Publications by authors named "Doris Koesling"

75 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

Nitric Oxide/Cyclic Guanosine Monophosphate Signaling via Guanylyl Cyclase Isoform 1 Mediates Early Changes in Synaptic Transmission and Brain Edema Formation after Traumatic Brain Injury.

J Neurotrauma 2021 Jun 16;38(12):1689-1701. Epub 2021 Feb 16.

Institute of Physiology, University Medical Center of the Johannes Gutenberg University Mainz, Mainz, Germany.

Traumatic brain injury (TBI) often induces structural damage, disruption of the blood-brain barrier (BBB), neurodegeneration, and dysfunctions of surviving neuronal networks. Nitric oxide (NO) signaling has been suggested to affect brain functions after TBI. The NO exhibits most of its biological effects by activation of the primary targets-guanylyl cyclases (NO-GCs), which exists in two isoforms (NO-GC1 and NO-GC2), and the subsequently produced cyclic guanosine monophosphate (cGMP). However, the specific function of the NO-NO-GCs-cGMP pathway in the context of brain injury is not fully understood. To investigate the specific role of the isoform NO-GC1 early after brain injuries, we performed an unilateral controlled cortical impact (CCI) in the somatosensory cortex of knockout mice lacking NO-GC1 and their wild-type (WT) littermates. Morphological and electrophysiological changes of cortical neurons located 500 μm distant from the lesion border were studied early (24 h) after TBI. The CCI-operated WT mice exhibited significant BBB disruption, an impairment of dendritic spine morphology, a reduced pre-synaptic glutamate release, and less neuronal activity in the ipsilateral cortical network. The impaired ipsilateral neuronal excitability was associated with increased A-type K currents () in the WT mice early after TBI. Interestingly, NO-GC1 KO mice revealed relatively less BBB rupture and a weaker brain edema formation early after TBI. Further, lack of NO-GC1 also prevented the impaired synaptic transmission and network function that were observed in TBI-treated WT mice. These data suggest that NO-GC1 signaling mediates early brain damage and the strength of ipsilateral cortical network in the early phase after TBI.
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http://dx.doi.org/10.1089/neu.2020.7364DOI Listing
June 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

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

Distinct functions of soluble guanylyl cyclase isoforms NO-GC1 and NO-GC2 in inflammatory and neuropathic pain processing.

Pain 2019 Mar;160(3):607-618

Pharmakologisches Institut für Naturwissenschaftler, Goethe-Universität, Frankfurt am Main, Germany.

A large body of evidence indicates that nitric oxide (NO)/cGMP signaling essentially contributes to the processing of chronic pain. In general, NO-induced cGMP formation is catalyzed by 2 isoforms of guanylyl cyclase, NO-sensitive guanylyl cyclase 1 (NO-GC1) and 2 (NO-GC2). However, the specific functions of the 2 isoforms in pain processing remain elusive. Here, we investigated the distribution of NO-GC1 and NO-GC2 in the spinal cord and dorsal root ganglia, and we characterized the behavior of mice lacking either isoform in animal models of pain. Using immunohistochemistry and in situ hybridization, we demonstrate that both isoforms are localized to interneurons in the spinal dorsal horn with NO-GC1 being enriched in inhibitory interneurons. In dorsal root ganglia, the distribution of NO-GC1 and NO-GC2 is restricted to non-neuronal cells with NO-GC2 being the major isoform in satellite glial cells. Mice lacking NO-GC1 demonstrated reduced hypersensitivity in models of neuropathic pain, whereas their behavior in models of inflammatory pain was normal. By contrast, mice lacking NO-GC2 exhibited increased hypersensitivity in models of inflammatory pain, but their neuropathic pain behavior was unaltered. Cre-mediated deletion of NO-GC1 or NO-GC2 in spinal dorsal horn neurons recapitulated the behavioral phenotypes observed in the global knockout. Together, these results indicate that cGMP produced by NO-GC1 or NO-GC2 in spinal dorsal horn neurons exert distinct, and partly opposing, functions in chronic pain processing.
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http://dx.doi.org/10.1097/j.pain.0000000000001440DOI Listing
March 2019

Modulation of Hyperpolarization-Activated Inward Current and Thalamic Activity Modes by Different Cyclic Nucleotides.

Front Cell Neurosci 2018 24;12:369. Epub 2018 Oct 24.

Institut für Physiologie I, Westfälische Wilhelms-Universität, Münster, Germany.

The hyperpolarization-activated inward current, I, plays a key role in the generation of rhythmic activities in thalamocortical (TC) relay neurons. Cyclic nucleotides, like 3',5'-cyclic adenosine monophosphate (cAMP), facilitate voltage-dependent activation of hyperpolarization-activated cyclic nucleotide-gated (HCN) channels by shifting the activation curve of I to more positive values and thereby terminating the rhythmic burst activity. The role of 3',5'-cyclic guanosine monophosphate (cGMP) in modulation of I is not well understood. To determine the possible role of the nitric oxide (NO)-sensitive cGMP-forming guanylyl cyclase 2 (NO-GC2) in controlling the thalamic I, the voltage-dependency and cGMP/cAMP-sensitivity of I was analyzed in TC neurons of the dorsal part of the lateral geniculate nucleus (dLGN) in wild type (WT) and NO-GC2-deficit (NO-GC2) mice. Whole cell voltage clamp recordings in brain slices revealed a more hyperpolarized half maximal activation (V) of I in NO-GC2 TC neurons compared to WT. Different concentrations of 8-Br-cAMP/8-Br-cGMP induced dose-dependent positive shifts of V in both strains. Treatment of WT slices with lyase enzyme (adenylyl and guanylyl cyclases) inhibitors (SQ22536 and ODQ) resulted in further hyperpolarized V. Under current clamp conditions NO-GC2 neurons exhibited a reduction in the I-dependent voltage sag and reduced action potential firing with hyperpolarizing and depolarizing current steps, respectively. Intrathalamic rhythmic bursting activity in brain slices and in a simplified mathematical model of the thalamic network was reduced in the absence of NO-GC2. In freely behaving NO-GC2 mice, delta and theta band activity was enhanced during active wakefulness (AW) as well as rapid eye movement (REM) sleep in cortical local field potential (LFP) in comparison to WT. These findings indicate that cGMP facilitates I activation and contributes to a tonic activity in TC neurons. On the network level basal cGMP production supports fast rhythmic activity in the cortex.
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http://dx.doi.org/10.3389/fncel.2018.00369DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6207575PMC
October 2018

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

Nitric oxide dependent signaling via cyclic GMP in dendritic cells regulates migration and T-cell polarization.

Sci Rep 2018 Jul 20;8(1):10969. Epub 2018 Jul 20.

Department of Experimental Pneumology, Medical Faculty, Ruhr University Bochum, 44780, Bochum, Germany.

Allergic airway inflammation is accompanied by excessive generation of nitric oxide (NO). Beside its detrimental activity due to the generation of reactive nitrogen species, NO was found to modulate immune responses by activating the NO-sensitive Guanylyl Cyclases (NO-GCs) thereby mediating the formation of the second messenger cyclic GMP (cGMP). To investigate the contribution of the key-enzyme NO-GC on the development of Th2 immunity in vivo, we sensitized knock-out (KO) mice of the major isoform NO-GC1 to the model allergen ovalbumin (OVA). The loss of NO-GC1 attenuates the Th2 response leading to a reduction of airway inflammation and IgE production. Further, in vitro-generated OVA-presenting DCs of the KO induce only a weak Th2 response in the WT recipient mice upon re-exposure to OVA. In vitro, these NO-GC1 KO BMDCs develop a Th1-polarizing phenotype and display increased cyclic AMP (cAMP) formation, which is known to induce Th1-bias. According to our hypothesis of a NO-GC1/cGMP-dependent regulation of cAMP-levels we further demonstrate activity of the cGMP-activated cAMP-degrading phosphodiesterase 2 in DCs. Herewith, we show that activity of NO-GC1 in DCs is important for the magnitude and bias of the Th response in allergic airway disease most likely by counteracting intracellular cAMP.
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http://dx.doi.org/10.1038/s41598-018-29287-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6054623PMC
July 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

Impact of the NO-Sensitive Guanylyl Cyclase 1 and 2 on Renal Blood Flow and Systemic Blood Pressure in Mice.

Int J Mol Sci 2018 Mar 23;19(4). Epub 2018 Mar 23.

Department of Nephrology, Medical Faculty, University Hospital Düsseldorf, Heinrich-Heine-University Düsseldorf, 40225 Düsseldorf, Germany.

Nitric oxide (NO) modulates renal blood flow (RBF) and kidney function and is involved in blood pressure (BP) regulation predominantly via stimulation of the NO-sensitive guanylyl cyclase (NO-GC), existing in two isoforms, NO-GC1 and NO-GC2. Here, we used isoform-specific knockout (KO) mice and investigated their contribution to renal hemodynamics under normotensive and angiotensin II-induced hypertensive conditions. Stimulation of the NO-GCs by -nitrosoglutathione (GSNO) reduced BP in normotensive and hypertensive wildtype (WT) and NO-GC2-KO mice more efficiently than in NO-GC1-KO. NO-induced increase of RBF in normotensive mice did not differ between the genotypes, but the respective increase under hypertensive conditions was impaired in NO-GC1-KO. Similarly, inhibition of endogenous NO increased BP and reduced RBF to a lesser extent in NO-GC1-KO than in NO-GC2-KO. These findings indicate NO-GC1 as a target of NO to normalize RBF in hypertension. As these effects were not completely abolished in NO-GC1-KO and renal cyclic guanosine monophosphate (cGMP) levels were decreased in both NO-GC1-KO and NO-GC2-KO, the results suggest an additional contribution of NO-GC2. Hence, NO-GC1 plays a predominant role in the regulation of BP and RBF, especially in hypertension. However, renal NO-GC2 appears to compensate the loss of NO-GC1, and is able to regulate renal hemodynamics under physiological conditions.
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http://dx.doi.org/10.3390/ijms19040967DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5979494PMC
March 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

Identification of a soluble guanylate cyclase in RBCs: preserved activity in patients with coronary artery disease.

Redox Biol 2018 04 8;14:328-337. Epub 2017 Sep 8.

Cardiovascular Research Laboratory, Division of Cardiology, Pneumology and Vascular Medicine, Medical Faculty, Heinrich Heine University, Moorenstraße 5, 40225 Düsseldorf, Germany; CARID, Cardiovascular Research Institute Düsseldorf, Medical Faculty, Heinrich Heine University, Moorensstraße 5, 40225 Düsseldorf, Germany.

Endothelial dysfunction is associated with decreased NO bioavailability and impaired activation of the NO receptor soluble guanylate cyclase (sGC) in the vasculature and in platelets. Red blood cells (RBCs) are known to produce NO under hypoxic and normoxic conditions; however evidence of expression and/or activity of sGC and downstream signaling pathway including phopshodiesterase (PDE)-5 and protein kinase G (PKG) in RBCs is still controversial. In the present study, we aimed to investigate whether RBCs carry a functional sGC signaling pathway and to address whether this pathway is compromised in coronary artery disease (CAD). Using two independent chromatographic procedures, we here demonstrate that human and murine RBCs carry a catalytically active αβ-sGC (isoform 1), which converts P-GTP into P-cGMP, as well as PDE5 and PKG. Specific sGC stimulation by NO+BAY 41-2272 increases intracellular cGMP-levels up to 1000-fold with concomitant activation of the canonical PKG/VASP-signaling pathway. This response to NO is blunted in α1-sGC knockout (KO) RBCs, but fully preserved in α2-sGC KO. In patients with stable CAD and endothelial dysfunction red cell eNOS expression is decreased as compared to aged-matched controls; by contrast, red cell sGC expression/activity and responsiveness to NO are fully preserved, although sGC oxidation is increased in both groups. Collectively, our data demonstrate that an intact sGC/PDE5/PKG-dependent signaling pathway exists in RBCs, which remains fully responsive to NO and sGC stimulators/activators in patients with endothelial dysfunction. Targeting this pathway may be helpful in diseases with NO deficiency in the microcirculation like sickle cell anemia, pulmonary hypertension, and heart failure.
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http://dx.doi.org/10.1016/j.redox.2017.08.020DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5975213PMC
April 2018

NO-Sensitive Guanylate Cyclase Isoforms NO-GC1 and NO-GC2 Contribute to Noise-Induced Inner Hair Cell Synaptopathy.

Mol Pharmacol 2017 10;92(4):375-388

Department of Otolaryngology, Head and Neck Surgery, Hearing Research Centre Tübingen, Molecular Physiology of Hearing, University of Tübingen, Tübingen (D.M., K.R., S.W., K.V., N.E., H.-S.G., U.Z., M.K., L.R.), Interfaculty Institute of Biochemistry, University of Tübingen, Tübingen (M.W., R.F.), Department of Pharmacology and Toxicology, University of Bochum, Bochum (E.M., D.K.), Bayer AG, Drug Discovery Pharma Research Centre Wuppertal, Wuppertal (P.S.), Department of Pharmacology, Toxicology and Clinical Pharmacy, Institute of Pharmacy, University of Tübingen, Tübingen (P.R.), and Department of Physiology, University of Würzburg, Würzburg (A.F.), Germany

Nitric oxide (NO) activates the NO-sensitive soluble guanylate cyclase (NO-GC, sGC) and triggers intracellular signaling pathways involving cGMP. For survival of cochlear hair cells and preservation of hearing, NO-mediated cascades have both protective and detrimental potential. Here we examine the cochlear function of mice lacking one of the two NO-sensitive guanylate cyclase isoforms [NO-GC1 knockout (KO) or NO-GC2 KO]. The deletion of NO-GC1 or NO-GC2 did not influence electromechanical outer hair cell (OHC) properties, as measured by distortion product otoacoustic emissions, neither before nor after noise exposure, nor were click- or noise-burst-evoked auditory brainstem response thresholds different from controls. Yet inner hair cell (IHC) ribbons and auditory nerve responses showed significantly less deterioration in NO-GC1 KO and NO-GC2 KO mice after noise exposure. Consistent with a selective role of NO-GC in IHCs, NO-GC 1 mRNA was found in isolated IHCs but not in OHCs. Using transgenic mice expressing the fluorescence resonance energy transfer-based cGMP biosensor cGi500, NO-induced elevation of cGMP was detected in real-time in IHCs but not in OHCs. Pharmacologic long-term treatment with a NO-GC stimulator altered auditory nerve responses but did not affect OHC function and hearing thresholds. Interestingly, NO-GC stimulation exacerbated the loss of auditory nerve response in aged animals but attenuated the loss in younger animals. We propose NO-GC2 and, to some degree, NO-GC1 as targets for early pharmacologic prevention of auditory fiber loss (synaptopathy). Both isoforms provide selective benefits for hearing function by maintaining the functional integrity of auditory nerve fibers in early life rather than at old age.
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http://dx.doi.org/10.1124/mol.117.108548DOI Listing
October 2017

Nitric oxide/cGMP signaling via guanylyl cyclase isoform 1 modulates glutamate and GABA release in somatosensory cortex of mice.

Neuroscience 2017 Sep 4;360:180-189. Epub 2017 Aug 4.

Institute of Physiology, UMC of the Johannes Gutenberg University Mainz, Duesbergweg 6, 55128 Mainz, Germany. Electronic address:

In hippocampus, two guanylyl cyclases (NO-GC1 and NO-GC2) are involved in the transduction of the effects of nitric oxide (NO) on synaptic transmission. However, the respective roles of the NO-GC isoforms on synaptic transmission are less clear in other regions of the brain. In the present study, we used knock-out mice deficient for the NO-GC1 isoform (NO-GC1 KO) to analyze its role in the glutamatergic and GABAergic neurotransmission at pyramidal neurons in layers II/III of somatosensory cortex. NO-GC1 KO slices revealed reduced frequencies of miniature excitatory- and inhibitory-postsynaptic currents, increased paired-pulse ratios and decreased input-output curves of evoked signals, which indicated the reduction of glutamate and GABA release in NO-GC1 KO mice. The functional changes in NO-GC1 KO mice were caused by the lack of cGMP as they were rescued to WT-like levels by the cGMP analog, 8-Br-PET-cGMP and conversely, mimicked by the NO-GC inhibitor, ODQ, in WT slices. In search of a cGMP target, two blockers of hyperpolarization-activated cyclic nucleotide-gated (HCN) channels (ZD7288 and DK-AH269) reduced glutamate release in WT to the level of NO-GC1 KO mice suggesting HCN channels as possible effectors for presynaptic cGMP enhancing the glutamate release probability. By blocking postsynaptic NMDA receptors, the NMDA receptor-dependent NO signal was shown to be linked to the effect of NO-GC1 on presynaptic GABA release. Of note, the balance between glutamatergic and GABAergic inputs at individual synapses remained unaltered in the NO-GC1 KO mice. In sum, our results indicate a role for cGMP generated by presynaptic localized NO-GC1 to adjust inhibitory and excitatory inputs at individual synapses in the somatosensory cortex.
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http://dx.doi.org/10.1016/j.neuroscience.2017.07.063DOI Listing
September 2017

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

Stimulators of the soluble guanylyl cyclase: promising functional insights from rare coding atherosclerosis-related GUCY1A3 variants.

Basic Res Cardiol 2016 07 24;111(4):51. Epub 2016 Jun 24.

Klinik für Herz- und Kreislauferkrankungen, Deutsches Herzzentrum München, Technische Universität München, Lazarettstr. 36, 80636, Munich, Germany.

Stimulators of the soluble guanylyl cyclase (sGC) are emerging therapeutic agents in cardiovascular diseases. Genetic alterations of the GUCY1A3 gene, which encodes the α1 subunit of the sGC, are associated with coronary artery disease. Studies investigating sGC stimulators in subjects with CAD and carrying risk-related variants in sGC are, however, lacking. Here, we functionally investigate the impact of coding GUCY1A3 variants on sGC activity and the therapeutic potential of sGC stimulators in vitro. In addition to a known loss-of-function variant, eight coding variants in GUCY1A3 were cloned and expressed in HEK 293 cells. Protein levels and dimerization capability with the β1 subunit were analysed by immunoblotting and co-immunoprecipitation, respectively. All α1 variants found in MI patients dimerized with the β1 subunit. Protein levels were reduced by 72 % in one variant (p < 0.01). Enzymatic activity was analysed using cGMP radioimmunoassay after stimulation with a nitric oxide (NO) donor. Five variants displayed decreased cGMP production upon NO stimulation (p < 0.001). The addition of the sGC stimulator BAY 41-2272 increased cGMP formation in all of these variants (p < 0.01). Except for the variant leading to decreased protein level, cGMP amounts reached the wildtype NO-induced level after addition of BAY 41-2272. In conclusion, rare coding variants in GUCY1A3 lead to reduced cGMP formation which can be rescued by a sGC stimulator in vitro. These results might therefore represent the starting point for discovery of novel treatment strategies for patients at risk with coding GUCY1A3 variants.
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http://dx.doi.org/10.1007/s00395-016-0570-5DOI Listing
July 2016

Proatherosclerotic Effect of the α1-Subunit of Soluble Guanylyl Cyclase by Promoting Smooth Muscle Phenotypic Switching.

Am J Pathol 2016 08 15;186(8):2220-2231. Epub 2016 Jun 15.

Institute for Integrative and Experimental Genomics, University of Lübeck, DZHK (German Centre for Cardiovascular Research), Partner Site Hamburg/Kiel/Lübeck, University Heart Centre Lübeck, Lübeck, Germany. Electronic address:

Soluble guanylate cyclase (sGC), a key enzyme of the nitric oxide signaling pathway, is formed as a heterodimer by various isoforms of its α and β subunit. GUCY1A3, encoding the α1 subunit, was identified as a risk gene for coronary artery disease and myocardial infarction, but its specific contribution to atherosclerosis remains unclear. This study sought to decipher the role of Gucy1a3 in atherosclerosis in mice. At age 32 weeks and after 20 weeks of standard or high-fat diet, Gucy1a3(-/-)/Ldlr(-/-) mice exhibited a significant reduction of the atherosclerotic plaque size at the aortic root and the aorta for high-fat diet animals as compared with Ldlr(-/-) control mice. Collagen content in plaques in the aortic root was reduced, suggesting an alteration of smooth muscle cell function. Proliferation and migration were reduced in Gucy1a3(-/-) primary aortic smooth muscle cells (AoSMCs), and proliferation was also reduced in human AoSMCs after inhibition of sGC by 1H-[1,2,4] oxadiazolo [4,3-a] quinoxalin-1-one. Gucy1a3 deficiency in AoSMCs prevents their phenotypic switching, as indicated by the differential expression of marker proteins. The inherited Gucy1a3(-/-) loss exerts an atheroprotective effect. We suggest that sGC activity promotes the phenotypic switching of smooth muscle cells from a contractile to a synthetic state, fostering the formation of atherosclerosis. Preventing this switch by sGC inhibition may provide a novel target in atherosclerotic disease.
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http://dx.doi.org/10.1016/j.ajpath.2016.04.010DOI Listing
August 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

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

Phosphodiesterase 5 attenuates the vasodilatory response in renovascular hypertension.

PLoS One 2013 15;8(11):e80674. Epub 2013 Nov 15.

Klinik für Nephrologie, Universitätsklinikum Heinrich-Heine-Universität Düsseldorf, Düsseldorf, Germany.

NO/cGMP signaling plays an important role in vascular relaxation and regulation of blood pressure. The key enzyme in the cascade, the NO-stimulated cGMP-forming guanylyl cyclase exists in two enzymatically indistinguishable isoforms (NO-GC1, NO-GC2) with NO-GC1 being the major NO-GC in the vasculature. Here, we studied the NO/cGMP pathway in renal resistance arteries of NO-GC1 KO mice and its role in renovascular hypertension induced by the 2-kidney-1-clip-operation (2K1C). In the NO-GC1 KOs, relaxation of renal vasculature as determined in isolated perfused kidneys was reduced in accordance with the marked reduction of cGMP-forming activity (80%). Noteworthy, increased eNOS-catalyzed NO formation was detected in kidneys of NO-GC1 KOs. Upon the 2K1C operation, NO-GC1 KO mice developed hypertension but the increase in blood pressures was not any higher than in WT. Conversely, operated WT mice showed a reduction of cGMP-dependent relaxation of renal vessels, which was not found in the NO-GC1 KOs. The reduced relaxation in operated WT mice was restored by sildenafil indicating that enhanced PDE5-catalyzed cGMP degradation most likely accounts for the attenuated vascular responsiveness. PDE5 activation depends on allosteric binding of cGMP. Because cGMP levels are lower, the 2K1C-induced vascular changes do not occur in the NO-GC1 KOs. In support of a higher PDE5 activity, sildenafil reduced blood pressure more efficiently in operated WT than NO-GC1 KO mice. All together our data suggest that within renovascular hypertension, cGMP-based PDE5 activation terminates NO/cGMP signaling thereby providing a new molecular basis for further pharmacological interventions.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0080674PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3829872PMC
August 2014

Dysfunctional nitric oxide signalling increases risk of myocardial infarction.

Nature 2013 Dec 10;504(7480):432-6. Epub 2013 Nov 10.

Charité Research Group on Geriatrics, Charité-Universitätsmedizin, 10117 Berlin, Germany.

Myocardial infarction, a leading cause of death in the Western world, usually occurs when the fibrous cap overlying an atherosclerotic plaque in a coronary artery ruptures. The resulting exposure of blood to the atherosclerotic material then triggers thrombus formation, which occludes the artery. The importance of genetic predisposition to coronary artery disease and myocardial infarction is best documented by the predictive value of a positive family history. Next-generation sequencing in families with several affected individuals has revolutionized mutation identification. Here we report the segregation of two private, heterozygous mutations in two functionally related genes, GUCY1A3 (p.Leu163Phefs*24) and CCT7 (p.Ser525Leu), in an extended myocardial infarction family. GUCY1A3 encodes the α1 subunit of soluble guanylyl cyclase (α1-sGC), and CCT7 encodes CCTη, a member of the tailless complex polypeptide 1 ring complex, which, among other functions, stabilizes soluble guanylyl cyclase. After stimulation with nitric oxide, soluble guanylyl cyclase generates cGMP, which induces vasodilation and inhibits platelet activation. We demonstrate in vitro that mutations in both GUCY1A3 and CCT7 severely reduce α1-sGC as well as β1-sGC protein content, and impair soluble guanylyl cyclase activity. Moreover, platelets from digenic mutation carriers contained less soluble guanylyl cyclase protein and consequently displayed reduced nitric-oxide-induced cGMP formation. Mice deficient in α1-sGC protein displayed accelerated thrombus formation in the microcirculation after local trauma. Starting with a severely affected family, we have identified a link between impaired soluble-guanylyl-cyclase-dependent nitric oxide signalling and myocardial infarction risk, possibly through accelerated thrombus formation. Reversing this defect may provide a new therapeutic target for reducing the risk of myocardial infarction.
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http://dx.doi.org/10.1038/nature12722DOI Listing
December 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

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

Postsynaptic NO/cGMP increases NMDA receptor currents via hyperpolarization-activated cyclic nucleotide-gated channels in the hippocampus.

Cereb Cortex 2014 Jul 28;24(7):1923-36. Epub 2013 Feb 28.

Institute of Physiology and Pathophysiology, University Medical Center of the Johannes-Gutenberg University, D-55128 Mainz, Germany and.

The nitric oxide (NO)/cyclic guanosine monophosphate (cGMP) signaling cascade participates in the modulation of synaptic transmission. The effects of NO are mediated by the NO-sensitive cGMP-forming guanylyl cyclases (NO-GCs), which exist in 2 isoforms with indistinguishable regulatory properties. The lack of long-term potentiation (LTP) in knock-out (KO) mice deficient in either one of the NO-GC isoforms indicates the contribution of both NO-GCs to LTP. Recently, we showed that the NO-GC1 isoform is located presynaptically in glutamatergic neurons and increases the glutamate release via hyperpolarization-activated cyclic nucleotide (HCN)-gated channels in the hippocampus. Electrophysiological analysis of hippocampal CA1 neurons in whole-cell recordings revealed a reduction of HCN currents and a hyperpolarizing shift of the activation curve in the NO-GC2 KOs associated with reduced resting membrane potentials. These features were mimicked in wild-type (WT) neurons with an NO-GC inhibitor. Analysis of glutamate receptors revealed a cGMP-dependent reduction of NMDA receptor currents in the NO-GC2 KO mice, which was mimicked in WT by HCN channel inhibition. Lowering extracellular Mg(2+) increased NMDA receptor currents in the NO-GC2 KO and allowed the induction of LTP that was absent at physiological Mg(2+). In sum, our data indicate that postsynaptic cGMP increases the N-methyl-D-aspartate (NMDA) receptor current by gating HCN channels and thereby is required for LTP.
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http://dx.doi.org/10.1093/cercor/bht048DOI Listing
July 2014
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