cAMP Phosphodiesterase Activity Research Articles

The cAMP signaling module regulates sperm motility in the liverwort Marchantia polymorpha

Adenosine 3',5'-cyclic monophosphate (cAMP) is a universal signaling molecule that acts as a second messenger in various organisms. It is well established that cAMP plays essential roles across the tree of life, although the function of cAMP in land plants has long been debated. We previously identified the enzyme with both adenylyl cyclase (AC) and cAMP phosphodiesterase (PDE) activity as the cAMP-synthesis/hydrolysis enzyme COMBINED AC with PDE (CAPE) in the liverwort Marchantia polymorpha. CAPE is conserved in streptophytes that reproduce with motile sperm; however, the precise function of CAPE is not yet known. In this study, we demonstrate that the loss of function of CAPE in M. polymorpha led to male infertility due to impaired sperm flagellar motility. We also found that two genes encoding the regulatory subunits of cAMP-dependent protein kinase (PKA-R) were also involved in sperm motility. Based on these findings, it is evident that CAPE and PKA-Rs act as a cAMP signaling module that regulates sperm motility in M. polymorpha. Therefore, our results have shed light on the function of cAMP signaling and sperm motility regulators in land plants. This study suggests that cAMP signaling plays a common role in plant and animal sperm motility.

A bioluminescent and homogeneous assay for monitoring GPCR-mediated cAMP modulation and PDE activity.

3',5'-Cyclic adenosine monophosphate (cAMP), the first identified second messenger, is implicated in diverse cellular processes involving cellular metabolism, cell proliferation and differentiation, apoptosis, and gene expression. cAMP is synthesized by adenylyl cyclase (AC), which converts ATP to cAMP upon activation of Gαs-protein coupled receptors (GPCRs) in most cases and hydrolyzed by cyclic nucleotide phosphodiesterases (PDEs) to 5'-AMP. Dysregulation of cAMP signaling is implicated in a wide range of pathophysiological conditions such as cardiovascular diseases, neurodegenerative and behavioral disorders, cancers, diabetes, obesity, cataracts, and others. Therefore, cAMP targeted therapies have been and are still undergoing intense investigation for the treatment of these and other diseases. This highlights the need for developing assays to detect and monitor cAMP levels. In this study, we show cAMP Lumit assay as a highly specific homogeneous bioluminescent assay suitable for high throughput screenings with a large assay window and a wide dynamic range for cAMP detection. We believe that this assay will aid and simplify drug discovery screening efforts for cAMP signaling targeted therapies.

Human Prune Regulates the Metabolism of Mammalian Inorganic Polyphosphate and Bioenergetics.

Inorganic polyphosphate (polyP) is an evolutionarily conserved and ubiquitous polymer that is present in all studied organisms. PolyP consists of orthophosphates (Pi) linked together by phosphoanhydride bonds. The metabolism of polyP still remains poorly understood in higher eukaryotes. Currently, only F0F1-ATP synthase, Nudt3, and Prune have been proposed to be involved in this metabolism, although their exact roles and regulation in the context of polyP biology have not been fully elucidated. In the case of Prune, in vitro studies have shown that it exhibits exopolyphosphatase activity on very short-chain polyP (up to four units of Pi), in addition to its known cAMP phosphodiesterase (PDE) activity. Here, we expand upon studies regarding the effects of human Prune (h-Prune) on polyP metabolism. Our data show that recombinant h-Prune is unable to hydrolyze short (13-33 Pi) and medium (45-160 Pi) chains of polyP, which are the most common chain lengths of the polymer in mammalian cells. Moreover, we found that the knockdown of h-Prune (h-Prune KD) results in significantly decreased levels of polyP in HEK293 cells. Likewise, a reduction in the levels of polyP is also observed in Drosophila melanogaster loss-of-function mutants of the h-Prune ortholog. Furthermore, while the activity of ATP synthase, and the levels of ATP, are decreased in h-Prune KD HEK293 cells, the expression of ATP5A, which is a main component of the catalytic subunit of ATP synthase, is upregulated in the same cells, likely as a compensatory mechanism. Our results also show that the effects of h-Prune on mitochondrial bioenergetics are not a result of a loss of mitochondrial membrane potential or of significant changes in mitochondrial biomass. Overall, our work corroborates the role of polyP in mitochondrial bioenergetics. It also demonstrates a conserved effect of h-Prune on the metabolism of short- and medium-chain polyP (which are the predominant chain lengths found in mammalian cells). The effects of Prune in polyP are most likely exerted via the regulation of the activity of ATP synthase. Our findings pave the way for modifying the levels of polyP in mammalian cells, which could have pharmacological implications in many diseases where dysregulated bioenergetics has been demonstrated.

Prune regulates the metabolism of mammalian inorganic polyphosphates and bioenergetics.

Inorganic polyphosphate (polyP) is an evolutionarily conserved, ubiquitous polymer that is present in all studied organisms. It has shown a preferred localization within the mitochondria and it is composed of multiple orthophosphates (Pi) linked together by high energy phosphoanhydride bonds, identical to those found in ATP. The metabolism of polyP is well described in bacteria and yeast, but has yet to be elucidated in higher eukaryotes. However, recent evidence suggests that the mammalian F0F1 ATP synthase could be involved in both the synthesis and hydrolysis of polyP. The mitochondrial localized protein h-Prune, the human ortholog of Drosophila Prune, exhibits short chain exopolyphosphatase activity in vitro, in addition to its known cAMP phosphodiesterase activity. Here, we studied the role of prune orthologs in polyP metabolism in both mammalian HEK293 cells and Drosophila melanogaster. Our data shows that purified h-Prune is unable to hydrolyze polyP with chain lengths spanning from 13-33 Pi long. We then used interfering RNA to knock down the expression of prune orthologs and found that the polyP levels were significantly reduced in both HEK293 and Drosophila melanogaster. In HEK293 cells, ATP levels were also significantly decreased by h-Prune knockdown, although mitochondrial membrane potential was not affected. This suggests that h-Prune could either directly or indirectly influence the activity of the F0F1 ATP synthase. Concordantly, we found that h-Prune knockdown significantly decreased ATP synthase activity to levels comparable to inhibition by Oligomycin A. Our results collectively demonstrate the downstream ability of prune to regulate cellular polyP levels and bioenergetics, although further studies are required to elucidate the exact mechanism of this pathway.

B-adrenergic/cAMP signaling in doxorubicin-induced cardiotoxicity

Doxorubicin (Dox) is one of the most commonly used chemotherapy but also one of the most cardiotoxic. As of today, only the iron chelator Dexrazoxane has been FDA and EMA approved as a cardioprotective agent for Dox but its usage is still controversial. Thus, there is an urgent need to identify new targets to develop an effective protection against Dox-induced cardiotoxicity (DIC) without compromising its anti-tumor action. Well-characterized DIC mechanisms include topoisomerase II inhibition, DNA damages, induction of ROS and bioenergetic distress that lead to cardiomyocyte death. Alternative mechanisms are emerging such as an alteration of the β-adrenergic system. Our objective here was to study DIC mechanisms with a specific focus on how Dox treatment affects the β-adrenergic/cAMP signaling cascade. We used FRET-based sensors for cAMP targeted to two different cellular compartments, cytosol and nucleus, and measured the cAMP response to the β-adrenergic agonist isoprenaline (Iso, 10 nM) in adult rat ventricular myocytes after 24 h treatment with 1 or 10 μM Dox. Basal cAMP concentration was measured by Elisa. Adenylyl cyclase (AC5 and AC6 isoforms) mRNA was measured by RT-qPCR and phosphodiesterase (PDE) isoform expression and activity by western blot and radioenzymatic assay, respectively. cAMP response to Iso was decreased by 25% at 1 μM and > 50% at 10 μM Dox in both cytosol and nucleus. The maximal response to 20 μM L858051 (a forskolin analog) + 200 μM IBMX (a PDE inhibitor) on top of Iso was also decreased by Dox. Dox treatment did not change basal cAMP level nor PDE activity, but decreased AC5 and AC6 mRNA and PDE4 protein expression. Doxorubicin treatment downregulates the β-adrenergic/cAMP cascade in ventricular myocytes by decreasing adenylyl cyclase expression and maximal activity. This may contribute to the Dox-induced cardiotoxicity mechanisms.

Reprioritization of biofilm metabolism is associated with nutrient adaptation and long-term survival of Haemophilus influenzae

Nontypeable Haemophilus influenzae (NTHI) is a human-restricted pathogen with an essential requirement for heme–iron acquisition. We previously demonstrated that microevolution of NTHI promotes stationary phase survival in response to transient heme–iron restriction. In this study, we examine the metabolic contributions to biofilm formation using this evolved NTHI strain, RM33. Quantitative analyses identified 29 proteins, 55 transcripts, and 31 metabolites that significantly changed within in vitro biofilms formed by RM33. The synthesis of all enzymes within the tryptophan and glycogen pathways was significantly increased in biofilms formed by RM33 compared with the parental strain. In addition, increases were observed in metabolite transport, adhesin production, and DNA metabolism. Furthermore, we observed pyruvate as a pivotal point in the metabolic pathways associated with changes in cAMP phosphodiesterase activity during biofilm formation. Taken together, changes in central metabolism combined with increased stores of nutrients may serve to counterbalance nutrient sequestration.

Luteolin suppresses inflammation through inhibiting cAMP-phosphodiesterases activity and expression of adhesion molecules in microvascular endothelial cells.

Luteolin, an anti-inflammatory ingredient found in the Chinese herb Folium perillae, can inhibit not only the cyclic adenosine monophosphate (cAMP)-phosphodiesterases (PDEs) activity of neutrophils, but also the expression of lymphocyte function-associated antigen-1 in neutrophils, both of which result in a decrease in the adhesion between neutrophils and microvascular endothelial cells. However, the effect of luteolin on the cAMP-PDEs activity and expression of adhesion molecules in endothelial cells are not clear. In the present study, primary rat pulmonary microvascular endothelial cells and a lipopolysaccharide-induced rat acute pneumonia model were used to explore the role of luteolin on cAMP-PDEs activity, expression of adhesion molecules, and leukocyte infiltration. We demonstrate that rat pulmonary microvascular endothelial cells expressed high levels of cAMP-PDEs, specifically PDE4, and further luteolin exhibited dose-dependent inhibition on the activity of cAMP-PDEs or PDE4 in endothelial cells. Luteolin also had a significant inhibitory effect on the expression of vascular cell adhesion molecule (VCAM)-1, but not intracellular cell adhesion molecule (ICAM)-1 in microvascular endothelial cells. Further, we show that luteolin decreased the levels of soluble ICAM-1 (sICAM-1), but not soluble E-selectin in the serum of rats subjected to acute pneumonia. We also show that luteolin treatment decreased the wet/dry weight ratio of lung tissue and reduced the total number of serum leukocytes in a dose-dependent manner in a rat acute pneumonia model. In conclusion, these results demonstrate that luteolin suppresses inflammation, at least in part, through inhibiting both cAMP-PDEs or PDE4 activity and the expression of VCAM-1 (in vitro) and sICAM-1 (in vivo) in endothelial cells.

An improved “ion pairing agent free” HPLC-RP method for testing cAMP Phosphodiesterase activity

Current HPLC methods for analyzing cAMP Phosphodiesterase activity (PDE) use salts, limiting the life of the columns. For this reason, we have developed an improved “ion pairing agent free” method, using a simple 150 mm C18-hydro column at 30 °C and two phases: (a) water with 0.1% acetic acid and (b) 85/15 w/w MeOH/tetrahydrofuran with 0.1% acetic acid. Using this method the peaks for cAMP and AMP were obtained with good resolution (R ≈ 1.35) and sensitivity (5·10−9 mols) in only 15 min. Moreover, the method was applied to the GMP/cGMP pair obtaining the same sensitivity and resolution (R ≈ 1.38). The precision and accuracy were tested and the method was verified with a Type IV Phosphodiesterase reaction, which produced AMP from cAMP. The method is cleaner and less aggressive, and represents an interesting alternative to currently used methods.

Multiple cAMP Phosphodiesterases Act Together to Prevent Premature Oocyte Meiosis and Ovulation.

Luteinizing hormone (LH) acts on the granulosa cells that surround the oocyte in mammalian preovulatory follicles to cause meiotic resumption and ovulation. Both of these responses are mediated primarily by an increase in cyclic adenosine monophosphate (cAMP) in the granulosa cells, and the activity of cAMP phosphodiesterases (PDEs), including PDE4, contributes to preventing premature responses. However, two other cAMP-specific PDEs, PDE7 and PDE8, are also expressed at high levels in the granulosa cells, raising the question of whether these PDEs also contribute to preventing uncontrolled activation of meiotic resumption and ovulation. With the use of selective inhibitors, we show that inhibition of PDE7 or PDE8 alone has no effect on the cAMP content of follicles, and inhibition of PDE4 alone has only a small and variable effect. In contrast, a mixture of the three inhibitors elevates cAMP to a level comparable with that seen with LH. Correspondingly, inhibition of PDE7 or PDE8 alone has no effect on meiotic resumption or ovulation, and inhibition of PDE4 alone has only a partial and slow effect. However, the fraction of oocytes resuming meiosis and undergoing ovulation is increased when PDE4, PDE7, and PDE8 are simultaneously inhibited. PDE4, PDE7, and PDE8 also function together to suppress the premature synthesis of progesterone and progesterone receptors, which are required for ovulation. Our results indicate that three cAMP PDEs act in concert to suppress premature responses in preovulatory follicles.

Silymarin Activates c-AMP Phosphodiesterase and Stimulates Insulin Secretion in a Glucose-Dependent Manner in HIT-T15 Cells.

Silymarin (SIL) is a flavonoid extracted from milk thistle seed that has been reported to decrease hyperglycemia in people with type 2 diabetes (T2D). However, it is not known whether SIL has direct secretory effects on β-cells. Using the β-cell line HIT-T15, SIL was shown to decrease intracellular peroxide levels and to augment glucose-stimulated insulin secretion (GSIS). However, the latter was observed using a concentration range of 25–100 µM, which was too low to affect endogenous peroxide levels. The stimulatory effect of SIL dissipated at higher concentrations (100–200 µM), and mild apoptosis was observed. The smaller concentrations of SIL also decreased cAMP phosphodiesterase activity in a Ca2+/calmodulin-dependent manner. The stimulatory effects of SIL on GSIS were inhibited by three different inhibitors of exocytosis, indicating that SIL’s mechanism of stimulating GSIS operated via closing β-cell K-ATP channels, and perhaps more distal sites of action involving calcium influx and G-proteins. We concluded that augmentation of GSIS by SIL can be observed at concentrations that also inhibit cAMP phosphodiesterase without concomitant lowering of intracellular peroxides.

Characterization of cAMP-phosphodiesterase activity in bovine seminal plasma.

The second messenger cyclic adenosine monophosphate (cAMP) has a central role in sperm physiology. Extracellular cAMP can be sequentially degraded into 5'AMP and adenosine by ecto-phosphodiesterases (ecto-PDE) and ecto-nucleotidases, a phenomenon called extracellular cAMP-adenosine pathway. As cAMP-adenosine pathway is involved in sperm capacitation, we hypothesize that extracellular PDEs are functionally present in seminal plasma. Exclusively measuring cAMP-PDE activity, total activity in bovine seminal plasma was 10.1±1.5 fmoles/min/μg. Using different family-specific PDE inhibitors, we showed that in seminal plasma, the major cAMP-PDE activity was papaverine sensitive (47.5%). These data support the presence of PDE10 in bovine seminal plasma and was further confirmed by western blot. In epididymal fluid, total cAMP-PDE activity was 48.2±14.8 fmoles/min/μg and we showed that the major cAMP-PDE activity was 3-isobutyl-methylxanthine insensitive and thus ascribed to PDE8 family. PDE10A mRNAs were found in the testis, epididymis, and seminal vesicles. cAMP-PDE activity is present in bovine seminal plasma and epididymal fluid. The results suggest a role for ecto-PDEs present in those fluids in the signaling pathways involved in sperm functions.

Dietary flavonoid luteolin attenuates uropathogenic Escherichia. Coli invasion of the urinary bladder.

Uropathogenic Escherichia coli (UPEC), the primary uropathogen, adhere to and invade bladder epithelial cells (BECs) to establish a successful urinary tract infection (UTI). Emerging antibiotic resistance requires novel nonantibiotic strategies. Our previous study indicated that luteolin attenuated adhesive and invasive abilities as well as cytotoxicity of UPEC on T24 BECs through down-regulating UPEC virulence factors. The aims of this study were to investigate the possible function of the flavonoid luteolin and the mechanisms by which luteolin functions in UPEC-induced bladder infection. Firstly, obvious reduction of UPEC invasion but not adhesion were observed in luteolin-pretreated 5637 and T24 BECs sa well as mice bladder via colony counting. The luteolin-mediated suppression of UPEC invasion was linked to elevated levels of intracellular cAMP induced by inhibiting the activity of cAMP-phosphodiesterases (cAMP-PDEs), which resulting activation of protein kinase A, thereby negatively regulating Rac1-GTPase-mediated actin polymerization. Furthermore, p38 MAPK was primarily and ERK1/2 was partially involved in luteolin-mediated suppression of UPEC invasion and actin polymerization, as confirmed with chemical activators of p38 MAPK and ERK1/2. These data suggest that luteolin can protect bladder epithelial cells against UPEC invasion. Therefore, luteolin or luteolin-rich products as dietary supplement may be beneficial to control the UPEC-related bladder infections, and cAMP-PDEs may be a therapy target for UTIs treatment. © 2016 BioFactors, 42(6):674-685, 2016.

Modulation of Polycystic Kidney Disease Severity by Phosphodiesterase 1 and 3 Subfamilies.

Aberrant intracellular calcium levels and increased cAMP signaling contribute to the development of polycystic kidney disease (PKD). cAMP can be hydrolyzed by various phosphodiesterases (PDEs). To examine the role of cAMP hydrolysis and the most relevant PDEs in the pathogenesis of PKD, we examined cyst development in Pde1- or Pde3-knockout mice on the Pkd2(-/WS25) background (WS25 is an unstable Pkd2 allele). These PDEs were selected because of their importance in cross-talk between calcium and cyclic nucleotide signaling (PDE1), control of cell proliferation and cystic fibrosis transmembrane conductance regulator (CFTR) -driven fluid secretion (PDE3), and response to vasopressin V2 receptor activation (both). In Pkd2(-/WS25) mice, knockout of Pde1a, Pde1c, or Pde3a but not of Pde1b or Pde3b aggravated the development of PKD and was associated with higher levels of protein kinase A-phosphorylated (Ser133) cAMP-responsive binding protein (P-CREB), activating transcription factor-1, and CREB-induced CRE modulator proteins in kidney nuclear preparations. Immunostaining also revealed higher expression of P-CREB in Pkd2(-/) (WS25);Pde1a(-/-), Pkd2(-) (/WS25);Pde1c(-/-), and Pkd2(-/) (WS25);Pde3a(-/-) kidneys. The cystogenic effect of desmopressin administration was markedly enhanced in Pkd2(-/WS25);Pde3a(-/-) mice, despite PDE3 accounting for only a small fraction of renal cAMP PDE activity. These observations show that calcium- and calmodulin-dependent PDEs (PDE1A and PDE1C) and PDE3A modulate the development of PKD, possibly through the regulation of compartmentalized cAMP pools that control cell proliferation and CFTR-driven fluid secretion. Treatments capable of increasing the expression or activity of these PDEs may, therefore, retard the development of PKD.

The Cyclic AMP-Vfr Signaling Pathway in Pseudomonas aeruginosa Is Inhibited by Cyclic Di-GMP.

The opportunistic human pathogen Pseudomonas aeruginosa expresses numerous acute virulence factors in the initial phase of infection, and during long-term colonization it undergoes adaptations that optimize survival in the human host. Adaptive changes that often occur during chronic infection give rise to rugose small colony variants (RSCVs), which are hyper-biofilm-forming mutants that commonly possess mutations that increase production of the biofilm-promoting secondary messenger cyclic di-GMP (c-di-GMP). We show that RSCVs display a decreased production of acute virulence factors as a direct result of elevated c-di-GMP content. Overproduction of c-di-GMP causes a decrease in the transcription of virulence factor genes that are regulated by the global virulence regulator Vfr. The low level of Vfr-dependent transcription is caused by a low level of its coactivator, cyclic AMP (cAMP), which is decreased in response to a high level of c-di-GMP. Mutations that cause reversion of the RSCV phenotype concomitantly reactivate Vfr-cAMP signaling. Attempts to uncover the mechanism underlying the observed c-di-GMP-mediated lowering of cAMP content provided evidence that it is not caused by inhibition of adenylate cyclase production or activity and that it is not caused by activation of cAMP phosphodiesterase activity. In addition to the studies of the RSCVs, we present evidence that the deeper layers of wild-type P. aeruginosa biofilms have high c-di-GMP levels and low cAMP levels. Our work suggests that cross talk between c-di-GMP and cAMP signaling pathways results in downregulation of acute virulence factors in P. aeruginosa biofilm infections. Knowledge about this cross-regulation adds to our understanding of virulence traits and immune evasion by P. aeruginosa in chronic infections and may provide new approaches to eradicate biofilm infections.

0116 : Phosphodiesterases regulate cAMP level in the mitochondria of adult cardiomyocytes

The heart is highly energy-dependent with most of its energy provided by mitochondria. Mitochondria also play a role in many essential cellular processes including metabolite synthesis, redox balance, calcium homeostasis and cell death. Therefore, maintaining a functional population of mitochondria is critical for cardiac function and identification of novel regulatory mechanisms. Recently, a soluble adenylyl cyclase (sAC) has been revealed to serve as a local source of the second messenger cAMP in the mitochondrial matrix in response to bicarbonate and calcium. PDE2A, a cGMP-stimulated cAMP-hydrolyzing phosphodiesterase (PDE), was also shown to be expressed in the mitochondrial matrix of liver and brain. To gain new insights into the control of mitochondrial pool of cAMP, we investigated the role of various isoforms of PDEs in isolated cardiac mitochondria. Western blot analysis of PDE protein expression revealed positive bands for PDE2A, 3A, 4A and 4B subtypes in cardiac mitochondria. Basal cAMP-degrading enzymatic activities were determined by radioenzymatic assay in cardiac mitochondrial lysates with 1μm cAMP as substrate. PDE2 represented the largest mitochondrial cAMP PDE activity (35% of total) and its activity was enhanced ~2-fold by 5μM cGMP and was inhibited by the PDE2 inhibitor, Bay 60-7550 (100nm). PDE3 and PDE4 activities represented, respectively, 30% and 25% of total, and they were inhibited by their respective inhibitors, cilostamide (1μm) and Ro 20- 1724 (10 μM). Accordingly, measurements by ELISA of mitochondrial cAMP levels confirmed that inhibition of PDE2, 3 and 4 families leads to an increase in cAMP level. In conclusion, at least three PDE families and four PDE subtypes are located in the cardiac mitochondrial matrix, participating to a local signaling pathway with sAC to control cAMP level. Our findings unravel a cAMP signaling cascade in cardiac mitochondria which may have implications for the metabolic control of cardiac function.

Levodopa-induced dyskinesias are associated with transient down-regulation of cAMP and cGMP in the caudate-putamen of hemiparkinsonian rats: Reduced synthesis or increased catabolism?

Second messenger cAMP and cGMP represent a key step in the action of dopamine that modulates directly or indirectly their synthesis. We aimed to verify whether levodopa-induced dyskinesias are associated with changes of the time course of levodopa/dopamine stimulated cAMP and cGMP levels, and/or with changes of their catabolism by phosphodiesterase activity in rats with experimental hemiparkinsonism. Microdialysis and tissue homogenates of the striatal tissues demonstrated that extracellular and intracellular cAMP/cGMP levels were lower in dyskinetic animals during the increasing phase of dyskinesias compared to eukinetic animals, but cAMP/cGMP levels increased in dyskinetic animals during the phase of decreasing and extinction of dyskinesias. Dyskinesias and the abnormal lowering of striatal cGMP and cAMP after levodopa were prevented by pretreatment with the multipotent drug amantadine, outlining the inverse relationship of cAMP/cGMP to dyskinesias. Moreover, dyskinetic animals showed higher striatal hydrolyzing cGMP-phosphodiesterase but not hydrolyzing cAMP-phosphodiesterase activity, suggesting that low cGMP but not cAMP levels could be due to increased catabolism. However, expressions of isozyme phosphodiesterase-1B and -10A highly and specifically located in the basal ganglia were not changed after levodopa in dyskinetic and eukinetic animals: accordingly, selective inhibitors of phosphodiesterase-1B and -10A were ineffective on levodopa dyskinesias. Therefore, the isozyme(s) expressing higher cGMP-phosphodiesterase activity in the striatum of dyskinetic animal should be determined. These observations suggest that dopamine-mediated processes of synthesis and/or degradation of cAMP/cGMP could be acutely impaired in levodopa dyskinesias, opening new ways to understanding physiopathology and treatment.

Phosphodiesterase DosP increases persistence by reducing cAMP which reduces the signal indole.

Persisters are bacteria that are highly tolerant to antibiotics due to their dormant state and are of clinical significance owing to their role in infections. Given that the population of persisters increases in biofilms and that cyclic diguanylate (c-di-GMP) is an intracellular signal that increases biofilm formation, we sought to determine whether c-di-GMP has a role in bacterial persistence. By examining the effect of 30 genes from Escherichia coli, including diguanylate cyclases that synthesize c-di-GMP and phosphodiesterases that breakdown c-di-GMP, we determined that DosP (direct oxygen sensing phosphodiesterase) increases persistence by over a thousand fold. Using both transcriptomic and proteomic approaches, we determined that DosP increases persistence by decreasing tryptophanase activity and thus indole. Corroborating this effect, addition of indole reduced persistence. Despite the role of DosP as a c-di-GMP phosphodiesterase, the decrease in tryptophanase activity was found to be a result of cyclic adenosine monophosphate (cAMP) phosphodiesterase activity. Corroborating this result, the reduction of cAMP via CpdA, a cAMP-specific phosphodiesterase, increased persistence and reduced indole levels similarly to DosP. Therefore, phosphodiesterase DosP increases persistence by reducing the interkingdom signal indole via reduction of the global regulator cAMP.

Modulation of 3',5'-cyclic AMP homeostasis in human platelets by coffee and individual coffee constituents.

3',5'-Cyclic AMP (cAMP) is one of the most important second messengers in mammalian cells, mediating a multitude of diverse cellular signalling responses. Its homeostasis is primarily regulated by adenylate cyclases and phosphodiesterases (PDE), the activities of which are partially dependent on the downstream events of adenosine receptor signalling. The present study was conducted to determine whether coffee constituents other than caffeine can influence the homeostasis of intracellular cAMP in vitro and in vivo by evaluating the effects of selected constituents present in coffee, coffee brews and coffee extracts on platelet PDE activity. In addition, to evaluate the potential effects of these constituents on platelet cAMP concentrations and PDE activity in humans, a 7-week pilot intervention study with eight subjects was conducted. The subjects consumed a regular commercial coffee and a low-caffeine coffee at a rate of 750ml/d for 2 weeks each. The in vivo results revealed a highly significant inhibition of PDE activity (P<0·001) after coffee intervention that was not directly dependent on the caffeine content of coffee. Although our in vitro and in vivo findings suggest that caffeine plays some role in the modulation of platelet cAMP status, other natural and roasting-associated compounds such as pyrazines and other currently unidentified species also appear to contribute significantly. In conclusion, moderate consumption of coffee can modulate platelet PDE activity and cAMP concentrations in humans, which may contribute to the putative beneficial health effects of coffee. Further detailed mechanistic investigations will be required to substantiate these beneficial effects and to elucidate the underlying mechanisms.

Phosphoinositide 3-Kinase γ Affects LPS-Induced Disturbance of Blood–Brain Barrier Via Lipid Kinase-Independent Control of cAMP in Microglial Cells

The breakdown of the blood-brain barrier (BBB) is a key event in the development of sepsis-induced brain damage. BBB opening allows blood-born immune cells to enter the CNS to provoke a neuroinflammatory response. Abnormal expression and activation of matrix metalloproteinases (MMP) was shown to contribute to BBB opening. Using different mouse genotypes in a model of LPS-induced systemic inflammation, our present report reveals phosphoinositide 3-kinase γ (PI3Kγ) as a mediator of BBB deterioration and concomitant generation of MMP by microglia. Unexpectedly, microglia expressing lipid kinase-deficient mutant PI3Kγ exhibited similar MMP regulation as wild-type cells. Our data suggest kinase-independent control of cAMP phosphodiesterase activity by PI3Kγ as a crucial mediator of microglial cell activation, MMP expression and subsequent BBB deterioration. The results identify the suppressive effect of PI3Kγ on cAMP as a critical mediator of immune cell functions.

Vasorelaxant effect of propentofylline in isolated equine digital veins

We evaluated the vasorelaxant effect of propentofylline (PPF), a methylxanthine derivative, and its mechanism of action in equine digital veins (EDVs). Cumulative concentration-response curves to PPF (1nM–300µM) were recorded in phenylephrine-precontracted EDV rings under different experimental conditions. PPF-induced relaxation was partially inhibited by endothelium removal, but was unaltered by CGS-15943 (an adenosine receptor antagonist; 3µM). PPF-induced relaxation was partially inhibited in the presence of L-NAME (a nitric oxide (NO) synthase inhibitor; 100µM), ODQ (an inhibitor of soluble guanylyl cyclase; 30µM) or Rp-8-Br-PET-cGMP-S (a protein kinase G inhibitor; 3µM). It was not modified by indomethacin (a non-selective cyclooxygenase (COX) inhibitor; 10µM), and was slightly potentiated by H-89 (a protein kinase A inhibitor; 2µM). In endothelium-intact EDVs, PPF-induced relaxation was associated with a 2.4- and 24.1-fold increase in the tissue cGMP and cAMP content respectively. PPF (100μM) did not shift the concentration–response curve to phenylephrine (1nM–300µM) but reduced the maximal effect. To investigate whether PPF can affect cAMP- and cGMP-induced relaxations, relaxation curves to forskolin (an activator of adenylate cyclase) and to sodium nitroprusside (SNP, a NO donor) were recorded in EDV rings pretreated with PPF (100µM). PPF only slightly potentiated the forskolin-induced relaxation without affecting the SNP-induced relaxation. We demonstrated that PPF-induced relaxation in EDVs is partially endothelium-dependent. The PPF-induced relaxation partially occurred via NO release and both cAMP and cGMP generation, through COX-independent mechanisms but could also result from the inhibition of cAMP-phosphodiesterase activity for the highest concentrations.