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The Concise Guide to PHARMACOLOGY 2023/24 is the sixth in this series of biennial publications. The Concise Guide provides concise overviews, mostly in tabular format, of the key properties of approximately 1800 drug targets, and nearly 6000 interactions with about 3900 ligands. There is an emphasis on selective pharmacology (where available), plus links to the open access knowledgebase source of drug targets and their ligands (https://www.guidetopharmacology.org/), which provides more detailed views of target and ligand properties. Although the Concise Guide constitutes almost 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/10.1111/bph.16180. Catalytic receptors 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, enzymes 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-2023, and supersedes data presented in the 2021/22, 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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Bases de Datos Farmacéuticas , Farmacología , Humanos , Ligandos , Receptores Acoplados a Proteínas G , Canales Iónicos/química , Receptores Citoplasmáticos y NuclearesRESUMEN
Chronic smoking causes dysfunction of vascular endothelial cells, evident as a reduction of flow-mediated dilation in smokers, but the role of nicotine is still controversial. Given the increasing use of e-cigarettes and other nicotine products, it appears essential to clarify this issue. We studied extracts from cigarette smoke (CSE) and vapor from e-cigarettes (EVE) and heated tobacco (HTE) for their effects on vascular relaxation, endothelial nitric oxide signaling, and the activity of soluble guanylyl cyclase. The average nicotine concentrations of CSE, EVE, and HTE were 164, 800, and 85 µM, respectively. At a dilution of 1:3, CSE almost entirely inhibited the relaxation of rat aortas and porcine coronary arteries to acetylcholine and bradykinin, respectively, while undiluted EVE, with a 15-fold higher nicotine concentration, had no significant effect. With about 50% inhibition at 1:2 dilution, the effect of HTE was between CSE and EVE. Neither extract affected endothelium-independent relaxation to an NO donor. At the dilutions tested, CSE was not toxic to cultured endothelial cells but, in contrast to EVE, impaired NO signaling and inhibited NO stimulation of soluble guanylyl cyclase. Our results demonstrate that nicotine does not mediate the impaired endothelium-dependent vascular relaxation caused by smoking.
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Cigarrillo Electrónico a Vapor , Sistemas Electrónicos de Liberación de Nicotina , Contaminación por Humo de Tabaco , Ratas , Animales , Porcinos , Nicotina/farmacología , Células Endoteliales , Óxido Nítrico , Guanilil Ciclasa Soluble , EndotelioRESUMEN
The occurrence of NO/cGMP signalling in cardiac cells is a matter of debate. Recent measurements with a FRET-based cGMP indicator in isolated cardiac cells revealed NO-induced cGMP signals in cardiac fibroblasts while cardiomyocytes were devoid of these signals. In a fibroblast/myocyte co-culture model though, cGMP formed in fibroblasts in response to NO entered cardiomyocytes via gap junctions. Here, we demonstrate gap junction-mediated cGMP transfer from cardiac fibroblasts to myocytes in intact tissue. In living cardiac slices of mice with cardiomyocyte-specific expression of a FRET-based cGMP indicator (αMHC/cGi-500), NO-dependent cGMP signals were shown to occur in myocytes, to depend on gap junctions and to be degraded mainly by PDE3. Stimulation of NO-sensitive guanylyl cyclase enhanced Forskolin- and Isoproterenol-induced cAMP and phospholamban phosphorylation. Genetic inactivation of NO-GC in Tcf21-expressing cardiac fibroblasts abrogated the synergistic action of NO-GC stimulation on Iso-induced phospholamban phosphorylation, identifying fibroblasts as cGMP source and substantiating the necessity of cGMP-transfer to myocytes. In sum, NO-stimulated cGMP formed in cardiac fibroblasts enters cardiomyocytes in native tissue where it exerts an inhibitory effect on cAMP degradation by PDE3, thereby increasing cAMP and downstream effects in cardiomyocytes. Hence, enhancing ß-receptor-induced contractile responses appears as one of NO/cGMP's functions in the non-failing heart.
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Corazón , Células Cultivadas , Animales , Ratones , Fibroblastos/metabolismo , GMP Cíclico/metabolismo , Óxido Nítrico/metabolismo , Células Musculares/metabolismo , AMP Cíclico/metabolismo , Fosfodiesterasas de Nucleótidos Cíclicos Tipo 3/metabolismo , Transducción de Señal , Supervivencia CelularRESUMEN
The enzyme, nitric oxide-sensitive guanylyl cyclase (NO-GC), is activated by binding NO to its prosthetic heme group and catalyzes the formation of cGMP. The NO-GC is primarily known to mediate vascular smooth muscle relaxation in the lung, and inhaled NO has been successfully used as a selective pulmonary vasodilator. In comparison, NO-GC's impact on the regulation of airway tone is less acknowledged and, most importantly, little is known about the issue that NO-GC signaling is accomplished by two isoforms: NO-GC1 and NO-GC2, implying the existence of distinct "cGMP pools." Herein, we investigated the functional role of the NO-GC isoforms in respiration by measuring lung function parameters of isoform-specific knockout (KO) mice using noninvasive and invasive techniques. Our data revealed the participation and ongoing influence of NO-GC1-derived cGMP in the regulation of airway tone by showing that respiratory resistance was enhanced in NO-GC1-KOs and increased more pronouncedly after the challenge with the bronchoconstrictor methacholine. The tissue resistance and stiffness of NO-GC1-KOs were also higher because of narrowed airways that cause tissue distortion. Contrariwise, NO-GC2-KOs displayed reduced tissue elasticity, elastic recoil, and airway reactivity to methacholine, which did not even increase in an ovalbumin model of asthma that induced hyperresponsiveness in NO-GC1-KOs. In addition, conscious NO-GC2-KOs showed a higher breathing rate with a shorter duration of inspiration and expiration time, which remained faster even in the presence of bronchoconstrictors that slow down breathing. Thus, we provide evidence of two distinct NO/cGMP pathways in airways, accomplished by either NO-GC1 or NO-GC2, adjusting differentially the airway reactivity.
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Broncoconstrictores , Guanilato Ciclasa , Animales , GMP Cíclico/metabolismo , Guanilato Ciclasa/metabolismo , Hemo , Cloruro de Metacolina/farmacología , Ratones , Ratones Noqueados , Óxido Nítrico/metabolismo , Ovalbúmina , Isoformas de Proteínas/metabolismo , Guanilil Ciclasa Soluble/metabolismo , VasodilatadoresRESUMEN
In the central nervous system, the nitric oxide (NO)/cyclic guanosine monophosphate (cGMP) signalling cascade has an established role in fine-tuning of synaptic transmission. In the present study, we asked which isoform of NO-sensitive guanylyl cyclase, NO-GC1 or NO-GC2, is responsible for generation of N-methyl-d-aspartate (NMDA)- and AMPA (α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid)-induced cGMP signals and which of the phosphodiesterases (PDEs) is responsible for degradation. To this end, we performed live cell fluorescence measurements of primary hippocampal neurons isolated from NO-GC isoform-deficient mice. Although both isoforms contributed to the NMDA- and AMPA-induced cGMP signals, NO-GC2 clearly played the predominant role. Whereas under PDE-inhibiting conditions the cGMP levels elicited by both glutamatergic ligands were comparable, NMDA-induced cGMP signals were clearly higher than the AMPA-induced ones in the absence of PDE inhibitors. Thus, AMPA-induced cGMP signals are more tightly controlled by PDE-mediated degradation than NMDA-induced signals. In addition, these findings are compatible with the existence of at least two different pools of cGMP in both of which PDE1 and PDE2-known to be highly expressed in the hippocampus-are mainly responsible for cGMP degradation. The finding that distinct pools of cGMP are equipped with different amounts of PDEs highlights the importance of PDEs for the shape of NO-induced cGMP signals in the central nervous system.
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N-Metilaspartato , Óxido Nítrico , Animales , GMP Cíclico/metabolismo , Hipocampo/metabolismo , Ratones , N-Metilaspartato/farmacología , Óxido Nítrico/metabolismo , Hidrolasas Diéster Fosfóricas/metabolismo , Isoformas de Proteínas/metabolismo , Ácido alfa-Amino-3-hidroxi-5-metil-4-isoxazol Propiónico/metabolismoRESUMEN
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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Bases de Datos Farmacéuticas , Farmacología , Humanos , Canales Iónicos , Ligandos , Receptores Citoplasmáticos y Nucleares , Receptores Acoplados a Proteínas GRESUMEN
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 in vivo 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 (IA) 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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Edema Encefálico/patología , Lesiones Traumáticas del Encéfalo/patología , Guanilato Ciclasa/metabolismo , Óxido Nítrico/metabolismo , Receptores de Superficie Celular/metabolismo , Transmisión Sináptica/fisiología , Animales , Edema Encefálico/etiología , Lesiones Traumáticas del Encéfalo/complicaciones , GMP Cíclico/metabolismo , Isoenzimas/metabolismo , Ratones , Ratones Noqueados , Transducción de Señal/fisiología , Corteza Somatosensorial/lesiones , Corteza Somatosensorial/patologíaRESUMEN
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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Sistema Cardiovascular/enzimología , Sistema Cardiovascular/patología , Guanilato Ciclasa/metabolismo , Óxido Nítrico/metabolismo , Angiotensina II , Animales , Aorta/patología , Fibrosis , Masculino , Ratones Endogámicos C57BL , Ratones Noqueados , ARN Mensajero/genética , ARN Mensajero/metabolismoRESUMEN
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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Canales de Calcio Tipo L/metabolismo , Corteza Cerebral/metabolismo , GMP Cíclico/metabolismo , Hipocampo/metabolismo , Óxido Nítrico/metabolismo , Ácido alfa-Amino-3-hidroxi-5-metil-4-isoxazol Propiónico/farmacología , Animales , Células Cultivadas , Corteza Cerebral/citología , Corteza Cerebral/efectos de los fármacos , Hipocampo/citología , Hipocampo/efectos de los fármacos , Ratones , Ratones de la Cepa 129 , Ratones Endogámicos C57BL , Ratones Transgénicos , Neuronas/efectos de los fármacos , Neuronas/metabolismo , Técnicas de Cultivo de ÓrganosRESUMEN
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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Aorta/efectos de los fármacos , Aorta/fisiología , Sistemas Electrónicos de Liberación de Nicotina , Endotelio Vascular/efectos de los fármacos , Endotelio Vascular/metabolismo , Aromatizantes/farmacología , Acroleína/análogos & derivados , Acroleína/farmacología , Animales , GMP Cíclico/metabolismo , Óxido Nítrico/metabolismo , Ratas , Ratas Sprague-Dawley , Vasodilatación/efectos de los fármacosRESUMEN
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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GMP Cíclico/metabolismo , Fibroblastos/metabolismo , Uniones Comunicantes/metabolismo , Miocitos Cardíacos/metabolismo , Óxido Nítrico/farmacología , Animales , Células Cultivadas , Técnicas de Cocultivo , GMP Cíclico/genética , Fibroblastos/citología , Fibroblastos/efectos de los fármacos , Uniones Comunicantes/efectos de los fármacos , Técnicas de Sustitución del Gen , Ratones , Miocardio/metabolismo , Miocitos Cardíacos/citología , Miocitos Cardíacos/efectos de los fármacosRESUMEN
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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Inflamación/enzimología , Neuralgia/enzimología , Isoformas de Proteínas/metabolismo , Guanilil Ciclasa Soluble/metabolismo , Animales , Modelos Animales de Enfermedad , Adyuvante de Freund/toxicidad , Ganglios Espinales/enzimología , Inflamación/inducido químicamente , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Proteínas Musculares/genética , Proteínas Musculares/metabolismo , Neuralgia/etiología , Dimensión del Dolor , Isoformas de Proteínas/genética , ARN Mensajero/metabolismo , Guanilil Ciclasa Soluble/genética , Médula Espinal/enzimología , Proteína 2 de Transporte Vesicular de Glutamato/metabolismo , Proteínas del Transporte Vesicular de Aminoácidos Inhibidores/metabolismoRESUMEN
The hyperpolarization-activated inward current, Ih, 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 Ih to more positive values and thereby terminating the rhythmic burst activity. The role of 3',5'-cyclic guanosine monophosphate (cGMP) in modulation of Ih 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 Ih, the voltage-dependency and cGMP/cAMP-sensitivity of Ih 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 (V1/2) of Ih in NO-GC2-/- TC neurons compared to WT. Different concentrations of 8-Br-cAMP/8-Br-cGMP induced dose-dependent positive shifts of V1/2 in both strains. Treatment of WT slices with lyase enzyme (adenylyl and guanylyl cyclases) inhibitors (SQ22536 and ODQ) resulted in further hyperpolarized V1/2. Under current clamp conditions NO-GC2-/- neurons exhibited a reduction in the Ih-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 Ih 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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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 40â¯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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Proteínas del Citoesqueleto/metabolismo , Músculo Liso Vascular/metabolismo , Óxido Nítrico/farmacología , Guanilil Ciclasa Soluble/metabolismo , Animales , Vasos Coronarios , Proteínas del Citoesqueleto/genética , Regulación Enzimológica de la Expresión Génica/efectos de los fármacos , Guanilil Ciclasa Soluble/genética , PorcinosRESUMEN
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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Encéfalo/metabolismo , GMP Cíclico/análisis , Guanilato Ciclasa/metabolismo , Óxido Nítrico/metabolismo , Animales , Células Cultivadas , GMP Cíclico/metabolismo , Transferencia Resonante de Energía de Fluorescencia/métodos , Ratones Noqueados , Neuroimagen/métodos , Neuronas , Células de PurkinjeRESUMEN
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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GMP Cíclico/metabolismo , Células Dendríticas/inmunología , Óxido Nítrico/metabolismo , Transducción de Señal , Animales , Movimiento Celular , Ratones , Ratones Noqueados , Ovalbúmina/inmunología , Linfocitos T/inmunología , Linfocitos T/fisiología , Balance Th1 - Th2RESUMEN
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.
Asunto(s)
GMP Cíclico/metabolismo , Transferencia Resonante de Energía de Fluorescencia , Animales , GMP Cíclico/biosíntesis , Guanilato Ciclasa/metabolismo , Humanos , Óxido Nítrico/metabolismo , Transducción de SeñalRESUMEN
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 S-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.
Asunto(s)
Guanilil Ciclasa Soluble/metabolismo , Animales , Presión Sanguínea/efectos de los fármacos , GMP Cíclico/metabolismo , Riñón/efectos de los fármacos , Riñón/metabolismo , Ratones , Ratones Noqueados , NG-Nitroarginina Metil Éster/metabolismo , Óxido Nítrico/metabolismo , Circulación Renal/efectos de los fármacos , S-Nitrosoglutatión/farmacología , Vasodilatación/efectos de los fármacosRESUMEN
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 EC50 value of â¼0.2 µM and maximal cGMP formation at 10 µM. By contrast, the effect of cinaciguat on 1H-[1,2,4]oxadiazolo-[4,3-a]quinoxalin-1-one-oxidized (ferric) sGC was moderate, reaching â¼10%-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.
Asunto(s)
Benzoatos/farmacología , Inhibidores Enzimáticos/farmacología , Imitación Molecular , Protoporfirinas/metabolismo , Guanilil Ciclasa Soluble/antagonistas & inhibidores , Vasodilatación/efectos de los fármacos , Animales , Aorta Torácica/fisiología , Bovinos , Vasos Coronarios/metabolismo , GMP Cíclico/metabolismo , Endotelio Vascular/efectos de los fármacos , Endotelio Vascular/enzimología , Activación Enzimática , Estabilidad de Enzimas , Pulmón/efectos de los fármacos , Pulmón/enzimología , Protoporfirinas/química , Ratas Sprague-Dawley , Guanilil Ciclasa Soluble/metabolismo , Porcinos , Vasodilatadores/farmacologíaRESUMEN
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 α1ß1-sGC (isoform 1), which converts 32P-GTP into 32P-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.