2′,3′-cAMP hydrolysis by metal-dependent phosphodiesterases containing DHH, EAL, and HD domains is non-specific: Implications for PDE screening

The ability of cytosolic lipopolysaccharide (LPS) to activate caspase-11–dependent nonclassical inflammasome is intricately controlled to avoid excessive inflammatory responses. However, very little is known about the regulatory role of various metabolic pathways in the control of caspase-11 activation. Here, we demonstrate that l-adrenaline can act on receptor ADRA2B to inhibit the activation of the caspase-11 inflammasome by cytosolic LPS or Escherichia coli infection in macrophages. l-adrenaline–induced cAMP production via the enzyme ADCY4 promotes protein kinase A (PKA) activation, which then blocks the caspase-11–mediated proteolytic maturation of interleukin-1β, gasdermin D (GSDMD) cleavage, and consequent DAMP release. Inhibition of PDE8A-mediated cAMP hydrolysis limits caspase-11 inflammasome activation and pyroptosis in macrophages. Consequently, pharmacological modulation of the ADRA2B-ADCY4-PDE8A-PKA axis, knockout of caspase-11 (Casp11−/−), or Gsdmd inactivation (GsdmdI105N/I105N) similarly protects against LPS-induced lethality in poly(I:C)-primed mice. Our results provide previously unidentified mechanistic insight into immune regulation by cAMP and represent a proof of concept that immunometabolism constitutes a potential therapeutic target in sepsis.

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Functional Proteomic Profiling of Phosphodiesterases Using SeraFILE Separations Platform

International Journal of Proteomics ◽

10.1155/2012/515372 ◽

2012 ◽

Vol 2012 ◽

pp. 1-8

Author(s):

Amita R. Oka ◽

Matthew P. Kuruc ◽

Ketan M. Gujarathi ◽

Swapan Roy

Keyword(s):

Biomarker Discovery ◽

Brain Homogenate ◽

Disease Diagnosis ◽

Bovine Brain ◽

Tissue Cell ◽

Kinetic Properties ◽

Proteomic Profiling ◽

Tissue Samples ◽

Camp Hydrolysis ◽

Few Data

Functional proteomic profiling can help identify targets for disease diagnosis and therapy. Available methods are limited by the inability to profile many functional properties measured by enzymes kinetics. The functional proteomic profiling approach proposed here seeks to overcome such limitations. It begins with surface-based proteome separations of tissue/cell-line extracts, using SeraFILE, a proprietary protein separations platform. Enzyme kinetic properties of resulting subproteomes are then characterized, and the data integrated into proteomic profiles. As a model, SeraFILE-derived subproteomes of cyclic nucleotide-hydrolyzing phosphodiesterases (PDEs) from bovine brain homogenate (BBH) and rat brain homogenate (RBH) were characterized for cAMP hydrolysis activity in the presence (challenge condition) and absence of cGMP. Functional profiles of RBH and BBH were compiled from the enzyme activity response to the challenge condition in each of the respective subproteomes. Intersample analysis showed that comparable profiles differed in only a few data points, and that distinctive subproteomes can be generated from comparable tissue samples from different animals. These results demonstrate that the proposed methods provide a means to simplify intersample differences, and to localize proteins attributable to sample-specific responses. It can be potentially applied for disease and nondisease sample comparison in biomarker discovery and drug discovery profiling.

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Abstract 049: Phosphodiesterase-3a Catalytic-domain Mutation and Hypertension

Hypertension ◽

10.1161/hyp.68.suppl_1.049 ◽

2016 ◽

Vol 68 (suppl_1) ◽

Author(s):

Sylvia Bähring ◽

Carolin Schächterle ◽

Atakan Aydin ◽

Enno Klussmann ◽

Friedrich C Luft

Keyword(s):

Blood Pressure ◽

Turnover Rate ◽

Catalytic Domain ◽

Hydrolytic Activity ◽

Hek293 Cells ◽

Michaelis Constant ◽

Salt Form ◽

Cysteine Substitution ◽

Camp Hydrolysis ◽

Kinetics Of

We recently discovered phosphodiesterase-3A (PDE3A) mutations causing a 50 mm Hg increase in blood pressure and stroke >50 years, as the first non-salt form of Mendelian genetic hypertension, autosomal-dominant hypertension with brachydactyly (HTNB). The mutations cause increased PDE3A phosphorylation and higher cAMP affinity. We now have found a completely different PDE3A mutation causing a similar syndrome in a New Zealand pedigree. The mutation resides in the enzyme’s catalytic domain, results in an arginine-to-cysteine substitution, and represents a more direct mechanism of PDE3A activation. For Michaelis-Menten kinetics of cAMP hydrolysis, we transfected HEK293 cells transiently expressing Flag-tagged versions of PDE3A1, PDE3A2, or PDE3A3 mutant vs. wildtype and stimulated with forskolin and phorbol-12-myristate-13-acetate (PMA) to enhance intrinsic phosphorylation. Vmax and Km (Michaelis constant) were calculated using GraphPad Prism software to reveal the maximum cAMP turnover rate at saturated substrate concentration and the affinity of cAMP to wildtype and mutated PDE3A1, PDE3A2 and PDE3A3. For PDE3A1 hydrolytic activity (triplicate), we observed: Vmax Km Wildtype 7.5 340 Wildtype+forskolin/PMA 7.2 203 Mutant 6.6 116 Mutant+forskolin/PMA 6.3 81 The dramatically lower Km of mutant PDE3A indicates a substantially greater affinity for cAMP consistent with gain-of-function. These data underscore the importance of PDE3A to high blood pressure by means of a different, novel genetic mechanism directly implicating the catalytic domain.

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Inotropic responses change during postnatal maturation in rabbit

AJP Heart and Circulatory Physiology ◽

10.1152/ajpheart.1988.255.2.h335 ◽

1988 ◽

Vol 255 (2) ◽

pp. H335-H342 ◽

Cited By ~ 2

Author(s):

M. Artman ◽

P. A. Kithas ◽

J. S. Wike ◽

S. J. Strada

Keyword(s):

Adenylate Cyclase ◽

Calcium Sensitivity ◽

Inotropic Response ◽

Phosphodiesterase Activity ◽

Camp Phosphodiesterase ◽

Tension Development ◽

Postnatal Maturation ◽

High Affinity ◽

Age Related ◽

Camp Hydrolysis

Inotropic response to four different types of pharmacological stimuli were compared in isolated right ventricular papillary muscles from newborn (24–48 h of age), immature (14–16 days), and adult (6–7 mo) rabbits. Forskolin, a direct activator of adenylate cyclase, produced a 12.5-fold increase in the maximal rate of tension development in the newborn group. The maximum response to isoproterenol was only 45% of the maximum forskolin response, suggesting incomplete physiological coupling of myocardial beta-adrenergic receptors to adenylate cyclase at birth. In contrast to the substantial inotropic response to agents that stimulate adenosine 3',5'-cyclic monophosphate (cAMP) generation (forskolin and isoproterenol), a selective inhibitor of cAMP hydrolysis (milrinone) was relatively ineffective in the newborn group. Sulmazole, a drug that enhances calcium sensitivity of the contractile proteins, produced its greatest inotropic effect in immature myocardium. Cytosolic high-affinity cAMP phosphodiesterase activity was partially purified from ventricular homogenates by anion-exchange chromatography. The kinetics of cAMP hydrolysis (Km and Vmax) and inhibitory potency of milrinone were comparable in each age group. Thus the age-related differences in inotropic responsiveness may not be attributable to postnatal changes in myocardial cytosolic high-affinity cAMP phosphodiesterase activity.

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Evidence for the presence of cyclic adenosine 3′,5′-monophosphate and its metabolism in loblolly pine (Pinustaeda L.) callus

Canadian Journal of Forest Research ◽

10.1139/x77-010 ◽

1977 ◽

Vol 7 (1) ◽

pp. 68-75 ◽

Cited By ~ 5

Author(s):

Richard H. Smeltzer ◽

Morris A. Johnson

Keyword(s):

Loblolly Pine ◽

Direct Relationship ◽

Defined Medium ◽

Callus Cultures ◽

Adenyl Cyclase ◽

Fresh Weight ◽

Camp Concentration ◽

Adenyl Cyclase Activity ◽

Stable Product ◽

Camp Hydrolysis

Evidence is presented suggesting the occurrence of cyclic adenosine 3′,5′-monophosphate (cAMP) and adenyl cyclase activity in loblolly pine (Pinustaeda L.) callus cultures grown aseptically on a defined medium. The presence of enzymes in the callus capable of catalyzing cAMP hydrolysis also was detected. Adenosine was the most stable product resulting from cAMP hydrolysis. A direct relationship was found between apparent cAMP concentration and the rate of fresh weight increase of dark-grown callus during a 6-week passage.

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Adenosine 3',5-cyclic monophosphate phosphodiesterase activity in granulosa cells from Booroola × Romney ewes with and without the F gene

Journal of Endocrinology ◽

10.1677/joe.0.1200287 ◽

1989 ◽

Vol 120 (2) ◽

pp. 287-293

Author(s):

K. P. McNatty ◽

D. A. Heath ◽

S. Lun ◽

N. L. Hudson

Keyword(s):

Granulosa Cells ◽

Maximum Velocity ◽

Camp Accumulation ◽

Camp Phosphodiesterase ◽

F Gene ◽

Specific Effects ◽

The Mean ◽

Camp Hydrolysis ◽

Camp Levels ◽

In Cells

ABSTRACT Granulosa cells from ovarian follicles (≥ 1 mm diameter) in Booroola ewes which are homozygous (FF) or heterozygous (F+) for the F gene have previously been shown to produce significantly more cAMP in response to FSH or LH than those from similar sized follicles in ewes without the F gene (++). The aim of these studies was to test whether these F gene-specific differences arose because of differences in cAMP-phosphodiesterase (cAMP-PDE) activity. In the first study using 1 μmol cAMP/1 as substrate, no F gene-specific effects were noted in cAMP-PDE activity in granulosa cells from small (1–2·5 mm diameter, n = 4 per genotype) or large (≥ 3 mm diameter, n = 4 per genotype) follicles from FF, F+ or ++ ewes, despite F gene-specific effects in FSH (1 μg/ml)and LH (0·1 μg/ml)-induced cAMP accumulation in these same cell preparations. The overall mean levels of cAMP-PDE across all genotypes in cells from small and large follicles were 0·47± 0·04 (s.e.m., n = 12) and 0·28 ± 0·03 pmol cAMP/106 cells per min respectively; the mean PDE activity in cells from small follicles was significantly (P < 0·05) higher compared with that in cells from large follicles. In a second study, granulosa cells from each genotype were pooled over all follicle sizes (≥ 1 mm diameter, one pool per genotype) and the rates of cAMP hydrolysis tested over a range of substrate concentrations (0–16 μmol/l) but no genespecific differences with respect to the Michaelis constant and maximum velocity were noted. In a third study, the rates of FSH-induced cAMP accumulation by granulosa cells from small and large follicles of FF and ++ ewes were examined in the presence of a PDE inhibitor, 1-methyl-3-isobutylxanthine (0·2 mmol/l). In this study FSH (1 μg/ml) stimulated significantly more cAMP in cells from FF compared with ++ ewes in both small and large follicles. In cells from small follicles, the mean ± s.e.m. FSHstimulated cAMP levels were 4·7 ± 0·8 (n = 6) for FF ewes and 1·8 ± 0·4 (n = 8) pmol/106 cells per h for ++ ewes (P < 0·025). In large follicles, the cAMP levels were 17·0 ± 3·5 (n = 6) for FF ewes and 6·3 ± 0·5 pmol/106 cells per h for ++ ewes (P < 0·05). Collectively, these data suggest that the F genespecific difference in cAMP synthesis is probably the result of an event(s) associated with its formation rather than its degradation. Journal of Endocrinology (1989) 120, 287–293

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Molecular cloning and transient expression in COS7 cells of a novel human PDE4B cAMP-specific phosphodiesterase, HSPDE4B3

Biochemical Journal ◽

10.1042/bj3280549 ◽

1997 ◽

Vol 328 (2) ◽

pp. 549-558 ◽

Cited By ~ 72

Author(s):

Elaine HUSTON ◽

Simon LUMB ◽

Annette RUSSELL ◽

Cath CATTERALL ◽

H. Annette ROSS ◽

...

Keyword(s):

High Speed ◽

Splice Variants ◽

Short Form ◽

Amino Acid Identity ◽

Open Reading Frame ◽

Cytosol Fraction ◽

Reading Frame ◽

Alternatively Spliced ◽

Camp Hydrolysis ◽

Rapid Amplification

5ʹ-Rapid amplification of cDNA ends, done on poly(A)+ RNA from human U87 cells, was used to identify 420 bp of novel 5ʹ sequence of a PDE4B cAMP-specific phosphodiesterase (PDE). This identified an open reading frame encoding a putative 721-residue ‘long-form’ PDE4B splice variant, which we term HSPDE4B3. HSPDE4B3 differs from the two known PDE4B forms by virtue of its unique 79-residue N-terminal region, compared with the unique N-terminal regions of 94 and 39 residues found in HSPDE4B1 and HSPDE4B2 respectively. In transfected COS7 cells the two long forms, HSPDE4B1 and HSPDE4B3, had molecular masses of approx. 104 and approx. 103 kDa respectively. Expressed in COS-7 cells, the three HSPDE4B isoforms were found in the high-speed supernatant (cytosol) fraction as well as both the high-speed pellet (P2) and low-speed pellet (P1) fractions. All isoforms showed similar Km values for cAMP hydrolysis (1.5-2.6 μM). The maximal activities of the soluble cytosolic activity of the two long forms were very similar, whereas that of the short form, HSPDE4B2, was approx. 4-fold higher. Particulate-associated HSPDE4B1 and HSPDE4B2 were less active (approx. 40%) than their cytosol forms, whereas particulate HSPDE4B3 was similar in activity to its cytosolic form. Particulate and cytosolic forms of HSPDE4B1 and HSPDE4B3 were similarly inhibited by rolipram {4-[3-(cyclopentoxyl)-4-methoxyphenyl]-2-pyrrolidone}, the selective inhibitor of PDE4 (IC50 0.05-0.1 μM), whereas particulate-associated HSPDE4B2 was profoundly (approx. 10-fold) more sensitive (IC50 0.02 μM) to rolipram inhibition than its cytosolic form (IC50 0.2 μM). The various particulate-associated HSPDE4B isoforms showed very different susceptibilities to solubilization with the detergent Triton X-100 and high NaCl concentration. A novel cDNA, called pRPDE74, was obtained by screening a rat olfactory lobe cDNA library. This contained an open reading frame encoding a 721-residue protein that showed approx. 96% amino acid identity with HSPDE4B3 and is proposed to reflect the rat homologue of this human enzyme and is thus called RNPDE4B3. Alternative splicing of mRNA generated from both the human and rat PDE4B genes produces long and short splice variants that have unique N-terminal splice regions. It is suggested that these alternatively spliced regions determine changes in the maximal catalytic activity of the isoforms, their susceptibility to inhibition by rolipram and mode of interaction with particulate fractions.

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Phosphodiesterase 4 in macrophages: relationship between cAMP accumulation, suppression of cAMP hydrolysis and inhibition of [3H]R-(—)-rolipram binding by selective inhibitors

Biochemical Journal ◽

10.1042/bj3180425 ◽

1996 ◽

Vol 318 (2) ◽

pp. 425-436 ◽

Cited By ~ 32

Author(s):

John J KELLY ◽

Peter J BARNES ◽

Mark A GIEMBYCZ

Keyword(s):

Binding Site ◽

Rank Order ◽

Smooth Muscle Relaxation ◽

Camp Accumulation ◽

Functional Responses ◽

Camp Content ◽

Phosphodiesterase 4 ◽

Ic50 Values ◽

Membrane Bound ◽

Camp Hydrolysis

A perplexing phenomenon identified in several tissues is the lack of correlation between inhibition of phosphodiesterase 4 (PDE4) and certain functional responses such as smooth muscle relaxation, gastric acid secretion and cAMP accumulation. Interpretation of these data is complicated further by the finding that function correlates with the ability of PDE4 inhibitors to displace [3H]rolipram [4-(3-cyclopentenyloxy-4-methoxyphenyl)-2-pyrrolidone] from a high-affinity site in rat brain that is apparently distinct from the catalytic centre of the enzyme. We have investigated this discrepancy by using guinea pig macrophages as a source of PDE4 and have confirmed that the ability of a limited range of structurally dissimilar PDE inhibitors (Org 20241, nitraquazone and the enantiomers of rolipram and benafentrine) to increase cAMP content did not correlate with their potency as inhibitors of partly purified PDE4, whereas a significant linear and rank order correlation was found when cAMP accumulation was related to the displacement of [3H]R-(-)-rolipram from a specific site identified in macrophage lysates. An explanation for these data emerged from the finding that the IC50 values and rank order of potency of these compounds for inhibition of partly purified PDE4 and the native (membrane-bound) form of the same enzyme were distinct. Similarly, no correlation was found when membrane-bound PDE4 was compared with the same enzyme that had been solubilized with Triton X-100. These unexpected results were attributable to a selective decrease in the potency of those inhibitors [nitraquazone, R-(-)- and S-(+)-rolipram] that interacted preferentially with the rolipram binding site. Indeed, if membrane-bound PDE4 was used as the enzyme preparation, excellent linear and rank order correlations between inhibition of cAMP hydrolysis, displacement of [3H]R-(-)-rolipram and cAMP accumulation were found, which improved further in the presence of the vanadyl (Vo)/2.GSH complex. Moreover, using Vo/2.GSH-treated membranes, the IC50 values of nitraquazone and the enantiomers of rolipram for the inhibition of PDE4 approached their affinity for the rolipram binding site. Collectively, these data suggest that the rolipram binding site and the catalytic domain on CPPDE4 might represent part of the same entity. In addition, these results support the concept that PDE4 can exist in different conformational states [Barnett, Manning, Cieslinski, Burman, Christensen and Torphy (1995) J. Pharmcol. Exp. Ther. 273, 674–679] and provide evidence that the cAMP content in macrophages is regulated primarily by a conformer of PDE4 for which rolipram has nanomolar affinity.

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Ammonia promotes accumulation of intracellular cAMP in differentiating amoebae of Dictyostelium discoideum

Development ◽

10.1242/dev.109.3.715 ◽

1990 ◽

Vol 109 (3) ◽

pp. 715-722

Author(s):

B.B. Riley ◽

S.L. Barclay

Keyword(s):

Dictyostelium Discoideum ◽

Active Species ◽

Spore Formation ◽

Intracellular Camp ◽

Simultaneous Exposure ◽

High Ph ◽

Camp Hydrolysis ◽

Extracellular Camp ◽

Camp Levels

We used sporogenous mutants of Dictyostelium discoideum to investigate the mechanism(s) by which exogenous NH4Cl and high ambient pH promote spore formation during in vitro differentiation. The level of NH4Cl required to optimize spore formation is correlated inversely with pH, indicating that NH3 rather than NH4+ is the active species. The spore-promoting activity of high ambient pH (without exogenous NH4Cl) was eliminated by the addition of an NH3-scavenging cocktail, suggesting that high pH promotes spore differentiation by increasing the ratio of NH3:NH4+ secreted into the medium by developing cells. High ammonia levels and high pH stimulated precocious accumulation of intracellular cAMP in both sporogenous and wild-type cells. In both treatments, peak cAMP levels equaled or exceeded control levels and were maintained for longer periods than in control cells. In contrast, ammonia strongly inhibited accumulation of extracellular cAMP without increasing the rate of extracellular cAMP hydrolysis, indicating that ammonia promotes accumulation of intracellular cAMP by inhibiting cAMP secretion. These results are consistent with previous observations that factors that raise intracellular cAMP levels increase spore formation. Lowering intracellular cAMP levels with caffeine or progesterone inhibited spore formation, but simultaneous exposure to these drugs and optimal concentrations of NH4Cl restored both cAMP accumulation and spore formation to normal levels. These data suggest that ammonia, which is a natural Dictyostelium morphogen, favors spore formation by promoting accumulation or maintenance of high intracellular cAMP levels.

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PDE2-mediated cAMP hydrolysis accelerates cardiac fibroblast to myofibroblast conversion and is antagonized by exogenous activation of cGMP signaling pathways

AJP Heart and Circulatory Physiology ◽

10.1152/ajpheart.00852.2013 ◽

2014 ◽

Vol 306 (8) ◽

pp. H1246-H1252 ◽

Cited By ~ 30

Author(s):

C. Vettel ◽

S. Lämmle ◽

S. Ewens ◽

C. Cervirgen ◽

J. Emons ◽

...

Keyword(s):

Atrial Natriuretic Peptide ◽

Sodium Nitroprusside ◽

Natriuretic Peptide ◽

Neonatal Rat ◽

Nitric Oxide Donor ◽

Signal Molecules ◽

Protective Mechanism ◽

Camp Hydrolysis ◽

Camp And Cgmp ◽

Atrial Natriuretic

Recent studies suggest that the signal molecules cAMP and cGMP have antifibrotic effects by negatively regulating pathways associated with fibroblast to myofibroblast (MyoCF) conversion. The phosphodiesterase 2 (PDE2) has the unique property to be stimulated by cGMP, which leads to a remarkable increase in cAMP hydrolysis and thus mediates a negative cross-talk between both pathways. PDE2 has been recently investigated in cardiomyocytes; here we specifically addressed its role in fibroblast conversion and cardiac fibrosis. PDE2 is abundantly expressed in both neonatal rat cardiac fibroblasts (CFs) and cardiomyocytes. The overexpression of PDE2 in CFs strongly reduced basal and isoprenaline-induced cAMP synthesis, and this decrease was sufficient to induce MyoCF conversion even in the absence of exogenous profibrotic stimuli. Functional stress-strain experiments with fibroblast-derived engineered connective tissue (ECT) demonstrated higher stiffness in ECTs overexpressing PDE2. In regard to cGMP, neither basal nor atrial natriuretic peptide-induced cGMP levels were affected by PDE2, whereas the response to nitric oxide donor sodium nitroprusside was slightly but significantly reduced. Interestingly, despite persistently depressed cAMP levels, both cGMP-elevating stimuli were able to completely prevent the PDE2-induced MyoCF phenotype, arguing for a double-tracked mechanism. In conclusion, PDE2 accelerates CF to MyoCF conversion, which leads to greater stiffness in ECTs. Atrial natriuretic peptide- and sodium nitroprusside-mediated cGMP synthesis completely reverses PDE2-induced fibroblast conversion. Thus PDE2 may augment cardiac remodeling, but this effect can also be overcome by enhanced cGMP. The redundant role of cAMP and cGMP as antifibrotic meditators may be viewed as a protective mechanism in heart failure.

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