Publications by authors named "Michael Russwurm"

55 Publications

THE CONCISE GUIDE TO PHARMACOLOGY 2021/22: Enzymes.

Br J Pharmacol 2021 Oct;178 Suppl 1:S313-S411

Keele University, Keele, UK.

The Concise Guide to PHARMACOLOGY 2021/22 is the fifth in this series of biennial publications. The Concise Guide provides concise overviews, mostly in tabular format, of the key properties of nearly 1900 human drug targets with an emphasis on selective pharmacology (where available), plus links to the open access knowledgebase source of drug targets and their ligands (www.guidetopharmacology.org), which provides more detailed views of target and ligand properties. Although the Concise Guide constitutes over 500 pages, the material presented is substantially reduced compared to information and links presented on the website. It provides a permanent, citable, point-in-time record that will survive database updates. The full contents of this section can be found at http://onlinelibrary.wiley.com/doi/bph.15542. Enzymes are one of the six major pharmacological targets into which the Guide is divided, with the others being: G protein-coupled receptors, ion channels, nuclear hormone receptors, catalytic receptors and transporters. These are presented with nomenclature guidance and summary information on the best available pharmacological tools, alongside key references and suggestions for further reading. The landscape format of the Concise Guide is designed to facilitate comparison of related targets from material contemporary to mid-2021, and supersedes data presented in the 2019/20, 2017/18, 2015/16 and 2013/14 Concise Guides and previous Guides to Receptors and Channels. It is produced in close conjunction with the Nomenclature and Standards Committee of the International Union of Basic and Clinical Pharmacology (NC-IUPHAR), therefore, providing official IUPHAR classification and nomenclature for human drug targets, where appropriate.
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http://dx.doi.org/10.1111/bph.15542DOI Listing
October 2021

Angiotensin II-Induced Cardiovascular Fibrosis Is Attenuated by NO-Sensitive Guanylyl Cyclase1.

Cells 2020 11 8;9(11). Epub 2020 Nov 8.

Institut für Pharmakologie und Toxikologie, Med. Fak. MA N1, Ruhr-Universität Bochum, 44780 Bochum, Germany.

In the NO/cGMP signaling cascade, relevant in the cardiovascular system, two NO-sensitive guanylyl cyclase (NO-GC) isoforms are responsible for NO-dependent cGMP generation. Here, the impact of the major NO-GC isoform, NO-GC1, on fibrosis development in the cardiovascular system was studied in NO-GC1-deficient mice treated with AngiotensinII (AngII), known to induce vascular and cardiac remodeling. Morphometric analysis of NO-GC1 KO's aortae demonstrated an enhanced increase of perivascular area after AngII treatment accompanied by a higher aortic collagen1 mRNA content. Increased perivascular fibrosis also occurred in cardiac vessels of AngII-treated NO-GC1 KO mice. In line, AngII-induced interstitial fibrosis was 32% more pronounced in NO-GC1 KO than in WT myocardia associated with a higher cardiac Col1 and other fibrotic marker protein content. In sum, increased perivascular and cardiac interstitial fibrosis together with the enhanced collagen1 mRNA content in AngII-treated NO-GC1-deficient mice represent an exciting manifestation of antifibrotic properties of cGMP formed by NO-GC1, a finding with great pharmaco-therapeutic implications.
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http://dx.doi.org/10.3390/cells9112436DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7695185PMC
November 2020

Phosphodiesterase 3A and Arterial Hypertension.

Circulation 2020 07 11;142(2):133-149. Epub 2020 Jun 11.

Leibniz-Forschingsinstitut für Molekulare Pharmakologie (FMP), Berlin, Germany (J.E., M.L., B.W.).

Background: High blood pressure is the primary risk factor for cardiovascular death worldwide. Autosomal dominant hypertension with brachydactyly clinically resembles salt-resistant essential hypertension and causes death by stroke before 50 years of age. We recently implicated the gene encoding phosphodiesterase 3A (); however, in vivo modeling of the genetic defect and thus showing an involvement of mutant PDE3A is lacking.

Methods: We used genetic mapping, sequencing, transgenic technology, CRISPR-Cas9 gene editing, immunoblotting, and fluorescence resonance energy transfer. We identified new patients, performed extensive animal phenotyping, and explored new signaling pathways.

Results: We describe a novel mutation within a 15 base pair (bp) region of the gene and define this segment as a mutational hotspot in hypertension with brachydactyly. The mutations cause an increase in enzyme activity. A CRISPR/Cas9-generated rat model, with a 9-bp deletion within the hotspot analogous to a human deletion, recapitulates hypertension with brachydactyly. In mice, mutant transgenic PDE3A overexpression in smooth muscle cells confirmed that mutant PDE3A causes hypertension. The mutant PDE3A enzymes display consistent changes in their phosphorylation and an increased interaction with the 14-3-3θ adaptor protein. This aberrant signaling is associated with an increase in vascular smooth muscle cell proliferation and changes in vessel morphology and function.

Conclusions: The mutated gene drives mechanisms that increase peripheral vascular resistance causing hypertension. We present 2 new animal models that will serve to elucidate the underlying mechanisms further. Our findings could facilitate the search for new antihypertensive treatments.
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http://dx.doi.org/10.1161/CIRCULATIONAHA.119.043061DOI Listing
July 2020

AMPA Induces NO-Dependent cGMP Signals in Hippocampal and Cortical Neurons via L-Type Voltage-Gated Calcium Channels.

Cereb Cortex 2020 04;30(4):2128-2143

Institute of Pharmacology and Toxicology, Ruhr-University Bochum, 44780 Bochum, Germany.

The nitric oxide (NO)/cGMP signaling cascade has an established role in synaptic plasticity. However, with conventional methods, the underlying cGMP signals were barely detectable. Here, we set out to confirm the well-known NMDA-induced cGMP increases, to test the impact of AMPA on those signals, and to identify the relevant phosphodiesterases (PDEs) using a more sensitive fluorescence resonance energy transfer (FRET)-based method. Therefore, a "knock-in" mouse was generated that expresses a FRET-based cGMP indicator (cGi-500) allowing detection of cGMP concentrations between 100 nM and 3 μM. Measurements were performed in cultured hippocampal and cortical neurons as well as acute hippocampal slices. In hippocampal and cortical neurons, NMDA elicited cGMP signals half as high as the ones elicited by exogenous NO. Interestingly, AMPA increased cGMP independently of NMDA receptors and dependent on NO synthase (NOS) activation. NMDA- and AMPA-induced cGMP signals were not additive indicating that both pathways converge on the level of NOS. Accordingly, the same PDEs, PDE1 and PDE2, were responsible for degradation of NMDA- as well as AMPA-induced cGMP signals. Mechanistically, AMPAR induced calcium influx through L-type voltage-gated calcium channels leading to NOS and finally NO-sensitive guanylyl cyclase activation. Our results demonstrate that in addition to NMDA also AMPA triggers endogenous NO formation and hence cGMP production.
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http://dx.doi.org/10.1093/cercor/bhz227DOI Listing
April 2020

Effects of flavoring compounds used in electronic cigarette refill liquids on endothelial and vascular function.

PLoS One 2019 9;14(9):e0222152. Epub 2019 Sep 9.

Department of Pharmacology and Toxicology, Institute of Pharmaceutical Sciences, Karl-Franzens-Universität Graz, Graz, Austria.

Electronic cigarette refill liquids are commercially provided with a wide variety of flavoring agents. A recent study suggested that several common flavors may scavenge nitric oxide (NO) and cause endothelial dysfunction. It was the aim of the present study to investigate the effects of these flavors on NO/cyclic GMP-mediated signaling and vascular relaxation. We tested the flavoring agents for effects on Ca2+-induced cGMP accumulation and NO synthase activation in cultured endothelial cells. NO scavenging was studied with NO-activated soluble guanylate cyclase and as NO release from a NO donor, measured with a NO electrode. Blood vessel function was studied with precontracted rat aortic rings in the absence and presence of acetylcholine or a NO donor. Cinnamaldehyde inhibited Ca2+-stimulated endothelial cGMP accumulation and NO synthase activation at ≥0.3 mM. Cinnamaldehyde and diacetyl inhibited NO-activated soluble guanylate cyclase with IC50 values of 0.56 (0.54-0.58) and 0.29 (0.24-0.36) mM, respectively, and caused moderate NO scavenging at 1 mM that was not mediated by superoxide anions. The other compounds did not scavenge NO at 1 mM. None of the flavorings interfered with acetylcholine-induced vascular relaxation, but they caused relaxation of pre-contracted aortas. The most potent compounds were eugenol and cinnamaldehyde with EC50 values of ~0.5 mM. Since the flavors did not affect endothelium-dependent vascular relaxation, NO scavenging by cinnamaldehyde and diacetyl does not result in impaired blood vessel function. Although not studied in vivo, the low potency of the compounds renders it unlikely that the observed effects are relevant to humans inhaling flavored vapor from electronic cigarettes.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0222152PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6733504PMC
March 2020

Mind the gap (junction): cGMP induced by nitric oxide in cardiac myocytes originates from cardiac fibroblasts.

Br J Pharmacol 2019 12 27;176(24):4696-4707. Epub 2019 Dec 27.

Institute of Pharmacology and Toxicology, Ruhr-University Bochum, Bochum, Germany.

Background And Purpose: The intracellular signalling molecule cGMP, formed by NO-sensitive GC (NO-GC), has an established function in the vascular system. Despite numerous reports about NO-induced cGMP effects in the heart, the underlying cGMP signals are poorly characterized.

Experimental Approach: Therefore, we analysed cGMP signals in cardiac myocytes and fibroblasts isolated from knock-in mice expressing a FRET-based cGMP indicator.

Key Results: Whereas in cardiac myocytes, none of the known NO-GC-activating substances (NO, GC activators, and GC stimulators) increased cGMP even in the presence of PDE inhibitors, they induced substantial cGMP increases in cardiac fibroblasts. As cardiac myocytes and fibroblasts are electrically connected via gap junctions, we asked whether cGMP can take the same route. Indeed, in cardiomyocytes co-cultured on cardiac fibroblasts, NO-induced cGMP signals were detectable, and two groups of unrelated gap junction inhibitors abolished these signals.

Conclusion And Implication: We conclude that NO-induced cGMP formed in cardiac fibroblasts enters cardiac myocytes via gap junctions thereby turning cGMP into an intercellular signalling molecule. The findings shed new light on NO/cGMP signalling in the heart and will potentially broaden therapeutic opportunities for cardiac disease.
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http://dx.doi.org/10.1111/bph.14835DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6965686PMC
December 2019

Modulation of nitric oxide-stimulated soluble guanylyl cyclase activity by cytoskeleton-associated proteins in vascular smooth muscle.

Biochem Pharmacol 2018 10 10;156:168-176. Epub 2018 Aug 10.

Institute of Pharmaceutical Sciences, Department of Pharmacology and Toxicology, University of Graz, Graz, Austria.

Soluble guanylyl cyclase (sGC, EC 4.6.1.2) is a key enzyme in the regulation of vascular tone. In view of the therapeutic interest of the NO/cGMP pathway, drugs were developed that either increase the NO sensitivity of the enzyme or activate heme-free apo-sGC. However, modulation of sGC activity by endogenous agents is poorly understood. In the present study we show that the maximal activity of NO-stimulated purified sGC is significantly increased by cytosolic preparations of porcine coronary arteries. Purification of the active principle by several chromatographic steps resulted in a protein mixture consisting of 100, 70, and 40 kDa bands on SDS polyacrylamide gel electrophoresis. The respective proteins were identified by LC-MS/MS as gelsolin, annexin A6, and actin, respectively. Further purification resulted in loss of activity, indicating an interaction of sGC with a protein complex rather than a single protein. The partially purified preparation had no effect on basal sGC activity or enzyme activation by the heme mimetic BAY 60-2770, suggesting a specific effect on the conformation of the NO-bound heterodimeric holoenzyme. Since the three proteins identified are all related to contractile elements of smooth muscle, our data suggest that regulation of vascular tone involves a modulatory interaction of sGC with the cytoskeleton.
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http://dx.doi.org/10.1016/j.bcp.2018.08.009DOI Listing
October 2018

cGMP Imaging in Brain Slices Reveals Brain Region-Specific Activity of NO-Sensitive Guanylyl Cyclases (NO-GCs) and NO-GC Stimulators.

Int J Mol Sci 2018 Aug 7;19(8). Epub 2018 Aug 7.

Interfakultäres Institut für Biochemie, University of Tübingen, 72076 Tübingen, Germany.

Impaired NO-cGMP signaling has been linked to several neurological disorders. NO-sensitive guanylyl cyclase (NO-GC), of which two isoforms-NO-GC1 and NO-GC2-are known, represents a promising drug target to increase cGMP in the brain. Drug-like small molecules have been discovered that work synergistically with NO to stimulate NO-GC activity. However, the effects of NO-GC stimulators in the brain are not well understood. In the present study, we used Förster/fluorescence resonance energy transfer (FRET)-based real-time imaging of cGMP in acute brain slices and primary neurons of cGMP sensor mice to comparatively assess the activity of two structurally different NO-GC stimulators, IWP-051 and BAY 41-2272, in the cerebellum, striatum and hippocampus. BAY 41-2272 potentiated an elevation of cGMP induced by the NO donor DEA/NO in all tested brain regions. Interestingly, IWP-051 potentiated DEA/NO-induced cGMP increases in the cerebellum and striatum, but not in the hippocampal CA1 area or primary hippocampal neurons. The brain-region-selective activity of IWP-051 suggested that it might act in a NO-GC isoform-selective manner. Results of mRNA in situ hybridization indicated that the cerebellum and striatum express NO-GC1 and NO-GC2, while the hippocampal CA1 area expresses mainly NO-GC2. IWP-051-potentiated DEA/NO-induced cGMP signals in the striatum of NO-GC2 knockout mice but was ineffective in the striatum of NO-GC1 knockout mice. These results indicate that IWP-051 preferentially stimulates NO-GC1 signaling in brain slices. Interestingly, no evidence for an isoform-specific effect of IWP-051 was observed when the cGMP-forming activity of whole brain homogenates was measured. This apparent discrepancy suggests that the method and conditions of cGMP measurement can influence results with NO-GC stimulators. Nevertheless, it is clear that NO-GC stimulators enhance cGMP signaling in the brain and should be further developed for the treatment of neurological diseases.
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http://dx.doi.org/10.3390/ijms19082313DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6122017PMC
August 2018

Measurement of cGMP-generating and -degrading activities and cGMP levels in cells and tissues: Focus on FRET-based cGMP indicators.

Nitric Oxide 2018 07 21;77:44-52. Epub 2018 Apr 21.

Pharmakologie und Toxikologie, Medizinische Fakultät, Ruhr-Universität Bochum, Bochum, Germany.

The intracellular messenger molecule cGMP has an established function in the regulation of numerous physiological events. Yet for the identification of further biological cGMP-mediated functions, precise information whether a cGMP response exists in a certain cell type or tissue is mandatory. In this review, the techniques to measure cGMP i.e. cGMP-formation, -degradation or levels are outlined and discussed. As a superior method to measure cGMP, the article focusses on FRET-based cGMP indicators, describes the different cGMP indicators and discusses their advantages and drawbacks. Finally, the successful applications of these cGMP indicators to measure cGMP responses in cells and tissues are outlined and summarized. Hopefully, with the availability of the FRET-based cGMP indicators, the knowledge about the cGMP responses in special cells or tissues is going to increase thereby allowing to assess further cGMP-mediated functional responses and possibly to address their pathophysiology with the available guanylyl cyclase activators, stimulators and PDE inhibitors.
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http://dx.doi.org/10.1016/j.niox.2018.04.006DOI Listing
July 2018

Irreversible Activation and Stabilization of Soluble Guanylate Cyclase by the Protoporphyrin IX Mimetic Cinaciguat.

Mol Pharmacol 2018 02 14;93(2):73-78. Epub 2017 Nov 14.

Department of Pharmacology and Toxicology, Karl-Franzens-Universität Graz, Graz, Austria (A.K., M.O., G.W., A.C.F.G., B.M., A.S.); and Department of Pharmacology and Toxicology, Ruhr University Bochum, Bochum, Germany (M.R., D.K.)

Belonging to the class of so-called soluble guanylate cyclase (sGC) activators, cinaciguat and BAY 60-2770 are interesting therapeutic tools for the treatment of various cardiovascular pathologies. The drugs are supposed to preferentially stimulate oxidized or heme-depleted, but not native sGC. Since this concept has been challenged by studies demonstrating complete relaxation of nondiseased vessels, this study was designed to reinvestigate the mode of action in greater detail. To this purpose, the effect of cinaciguat was studied on vessel tone of porcine coronary arteries and rat thoracic aortas. Organ bath studies showed that the compound caused time- and concentration-dependent relaxation of precontracted vessels with a maximal effect observed at 90 minutes. The dilatory response was not affected by extensive washout of the drug. Cinaciguat-induced vasodilation was associated with a time- and concentration-dependent increase of cGMP levels. Experiments with purified sGC in the presence of Tween 20 showed that cinaciguat activates the heme-free enzyme in a concentration-dependent manner with an EC value of ∼0.2 M and maximal cGMP formation at 10 M. By contrast, the effect of cinaciguat on 1-[1,2,4]oxadiazolo-[4,3-]quinoxalin-1-one-oxidized (ferric) sGC was moderate, reaching ∼10%-15% of maximal activity. Dilution experiments of cinaciguat/Tween 20-preincubated sGC revealed the irreversible character of the drug. Assuming a sensitive balance between heme-free, ferric, and nitric oxide-sensitive ferrous sGC in cells and tissues, we propose that cinaciguat by virtue of its irreversible mode of action is capable of shifting this equilibrium toward the heme-free apo-sGC species.
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http://dx.doi.org/10.1124/mol.117.109918DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5916872PMC
February 2018

Physiological Functions of NO-Sensitive Guanylyl Cyclase Isoforms.

Curr Med Chem 2016 ;23(24):2653-2665

Abteilung für Pharmakologie, Medizinische Fakultät, Ruhr-Universität Bochum, Universitätsstr. 150, 44780 Bochum, Germany.

NO-sensitive guanylyl cyclase (NO-GC) acts as the receptor for nitric oxide and by the increase in cGMP executes most of the NO effects in the cardiovascular and neuronal system. Two isoforms of NO-GC exist whose existence has not been paid much attention to probably because they reveal comparable regulatory and catalytic properties and therefore cannot be differentiated in vivo. Analysis of mice in which either one of the isoforms has been genetically deleted unequivocally establishes the coexpression of NO-GC1 and NOGC2 in any tissue tested to date with the exception of platelets. In tissues other than brain and platelets, no particular function could be ascribed to a specific NO-GC isoform so far. In contrast, NO-GC1 and NO-GC2 serve different functions in the central nervous system. With NO-GC1`s presynaptic role and NO-GC2`s postsynaptic action, two NO/cGMP pathways have been shown to exist that enhance the strength of synaptic transmission on either side of the synaptic cleft.
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http://dx.doi.org/10.2174/0929867323666160812145050DOI Listing
February 2017

Formation of Nitric Oxide by Aldehyde Dehydrogenase-2 Is Necessary and Sufficient for Vascular Bioactivation of Nitroglycerin.

J Biol Chem 2016 Nov 27;291(46):24076-24084. Epub 2016 Sep 27.

From the Institute of Pharmaceutical Sciences, Department of Pharmacology and Toxicology, University of Graz, A-8010 Graz, Austria,

Aldehyde dehydrogenase-2 (ALDH2) catalyzes vascular bioactivation of the antianginal drug nitroglycerin (GTN), resulting in activation of soluble guanylate cyclase (sGC) and cGMP-mediated vasodilation. We have previously shown that a minor reaction of ALDH2-catalyzed GTN bioconversion, accounting for about 5% of the main clearance-based turnover yielding inorganic nitrite, results in direct NO formation and concluded that this minor pathway could provide the link between vascular GTN metabolism and activation of sGC. However, lack of detectable NO at therapeutically relevant GTN concentrations (≤1 μm) in vascular tissue called into question the biological significance of NO formation by purified ALDH2. We addressed this issue and used a novel, highly sensitive genetically encoded fluorescent NO probe (geNOp) to visualize intracellular NO formation at low GTN concentrations (≤1 μm) in cultured vascular smooth muscle cells (VSMC) expressing an ALDH2 mutant that reduces GTN to NO but lacks clearance-based GTN denitration activity. NO formation was compared with GTN-induced activation of sGC. The addition of 1 μm GTN to VSMC expressing either wild-type or C301S/C303S ALDH2 resulted in pronounced intracellular NO elevation, with maximal concentrations of 7 and 17 nm, respectively. Formation of GTN-derived NO correlated well with activation of purified sGC in VSMC lysates and cGMP accumulation in intact porcine aortic endothelial cells infected with wild-type or mutant ALDH2. Formation of NO and cGMP accumulation were inhibited by ALDH inhibitors chloral hydrate and daidzin. The present study demonstrates that ALDH2-catalyzed NO formation is necessary and sufficient for GTN bioactivation in VSMC.
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http://dx.doi.org/10.1074/jbc.M116.752071DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5104933PMC
November 2016

Scavenging of nitric oxide by hemoglobin in the tunica media of porcine coronary arteries.

Nitric Oxide 2016 Apr 22;54:8-14. Epub 2016 Jan 22.

Institute of Pharmaceutical Sciences, Department of Pharmacology and Toxicology, University of Graz, Austria. Electronic address:

Scavenging of nitric oxide (NO) often interferes with studies on NO signaling in cell-free preparations. We observed that formation of cGMP by NO-stimulated purified soluble guanylate cyclase (sGC) was virtually abolished in the presence of cytosolic preparations of porcine coronary arteries, with the scavenging activity localized in the tunica media (smooth muscle layer). Electrochemical measurement of NO release from a donor compound and light absorbance spectroscopy showed that cytosolic preparations contained a reduced heme protein that scavenged NO. This protein, which reacted with anti-human hemoglobin antibodies, was efficiently removed from the preparations by haptoglobin affinity chromatography. The cleared cytosols showed only minor scavenging of NO according to electrochemical measurements and did not decrease cGMP formation by NO-stimulated sGC. In contrast, the column flow-through caused a nearly 2-fold increase of maximal sGC activity (from 33.1 ± 1.6 to 54.9 ± 2.2 μmol × min(-1) × mg(-1)). The proteins retained on the affinity column were identified as hemoglobin α and β subunits. The results indicate that hemoglobin, presumably derived from vasa vasorum erythrocytes, is present and scavenges NO in preparations of porcine coronary artery smooth muscle. Selective removal of hemoglobin-mediated scavenging unmasked stimulation of maximal NO-stimulated sGC activity by a soluble factor expressed in vascular tissue.
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http://dx.doi.org/10.1016/j.niox.2016.01.005DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5933522PMC
April 2016

Angiotensin II-Induced Hypertension Is Attenuated by Reduction of Sympathetic Output in NO-Sensitive Guanylyl Cyclase 1 Knockout Mice.

J Pharmacol Exp Ther 2016 Jan 11;356(1):191-9. Epub 2015 Nov 11.

Institut für Pharmakologie, Ruhr-Universität Bochum, Bochum, Germany (K.B., M.R., D.K., E.M.) and Klinik für Nephrologie, Universitätsklinikum Düsseldorf, Heinrich-Heine-Universität Düsseldorf, Germany (J.S., S.A.P.)

In the regulation of vascular tone, the dilatory nitric oxide (NO)/cGMP pathway balances vasoconstriction induced by the renin-angiotensin and sympathetic nervous systems. NO-induced cGMP formation is catalyzed by two guanylyl cyclases (GC), NO-sensitive guanylyl cyclase 1 (NO-GC1) and NO-GC2, with indistinguishable enzymatic properties. In vascular smooth muscle cells, NO-GC1 is the major isoform and is responsible for more than 90% of cGMP formation. Despite reduced vasorelaxation, NO-GC1-deficient mice are not hypertensive. Here, the role of NO-GC1 in hypertension provoked by contractile agonists angiotensin II (Ang II) and norepinephrine (NE) was evaluated in NO-GC1-deficient mice. Hypertension induced by chronic Ang II treatment did not differ between wild-type (WT) and NO-GC1 knockout mice (KO). Also, attenuation of NO-dependent aortic relaxation induced by the Ang II treatment was similar in both genotypes and was most probably attributable to an increase of phosphodiesterase 1 expression. Analysis of plasma NE content-known to be influenced by Ang II-revealed lower NE in the NO-GC1 KO under Ang II-treated- and nontreated conditions. The finding indicates reduced sympathetic output and is underlined by the lower heart rate in the NO-GC1 KO. To find out whether the lack of higher blood pressure in the NO-GC1 KO is a result of reduced sympathetic activity counterbalancing the reduced vascular relaxation, mice were challenged with chronic NE application. As the resulting blood pressure was higher in the NO-GC1 KO than in WT, we conclude that the reduced sympathetic activity in the NO-GC1 KO prevents hypertension and postulate a possible sympatho-excitatory action of NO-GC1 counteracting NO-GC1's dilatory effect in the vasculature.
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http://dx.doi.org/10.1124/jpet.115.227728DOI Listing
January 2016

Cyclic GMP in Vascular Relaxation: Export Is of Similar Importance as Degradation.

Arterioscler Thromb Vasc Biol 2015 Sep 23;35(9):2011-9. Epub 2015 Jul 23.

From the Institut für Pharmakologie und Toxikologie, Medizinische Fakultät, Ruhr-Universität Bochum, Bochum, Germany.

Objective: In the vascular system, cyclic GMP (cGMP) in smooth muscle cells plays an important role for blood vessel relaxation. Intracellular concentrations of cGMP are thought to be determined by the action of cGMP-generating guanylyl cyclases (sensitive to nitric oxide or natriuretic peptides) and cGMP-degrading phosphodiesterases (PDE1, PDE3, and PDE5). Because functionally relevant cGMP elevations are not accessible to conventional methods, we applied real-time imaging with a fluorescence resonance energy transfer (FRET)-based cGMP indicator to follow nitric oxide- and natriuretic peptide-induced cGMP signals in living smooth muscle cells and analyzed the contribution of the miscellaneous cGMP-generating and cGMP-degrading enzymes.

Approach And Results: By comparison of cGMP signals in living smooth muscle cells and vascular relaxation of aortic strips in organ bath experiments, we show for the first time that FRET-based cGMP indicators permit the measurement of functionally relevant cGMP signals. PDE5 was the major cGMP phosphodiesterase responsible for reducing nitric oxide- and natriuretic peptide-induced cGMP signals. In contrast, PDE3-involved in the degradation of lower cGMP concentrations-displayed a preference for natriuretic peptide-stimulated cGMP. Unexpectedly, we found that cGMP is transported out of the cells by the ABC transporter multidrug resistance-associated protein 4 and this export turned out to be of similar importance for intracellular cGMP signals as degradation by PDE5. Functionally, inhibition of cGMP export enhanced vascular relaxation as much as inhibition of PDE5.

Conclusions: The findings indicate that cGMP export out of smooth muscle cells is a key player in the regulation of smooth muscle cGMP signals and blood vessel relaxation.
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http://dx.doi.org/10.1161/ATVBAHA.115.306133DOI Listing
September 2015

Sildenafil Does Not Prevent Heart Hypertrophy and Fibrosis Induced by Cardiomyocyte Angiotensin II Type 1 Receptor Signaling.

J Pharmacol Exp Ther 2015 Sep 8;354(3):406-16. Epub 2015 Jul 8.

Department of Pharmacology, Toxicology and Clinical Pharmacy, Institute of Pharmacy, University of Tübingen, Tübingen, Germany (J.S., V.S., N.B., R.L.); Institute of Experimental Cardiovascular Research, University Medical Center Hamburg-Eppendorf, Hamburg, Germany (H.S., V.O.N.); Physiologisches Institut I, Universität Würzburg, Würzburg, Germany (S.D., A.F.); Institut für Pharmakologie und Toxikologie, Ruhr-Universität Bochum, Bochum, Germany (M.R.); Internal Medicine III, Cardiology and Cardiovascular Medicine, University Hospital Tübingen, Tübingen, Germany (M.G.); Laboratory of Cardiac Development and Differentiation, Department of Biochemistry, Microbiology, and Immunology, University of Ottawa, Ottawa, Ontario, Canada (M.N.); and Institut de Recherches Cliniques de Montréal, Montreal, Quebec, Canada (M.N.)

Analyses of several mouse models imply that the phosphodiesterase 5 (PDE5) inhibitor sildenafil (SIL), via increasing cGMP, affords protection against angiotensin II (Ang II)-stimulated cardiac remodeling. However, it is unclear which cell types are involved in these beneficial effects, because Ang II may exert its adverse effects by modulating multiple renovascular and cardiac functions via Ang II type 1 receptors (AT1Rs). To test the hypothesis that SIL/cGMP inhibit cardiac stress provoked by amplified Ang II/AT1R directly in cardiomyocytes (CMs), we studied transgenic mice with CM-specific overexpression of the AT1R under the control of the α-myosin heavy chain promoter (αMHC-AT1R(tg/+)). The extent of cardiac growth was assessed in the absence or presence of SIL and defined by referring changes in heart weight to body weight or tibia length. Hypertrophic marker genes, extracellular matrix-regulating factors, and expression patterns of fibrosis markers were examined in αMHC-AT1R(tg/+) ventricles (with or without SIL) and corroborated by investigating different components of the natriuretic peptide/PDE5/cGMP pathway as well as cardiac functions. cGMP levels in heart lysates and intact CMs were measured by competitive immunoassays and Förster resonance energy transfer. We found higher cardiac and CM cGMP levels and upregulation of the cGMP-dependent protein kinase type I with AT1R overexpression. However, even a prolonged SIL treatment regimen did not limit the progressive CM growth, fibrosis, or decline in cardiac functions in the αMHC-AT1R(tg/+) model, suggesting that SIL does not interfere with the pathogenic actions of amplified AT1R signaling in CMs. Hence, the cardiac/noncardiac cells involved in the cross-talk between SIL-sensitive PDE activity and Ang II/AT1R still need to be identified.
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http://dx.doi.org/10.1124/jpet.115.226092DOI Listing
September 2015

Phosphodiesterase 10A Is Tethered to a Synaptic Signaling Complex in Striatum.

J Biol Chem 2015 May 11;290(19):11936-47. Epub 2015 Mar 11.

From the Institut für Pharmakologie und Toxikologie, Medizinische Fakultät, Ruhr-Universität-Bochum, 44780 Bochum, Germany

Phosphodiesterase 10A (PDE10A) is a dual substrate PDE that can hydrolyze both cGMP and cAMP. In brain, PDE10A is almost exclusively expressed in the striatum. In several studies, PDE10A has been implicated in regulation of striatal output using either specific inhibitors or PDE10A knock-out mice and has been suggested as a promising target for novel antipsychotic drugs. In striatal medium spiny neurons, PDE10A is localized at the plasma membrane and in dendritic spines close to postsynaptic densities. In the present study, we identify PDE10A as the major cAMP PDE in mouse striatum and monitor PKA-dependent PDE10A phosphorylation. With recombinantly expressed PDE10A we demonstrate that phosphorylation does not alter PDE10A activity. In striatum, PDE10A was found to be associated with the A kinase anchoring protein AKAP150 suggesting the existence of a multiprotein signaling complex localizing PDE10A to a specific functional context at synaptic membranes. Furthermore, the cAMP effector PKA, the NMDA receptor subunits NR2A and -B, as well as PSD95, were tethered to the complex. In agreement, PDE10A was almost exclusively found in multiprotein complexes as indicated by migration in high molecular weight fractions in size exclusion chromatography. Finally, affinity of PDE10A to the signaling complexes formed around AKAP150 was reduced by PDE10A phosphorylation. The data indicate that phosphorylation of PDE10 has an impact on the interaction with other signaling proteins and adds an additional line of complexity to the role of PDE10 in regulation of synaptic transmission.
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http://dx.doi.org/10.1074/jbc.M114.595769DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4424333PMC
May 2015

Transgenic mice for cGMP imaging.

Circ Res 2013 Aug 25;113(4):365-71. Epub 2013 Jun 25.

Interfakultäres Institut für Biochemie, Universität Tübingen, Tübingen, Germany.

Rationale: Cyclic GMP (cGMP) is an important intracellular signaling molecule in the cardiovascular system, but its spatiotemporal dynamics in vivo is largely unknown.

Objective: To generate and characterize transgenic mice expressing the fluorescence resonance energy transfer-based ratiometric cGMP sensor, cGMP indicator with an EC50 of 500 nmol/L (cGi500), in cardiovascular tissues.

Methods And Results: Mouse lines with smooth muscle-specific or ubiquitous expression of cGi500 were generated by random transgenesis using an SM22α promoter fragment or by targeted integration of a Cre recombinase-activatable expression cassette driven by the cytomegalovirus early enhancer/chicken β-actin/β-globin promoter into the Rosa26 locus, respectively. Primary smooth muscle cells isolated from aorta, bladder, and colon of cGi500 mice showed strong sensor fluorescence. Basal cGMP concentrations were < 100 nmol/L, whereas stimulation with cGMP-elevating agents such as 2-(N,N-diethylamino)-diazenolate-2-oxide diethylammonium salt (DEA/NO) or the natriuretic peptides, atrial natriuretic peptide, and C-type natriuretic peptide evoked fluorescence resonance energy transfer changes corresponding to cGMP peak concentrations of ≈ 3 µmol/L. However, different types of smooth muscle cells had different sensitivities of their cGMP responses to DEA/NO, atrial natriuretic peptide, and C-type natriuretic peptide. Robust nitric oxide-induced cGMP transients with peak concentrations of ≈ 1 to > 3 µmol/L could also be monitored in blood vessels of the isolated retina and in the cremaster microcirculation of anesthetized mice. Moreover, with the use of a dorsal skinfold chamber model and multiphoton fluorescence resonance energy transfer microscopy, nitric oxide-stimulated vascular cGMP signals associated with vasodilation were detected in vivo in an acutely untouched preparation.

Conclusions: These cGi500 transgenic mice permit the visualization of cardiovascular cGMP signals in live cells, tissues, and mice under normal and pathological conditions or during pharmacotherapy with cGMP-elevating drugs.
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http://dx.doi.org/10.1161/CIRCRESAHA.113.301063DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3896241PMC
August 2013

Potent inhibition of aldehyde dehydrogenase-2 by diphenyleneiodonium: focus on nitroglycerin bioactivation.

Mol Pharmacol 2013 Sep 21;84(3):407-14. Epub 2013 Jun 21.

Department of Pharmacology and Toxicology, Karl-Franzens-Universität Graz, Austria.

Aldehyde dehydrogenase-2 (ALDH2) catalyzes vascular bioactivation of the antianginal drug nitroglycerin (GTN) to yield nitric oxide (NO) or a related species that activates soluble guanylate cyclase (sGC), resulting in cGMP-mediated vasodilation. Accordingly, established ALDH2 inhibitors attenuate GTN-induced vasorelaxation in vitro and in vivo. However, the ALDH2 hypothesis has not been reconciled with early studies demonstrating potent inhibition of the GTN response by diphenyleneiodonium (DPI), a widely used inhibitor of flavoproteins, in particular NADPH oxidases. We addressed this issue and investigated the effects of DPI on GTN-induced relaxation of rat aortic rings and the function of purified ALDH2. DPI (0.3 µM) inhibited the high affinity component of aortic relaxation to GTN without affecting the response to NO, indicating that the drug interfered with GTN bioactivation. Denitration and bioactivation of 1-2 µM GTN, assayed as 1,2-glycerol dinitrate formation and activation of purified sGC, respectively, were inhibited by DPI with a half-maximally active concentration of about 0.2 µM in a GTN-competitive manner. Molecular modeling indicated that DPI binds to the catalytic site of ALDH2, and this was confirmed by experiments showing substrate-competitive inhibition of the dehydrogenase and esterase activities of the enzyme. Our data identify ALDH2 as highly sensitive target of DPI and explain inhibition of GTN-induced relaxation by this drug observed previously. In addition, the data provide new evidence for the essential role of ALDH2 in GTN bioactivation and may have implications to other fields of ALDH2 research, such as hepatic ethanol metabolism and cardiac ischemia/reperfusion injury.
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http://dx.doi.org/10.1124/mol.113.086835DOI Listing
September 2013

Visualization of cGMP with cGi biosensors.

Methods Mol Biol 2013 ;1020:89-120

Interfakultäres Institut für Biochemie, Universität Tübingen, Tübingen, Germany.

Cyclic guanosine 3'-5'-monophosphate (cGMP) is an important signaling molecule in physiology, pathophysiology, and pharmacological therapy. It has been proposed that the functional outcome of an increase of cGMP in a given cell largely depends on the existence of global versus local cGMP pools. The recent development of genetically encoded fluorescent biosensors for cGMP is a major technical advance in order to monitor the spatiotemporal dynamics and compartmentalization of cGMP signals in living cells. Here we give an overview of the available cGMP sensors and how they can be used to visualize cGMP. The focus is on the fluorescence resonance energy transfer (FRET)-based cGi-type sensors (Russwurm et al., Biochem J 407:69-77, 2007), which are currently among the most useful tools for cGMP imaging in cells, tissues, and living organisms. We present detailed protocols that cover the entire imaging experiment, from the isolation of primary cells from cGi-transgenic mice and adenoviral expression of cGi sensors to the description of the setup required to record FRET changes in single cells and tissues. In-cell calibration of sensors and data evaluation is also described in detail and the limitations and common pitfalls of cGMP imaging are discussed. Specifically, we outline the use of FRET microscopy to visualize cGMP in murine smooth muscle cells (from aorta, bladder, and colon) and cerebellar granule neurons expressing cGi sensors. Most of the protocols can be easily adapted to other cell types and cGMP indicators and can be used as general guidelines for cGMP imaging in living cells, tissues and, eventually, whole organisms.
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http://dx.doi.org/10.1007/978-1-62703-459-3_6DOI Listing
December 2013

NO/cGMP: the past, the present, and the future.

Methods Mol Biol 2013 ;1020:1-16

Pharmakologie und Toxikologie Medizinische, Fakultät, Ruhr-Universität Bochum, Bochum, Germany.

The NO/cGMP signalling cascade participates in the regulation of physiological parameters such as smooth muscle relaxation, inhibition of platelet aggregation, and neuronal transmission. cGMP is formed in response to nitric oxide (NO) by NO-sensitive guanylyl cyclases that exist in two isoforms (NO-GC1 and NO-GC2). Much has been learned about the regulation of NO-GC; however the precise role of cGMP in complex physiological and especially in pathophysiological settings and its alteration by biological factors needs to be established. Despite reports on a variety of cGMP-independent NO effects, KO mice with a complete lack of NO-GC provide evidence that the vasorelaxing and platelet-inhibiting effects of NO are solely mediated by NO-GC. Isoform-specific KOs demonstrate that low cGMP increases are sufficient to induce smooth muscle relaxation and that either NO-GC isoform is sufficient in most instances outside the central nervous system. In the neuronal system, however, the NO-GC isoforms obviously serve distinct functions as both isoforms are required for long-term potentiation and NO-GC1 was shown to enhance glutamate release in excitatory neurons in the hippocampal CA1 region by gating HCN channels. Future studies have to clarify the role of NO-GC2, to show whether HCN channels are general targets of cGMP in the nervous system and whether the NO/cGMP signalling cascade participates in synaptic transmission in other brain regions.
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http://dx.doi.org/10.1007/978-1-62703-459-3_1DOI Listing
December 2013

Activation of PDE10 and PDE11 phosphodiesterases.

J Biol Chem 2012 Jan 21;287(2):1210-9. Epub 2011 Nov 21.

Institut für Pharmakologie und Toxikologie, Medizinische Fakultät, Ruhr-Universität Bochum, 44780 Bochum, Germany.

The most recently identified cyclic nucleotide phosphodiesterases, PDE10 and PDE11, contain a tandem of so-called GAF domains in their N-terminal regulatory regions. In PDE2 and PDE5, the GAF domains mediate cGMP stimulation; however, their function in PDE10 and PDE11 remains controversial. Although the GAF domains of PDE10 mediate cAMP-induced stimulation of chimeric adenylyl cyclases, cAMP binding did not stimulate the PDE10 holoenzyme. Comparable data about cGMP and the PDE11 GAF domains exist. Here, we identified synthetic ligands for the GAF domains of PDE10 and PDE11 to reduce interference of the GAF ligand with the catalytic reaction of PDE. With these ligands, GAF-mediated stimulation of the PDE10 and PDE11 holoenzymes is demonstrated for the first time. Furthermore, PDE10 is shown to be activated by cAMP, which paradoxically results in potent competitive inhibition of cGMP turnover by cAMP. PDE11, albeit susceptible to GAF-dependent stimulation, is not activated by the native cyclic nucleotides cAMP and cGMP. In summary, PDE11 can be stimulated by GAF domain ligands, but its native ligand remains to be identified, and PDE10 is the only PDE activated by cAMP.
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http://dx.doi.org/10.1074/jbc.M111.263806DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3256904PMC
January 2012

A phosphodiesterase 2A isoform localized to mitochondria regulates respiration.

J Biol Chem 2011 Sep 1;286(35):30423-30432. Epub 2011 Jul 1.

Physiological Chemistry, Ruhr-University Bochum, 44780 Bochum, Germany; Department of Biochemistry, University of Bayreuth, 95447 Bayreuth, Germany. Electronic address:

Mitochondria are central organelles in cellular energy metabolism, apoptosis, and aging processes. A signaling network regulating these functions was recently shown to include soluble adenylyl cyclase as a local source of the second messenger cAMP in the mitochondrial matrix. However, a mitochondrial cAMP-degrading phosphodiesterase (PDE) necessary for switching off this cAMP signal has not yet been identified. Here, we describe the identification and characterization of a PDE2A isoform in mitochondria from rodent liver and brain. We find that mitochondrial PDE2A is located in the matrix and that the unique N terminus of PDE2A isoform 2 specifically leads to mitochondrial localization of this isoform. Functional assays show that mitochondrial PDE2A forms a local signaling system with soluble adenylyl cyclase in the matrix, which regulates the activity of the respiratory chain. Our findings complete a cAMP signaling cascade in mitochondria and have implications for understanding the regulation of mitochondrial processes and for their pharmacological modulation.
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http://dx.doi.org/10.1074/jbc.M111.266379DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3162402PMC
September 2011

Site-directed mutagenesis of aldehyde dehydrogenase-2 suggests three distinct pathways of nitroglycerin biotransformation.

Mol Pharmacol 2011 Aug 2;80(2):258-66. Epub 2011 May 2.

Department of Pharmacology and Toxicology, Karl-Franzens-Universität Graz, Universitätsplatz 2, A-8010, Austria.

To elucidate the mechanism underlying reduction of nitroglycerin (GTN) to nitric oxide (NO) by mitochondrial aldehyde dehydrogenase (ALDH2), we generated mutants of the enzyme lacking the cysteines adjacent to reactive Cys302 (C301S and C303S), the glutamate that participates as a general base in aldehyde oxidation (E268Q) or combinations of these residues. The mutants were characterized regarding acetaldehyde dehydrogenation, GTN-triggered enzyme inactivation, GTN denitration, NO formation, and soluble guanylate cyclase activation. Lack of the cysteines did not affect dehydrogenase activity but impeded GTN denitration, aggravated GTN-induced enzyme inactivation, and increased NO formation. A triple mutant lacking the cysteines and Glu268 catalyzed sustained formation of superstoichiometric amounts of NO and exhibited slower rates of inactivation. These results suggest three alternative pathways for the reaction of ALDH2 with GTN, all involving formation of a thionitrate/sulfenyl nitrite intermediate at Cys302 as the initial step. In the first pathway, which predominates in the wild-type enzyme and reflects clearance-based GTN denitration, the thionitrate apparently reacts with one of the adjacent cysteine residues to yield nitrite and a protein disulfide. The predominant reaction catalyzed by the single and double cysteine mutants requires Glu268 and results in irreversible enzyme inactivation. Finally, combined lack of the cysteines and Glu268 shifts the reaction toward formation of the free NO radical, presumably through homolytic cleavage of the sulfenyl nitrite intermediate. Although the latter reaction accounts for less than 10% of total turnover of GTN metabolism catalyzed by wild-type ALDH2, it is most likely essential for vascular GTN bioactivation.
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http://dx.doi.org/10.1124/mol.111.071704DOI Listing
August 2011

Crystal structure of the GAF-B domain from human phosphodiesterase 5.

Proteins 2011 May 21;79(5):1682-7. Epub 2011 Mar 21.

Department of Pharmacology and Toxicology, Ruhr-University Bochum, Germany.

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http://dx.doi.org/10.1002/prot.22989DOI Listing
May 2011

Bioactivation of pentaerythrityl tetranitrate by mitochondrial aldehyde dehydrogenase.

Mol Pharmacol 2011 Mar 14;79(3):541-8. Epub 2010 Dec 14.

Department of Pharmacology and Toxicology, Karl-Franzens-Universität Graz, Graz, Austria.

Mitochondrial aldehyde dehydrogenase (ALDH2) contributes to vascular bioactivation of the antianginal drugs nitroglycerin (GTN) and pentaerythrityl tetranitrate (PETN), resulting in cGMP-mediated vasodilation. Although continuous treatment with GTN results in the loss of efficacy that is presumably caused by inactivation of ALDH2, PETN does not induce vascular tolerance. To clarify the mechanisms underlying the distinct pharmacological profiles of GTN and PETN, bioactivation of the nitrates was studied with aortas isolated from ALDH2-deficient and nitrate-tolerant mice, isolated mitochondria, and purified ALDH2. Pharmacological inhibition or gene deletion of ALDH2 attenuated vasodilation to both GTN and PETN to virtually the same degree as long-term treatment with GTN, whereas treatment with PETN did not cause tolerance. Purified ALDH2 catalyzed bioactivation of PETN, assayed as activation of soluble guanylate cyclase (sGC) and formation of nitric oxide (NO). The EC(50) value of PETN for sGC activation was 2.2 ± 0.5 μM. Denitration of PETN to pentaerythrityl trinitrate was catalyzed by ALDH2 with a specific activity of 9.6 ± 0.8 nmol · min(-1) · mg(-1) and a very low apparent affinity of 94.7 ± 7.4 μM. In contrast to GTN, PETN did not cause significant inactivation of ALDH2. Our data suggest that ALDH2 catalyzes bioconversion of PETN in two distinct reactions. Besides the major denitration pathway, which occurs only at high PETN concentrations, a minor high-affinity pathway may reflect vascular bioactivation of the nitrate yielding NO. The very low rate of ALDH2 inactivation, presumably as a result of low affinity of the denitration pathway, may at least partially explain why PETN does not induce vascular tolerance.
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http://dx.doi.org/10.1124/mol.110.069138DOI Listing
March 2011

Activation of PDE2 and PDE5 by specific GAF ligands: delayed activation of PDE5.

Br J Pharmacol 2010 Dec;161(7):1645-60

Institut für Pharmakologie und Toxikologie, Medizinische Fakultät, Ruhr-Universität-Bochum, Bochum, Germany.

Background And Purpose: By controlling intracellular cyclic nucleotide levels, phosphodiesterases (PDE) serve important functions within various signalling pathways. The PDE2 and PDE5 families are allosterically activated by their substrate cGMP via regulatory so-called GAF domains. Here, we set out to identify synthetic ligands for the GAF domains of PDE2 and PDE5.

Experimental Approach: Using fluorophore-tagged, isolated GAF domains of PDE2 and PDE5, promising cGMP analogues were selected. Subsequently, the effects of these analogues on the enzymatic activity of PDE2 and PDE5 were analysed.

Key Results: The PDE2 ligands identified, 5,6-DM-cBIMP and 5,6-DCl-cBIMP, caused pronounced, up to 40-fold increases of the cAMP- and cGMP-hydrolysing activities of PDE2. The ligand for the GAF domains of PDE5, 8-Br-cGMP, elicited a 20-fold GAF-dependent activation and moreover revealed a time-dependent increase in PDE5 activity that occurred independently of a GAF ligand. Although GAF-dependent PDE5 activation was fast at high ligand concentrations, it was slow at physiologically relevant cGMP concentrations; PDE5 reached its final catalytic rates at 1µM cGMP after approximately 10min.

Conclusions And Implications: We conclude that the delayed activation of PDE5 is required to shape biphasic, spike-like cGMP signals. Phosphorylation of PDE5 further enhances activity and conserves PDE5 activation, thereby enabling PDE5 to act as a molecular memory balancing cGMP responses to nitric oxide or natriuretic peptide signals.
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http://dx.doi.org/10.1111/j.1476-5381.2010.00977.xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3010573PMC
December 2010

Decreased renal corin expression contributes to sodium retention in proteinuric kidney diseases.

Kidney Int 2010 Oct 7;78(7):650-9. Epub 2010 Jul 7.

Institute of Anatomy, Charité Universitätsmedizin, Berlin, Germany.

Patients with proteinuric kidney diseases often have symptoms of salt and water retention. It has been hypothesized that dysregulated sodium absorption is due to increased proteolytic cleavage of epithelial sodium channels (ENaCs) and increased Na,K-ATPase expression. Microarray analysis identified a reduction in kidney corin mRNA expression in rat models of puromycin aminonucleoside-induced nephrotic syndrome and acute anti-Thy1 glomerulonephritis (GN). As atrial natriuretic peptide (ANP) resistance is a mechanism accounting for volume retention, we analyzed the renal expression and function of corin; a type II transmembrane serine protease that converts pro-ANP to active ANP. Immunohistochemical analysis found that corin colocalized with ANP. The nephrotic and glomerulonephritic models exhibited concomitant increased pro-ANP and decreased ANP protein levels in the kidney consistent with low amounts of corin. Importantly, kidneys from corin knockout mice had increased amounts of renal β-ENaC and its activators, phosphodiesterase (PDE) 5 and protein kinase G II, when compared to wild-type mice. A similar expression profile was also found in cell culture suggesting the increase in PDE5 and kinase G II could account for the increase in β-ENaC seen in nephrotic syndrome and GN. Thus, we suggest that corin might be involved in the salt retention seen in glomerular diseases.
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http://dx.doi.org/10.1038/ki.2010.197DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4433746PMC
October 2010

Characterization of the East Asian variant of aldehyde dehydrogenase-2: bioactivation of nitroglycerin and effects of Alda-1.

J Biol Chem 2010 Jan 11;285(2):943-52. Epub 2009 Nov 11.

Department of Pharmacology and Toxicology, Karl-Franzens-Universität Graz, 8010 Graz, Austria

The East Asian variant of mitochondrial aldehyde dehydrogenase (ALDH2) exhibits significantly reduced dehydrogenase, esterase, and nitroglycerin (GTN) denitrating activities. The small molecule Alda-1 was reported to partly restore low acetaldehyde dehydrogenase activity of this variant. In the present study we compared the wild type enzyme (ALDH2*1) with the Asian variant (ALDH2*2) regarding GTN bioactivation and the effects of Alda-1. Alda-1 increased acetaldehyde oxidation by ALDH2*1 and ALDH2*2 approximately 1.5- and 6-fold, respectively, and stimulated the esterase activities of both enzymes to similar extent as the coenzyme NAD. The effect of NAD was biphasic with pronounced inhibition occurring at > or = 5 mM. In the presence of 1 mM NAD, Alda-1 stimulated ALDH2*2-catalyzed ester hydrolysis 73-fold, whereas the NAD-stimulated activity of ALDH2*1 was inhibited because of 20-fold increased inhibitory potency of NAD in the presence of the drug. Although ALDH2*2 exhibited 7-fold lower GTN denitrating activity and GTN affinity than ALDH2*1, the rate of nitric oxide formation was only reduced 2-fold, and soluble guanylate cyclase (sGC) activation was more pronounced than with wild type ALDH2 at saturating GTN. Alda-1 caused slight inhibition of GTN denitration and did not increase GTN-induced sGC activation in the presence of either variant. The present results indicate that Alda-1 stimulates established ALDH2 activities by improving NAD binding but does not improve the GTN binding affinity of the Asian variant. In addition, our data revealed an unexpected discrepancy between GTN reductase activity and sGC activation, suggesting that GTN denitration and bioactivation may reflect independent pathways of ALDH2-catalyzed GTN biotransformation.
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http://dx.doi.org/10.1074/jbc.M109.014548DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2801295PMC
January 2010

Mechanisms underlying activation of soluble guanylate cyclase by the nitroxyl donor Angeli's salt.

Mol Pharmacol 2009 Nov 31;76(5):1115-22. Epub 2009 Aug 31.

Department of Pharmacology and Toxicology, Karl-Franzens-Universität Graz, Graz, Austria.

Nitroxyl (HNO) may be formed endogenously by uncoupled nitric-oxide (NO) synthases, enzymatic reduction of NO or as product of vascular nitroglycerin bioactivation. The established HNO donor Angeli's salt (trioxodinitrate, AS) causes cGMP-dependent vasodilation through activation of soluble guanylate cyclase (sGC). We investigated the mechanisms underlying this effect using purified sGC and cultured endothelial cells. AS (up to 0.1 mM) had no significant effect on sGC activity in the absence of superoxide dismutase (SOD) or dithiothreitol (DTT). In the presence of SOD, AS caused biphasic sGC activation (apparent EC(50) approximately 10 nM, maximum at 1 microM) that was accompanied by the formation of NO. DTT (2 mM) inhibited the effects of <10 microM AS but led to sGC activation and NO release at 0.1 mM AS even without SOD. AS had no effect on ferric sGC, excluding activation of the oxidized enzyme by HNO. The NO scavenger carboxy-PTIO inhibited endothelial cGMP accumulation induced by AS in the presence but not in the absence of SOD (EC(50) approximately 50 nM and approximately 16 microM, respectively). Carboxy-PTIO (0.1 mM) inhibited the effect of
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http://dx.doi.org/10.1124/mol.109.059915DOI Listing
November 2009
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