Gasotransmitters: Physiology and Pathophysiology

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2 Gasotransmitters: Physiology and Pathophysiology

3 Anton Hermann Guzel F. Sitdikova Thomas M. Weiger Editors Gasotransmitters: Physiology and Pathophysiology 123

4 Editors Prof. Dr. Anton Hermann Department of Cell Biology Division of Cellular and Molecular Neurobiology University of Salzburg Salzburg Austria A.o. Prof. Dr. Thomas M. Weiger Department of Cell Biology Division of Cellular and Molecular Neurobiology University of Salzburg Salzburg Austria Prof. Dr. Guzel F. Sitdikova Department of Human and Animal Physiology Kazan Federal University Kazan Russia ISBN ISBN (ebook) DOI / Springer Heidelberg New York Dordrecht London Library of Congress Control Number: Ó Springer-Verlag Berlin Heidelberg 2012 This work is subject to copyright. All rights are reserved by the Publisher, whether the whole or part of the material is concerned, specifically the rights of translation, reprinting, reuse of illustrations, recitation, broadcasting, reproduction on microfilms or in any other physical way, and transmission or information storage and retrieval, electronic adaptation, computer software, or by similar or dissimilar methodology now known or hereafter developed. Exempted from this legal reservation are brief excerpts in connection with reviews or scholarly analysis or material supplied specifically for the purpose of being entered and executed on a computer system, for exclusive use by the purchaser of the work. Duplication of this publication or parts thereof is permitted only under the provisions of the Copyright Law of the Publisher s location, in its current version, and permission for use must always be obtained from Springer. Permissions for use may be obtained through RightsLink at the Copyright Clearance Center. Violations are liable to prosecution under the respective Copyright Law. The use of general descriptive names, registered names, trademarks, service marks, etc. in this publication does not imply, even in the absence of a specific statement, that such names are exempt from the relevant protective laws and regulations and therefore free for general use. While the advice and information in this book are believed to be true and accurate at the date of publication, neither the authors nor the editors nor the publisher can accept any legal responsibility for any errors or omissions that may be made. The publisher makes no warranty, express or implied, with respect to the material contained herein. Printed on acid-free paper Springer is part of Springer Science+Business Media (

5 Preface From ancient Greek philosophers the idea evolved that an animalic spirit called pneuma pervades through our body acting primarily at the brain, the heart, and the liver. As a vital force it allowed the various parts of the body to communicate with each other. This idea survived several 100 years longer than most of our present hypothesis. It came as a great surprise to the scientific community when researchers discovered that poisonous gases, endogenously produced by a great variety of living organisms from bacteria to men, exert important intra- and intercellular tasks kind of reviving the pneuma idea. Since the epochal discovery of the radical nitric oxide (NO) as gaseous signaling molecule two other gases carbon monoxide (CO) and hydrogen sulfide (H 2 S) have been found to be also involved in a plethora of physiological and pathophysiological functions. The gases, now a family of at least three, have been termed gasotransmitters. A definition of molecules to be classified as gasotransmitters is given by Untereiner et al. in their chapter. The most prominent features that characterize the gases and discriminate them from classical transmitters are their amphiphilic chemical nature that allows them to diffuse in the cytosol as well as through lipid membranes which prevents them from being stored in vesicles. The gases, because of their small size and hence high diffusion rate can, immediately after they are released, act in autocrine or paracrine fashion and in contrast to the classical transmitters are not localized to specific synaptic sites. Since the gases may affect many cells in their vicinity this function has been called volume signalling. Of course, this kind of signaling is not as punctual in targeting postsynaptic cells as it is the case with classical synaptic transmission and its action is highly dependent on the concentration gradient within the tissue. Volume signaling is particularly interesting in the brain where thousands or millions of synaptic contacts may be affected. The implication of such signals on nervous function and information processing in the central nervous system, however, remains to be investigated in detail. In experiments using knockout animals it could be shown for all gasotransmitters that after elimination of appropriate enzymes the gases are no longer produced and the expected pathophysiological modifications developed, v

6 vi Preface i.e., metabolic or erectile dysfunctions, or high blood pressure. Since the book on Signal Transduction and the Gasotransmitters: NO, CO, and H 2 S in Biology and Medicine, has been issued by Rui Wang (2004) an impressive amount of data has been collected and a great wealth of further information is distributed in the literature. We have asked distinguished colleagues in the field to summarize and review important biological, pharmacological, and medical functions on gasotransmitters and their implications. The authors were asked in particular to critically review the literature from their point of view and to ask questions and even speculate on new vistas. Ulrich Förstermann and Huige Li in their chapter Nitric Oxide: Biological Synthesis and Functions summarize principles of NO biosynthesis, regulatory mechanisms, and a large array of physiological and pathophysiological functions. NO, due to its highly reactive chemical nature, is also capable of destroying parasites and tumor cells; however, in high concentrations it exhibits a Janus face contributing to processes such as neurodegeneration, inflammation, and tissue damage. Ashley Untereiner et al. describe in their chapter The role of CO as a gasotransmitter in cardiovascular and metabolic regulation the production, physiological functions, such as in proliferation and apoptosis, pathophysiological actions of CO, as in diabetes, vascular diseases, hypertension, atherosclerosis, or myocardial infarction. In their final sections they summarize cellular and molecular mechanisms of CO effects including ion channel and receptor signaling and discuss the interaction of CO with other gases. Hideo Kimura in his chapter on Physiological and pathophysiological functions of hydrogen sulfide, after introducing some basic properties and the amphipathic chemistry of H 2 S, its free and bound conditions, describes some detection methods and the endogenous enzymatic production of the gas. Furthermore, physiological functions, such as synaptic modulation in the brain and in the retina, in smooth muscle relaxation, its cytoprotective and pathophysiological roles in particular in ethylmalonic encephalopathy, in Down s syndrome or in vascular dysfunctions, and some therapeutic implications are covered. In the chapter by Hanjing Peng et al. on Methods for detection of gasotransmitters a great deal of chemical and technical details are summarized. Various new techniques and chemoprobes for measuring all three gases, their applicability to biological systems, and their advantages and limitations are discussed. Electrochemical measurements appear most sensitive and allow for determination of temporal concentration changes, whereas fluorescent probes are favorable for spatial monitoring in living cells. Guzel Sitdikova and Andrey Zefirov specialized in their chapter Gasotransmitters on the regulation of neuromuscular transmission. All three gases are produced in the central nervous system in response to neural excitation and modulate neurotransmitter release and are involved in the regulation of synaptic plasticity affecting pre- or postsynaptic sites by different mechanisms. The authors summarize the literature and present own data concerning the effects and

7 Preface vii mechanisms of the transmitter gases in the peripheral nervous system focusing on neuromuscular synapses. Finally, Anton Hermann et al. focus on Ca 2+ activated BK channel modulation by gasotransmitters. These ion channels, which are present in a large variety of cells and organs, are prominent targets of the gases. The structure and functions of these channels and their pharmacology and posttranslational modifications are described. BK channel modulation through gasotransmitters and their implication for physiology and pathophysiology are highlighted. The advent of gasotransmitters has profoundly changed our way of thinking about biosynthesis, liberation, storage, and action mechanisms by cellular signaling. The gases will certainly play an increasingly important role to understand how cellular signaling is modulated and fine-tuned, particularly in the brain. The investigation of the interaction of NO, CO, and H 2 S is still at its infancy! More knowledge is needed concerning the metabolic products of gasotransmitters, in particular of NO and H 2 S, and the functions of some related molecules, such as nitrosonium cation (NO + ), which is isoelectronic with CO or the hyponitrite anion (NO - ). Future studies will have to probe into further details of their physiology, pathophysiology, and pharmacology. The development of drugs containing specific active ingredients with little or no side effects to manipulate the ana-/ metabolism of gasotransmitters or their targets could be of an interesting and probably fruitful pharmacological task. Anton Hermann Guzel Sitdikova Thomas Weiger

8 Contents 1 Nitric Oxide: Biological Synthesis and Functions Ulrich Förstermann and Huige Li 2 The Role of Carbon Monoxide as a Gasotransmitter in Cardiovascular and Metabolic Regulation Ashley A. Untereiner, Lingyun Wu and Rui Wang 3 Physiological and Pathophysiological Functions of Hydrogen Sulfide Hideo Kimura 4 Methods for the Detection of Gasotransmitters Hanjing Peng, Weixuan Chen and Binghe Wang 5 Gasotransmitters in Regulation of Neuromuscular Transmission Guzel F. Sitdikova and Andrey L. Zefirov 6 Modulated by Gasotransmitters: BK Channels Anton Hermann, Guzel F. Sitdikova and Thomas M. Weiger Index ix

9 Chapter 1 Nitric Oxide: Biological Synthesis and Functions Ulrich Förstermann and Huige Li Abstract The pluripotent gaseous messenger molecule nitric oxide (NO) controls vital functions such as neurotransmission or vascular tone (via activation of soluble guanylyl cyclase), gene transcription, mrna translation (via iron-responsive elements), and post-translational modifications of proteins (via ADP-ribosylation). In higher concentrations, NO is capable of destroying parasites and tumor cells by inhibiting iron-containing enzymes or directly interacting with the DNA of these cells. In view of this multitude of functions of NO, it is important to understand the mechanisms by which cells accomplish and regulate the production of this molecule. In mammals, three isozymes of NO synthase (NOS; L-arginine, NADPH:oxygen oxidoreductases, nitric oxide forming; EC ) have been identified. These isoforms are referred to as neuronal n NOS (or NOS I), inducible i NOS (or NOS II), and endothelial e NOS (or NOS III). In pathophysiology, massive amounts of NO produced by hyperactive nnos or highly expressed inos can contribute to processes such as neurodegeneration, inflammation, and tissue damage. This chapter will describe principles of NO biosynthesis, regulatory mechanisms controlling the production of this molecule, and the large array of (physiologic and pathophysiologic) functions that Mother Nature has assigned to this small messenger molecule. Keywords (6R)-5,6,7,8-tetrahydrobiopterin Glutathione L-arginine Asymmetric dimethyl-l-arginine NADPH oxidase Peroxynitrite U. Förstermann (&) H. Li Department of Pharmacology, Johannes Gutenberg University Medical Center, Obere Zahlbacher Strasse 67, Mainz, Germany ulrich.forstermann@uni-mainz.de A. Hermann et al. (eds.), Gasotransmitters: Physiology and Pathophysiology, DOI: / _1, Ó Springer-Verlag Berlin Heidelberg

10 2 U. Förstermann and H. Li Abbreviations AMP Adenosine monophosphate ADMA Asymmetric dimethyl-l-arginine ADP Adenosine diphosphate Ala Alanine Akt Serine/threonine kinase (= protein kinase B) AMPK AMP-activated protein kinase Asp Aspartate AVE fluoro-N-indan-2-yl-benzamide (enos expression enhancer) BH 2 Quinonoid 6,7-[8H]-H 2 -biopterin. BH 3 Trihydrobiopterin radical BH. 3 H + Trihydropterin radical cation protonated at N5 BH 4 (6R)-5,6,7,8-tetrahydro-L-biopterin CAPON Carboxy-terminal PDZ ligand of nnos CaM Calmodulin CaMK Ca 2+ /Calmodulin-dependent protein kinase cyclic GMP Cyclic guanosine monophosphate DDAH Dimethylarginine dimethylaminohydrolase Dexras1 A small monomeric G protein found predominantly in brain enos Endothelial nitric oxide synthase FAD Flavin adenine dinucleotide FMN Flavin mononucleotide GLGF Glycine, leucine, glycine, phenylalanine motif H 2 O 2 Hydrogen peroxide hsp90 Heat shock protein 90 hsp70 Heat shock protein 70 inos Inducible nitric oxide synthase LLC-PK 1 Porcine kidney tubular epithelial cells L-NMMA N G -monomethyl-l-arginine LPS Bacterial lipopolysaccharide mrna Messenger ribonucleic acid NAP110 NOS-associated protein 110 kda NADPH Reduced nicotinamide adenine dinucleotide phosphate NMDA N-methyl-D-aspartate nnos Neuronal nitric oxide synthase NO Nitric oxide NOS Nitric oxide synthase NOSIP Nitric oxide synthase interacting protein -. O 2 Superoxide anion ONOO - Peroxynitrite PARP Poly(ADP-ribose)polymerase PDZ Postsynaptic density protein 95/discs large/zo-1 homology domain PIN Protein inhibitor of nnos PFK-M Phosphofructokinase (muscle type)

11 1 Nitric Oxide: Biological Synthesis and Functions 3 PKA PKC PRMT Ser SMTC ROS Thr Tyr VEGF Protein kinase A Protein kinase C Protein arginine N-methyltransferase Serine S-methyl-L-thiocitrulline (inhibitor of nnos) Reactive oxygen species Threonine Tyrosine Vascular endothelial growth factor 1.1 Introduction Nitric oxide (NO) is a gaseous messenger molecule, that controls servoregulatory functions such as neurotransmission (O Dell et al. 1991; Schuman and Madison 1991) or vascular tone (Rapoport et al. 1983; Förstermann et al. 1986) (by stimulating NO sensitive guanylyl cyclase), regulates gene transcription (Khan et al. 1996;Gudietal.1999)and mrna translation (for example by binding to iron-responsive elements) (Pantopoulos and Hentze 1995; Liu et al. 2002), and produces post-translational modifications of proteins (for example by ADP-ribosylation) (Pozdnyakov et al. 1993; Brune et al. 1994). An important mode of inactivation of NO is its reaction with superoxide anion (O 2 -. )to form the potent oxidant peroxynitrite (ONOO - ). This compound can cause oxidative damage, nitration, and S-nitrosylation of biomolecules including proteins, lipids, and DNA (Mikkelsen and Wardman 2003; Lee et al. 2003). Nitrosative stress by ONOO - has been implicated in DNA single strand breakage, followed by poly(adp-ribose) polymerase (PARP) activation (Ridnour et al. 2004). NO synthases (NOS; L-arginine, NADPH: oxygen oxidoreductases, nitric oxide forming; EC ) are phylogenetically old enzymes and exist in organisms as low as nematodes, protozoa, and even in plants. Three different mammalian isoforms of the enzyme have been identified: neuronal n NOS (or NOS I), inducible i NOS (or NOS II), and endothelial e NOS (or NOS III) (Förstermann et al. 1994; Förstermann and Sessa 2011; Fig. 1.1). 1.2 Three Mammalian Isoforms of NOS Basic Characteristics of the Three Isozymes nnos is a low output enzyme that is constitutively expressed in neurons and some other cell types. inos is a high output enzyme whose expression can be induced by cytokines and other agents in almost any cell type. Its activity is largely Ca 2+ -independent. enos is also a low output enzyme that is constitutively expressed in

12 4 U. Förstermann and H. Li Oxygenase Domain Reductase Domain GLGF (PDZ) nnos (size: 161 kda) Heme Fe L-Arg BH 4 CaM FMN FAD NADPH pyro iso rib ade 58% Sequence identity (aa) inos (size: 131 kda) Heme Fe L-Arg BH 4 enos (size: 133 kda) 52% Sequence identity (aa) palm myr Heme Fe L-Arg BH 4 57% Sequence identity (aa) Fig. 1.1 Schematic diagram of the spatial relationships between the three isoforms of NOS. Alignment of the deduced amino acid sequences of the three cloned NOS isoforms demonstrates % sequence identity among the enzymes. All NOS isoforms consist of a reductase domain and an oxygenase domain. The reductase domain of the enzymes shows about 35 % sequence identity with cytochrome P 450 reductase, and this enzyme shares the cofactor binding regions of the NOS for reduced nicotinamide adenine dinucleotide phosphate (NADPH), flavin adenine dinucleotide (FAD), and flavin mononucleotide (FMN). Consensus sequences for the binding of the cosubstrate NADPH (adenine and ribose), FAD (isoalloxazine and pyrophosphate), FMN, calmodulin (CaM), and heme are indicated. The proximal part of the N-terminal oxygenase domain (black box) shows % sequence identity among the three NOS isoforms and contains the closely linked binding sites for L-arginine (L-Arg) and (6R)-5,6,7,8-tetrahydro-L-biopterin (BH 4 ). The dominant splice form of nnos depicted here (nnosa) contains an N-terminal tail that includes a GLGF (glycine, leucine, glycine, phenylalanine) motif or PDZ (postsynaptic density protein 95/ discs large/zo-1 homology) domain. This motif targets nnos to other cytoskeletal proteins in brain and skeletal muscle. enos contains N-terminal myristoylation (myr) and palmitoylation (palm) sites. These lipid anchors contribute to the membrane localization of enos endothelial cells and few other cell types. The nature of nnos and enos as low output enzymes and inos as a high output enzyme depends not so much on the conversion rate of the different isozymes, but rather reflects the short-lasting, pulsatile, Ca 2+ - activated NO production of nnos and enos versus the continuous, Ca 2+ -independent NO production by inos (Förstermann et al. 1994). All isoforms are well conserved across mammalian species ([90 % amino acid identity for nnos and enos, [80 % for inos). Within the human species, amino acid sequences of the three NOS isoforms share % identity (Fig. 1.1). Despite the similarities between nnos and enos, nnos, has a sixfold higher maximal catalytic activity than enos (Ilagan et al. 2008). This may become relevant under pathologic conditions when a maximal and sustained activation on nnos contributes to neurotoxicity.

13 1 Nitric Oxide: Biological Synthesis and Functions Structural and Mechanistic Aspects of NO Synthesis L-arginine is the substrate for all isoforms of NOS; molecular oxygen and reduced nicotinamide adenine dinucleotide phosphate (NADPH) are cosubstrates. Flavin adenine dinucleotide (FAD), flavin mononucleotide (FMN), and (6R)-5,6,7,8-tetrahydro-L-biopterin (BH 4 ) are cofactors of all isozymes. All NOS enzymes are homodimers. Functionally, the C-terminal reductase domain of one monomer (that binds NADPH, FMN, and FAD) is linked to the N-terminal oxygenase domain of the other monomer (Fig. 1.2). This oxygenase domain carries a prosthetic heme group. The oxygenase domain also binds BH 4, molecular oxygen, and the substrate L-arginine (Crane et al. 1998; Alderton et al. 2001). Sequences located near the cysteine ligand of the heme are apparently also involved in L-arginine and BH 4 binding; in fact, the prosthetic heme group interacts with the BH 4 binding domain and is also part of the amino acid substrate site (Nishimura et al. 1995). All three NOS isoforms show a zinc thiolate cluster formed by a zinc ion that is tetrahedrally coordinated to two CXXXXC motifs (one contributed by each monomer) at the NOS dimer interface (Hemmens et al. 2000; Raman et al. 1998; Li et al. 1999; Miller et al. 1999). Chemical removal of Zn from NOS or the possibility of expressing a zinc-deficient NOS that remained catalytically active, demonstrated that the zinc in NOS has a structural rather than a catalytic function. All NOS isozymes catalyze flavin-mediated electron transfer from the C-terminally bound NADPH to the heme on the N-terminus (Fig. 1.2). In nnos and enos, calmodulin (CaM) binding to the enzymes is brought about by an increase in intracellular Ca 2+ (half-maximal activity between 200 and 400 nm). This leads to an enhanced binding of CaM to the enzyme, which in turn displaces an auto-inhibitory loop and facilitates the flow of electrons from NADPH in the reductase domain to the heme in the oxygenase domain (Hemmens and Mayer 1998). In inos, calmodulin already binds at low intracellular Ca 2+ concentrations (below 40 nm) due to a different amino acid structure of the calmodulin binding site (Cho et al. 1992). Therefore, inos activity is largely Ca 2+ -independent. At the heme, the electrons are utilized to reduce and activate O 2. In order to synthesize NO, the enzyme needs to cycle twice. In a first step, NOS hydroxylates L-arginine to N x -hydroxy-l-arginine (which remains largely bound to the enzyme). In a second step, NOS oxidizes N-hydroxy-L-arginine to citrulline and NO (Noble et al. 1999; Stuehr et al. 2001;Fig.1.2). The NO formed by NOS can act on a number of target enzymes and proteins. The most important physiologic pathway stimulated by NO is the activation of soluble guanylyl cyclase and the generation of cyclic GMP (Rapoport et al. 1983; Furchgott et al. 1984; Förstermann et al. 1986; Knowles et al. 1989; Garthwaite 1991) The Neuronal Isoform of NO Synthase The neuronal isoform of NO synthase (nnos) has first been found constitutively expressed in specific neurons of the central nervous system. The activity of the enzyme is regulated by Ca 2+ and calmodulin. In addition to brain tissue, nnos has

14 6 U. Förstermann and H. Li NADP + + H + NADPH Reductase Domains e - e - FAD FMN NADP + + H + NADPH FMN FAD e - e - O 2 O 2 -. Monomer 2 Monomer 1 Oxygenase Domains (a) NH 2 Fig. 1.2 a Basic structure of NOS and scheme of NOS catalysis. All NOS enzymes are primarily c synthesized as monomers. Each subunit consists of a reductase domain and an oxygenase domain. Monomers and even isolated reductase domains are able to transfer electrons from NADPH to the flavins FAD and FMN, and have a limited capacity to reduce molecular oxygen to superoxide (O -. 2 ). Monomers and isolated reductase domains can bind calmodulin (CaM), which stimulates the electron transfer within the reductase domain. However, monomers are unable to bind the cofactor (6R-)5,6,7,8-tetrahydrobiopterin (BH 4 ) or the substrate L-arginine and cannot catalyze NO production. b The presence of heme allows for NOS dimerization; in fact, heme is the only cofactor that is absolutely required for the formation of active NOS dimers. Heme is also essential for the interaction between reductase and oxygenase domains and for the interdomain electron transfer from the flavins to the heme of the opposite monomer. NADPH oxidation rates are significantly enhanced in heme-containing substrate-free NOS dimers compared with monomers, consistent with a more effective O -. 2 production. All NOS isoforms show a zinc thiolate cluster formed by a zinc ion that is tetrahedrally coordinated to two CXXXXC motifs (one contributed by each monomer) at the NOS dimer interface. This zinc in NOS seems to have structural rather than catalytic function. c When sufficient substrate L-arginine and cofactor BH 4 are present, intact NOS dimers couple their heme- and O 2 -reduction to the synthesis of NO. L-citrulline is formed as the byproduct; N x -hydroxy-l-arginine is an intermediate product in the reaction. Note the cooperative binding of L-arginine and BH 4 to the enzyme been identified by immunohistochemistry in peripheral nitrergic nerves, in epithelial cells of various organs, in kidney macula densa cells, in pancreatic islet cells, in spinal cord, in sympathetic ganglia and adrenal glands, and in vascular smooth muscle (Förstermann et al. 1994). In mammalians, the largest source of nnos in terms of tissue mass is skeletal muscle (Nakane et al. 1993; Förstermann et al. 1994).

15 O 1 Nitric Oxide: Biological Synthesis and Functions 7 NADP + + H + NADPH FAD O 2 FMN Reductase Domains FMN NADP + + H + NADPH e - FAD e - CaM CaM Heme Fe Heme Fe Zn O 2 O 2 -. Homodimer Oxygenase Domains (b) NADPH NADP + + H + FMN O 2 Reductase Domains NADP + + H + NADPH FAD FMN e - FAD e - O 2 L-Arg O CaM Heme L-Arg Fe BH 4 Zn CaM Heme Fe BH 4 L-Arg - O O 2 L-Cit NO NO L-Cit Homodimer Oxygenase Domains (c) Fig. 1.2 continued