indicating the use of specific PDE inhibitors or activators that may have therapeutic potential in CI.

A proof of concept phase II study with the PDE-4 inhibitor roflumilast in patients with mild cognitive impairment or mild Alzheimer’s disease dementia (ROMEMA): study protocol of a double-blind, randomized, placebo-controlled, between-subjects trial

BackgroundResearch into the neurobiological underpinnings of learning and memory has demonstrated the cognitive-enhancing effects associated with diverse classes of phosphodiesterase (PDE) inhibitors. Specific PDE inhibitors have been identified to improve neuronal communication through selective inhibition of PDE activity. Roflumilast, a PDE4 inhibitor, has demonstrated efficacy in enhancing episodic memory in healthy adults and elderly participants with pronounced memory impairment, indicative of amnestic mild cognitive impairment (aMCI). In alignment with these findings, the present protocol aims to provide a proof of concept phase II of the potential of roflumilast to aid patients diagnosed with (a)MCI or mild Alzheimer’s disease (AD) dementia.MethodsThe study will be conducted according to a double-blind, randomized placebo-controlled, between-subjects design. Participants with (a)MCI and mild AD dementia will be recruited through the Memory Clinic at the Maastricht University Medical Centre + (MUMC +) in Maastricht, the Netherlands, alongside outreach through regional hospitals, and social media. The study will have three arms: placebo, 50 μg roflumilast, and 100 μg roflumilast, with a treatment duration of 24 weeks. The primary outcome measure will focus on the assessment of episodic memory, as evaluated through participants’ performance on the 15-word Verbal Learning Task (VLT). Our secondary objectives are multifaceted, including an exploration of various cognitive domains. In addition, insights into the well-being and daily functioning of participants will be investigated through interviews with both the participants and their (informal) caregivers, we are interested in the well-being and daily functioning of the participants.DiscussionThe outcomes of the present study aim to elucidate the significance of the PDE4 inhibition mechanism as a prospective therapeutic target for enhancing cognitive function in individuals with (a)MCI and mild AD dementia. Identifying positive effects within these patient cohorts could extend the relevance of this treatment to encompass a broader spectrum of neurological disorders.Trial registrationThe Medical Ethics Committee of MUMC + granted ethics approval for the 4th version of the protocol on September 10th, 2020. The trial was registered at the European Drug Regulatory Affairs Clinical Trials (EudraCT) registered on the 19th of December 2019 (https://www.clinicaltrialsregister.eu/ctr-search/trial/2019-004959-36/NL) and ClinicalTrial.gov (NCT04658654, https://clinicaltrials.gov/study/NCT04658654?intr=roflumilast&cond=mci&rank=1) on the 8th of December 2020. The Central Committee on Research Involving Human Subjects (CCMO) granted approval on the 30th of September 2020.

Theophylline derivatives promote primordial follicle activation via cAMP-PI3K/Akt pathway and ameliorate fertility deficits in naturally aged mice.

In elderly women and patients with premature ovarian insufficiency (POI), activating their remaining dormant primordial follicles in vivo is challenging. In this study, we found that phosphodiesterase (PDE) subtypes were expressed mainly in primordial follicle oocytes. The specific PDE inhibitors and theophylline derivatives (aminophylline, dyphylline, and enprofylline) activated primordial follicles in neonatal mice by ovary culture and intraperitoneal injection. These inhibitors also increased the levels of ovarian cyclic adenosine monophosphate (cAMP) and oocyte phosphorylated protein kinase B (p-Akt). The blockade of gap junctions using carbenoxolone (CBX) increased the levels of ovarian cAMP and pre-granulosa cell phosphorylated mammalian target of rapamycin (p-mTOR), suggesting that oocyte PDEs hydrolyze cAMP from pre-granulosa cells through gap junctions to maintain primordial follicle dormancy. Importantly, oral aminophylline improved ovulated oocyte quantity and quality, and increased offspring numbers in naturally aged mice. In addition, theophylline derivatives also activated human primordial follicles and increased p-Akt levels. Thus, theophylline derivatives activate primordial follicles by accumulating cAMP levels and activating phosphatidylinositol 3-kinase (PI3K)/Akt pathway in oocytes, and oral aminophylline increased fertility in naturally aged female mice by improving ovulated oocyte quantity and quality. As oral medications, theophylline derivatives may be used to improve fertility in elderly women and patients with POI.

Phosphodiesterase 7B regulates prostanoid‐ but not ß‐adrenergic‐stimulated cAMP levels in primary human airway smooth muscle cells

Human airway smooth muscle (HASM) cells contain G‐protein coupled receptor (GPCR) signal transduction pathways that are organized in the plasma membrane and form cAMP signaling compartments. One such compartment contains ß2‐adrenergic receptors (ß2AR) coupled to adenylyl cyclase (AC)6 and is principally localized in lipid rafts. Another compartment expresses E prostanoid receptors 2 and 4 (EP2/4) coupled to AC2 and is restricted to non‐raft regions of the plasma membrane. Various phosphodiesterase (PDE) isozymes modulate cyclic AMP (cAMP) levels in response to these pathways. Non‐specific or PDE‐family specific PDE inhibitors have been used to treat asthma and chronic obstructive pulmonary disease with limited success, in part due to expression of these PDE isoforms throughout the body. Drugs that inhibit specific PDE isozymes might produce therapeutic effects in airways with fewer off‐target effects. RNA‐sequencing data in HASM cells indicates that PDE7B is the third most highly expressed PDE isoform and includes expression of both the PDE7B‐201 and PDE7B‐203 splice variants. We sought to characterize the role of PDE7B in the lipid raft and non‐raft cAMP signaling compartments. We used siRNA to knock down both PDE7B splice variants, confirmed protein reduction via Western Blot analysis, and then observed live cells’ real‐time cAMP levels in response to either isoproterenol (ßAR agonist) or PGE2 (EP agonist). The cADDis‐FMP biosensor, which localizes in lipid raft regions, did not report altered cAMP dynamics in response to ß2AR or EPR agonists following PDE7B knockdown. However, the cADDis‐FS15 biosensor, which reports cAMP levels in non‐raft plasma membrane regions, reported significant increases in cAMP levels stimulated by EPR agonists but no change in cAMP levels stimulated by ß2AR agonist. PDE7B may play a unique role in regulating non‐raft associated EP2/4 signaling without altering cAMP levels in other areas of the cell, thereby presenting a novel therapeutic target for pulmonary diseases.

Cilostazol increases adenosine plasma concentration in patients with acute coronary syndrome

Cilostazol is a specific and strong inhibitor of phosphodiesterase (PDE) type III which can suppress the platelet aggregation by increasing cyclic adenosine monophosphate (cAMP) levels. The clinical benefit of cilostazol in ACS patients suggested that the drug may have non-platelet-directed properties. Some in vitro and animal studies also indicated that the 'pleiotropic' properties of cilostazol might be related to the interaction with adenosine metabolism. Adenosine is an important regulatory metabolite and an inhibitor of platelet activation. However, no human study has been conducted to determine whether cilostazol could increase the adenosine plasma concentration in vivo. As a result, this study aimed to investigate the impact of cilostazol on adenosine plasma concentration (APC) in acute coronary syndrome (ACS) patients. We prospectively analysed 149 ACS patients undergoing percutaneous coronary intervention (PCI) with drug-eluting stents. The included patients were divided into two groups according to the presence (cilostazol group, n=64) or absence (aspirin group, n=85) of aspirin intolerance. The inhibition of platelet aggregation (IPA), APC and cAMP concentration was measured. Patient characteristics, medications and 30-day clinical outcomes were examined. Patients receiving cilostazol had a significantly higher adenosine and cAMP plasma concentration than patients receiving aspirin (3.00±0.67 vs 2.56±0.74mol/L, P<.001; 28.10±14.74 vs 20.48±11.35pmol/mL, P=.0014). Cilostazol was associated with a higher inhibition rate of ADP induced platelet aggregation than aspirin (63.35±26.71 vs 52.2±28.35, P=.036). The plasma levels of adenosine and cAMP showed a positive correlation with ADP induced platelet aggregation. Cilostazol increases adenosine concentration compared with aspirin. Its potent antiplatelet effect in ACS patients may be partly mediated by adenosine.

Diabetic Theory in Anti-Alzheimer’s Drug Research and Development. Part 2: Therapeutic Potential of cAMP-Specific Phosphodiesterase Inhibitors

Alzheimer's disease (AD) is one of the most prevalent age-related neurodegenerative disease that affects the cognition, behavior, and daily activities of individuals. Studies indicate that this disease is characterized by several pathological mechanisms, including the accumulation of amyloid-beta peptide, hyperphosphorylation of tau protein, impairment of cholinergic neurotransmission, and increase in inflammatory responses within the central nervous system. Chronic neuroinflammation associated with AD is closely related to disturbances in metabolic processes, including insulin release and glucose metabolism. As AD is also called type III diabetes, diverse compounds having antidiabetic effects have been investigated as potential drugs for its symptomatic and disease-modifying treatment. In addition to insulin and oral antidiabetic drugs, scientific attention has been paid to cyclic-3',5'-adenosine monophosphate (cAMP)-specific phosphodiesterase (PDE) inhibitors that can modulate the concentration of glucose and related hormones and exert beneficial effects on memory, mood, and emotional processing. In this review, we present the most recent reports focusing on the involvement of cAMP-specific PDE4, PDE7, and PDE8 in glycemic and inflammatory response controls as well as the potential utility of the PDE inhibitors in the treatment of AD. Besides the results of in vitro and in vivo studies, the review also presents recent reports from clinical trials.

The Association Between Phosphodiesterase-5 Inhibitors and Colorectal Cancer in a National Cohort of Patients.

INTRODUCTION:To examine the association between phosphodiesterase-5 (PDE-5) inhibitor use and incidence of colorectal cancer among patients with erectile dysfunction treated in the Veterans Affairs (VA) Healthcare System.METHODS:A retrospective cohort study using the Veterans Affairs Informatics and Computing Infrastructure was conducted, with data spanning January 2001–December 2016. Patients were followed up from index until (i) the first diagnosis of colorectal cancer, (ii) death, or (iii) the end of study period. Statistical analyses evaluated demographics and baseline characteristics between cohorts (PDE-5 exposed or not) and the effect of additional dosages of each specific PDE-5 inhibitor using adjusted multivariate Cox proportional hazards models.RESULTS:A total of 221,538 patients met the study inclusion criteria, 192,691 patients in the PDE-5 cohort and 29,227 patients in the never use PDE-5 cohort. The multivariate Cox proportional hazards model results revealed that the those who had any exposure to a PDE-5 inhibitor have an 18% lower hazard of colorectal cancer (adjusted hazard ratio [HR] = 0.816, 95% confidence interval [CI] = 0.754–0.882). For each additional 100-mg dosage of sildenafil and 10-mg dosage of tadalafil, the hazard of colorectal cancer is reduced by 2.4% (adjusted HR = 0.976, 95% CI = 0.973–0.979) and 1.7% (adjusted HR = 0.983, 95% CI = 0.972–0.996), respectively.DISCUSSION:PDE-5 inhibitor usage in patients with erectile dysfunction is associated with a lower hazard of colorectal cancer compared with patients not exposed to PDE-5 inhibitors.

Atrial Natriuretic Peptide Prevented Lipid‐Induced Kidney Injuries by Inhibiting Endoplasmic Reticulum Stress

Hyperlipidemia plays an important role in the progression of kidney injury, likely due to renal lipotoxicity. The purpose of the present study was to investigate whether atrial natriuretic peptide (ANP) prevented lipid‐induced injury through inhibiting endoplasmic reticulum (ER) stress and apoptosis in the kidney. In human proximal tubule HK2 cells, saturated fatty acid palmitic acid (PA) treatment (0.4mM) for 24h markedly increased protein abundance of two ER stress makers BiP (3.7‐folds of controls) and CHOP (5.2‐folds of controls), which was associated with upregulated cleaved‐caspase 3 expression (3.2‐folds of controls) and decreased ratio of Bcl2/Bax (0.76‐folds of controls), two markers of apoptosis. ANP treatment (10pM) markedly decreased protein abundance of BiP (2.8‐folds of controls), CHOP (3.7‐folds of controls), and cleaved‐caspase 3 (1.7‐folds of controls), and increased the ratio of Bcl2/Bax (1.2‐folds of controls) in HK2 cells treated by PA. Co‐treatment with ANP and LBQ657, a neprilysin inhibitor preventing hydrolysis of ANP, showed better protective effects than ANP treatment alone in HK2 cells treated with PA. ANP is known to stimulate intracellular cGMP‐PKG signaling pathway, the role of cGMP and PKG in ANP‐induced protection after PA treatment in HK2 cells was thus investigated. Sildenafil, an inhibitor of phosphodiesterase‐5 (PDE5), and exogenous 8‐Br‐cGMP markedly suppressed PA‐induced ER stress and apoptosis, whereas KT‐5823, a selective cGMP‐dependent PKG inhibitor, blocked protection of ANP or cGMP in HK2 cells treated with PA. A synthesized, specific PDE5 inhibitor LW1646 showed slightly better prevention than sildenafil on PA‐induced HK2 cell injury. ANP attenuated PA‐induced upregulation of inositol 1,4,5‐triphate receptor (IP3R) that is involved in calcium efflux from ER to cytoplasm. ANP decreased PA‐induced intracellular calcium oscillation, prevented PA‐induced mitochondrial depolarization, and promoted mitochondrial biogenesis in HK2 cells. Compared with controls, in mice fed with high‐fat diet for 8wk, the protein expression of BiP and CHOP in the kidney cortex showed 2.8‐fold and 2.3‐fold increases, respectively, which was markedly prevented by sildenafil (15mg/kg·BW/day, gavage). In conclusion, these findings demonstrated that ANP inhibited lipid‐induced ER stress and apoptosis, likely via activating the cGMP‐PKG signaling pathway and maintaining mitochondrial function, it may be a potentially therapeutic target for lipid‐induced kidney injury.

Effects of Specific Inhibitor of Phosphodiesterase 7 at the Late Stage of Long-Term Potentiation in Murine Hippocampal Slices.

Second messengers cAMP and cGMP play an important role in synaptic plasticity and memory consolidation. The inhibitors of phosphodiesterases, enzymes hydrolyzing these cyclic nucleotides, are actively studied as potential drugs for the treatment of various cognitive disorders and depression. We studied the effects of a new inhibitor of phosphodiesterase 7 AGF2.20 on the formation of long-term potentiation in hippocampal slices. Administration of AGF2.20 (10 nM) in 90 min after weak tetanization prevented a decrease in the amplitude of excitatory post-synaptic potentials and stabilized long-term potentiation. These data attest to the involvement of phosphodiesterase 7 in the development of synaptic plasticity in the hippocampus. The inhibitor AGF2.20 is considered for the further analysis as a promising substance for the treatment of cognitive impairments.

Study on the clinical efficacy of specific phosphodiesterase inhibitor in patients with pulmonary hypertension due to left heart disease.

Pulmonary hypertension due to left heart disease (PH-LHD) is caused by left ventricular (LV) systolic and/or diastolic dysfunction and left heart valve disease. LV diseases lead to left ventricular filling pressure increases, pulmonary venous obstruction and pulmonary venous pressure increases, and thus to secondary PH. Exercise tolerance is lower and fatality rates are higher in patients with PH-LHD than those in subjects with normal pulmonary arterial pressure. In spite of the progress in the study of the mechanisms of PH-LHD in recent years, no specific treatment is currently available. The efficacy and safety of targeted therapies for pulmonary arterial hypertension remain to be fully established. In the present study, PH-LHD patients were treated with milrinone injection. It was concluded that milrinone significantly reduces pulmonary artery systolic pressure (PASP) in patients with PH-LHD, and significantly improves the cardiac structure, cardiac function and biochemical indexes. PASP was significantly correlated with the left atrial diameter, LV end diastolic diameter, LV ejection fraction, tricuspid annular plane systolic excursion, right ventricular fractional area change, N-terminal pro-B-type natriuretic peptide and hypersensitive C-reactive protein.

Enzymatic Formation of a Skipped Methyl‐Substituted Octaprenyl Side Chain of Longestin (KS‐505a): Involvement of Homo‐IPP as a Common Extender Unit

AbstractLongestin (KS‐505a), a specific inhibitor of phosphodiesterase, is a meroterpenoid that consists of a unique octacyclic terpene skeleton with branched methyl groups at unusual positions (C1 and C12). Biochemical analysis of Lon23, a methyltransferase involved in the biosynthesis of longestin, demonstrated that it methylates homoisopentenyl diphosphate (homo‐IPP) to afford (3Z)‐3‐methyl IPP. This compound, along with IPP, is selectively accepted as extender units by Lon22, a geranylgeranyl diphosphate (GGPP) synthase homologue, to yield dimethylated GGPP (dmGGPP). The absolute configuration of dmGGPP was determined to be (4R,12R) by degradation and chiral GC analysis. These findings allowed us to propose an enzymatic sequence for key steps of the biosynthetic pathway of the unusual homoterpenoid longestin.

Enzymatic Formation of a Skipped Methyl-Substituted Octaprenyl Side Chain of Longestin (KS-505a): Involvement of Homo-IPP as a Common Extender Unit.

Longestin (KS-505a), a specific inhibitor of phosphodiesterase, is a meroterpenoid that consists of a unique octacyclic terpene skeleton with branched methyl groups at unusual positions (C1 and C12). Biochemical analysis of Lon23, a methyltransferase involved in the biosynthesis of longestin, demonstrated that it methylates homoisopentenyl diphosphate (homo-IPP) to afford (3Z)-3-methyl IPP. This compound, along with IPP, is selectively accepted as extender units by Lon22, a geranylgeranyl diphosphate (GGPP) synthase homologue, to yield dimethylated GGPP (dmGGPP). The absolute configuration of dmGGPP was determined to be (4R,12R) by degradation and chiral GC analysis. These findings allowed us to propose an enzymatic sequence for key steps of the biosynthetic pathway of the unusual homoterpenoid longestin.

Flavonoid glycosides isolated from Epimedium brevicornum and their estrogen biosynthesis-promoting effects

Epimedium brevicornum Maxim has a long history of use in the treatment of estrogen deficiency-related diseases. However, the chemical constituents and mechanism of action of this medicinal plant are not fully understood. In the present study, we isolated four new isoprenylated flavonoid glycosides, as well as 16 known flavonoids (13 isoprenylated flavonoids), from this plant. The chemical structures of the new flavonoid glycosides were elucidated by extensive spectroscopic analysis. The new compounds 1–4 were potent promoters of estrogen biosynthesis in human ovarian granulosa-like KGN cells. ZW1, an isoprenylated flavonoid analogue and a specific inhibitor of phosphodiesterase 5 (PDE5), was synthesized and used to explore the mechanism of the isoprenylated analogues on estrogen biosynthesis. ZW1 treatment increased estrogen production by upregulation of aromatase mRNA and protein expression. ZW1 increased the phosphorylation of cAMP response element-binding protein (CREB). Further study showed that the inhibition of PDE5 by ZW1 increased estrogen biosynthesis partly through suppression of phosphodiesterase 3 (PDE3). Our results suggested that the isoprenylated flavonoids from E. brevicornum may produce beneficial health effects through the promotion of estrogen biosynthesis. PDE5 warrants further investigation as a new therapeutic target for estrogen biosynthesis in the prevention and treatment of estrogen-deficiency related diseases.

Cyclic nucleotide phosphodiesterases in human spermatozoa and seminal fluid: Presence of an active PDE10A in human spermatozoa

BackgroundCyclic adenosine monophosphate (cAMP) plays a crucial role as a signaling molecule for sperm functions such as capacitation, motility and acrosome reaction. It is well known that cAMP degradation by phosphodiesterase (PDE) enzyme has a major impact on sperm functions. The present study was undertaken to characterize cAMP-PDE activity in human semen. MethodscAMP-PDE activity was measured in human sperm and seminal plasma using family specific PDE inhibitors. Three sperm fractionation methods were applied to assess cAMP-PDE activity in spermatozoa. Western blots were used to validate the presence of specific family in sperm and seminal plasma. ResultsUsing three sperm fractionation methods, we demonstrated that in human sperm, the major cAMP-PDE activity is papaverine-sensitive and thus ascribed to PDE10. In seminal plasma, total cAMP-PDE activity was 1.14±0.39fmol of cAMP hydrolyzed per minute per μg of protein. Using specific inhibitors, we showed that the major cAMP-PDE activity found in human seminal plasma is ascribed to PDE4 and PDE11. Western blot analysis, immunoprecipitation with a specific monoclonal antibody, and mass spectrometry confirmed the presence of PDE10 in human spermatozoa. ConclusionThis study provides the first demonstration of the presence of functional PDE10 in human spermatozoa and functional PDE4 and PDE11 in human seminal plasma. General significanceSince the contribution of cyclic nucleotides in several sperm functions is well known, the finding that PDE10 is an active enzyme in human spermatozoa is novel and may lead to new insight into fertility.

Surface plasmon resonance biosensor assay for the analysis of small-molecule inhibitor binding to human and parasitic phosphodiesterases

In the past decade, surface plasmon resonance (SPR) biosensor-based technology has been exploited more and more to characterize the interaction between drug targets and small-molecule modulators. Here, we report the successful application of SPR methodology for the analysis of small-molecule binding to two therapeutically relevant cAMP phosphodiesterases (PDEs), Trypanosoma brucei PDEB1 which is implicated in African sleeping sickness and human PDE4D which is implicated in a plethora of disease conditions including inflammatory pulmonary disorders such as asthma, chronic obstructive pulmonary disease and central nervous system (CNS) disorders. A protocol combining the use of directed capture using His-tagged PDE_CDs with covalent attachment to the SPR surface was developed. This methodology allows the determination of the binding kinetics of small-molecule PDE inhibitors and also allows testing their specificity for the two PDEs. The SPR-based assay could serve as a technology platform for the development of highly specific and high-affinity PDE inhibitors, accelerating drug discovery processes.

Impact on monoclonal antibody production in murine hybridoma cell cultures of adenosine receptor antagonists and phosphodiesterase inhibitors.

The effects of different adenosine receptor antagonists and cyclic nucleotide phosphodiesterase (PDE) inhibitors on monoclonal antibody (mAb) titer and cell viability of murine hybridoma cells in culture were measured as part of our investigations to discover additives that enhance mAb production. Specific adenosine receptor antagonists and PDE inhibitors were found to enhance or decrease the titer of immunoglobulin G1 (IgG1) mAbs relative to negative controls, depending on the specific compound and cell line employed. The observed enhancements or decreases in IgG1 mAb titer appeared to be mainly due to an increase or decrease in specific productivity rates (ngmAb/cell), respectively. The different effects of the selective adenosine antagonists suggest that antagonism at the level of the adenosine A2A and A1 or the adenosine A3 receptors result in either enhancement or suppression of IgG1 mAb production by hybridoma cells. Overall, these studies have identified hitherto unknown activities of specific adenosine antagonists and PDE inhibitors which indicate they may have valuable roles as cell culture additives in industrial biomanufacturing processes designed to enhance the yields of mAbs or other recombinant proteins produced by mammalian cell culture procedures.

Regulation of cAMP Intracellular Levels in Human Platelets Stimulated by 2-Arachidonoylglycerol.

We demonstrated that in human platelets the endocannabinoid 2-arachidonoylglycerol (2-AG) decreased dose- and time-dependently cAMP intracellular levels. No effect on cAMP decrease induced by 2-AG was observed in the presence of the adenylate cyclase inhibitor SQ22536 as well in platelets pretreated with the thromboxane A2 receptor antagonist, SQ29548 or with aspirin, inhibitor of arachidonic acid metabolism through the cyclooxygenase pathway. An almost complete recovering of cAMP level was measured in platelets pretreated with the specific inhibitor of phosphodiesterase (PDE) 3A, milrinone. In platelets pretreated with LY294002 or MK2206, inhibitors of PI3K/AKT pathway, and with U73122, inhibitor of phospholipase C pathway, only a partial prevention was shown. cAMP intracellular level depends on synthesis by adenylate cyclase and hydrolysis by PDEs. In 2-AG-stimulated platelets adenylate cyclase activity seems to be unchanged. In contrast PDEs appear to be involved. In particular PDE3A was specifically activated, as milrinone reversed cAMP reduction by 2-AG. 2-AG enhanced PDE3A activity through its phosphorylation. The PI3K/AKT pathway and PKC participate to this PDE3A phosphorylation/activation mechanism as it was greatly inhibited by platelet pretreatment with LY294002, MK2206, U73122, or the PKC specific inhibitor GF109203X. Taken together these data suggest that 2-AG potentiates its power of platelet agonist reducing cAMP intracellular level.

Targeting cyclic nucleotides in CNS drug discovery: a translational approach into cognition enhancement

Cyclic adenosine monophosphate (cAMP) and/or cyclic guanosine monophosphate (cGMP) have been suggested to play specific roles in processes of memory. These cyclic nucleotides are hydrolyzed by specific enzymes, i.e. phosphodiesterases (PDEs). Thus, selective PDE inhibitors preventing the breakdown of cAMP and/or cGMP could improve memory. Studies with different timing of treatment with specific PDE inhibitors indicated that distinct underlying signaling pathways for early and late consolidation processes exist corresponding to specific time-windows for memory consolidation into long-term memory. There is evidence that the underlying mechanisms of PDE inhibition on the observed behavioral effects are independent of possible cerebrovascular effects. Most likely the underlying mechanisms are a cGMP/PKG pathway for early consolidation processes and a cAMP/PKA pathway for late consolidation processes. In addition, the early-phase cGMP/PKG signaling actually requires late-phase cAMP/PKA-signaling in long-term memory formation. Recently, the effects of specific PDE inhibitors are explored on other cognitive domains including acquisition processes/short-term memory and information processing. It will be shown that elevation of central cGMP levels or cAMP levels after treatment with a specific PDE inhibitor both improve acquisition processes/short-term memory. The effects of specific PDE inhibitors on information processing by using a sensory gating paradigm indicate that elevation of cGMP together with cAMP using a specific PDE inhibitor improves basic information processing, whereas elevation of cGMP alone has no effect. In a translational approach we also investigated the effect of PDE5 inhibition on cognition in humans. However, in contrast to studies including rodents and monkeys, PDE5 inhibition had no effect on cognition including memory processes in humans. Yet as sensory gating was also not affected this could underlie the translational value of this measure. It is clear that the transition of a drug from preclinical to clinical creates translational hurdles. Within the context as described above, the latest results of specific PDE inhibitors on cognitive processes will be presented and its implications will be discusses for finding and testing new cognition enhancers.

Evaluation of Modified Borg Dyspnoea Score and 6-Minute Walk Test in Pulmonaryarterial Hypertensionpatients Recievingphosphodiesterase Inhibitors

Introduction : Pulmonary Arterial Hypertension (PAH) is a progressive condition that results in right heart failure and death1. Patients with PAH who do not receive disease targeted therapy have a poor quality of life and high mortality rates with a median survival of less than three years from diagnosis2,3. In the last few years, oral Sildenafil Citrate (a specific phosphodiesterase-5 inhibitor which is widely used in the treatment of erectile dysfunction), has shownpromising results as a novel oral monotherapy in the treatment of PAH. Methodology: A prospective open label randomized study was conducted over a period of 8 months in hospitalized patients who visited the in- patient block in a tertiary care hospital in Nilgris. Simple Randomization technique was adopted to allocate the patients into three studygroups. At the baseline Visit, Modified Borg Dyspnea Scale (MBDS) and 6 MWT(6 Minute Walk Test) were evaluated to confirm study criteria and were allotted into 3 doses based on their severity upon Right Ventricular Systolic Pressure. The same parameters were assessed on each visit for all the patients throughout thestudy period. Results and Discussion: In our study, Sildenafil Citrate showed an improvement in MBDS and 6 MWT after 8 months of therapy which were assessed using Kruskal-Wallis non-parametric test. A remarkable improvement in6MWT from 171.88±52.4 metres to 337.12±71.84 metres was observed. There was an improvement in dyspnea by Borg scale in all study groups. Cor-pulmonale patients were more benefited (p<0.001) whereas patients with Atrial Septal Defect (ASD) and Rheumatic Heart Disease (RHD) also improved significantly (p<0.05). Conclusion: Sildenafil has been reported to be welltolerated among the patient population. The patients who were slightly affected at baseline with moderate to severe dyspnoea became slight grade which.

AN IMPROVED METHOD FOR THE FRET-BASED DETECTION OF CYCLIC NUCLEOTIDE SIGNALLING IN PRIMARY ADULT MOUSE CARDIOMYOCYTES

Primary adult murine cardiomyocytes are an important tool for the investigation of cardiovascular physiology, especially with the development of transgenic mouse models. Fluorescence Resonance Energy Transfer (FRET) based probes have contributed greatly to the understanding of subcellular cAMP signalling. However, the transfer of genes encoding such probes into adult murine cardiomyocytes has proved difficult. Here we present a method for the effective adenovirus-mediated transduction of FRET biosensors that allow the investigation of cAMP signalling in adult murine cardiomyocytes in under 24 hours in culture. Furthermore we show that the contractility of adult murine cardiomyocytes in culture is not impaired by adenoviral gene transfer of FRET biosensors. Combined with a microscopy chamber perfusion system, that allows the removal of pharmacological agents such as specific inhibitors of phosphodiesterases (PDEs), this method allows the cost-effective, reproducible and rapid investigation of sub-cellular cAMP signalling and hence PDE activity in adult murine cardiomyocytes.