Total PDE Activity Research Articles

Development of TRIB3-Based Therapy as a Gene-Independent Approach to Treat Retinal Degenerative Disorders.

Inherited retinal degeneration (RD) constitutes a heterogeneous group of genetic retinal degenerative disorders. The molecular mechanisms underlying RD encompass a diverse spectrum of cellular signaling, with the unfolded protein response (UPR) identified as a common signaling pathway chronically activated in degenerating retinas. TRIB3 has been recognized as a key mediator of the PERK UPR arm, influencing various metabolic pathways, such as insulin signaling, lipid metabolism, and glucose homeostasis, by acting as an AKT pseudokinase that prevents the activation of the AKT → mTOR axis. This study aimed to develop a gene-independent approach targeting the UPR TRIB3 mediator previously tested by our group using a genetic approach in mice with RD. The goal was to validate a therapeutic approach targeting TRIB3 interactomes through the pharmacological targeting of EGFR-TRIB3 and delivering cell-penetrating peptides targeting TRIB3 → AKT. The study employed rd10 and P23H RHO mice, with afatinib treatment conducted in p15 rd10 mice through daily intraperitoneal injections. P15 P23H RHO mice received intraocular injections of cell-penetrating peptides twice at a 2-week interval. Our study revealed that both strategies successfully targeted TRIB3 interactomes, leading to an improvement in scotopic A- and B-wave ERG recordings. Additionally, the afatinib-treated mice manifested enhanced photopic ERG amplitudes accompanied by a delay in photoreceptor cell loss. The treated rd10 retinas also showed increased PDE6β and RHO staining, along with an elevation in total PDE activity in the retinas. Consequently, our study demonstrated the feasibility of a gene-independent strategy to target common signaling in degenerating retinas by employing a TRIB3-based therapeutic approach that delays retinal function and photoreceptor cell loss in two RD models.

PDE4B and PDE4D differentially regulate cardiac pacemaker activity

Abstract Funding Acknowledgements Type of funding sources: Public grant(s) – National budget only. Main funding source(s): Agence Nationale de la Recherche (ANR) Background Heart rate (HR) is generated by spontaneous electrical activity in the sinoatrial node (SAN) and is modulated by the autonomic nervous system. During sympathetic stimulation, the activation of β-adrenergic receptors (βAR) increases cAMP levels, leading to positive chronotropic effect. Among the 6 cardiac cAMP-PDE families, PDE4 is critical for controlling excitation-contraction coupling (ECC) during β-stimulation in atrial and ventricular myocytes. PDE4 may also be important for automaticity. 3 genes encode for PDE4 in the heart (pde4a, 4b, 4d). We propose to investigate their respective contribution to the regulation of pacemaker activity. Methods Total PDE activity in mouse SAN was determined as the cAMP-hydrolytic activity measured in the absence of PDE inhibitor and the fraction corresponding to PDE4 activity was assessed by including the PDE4 inhibitor Ro-20-1724. The in vitro pacemaker activity was assessed by measuring spontaneous Ca2+ transients in Fluo4-loaded-SAN tissue from wild-type (WT) and PDE4KO mice. Images were obtained using confocal microscopy. Telemetry EKG was recorded in conscious mice in control (CTRL) conditions, after pharmacological denervation with atropine (1 mg/kg) and propranolol (2 mg/kg) and after β-stimulation with isoproterenol (ISO, 1.5 mg/kg). HR variability was evaluated by calculating the SDNN (standard deviation of RR intervals) parameter. Results Ro-20-1724 (10 µM) increased beating rate of intact SAN and increased PKA-phosphorylation of key ECC actors (ryanodine receptor, phospholamban and contractile proteins). PDE4 activity was found to account for 60% of total cAMP-PDE activity in SAN. PDE4A, 4B and 4D isoforms were found to be expressed in mouse SAN. In PDE4BKO SAN, the effect of ISO on SAN beating rate was higher than in WT. Ablation of PDE4D induced decreased beating rate in CTRL and ISO conditions and increased Ca2+ spark frequency compared to WT SAN. In vivo, PDE4BKO and PDE4DKO mice displayed increased resting HR during day and night. HR variability was decreased in PDE4BKO, but not in PDE4DKO mice during the day, and decreased in both genotypes at night compared to WT mice. After atropine + propranolol denervation, the rhythmic phenotype was only maintained in PDE4BKO but not in PDE4DKO mice. The response to β-AR stimulation with ISO was higher in PDE4BKO than in PDE4DKO. In addition, under ISO we observed an increased number of premature beats and atrioventricular blocks in PDE4DKO, but not in PDE4BKO mice. Conclusion PDE4B and PDE4D differentially regulate cardiac pacemaker activity. While PDE4B clearly controls intrinsic SAN automaticity, PDE4D might be important for ANS-mediated regulation of HR and conduction.

PDE4B and PDE4D differentially regulate cardiac pacemaker activity

During sympathetic stimulation, the activation of β-adrenergic receptors (βAR) increases cAMP levels, leading to positive chronotropic effect. Among the 6 cardiac cAMP-PDE families, PDE4 is critical for controlling excitation-contraction coupling (ECC) during β-stimulation in atrial and ventricular myocytes. PDE4 may also be important for automaticity. Three genes encode for PDE4 in the heart (pde4a, 4b, 4d). To investigate the respective contribution of PDE4 isoforms to the regulation of pacemaker activity. Total PDE activity in mouse SAN was determined as the cAMP-hydrolytic activity measured in the absence of PDE inhibitor and the fraction corresponding to PDE4 activity was assessed by including the PDE4 inhibitor Ro-20-1724. The in vitro pacemaker activity was assessed by measuring spontaneous Ca 2+ transients in Fluo4-loaded-SAN tissue from Wild-type (WT) and PDE4KO using confocal microscopy. Telemetry EKG was recorded in conscious mice in control (CTRL) conditions, after pharmacological denervation (atropine + propranolol) and after isoproterenol (ISO). PDE4 activity represents 60% of total cAMP-PDE activity in SAN. PDE4A, 4B and 4D isoforms are expressed in mouse SAN. In PDE4BKO, the effect of ISO on SAN beating rate was higher than in WT. Ablation of PDE4D induced decreased beating rate in CTRL and ISO conditions and increased Ca 2+ spark frequency compared to WT. In vivo, PDE4BKO and PDE4DKO mice displayed increased resting HR during day and night. After pharmacological denervation, the rhythmic phenotype was only maintained in PDE4BKO but not in PDE4DKO. The response to ISO was higher in PDE4BKO than in PDE4DKO. In addition, ISO provoked more premature beats and atrioventricular blocks in PDE4DKO than in PDE4BKO mice. PDE4B controls intrinsic SAN automaticity and PDE4D might be important for autonomic nervous system-mediated regulation of HR and conduction.

Type 4 Phosphodiesterase regulates cardiac pacemaker function

Numerous epidemiological and clinical studies have revealed a positive correlation between heart rate (HR) and cardiovascular morbimortality. The autonomic nervous system is the major extracardiac determinant of HR. During sympathetic stimulation, the activation of β-adrenergic receptors (βAR) induces an increase in cAMP levels, leading to positive chronotropic effect. Among the 5 cardiac cAMP-PDE families, PDE4 is critical for controlling excitation-contraction coupling (ECC) during β-stimulation in atrial and ventricular cells. PDE4 may also be important for automaticity. Three genes encode for PDE4: pde4a , 4b , 4d . Their respective contribution to the regulation of pacemaker activity remains unknown. Total PDE activity was determined in mouse sinoatrial node (SAN) tissue as the cAMP-hydrolytic activity measured in the absence of PDE inhibitor and the fraction corresponding to PDE4 activity was assessed by including the PDE4 inhibitor Ro-201724. The in vitro pacemaker activity was assessed by measuring spontaneous Ca 2+ transients in Fluo4-loaded-SAN tissue. Images were obtained using confocal microscopy. Ro-20-1724 increased beating rate of intact SAN and increased PKA-phosphorylation of key ECC actors (ryanodine receptor, phospholamban and contractile proteins). PDE4 activity was found to account for 60% of total cAMP-PDE activity in SAN ( n = 3). PDE4A, 4B and 4D isoforms were found to be expressed in mouse SAN ( n = 5 independent experiments). In 4D-, but not in 4BKO mice, Ca 2+ homeostasis was altered in control conditions (ctrl) and after β-stimulation with isoprenaline (iso). Indeed, ablation of PDE4D induced decreased beating rate (ctrl: 1.00 ± 0.08 s −1 vs. 1.57 ± 0.05 s −1 ; iso: 1.71 ± 0.17 s −1 vs. 2.39 ± 0.08 s −1 , P < 0.0001) and increased Ca 2+ spark frequency (ctrl: 15.9 ± 5.2 vs. 1.9 ± 0.4 sparks/s/100 μm; iso: 22.9 ± 7.1 vs. 0.6 ± 0.2 sparks/s/100 μm, P < 0.0001) ( Fig. 1 ). PDE4 controls pacemaker function and PDE4D ablation strongly perturbs normal SAN activity.

PDE4 regulates cardiac pacemaker function

Numerous epidemiological and clinical studies have revealed a positive correlation between heart rate (HR) and cardiovascular morbimortality. The autonomic nervous system is the major extracardiac determinant of HR. During sympathetic stimulation, the activation of β-adrenergic receptors (βAR) induces an increase in cAMP levels, leading to positive chronotropic effect. Among the 5 cardiac cAMP-PDE families, PDE4 is critical for controlling excitation-contraction coupling (ECC) during β-stimulation in atrial and ventricular cells. PDE4 may also be important for automaticity. 3 genes encode for PDE4: pde4a,4b,4d. Their respective contribution to the regulation of pacemaker activity remains unknown. Total PDE activity was determined in mouse sinoatrial node (SAN) tissue as the cAMP-hydrolytic activity measured in the absence of PDE inhibitor and the fraction corresponding to PDE4 activity was assessed by including the PDE4 inhibitor Ro-201724. The in vitro pacemaker activity was assessed by measuring spontaneous Ca2+ transients in Fluo4-loaded-SAN tissue. Images were obtained using confocal microscopy. Ro-20-1724 increased beating rate of intact SAN and increased PKA-phosphorylation of key ECC actors (ryanodine receptor, phospholamban and contractile proteins). PDE4 activity was found to account for 60% of total cAMP-PDE activity in SAN ( n = 3). PDE4A, 4B and 4D isoforms were found to be expressed in mouse SAN ( n = 5 independent experiments). In 4D-, but not in 4BKO mice, Ca2+ homeostasis was altered in control conditions (ctrl) and after β-stimulation with isoprenaline (iso). Indeed, ablation of PDE4D induced decreased beating rate (ctrl: 1.00 ± 0.08s-1 vs. 1.57 ± 0.05s-1; iso: 1.71 ± 0.17s-1 vs. 2.39 ± 0.08s-1, P < 0.0001) and increased Ca2+ spark frequency (ctrl: 15.9 ± 5.2 vs. 1.9 ± 0.4sparks/s/100 μm; iso: 22.9 ± 7.1 vs. 0.6 ± 0.2sparks/s/100 μm, P < 0.0001) ( Fig. 1 ). PDE4 controls pacemaker function in mice and PDE4D ablation strongly perturbs normal SAN activity.

Linderane Suppresses Hepatic Gluconeogenesis by Inhibiting the cAMP/PKA/CREB Pathway Through Indirect Activation of PDE 3 via ERK/STAT3.

The role of phosphodiesterase 3 (PDE3), a cyclic AMP (cAMP)-degrading enzyme, in modulating gluconeogenesis remains unknown. Here, linderane, a natural compound, was found to inhibit gluconeogenesis by activating hepatic PDE3 in rat primary hepatocytes. The underlying molecular mechanism and its effects on whole-body glucose and lipid metabolism were investigated. The effect of linderane on gluconeogenesis, cAMP content, phosphorylation of cAMP-response element-binding protein (CREB) and PDE activity were examined in cultured primary hepatocytes and C57BL/6J mice. The precise mechanism by which linderane activates PDE3 and inhibits the cAMP pathway was explored using pharmacological inhibitors. The amelioration of metabolic disorders was observed in ob/ob mice. Linderane inhibited gluconeogenesis, reduced phosphoenolpyruvate carboxykinase (Pck1) and glucose-6-phosphatase (G6pc) gene expression, and decreased intracellular cAMP concentration and CREB phosphorylation in rat primary hepatocytes under both basal and forskolin-stimulated conditions. In rat primary hepatocytes, it also increased total PDE and PDE3 activity but not PDE4 activity. The suppressive effect of linderane on the cAMP pathway and gluconeogenesis was abolished by the non-specific PDE inhibitor 3-isobutyl-1-methylxanthine (IBMX) and the specific PDE3 inhibitor cilostazol. Linderane indirectly activated PDE3 through extracellular regulated protein kinase 1/2 (ERK1/2) and signal transducer and activator of transcription 3 (STAT3) activation. Linderane improved glucose and lipid metabolism after chronic oral administration in ob/ob mice. Our findings revealed linderane as an indirect PDE3 activator that suppresses gluconeogenesis through cAMP pathway inhibition and has beneficial effects on metabolic syndromes in ob/ob mice. This investigation highlighted the potential for PDE3 activation in the treatment of type 2 diabetes.

Type 4 phosphodiesterases control cardiac pacemaker activity

Numerous epidemiological and clinical studies have revealed a positive correlation between heart rate (HR) and cardiovascular morbimortality. The autonomic nervous system is the major extracardiac determinant of HR. During sympathetic stimulation, the activation of β-adrenergic receptors (βAR) induces an increase in cAMP levels, leading to a positive chronotropic effect. Among the 5 cAMP-PDE families expressed in the heart, PDE4 is critical for controlling excitation-contraction coupling (ECC) during βAR stimulation in atrial and ventricular cells. PDE4 may also be important for automaticity. 3 genes encode for cardiac PDE4s: pde4a, pde4b and pde4d. Their respective contribution to the regulation of pacemaker activity remains ill defined. The total enzymatic PDE activity was determined in mouse sinoatrial node (SAN) tissue as the cAMP hydrolytic activity measured in the absence of PDE inhibitor and the fraction corresponding to PDE4 activity was assessed by including the PDE4 inhibitor Ro-20-1724 (10 μM). The in vitro pacemaker activity was assessed by measuring the spontaneous Ca 2+ transients in Fluo4-loaded-SAN intact tissue. Images were obtained using confocal microscopy. Ro-20-1724 increased the beating rate of intact mouse SAN and increased PKA-phosphorylation levels of key ECC actors (ryanodine receptor, phospholamban). PDE4 enzymatic activity was found to account for 60% of the total cAMP-PDE activity in SAN. The 3 isoforms PDE4A, 4B and 4D were found to be expressed in mouse SAN. In PDE4D−, but not in PDE4B− deficient mice, Ca 2+ homeostasis was altered in control conditions and after βAR stimulation. Indeed, ablation of PDE4D induced a decreased beating rate and an increased Ca 2+ spark frequency in control and βAR-stimulated conditions. Our results reveal that PDE4 controls pacemaker function in mice and that PDE4D ablation strongly perturbs normal SAN activity.

Regulation of Cardiac Pacemaker Activity by PDE4 Isoforms

Background Numerous epidemiological and clinical studies have revealed a positive correlation between heart rate (HR) and cardiovascular morbimortality. The autonomic nervous system is the major extracardiac determinant of HR. During sympathetic stimulation, the activation of β-adrenergic receptors (βAR) induces an increase in cAMP levels, leading to a positive chronotropic effect. Among the 5 cAMP-PDE families expressed in the heart, PDE4 is critical for controlling excitation-contraction coupling (ECC) during βAR stimulation in atrial and ventricular cells. PDE4 may also be important for automaticity. 3 genes encode for cardiac PDE4s: pde4a, pde4b and pde4d. Their respective contribution to the regulation of pacemaker activity remains ill-defined. Methods The total enzymatic PDE activity was determined in mouse sinoatrial node (SAN) tissue as the cAMP hydrolytic activity measured in the absence of PDE inhibitor and the fraction corresponding to PDE4 activity was assessed by including the PDE4 inhibitor Ro-20-1724 (10 µM). The in vitro pacemaker activity was assessed by measuring the spontaneous Ca2+ transients in Fluo4-loaded-SAN intact tissue. Images were obtained using confocal microscopy. Results Ro-20-1724 increased the beating rate of intact mouse SAN and increased PKA-phosphorylation levels of key ECC actors (ryanodine receptor, phospholamban). PDE4 enzymatic activity was found to account for 60% of the total cAMP-PDE activity in SAN. The 3 isoforms PDE4A, 4B and 4D were found to be expressed in mouse SAN. In PDE4D-, but not in PDE4B-deficient mice, Ca2+ homeostasis was altered in control conditions and after βAR stimulation. Indeed, ablation of PDE4D induced a decreased beating rate and an increased Ca2+ spark frequency in control and βAR-stimulated conditions. Conclusion Our preliminary results reveal that PDE4 controls pacemaker function in mice and that PDE4D ablation strongly perturbs normal SAN activity.

0128 : Regulation of pacemaker activity by PDE4 isoforms

Numerous epidemiological and clinical studies have revealed a positive correlation between heart rate (HR) and cardiovascular morbimortality. The autonomic nervous system is the major extracardiac determinant of HR. During sympathetic stimulation, the activation of β- adrenergic receptors (βAR) induces an increase in cAMP levels, leading to a chronotopic effect. Among the 5 cAMP-PDE families expressed in the heart, PDE4 is critical for controlling excitation-contraction coupling (ECC) during βAR stimulation in atrial and ventricular cells. PDE4 may also be important for automaticity. 3 genes encode for cardiac PDE4s : pde4a, pde4b and pde4d . Their respective contribution to the regulation of pacemaker activity remains ill-defined. The total enzymatic PDE activity was determined in mouse sinoatrial node (SAN) tissue as the cAMP hydrolytic activity measured in the absence of PDE inhibitor and the fraction corresponding to PDE4 activity was assessed by including the PDE4 inhibitor Ro-20-1724. The protein expression of PDE4A, 4B and 4D was evaluated by Western blot using specific antibodies for each variant. The in vitro pacemaker activity was assessed by measuring the spontaneous Ca 2+ transients in Fluo4-loaded-SAN intact tissue. Images were obtained using confocal microscopy. Inhibition of PDE4 by Ro-20-1724 increased the beating rate of intact mouse SAN. PDE4 inhibition also altered Ca 2+ homeostasis, as shown by the presence of irregular Ca 2+ transients and increased PKA-phosphorylation of key ECC actors (ryanodine receptor, phospholamban) after treatment with Ro-20-1724. PDE4 enzymatic activity was found to account for 60% of the total cAMP-PDE activity in SAN. The 3 isoforms PDE4A, 4B and 4D were found to be expressed in mouse SAN. Our preliminary results reveal the involvement of PDE4 in the control of pacemaker activity in SAN. The characterization of pacemaker activity in PDE4B and PDE4D-null mice is under investigation. The author hereby declares no conflict of interest

Profiling of cAMP and cGMP phosphodiesterases in isolated ventricular cardiomyocytes from human hearts: Comparison with rat and guinea pig

AimsPhosphodiesterases (PDEs) are key enzymes controlling cAMP and cGMP levels and spatial distribution within cardiomyocytes. Despite the clinical importance of several classes of PDE inhibitor there has not been a complete characterization of the PDE profile within the human cardiomyocyte, and no attempt to assess which species might best be used to model this for drug evaluation in heart disease. Main methodsVentricular cardiomyocytes were isolated from failing human hearts of patients with various etiologies of disease, and from rat and guinea pig hearts. Expression of PDE isoforms was determined using RT-PCR. cAMP- and cGMP-PDE hydrolytic activity was determined by scintillation proximity assay, before and after treatment with PDE inhibitors for PDEs 1, 2, 3, 4, 5 and 7. Functional effects of cAMP PDEi were determined on the contraction of single human, rat and guinea pig cardiomyocytes. Key findingsThe presence and activity of PDE5 were confirmed in ventricular cardiomyocytes from failing and hypertrophied human heart, as well as PDE3, with ventricle-specific results for PDE4 and a surprisingly large contribution from PDE1 for hydrolysis of both cAMP and cGMP. The total PDE activity of human cardiomyocytes, and the profile of inhibition by PDE1, 3, 4, and 5 inhibitors, was modelled well in guinea pig but not rat cardiomyocytes. SignificanceOur results provide the first full characterisation of human cardiomyocyte PDE isoforms, and suggest that guinea pig myocytes provide a better model than rat for PDE levels and activity.

Conserved expression and functions of PDE4 in rodent and human heart

PDE4 isoenzymes are critical in the control of cAMP signaling in rodent cardiac myocytes. Ablation of PDE4 affects multiple key players in excitation–contraction coupling and predisposes mice to the development of heart failure. As little is known about PDE4 in human heart, we explored to what extent cardiac expression and functions of PDE4 are conserved between rodents and humans. We find considerable similarities including comparable amounts of PDE4 activity expressed, expression of the same PDE4 subtypes and splicing variants, anchoring of PDE4 to the same subcellular compartments and macromolecular signaling complexes, and downregulation of PDE4 activity and protein in heart failure. The major difference between the species is a fivefold higher amount of non-PDE4 activity in human hearts compared to rodents. As a consequence, the effect of PDE4 inactivation is different in rodents and humans. PDE4 inhibition leads to increased phosphorylation of virtually all PKA substrates in mouse cardiomyocytes, but increased phosphorylation of only a restricted number of proteins in human cardiomyocytes. Our findings suggest that PDE4s have a similar role in the local regulation of cAMP signaling in rodent and human heart. However, inhibition of PDE4 has ‘global’ effects on cAMP signaling only in rodent hearts, as PDE4 comprises a large fraction of the total cardiac PDE activity in rodents but not in humans. These differences may explain the distinct pharmacological effects of PDE4 inhibition in rodent and human hearts.Electronic supplementary materialThe online version of this article (doi:10.1007/s00395-010-0138-8) contains supplementary material, which is available to authorized users.

Protein kinase B/Akt phosphorylation of PDE3A and its role in mammalian oocyte maturation

cGMP-inhibited cAMP phosphodiesterase 3A (PDE3A) is expressed in mouse oocytes, and its function is indispensable for meiotic maturation as demonstrated by genetic ablation. Moreover, PDE3 activity is required for insulin/insulin-like growth factor-1 stimulation of Xenopus oocyte meiotic resumption. Here, we investigated the cAMP-dependent protein kinase B (PKB)/Akt regulation of PDE3A and its impact on oocyte maturation. Cell-free incubation of recombinant mouse PDE3A with PKB/Akt or cAMP-dependent protein kinase A catalytic subunits leads to phosphorylation of the PDE3A protein. Coexpression of PDE3A with constitutively activated PKB/Akt (Myr-Akt) increases PDE activity as well as its phosphorylation state. Injection of pde3a mRNA potentiates insulin-dependent maturation of Xenopus oocytes and rescues the phenotype of pde3(-/-) mouse oocytes. This effect is greatly decreased by mutation of any of the PDE3A serines 290-292 to alanine in both Xenopus and mouse. Microinjection of myr-Akt in mouse oocytes causes in vitro meiotic maturation and this effect requires PDE3A. Collectively, these data indicate that activation of PDE3A by PKB/Akt-mediated phosphorylation plays a role in the control of PDE3A activity in mammalian oocytes.

Allergen challenge alters lymphocyte phosphodiesterase activity in horses with heaves

Heaves is an allergic airway disease in horses characterised by reversible airway obstruction, bronchial hyperresponsiveness and airway inflammation associated with a Th 2 response. Cyclic nucleotide-dependent signalling pathways can regulate lymphocyte function. In this study, we examined lymphocyte PDE activity comparing horses with heaves to healthy control animals. Total PDE activity and the effects of isoenzyme selective inhibitors were measured before, 5 and 24 h after the start of a 7 h allergen challenge. Allergen challenge had no effect on either total cAMP PDE activity or its inhibition by the PDE4 selective inhibitor, rolipram, and the non-selective PDE inhibitor, theophylline. In contrast, the PDE3 selective inhibitor, quazinone, caused significantly greater inhibition of cAMP PDE activity before challenge in the heaves susceptible group. Additionally, total cGMP PDE activity was significantly lower 24 h after the start of challenge in the heaves affected group (11±2 and 21±3 pmol/min/mg for heaves and control animals, respectively) and the PDE5 selective inhibitor, zaprinast, caused significantly less inhibition in the heaves group at this time point. The functional significance of these findings was explored by examining the effect of PDE3, PDE4 and PDE5 selective inhibitors on mitogen-induced mononuclear cell proliferation before and 24 h after the start of allergen challenge. Proliferation decreased after challenge in the heaves group (stimulation index=328±110 and 200±72 before and after challenge, respectively) whilst remaining constant in the control group (stimulation index=161±13 and 183±45 before and after challenge, respectively). However, all three PDE inhibitors caused a similar amount of inhibition at each time point and the effect of a combination of a PDE3 and a PDE5 inhibitor was simply additive in both groups. These results suggest differences in the control of lymphocyte PDE activity in horses with heaves.

Oviduct cells express the cyclic AMP-adenosine pathway.

The extracellular cAMP-adenosine pathway refers to the local production of adenosine mediated by cAMP egress into the extracellular space, conversion of cAMP to AMP by ectophosphodiesterase (PDE), and the metabolism of AMP to adenosine by ecto-5'-nucleotidase. The goal of this study was to assess whether the cAMP-adenosine pathway is expressed in oviduct cells. Studies were conducted in cultured bovine oviduct cells (mixed cultures of fibroblasts and epithelial cells, 1:1 ratio). Confluent monolayers of oviduct cells were exposed to cAMP (0.01-100 micromol/L) in the presence and absence of 3-isobutyl-1-methylxanthine (IBMX, 1 mmol/L, an inhibitor of both extracellular and intracellular PDE activity), 1,3-dipropyl-8-p-sulfophenylxanthine (DPSPX, 100 micromol/L, a xanthine that can inhibit extracellular or ecto-PDE activity at high concentrations), or alpha,beta-methylene-adenosine-5'-diphosphate (AMPCP, 100 micromol/L, an ecto-5'-nucleotidase inhibitor) for 0-60 min. The medium was then sampled and assayed for AMP, adenosine, and inosine. Addition of exogenous cAMP to oviduct cells increased extracellular levels of AMP, adenosine, and inosine in a concentration- and time-dependent manner. This effect was attenuated by blockade of total (extracellular and intracellular) PDE activity (IBMX), ecto-PDE activity (DPSPX), or ecto-5'-nucleotidase (AMPCP). The functional relevance of the cAMP-adenosine pathway is supported by the findings that treatment with adenylyl cyclase stimulants (forskolin plus isoproterenol) resulted in the egress of cAMP (97% extracellular) into the extracellular space and its conversion into adenosine. The extracellular cAMP-adenosine pathway exists in oviduct cells and may play an important role in regulating the biology and physiology of the oviduct. This pathway also may play a critical role in regulating sperm function, fertilization, and early embryo development.

Identification and quantification of phosphodiesterase 4 subtypes in CD4 and CD8 lymphocytes from healthy and asthmatic subjects.

In the present study, for the first time, PDE4 subtypes were identified and semi-quantified in both CD4 and CD8 lymphocytes from healthy and asthmatic individuals. CD4 and CD8 lymphocytes from healthy and mild asymptomatic asthmatic subjects (receiving beta-agonist therapy only) were isolated from peripheral venous blood using appropriate antibody coated paramagnetic beads. PDE4 subtypes and beta-actin were identified by digoxigenin (DIG)-labelling reverse transcriptase-polymerase chain reaction and semi-quantified by DIG-detection enzyme-linked immunosorbance assay. In CD4 and CD8 lymphocytes PDE4A, PDE4B and PDE4D were detected, with no significant differences observed between healthy and asthmatic groups. In CD8 lymphocytes, enzyme subtype expression was lower and showed more intersubject variability. In functional studies investigating the effects of various PDE inhibitors on PHA-induced proliferation of mononuclear cells from healthy and asthmatic subjects, CDP840 (0.03 - 10 microM), rolipram (0.1 - 10 microM) and theophylline (10 microM - 1 mM) inhibited PHA-induced proliferation of mononuclear cells from healthy and asthmatic subjects in a concentration-dependent manner, although no significant difference was observed between the groups investigated. In additional studies, total monocyte cyclic AMP PDE activity was investigated in cells isolated from asthmatic subjects both prior to and 24 h after allergen challenge. Total monocyte cyclic AMP PDE activity remained unaffected following challenge of asthmatic subjects with either house dust mite or cat dander and was inhibited in a concentration-dependent manner by rolipram (0.01 - 100 microM) both before and after allergen challenge.

Nephron distribution of total low Km cyclic AMP phosphodiesterase in mouse, rat and rabbit kidney.

The activity of cAMP degradation enzyme, cAMP phosphodiesterase (cAMP PDE), in renal tubules is a critically important factor in determining cellular cAMP levels, particularly in response to hormones. In this study we examine the nephron distribution of cAMP PDE activity in the mouse, rat and rabbit kidney and important cellular regulators of cAMP PDE, namely calmodulin and adenosine triphosphate (ATP). We assayed total low Km cAMP PDE in microdissected tubule segments, using 10(-6) M (3H) cAMP as a substrate. Activities were expressed in fentomol cAMP hydrolyzed per minute per mm tubular length or per one glomerulus. The content of ATP was measured in outer medullary collecting duct and medullary thick ascending limb of Henle's loop with microbioluminescence assay using firefly luciferase. In mouse kidney, cAMP PDE was significantly higher in all tubular segments compared to glomerulus. Proximal convoluted tubule, proximal straight tubule, medullary thick ascending limb of Henle's loop (mTAL), and outer medullary collecting duct (OMCD) had intermediated activity. Greater cAMP PDE activity was detected in cortical ascending limb of Henle's loop (cTAL), cortical collecting duct and in distal convoluted tubule (DCT). The highest activity was found in connecting tubules. In rat, nephron distribution of cAMP PDE activities was similar to mouse, except that activity in glomeruli was higher than in mouse glomeruli. In rabbit, nephron distribution of cAMP PDE activities was different from those of mouse and rat. There was no single prominent segment with high cAMP PDE activity. DCT and cTAL showed low enzyme activity. Overall, the highest cAMP PDE activities were measured in the mouse and the lowest were measured in the rabbit nephrons, with those of rat nephron showing an intermediate activity. The maximum effective dose of the calmodulin antagonist, trifluoperazine (200 microM), inhibited cAMP PDE in all nephron segments from the rat kidney. However, there is no statistical significance of its inhibition among nephron segments. In OMCD and mTAL of the rat kidney, cAMP PDE activity was inhibited by ATP (5 mM to approximately 10 mM) which is far beyond the physiological concentartion of ATP in normal epithelial cell. Actual determinations of ATP in mTAL and OMCD were 0.1 mM and 0.17 mM, respectively. These observations show that distal segments of tubules have more active catabolism of cAMP than proximal segments. cAMP PDE in each nephron segment appear to be almost equally dependent on trifluoperazine-sensitive pathway that may reflect the Ca2+-calmodulin system. Cellular concentration of ATP might not be involved in the regulation of the total low Km cAMP PDE activity in rat mTAL and OMCD.

The Human Cyclic AMP-specific Phosphodiesterase PDE-46 (HSPDE4A4B) Expressed in Transfected COS7 Cells Occurs as Both Particulate and Cytosolic Species That Exhibit Distinct Kinetics of Inhibition by the Antidepressant Rolipram

Transfection of COS7 cells with a plasmid encoding the human cyclic AMP-specific PDE4A phosphodiesterase PDE-46 (HSPDE4A4B) led to the expression of a rolipram-inhibited PDE4 activity, which contributed approximately 96% of the total COS cell PDE activity. A fusion protein was generated which encompassed residues (788-886) at the extreme C terminus of PDE-46 and was used to generate an antiserum that detected PDE-46 in transfected COS7 cells. Immunoblotting studies identified PDE-46 as a approximately 125-kDa species that was associated with both the soluble and particulate fractions. The relative Vmax of particulate PDE-46 was approximately 56% that of cytosolic PDE-46. Particulate PDE-46 was not solubilized using Triton X-100 or high NaCl concentrations. Immunofluorescence analysis by laser scanning confocal microscopy showed that PDE-46 was located at discrete margins of the cell, indicative of association with membrane cortical regions. The human PDE4A species, h6.1 (HSPDE4A4C), which lacks the N-terminal extension of PDE-46, was found as an entirely soluble species when expressed in COS7 cells. h6.1 was shown to have an approximately 11-fold higher Vmax relative to that of PDE-46. In dose-response studies rolipram inhibited particulate PDE-46 at much lower concentrations (IC50 = 0. 195 microM) than those needed to inhibit the cytosolic enzyme (IC50 = 1.6 microM). The basis of this difference lay in the fact that rolipram served as a simple competitive inhibitor of the cytosol enzyme (Ki = 1.6 microM) but as a partial competitive inhibitor of the particulate enzyme (Ki = 0.037 microM; Ki' = 2.3 microM). Particulate PDE-46 thus showed a approximately 60-fold higher affinity for rolipram than cytosolic PDE-46.

Selective inhibition of cGMP-inhibited and cGMP-noninhibited cyclic nucleotide phosphodiesterases and relaxation of rat aorta

In the supernatant (50,000 g, 1 hr) fraction from rat aortic smooth muscle homogenates, approximately 50% of total cAMPE PDE activity was inhibited by OPC 3911 (3 μM), while approximately 20% was inhibited by rolipram (30 μM). A cGMP-inhibited cyclic nucleotide phosphodiesterase (cGI-PDE) was further purified using DEAE chromatography followed by affinity chromatography on the N-(2-isothiocyanato)ethyl derivative of cilostamide conjugated to aminoethyl agarose (CIT-agarose). OPC 3911, CI-930, and milrinone, but not rolipram, were potent and selective inhibitors of this enzyme. The PDE-activity in the CIT-agarose flow through fraction (RI-PDE), however, was inhibited potently by rolipram, but not by cGMP, OPC 3911, CI-930 or milrinone. Functional studies showed that OPC 3911, CI-930, and milrinone were potent relaxants of contracted rat aorta. Rolipram had little relaxant effect. When OPC 3911 or milrinone was combined with rolipram more than additive effects on aortic relaxation and cAMP content were obtained. OPC 3911 combined with milrinone had only additive effects. These results demonstrate the presence of a cGI-PDE in rat aortic smooth muscle, and that inhibition of this isozyme may be of primary importance for the relaxant effects of OPC 3911, CI-930, and milrinone. A RI-PDE activity was also found, but it appeared to be less important for modulation of vascular tone unless the cGI-PDE was already inhibited. This may explain the synergistic relaxant effects observed when both PDE-isozymes were inhibited.