RESUMEN
The combination of lineage tracing and immunohistochemistry has helped to identify subpopulations and fate of hepatic stellate cells (HSC) in murine liver. HSC are sinusoidal pericytes that act as myofibroblast precursors after liver injury. Single cell RNA sequencing approaches have recently helped to differentiate central and portal HSC. A specific Cre line to lineage trace portal HSC has not yet been described. We used three Cre lines (Lrat-Cre, PDGFRß-CreERT2 and SMMHC-CreERT2) known to label mesenchymal cells including HSC in combination with a tdTomato-expressing reporter. All three Cre lines labeled populations of HSC as well as smooth muscle cells (SMC). Using the SMMHC-CreERT2, we identified a subtype of HSC in the periportal area of the hepatic lobule (termed zone 1-HSC). We lineage traced tdTomato-expressing zone 1-HSC over 1 year, described fibrotic behavior in two fibrosis models and investigated their possible role during fibrosis. This HSC subtype resides in zone 1 under healthy conditions; however, zonation is disrupted in preclinical models of liver fibrosis (CCl4 and MASH). Zone 1-HSC do not transform into αSMA-expressing myofibroblasts. Rather, they participate in sinusoidal capillarization. We describe a novel subtype of HSC restricted to zone 1 under physiological conditions and its possible function after liver injury. In contrast to the accepted notion, this HSC subtype does not transform into αSMA-positive myofibroblasts; rather, zone 1-HSC adopt properties of capillary pericytes, thereby participating in sinusoidal capillarization.
Asunto(s)
Células Estrelladas Hepáticas , Cirrosis Hepática , Miofibroblastos , Animales , Células Estrelladas Hepáticas/metabolismo , Células Estrelladas Hepáticas/patología , Miofibroblastos/metabolismo , Miofibroblastos/patología , Ratones , Cirrosis Hepática/patología , Cirrosis Hepática/metabolismo , Hígado/patología , Hígado/metabolismo , Pericitos/metabolismo , Pericitos/patología , Linaje de la Célula , Masculino , Diferenciación Celular , Modelos Animales de Enfermedad , Ratones Endogámicos C57BLRESUMEN
Bacterial infection is a crucial complication in implant restoration, in particular in permanent skin-penetrating implants. Therein, the resulting gap between transcutaneous implant and skin represents a permanent infection risk, limiting the field of application and the duration of application. To overcome this limitation, a tight physiological connection is required to achieve a biological and mechanical welding for a long-term stable closure including self-healing probabilities. This study describes a new approach, wherein the implant is connected covalently to a highly porous electrospun fleece featuring physiological dermal integration potential. The integrative potential of the scaffold is shown in vitro and confirmed in vivo, further demonstrating tissue integration by neovascularization, extracellular matrix formation, and prevention of encapsulation. To achieve a covalent connection between fleece and implant surface, self-initiated photografting and photopolymerization of hydroxyethylmethacrylate is combined with a new crosslinker (methacrylic acid coordinated titanium-oxo clusters) on proton-abstractable implant surfaces. For implant modification, the attached fleece is directed perpendicular from the implant surface into the surrounding dermal tissue. First in vitro skin implantations demonstrate the implants' dermal integration capability as well as wound closure potential on top of the fleece by epithelialization, establishing a bacteria-proof and self-healing connection of skin and transcutaneous implant.
Asunto(s)
Biomimética , Prótesis e Implantes , Humanos , Piel , Titanio , Neovascularización Patológica , Propiedades de SuperficieRESUMEN
Actin binding proteins are of crucial importance for the spatiotemporal regulation of actin cytoskeletal dynamics, thereby mediating a tremendous range of cellular processes. Since their initial discovery more than 30 years ago, the enabled/vasodilator-stimulated phosphoprotein (Ena/VASP) family has evolved as one of the most fascinating and versatile family of actin regulating proteins. The proteins directly enhance actin filament assembly, but they also organize higher order actin networks and link kinase signaling pathways to actin filament assembly. Thereby, Ena/VASP proteins regulate dynamic cellular processes ranging from membrane protrusions and trafficking, and cell-cell and cell-matrix adhesions, to the generation of mechanical tension and contractile force. Important insights have been gained into the physiological functions of Ena/VASP proteins in platelets, leukocytes, endothelial cells, smooth muscle cells and cardiomyocytes. In this review, we summarize the unique and redundant functions of Ena/VASP proteins in cardiovascular cells and discuss the underlying molecular mechanisms.
Asunto(s)
Actinas , Células Endoteliales , Actinas/metabolismo , Células Endoteliales/metabolismo , Proteínas de Microfilamentos/metabolismo , Fosfoproteínas/metabolismoRESUMEN
BACKGROUND: The origin of αSMA-positive myofibroblasts, key players within organ fibrosis, is still not fully elucidated. Pericytes have been discussed as myofibroblast progenitors in several organs including the lung. METHODS: Using tamoxifen-inducible PDGFRß-tdTomato mice (PDGFRß-CreERT2; R26tdTomato) lineage of lung pericytes was traced. To induce lung fibrosis, a single orotracheal dose of bleomycin was given. Lung tissue was investigated by immunofluorescence analyses, hydroxyproline collagen assay and RT-qPCR. RESULTS: Lineage tracing combined with immunofluorescence for nitric oxide-sensitive guanylyl cyclase (NO-GC) as marker for PDGFRß-positive pericytes allows differentiating two types of αSMA-expressing myofibroblasts in murine pulmonary fibrosis: (1) interstitial myofibroblasts that localize in the alveolar wall, derive from PDGFRß+ pericytes, express NO-GC and produce collagen 1. (2) intra-alveolar myofibroblasts which do not derive from pericytes (but express PDGFRß de novo after injury), are negative for NO-GC, have a large multipolar shape and appear to spread over several alveoli within the injured areas. Moreover, NO-GC expression is reduced during fibrosis, i.e., after pericyte-to-myofibroblast transition. CONCLUSION: In summary, αSMA/PDGFRß-positive myofibroblasts should not be addressed as a homogeneous target cell type within pulmonary fibrosis.
Asunto(s)
Fibrosis Pulmonar , Ratones , Animales , Fibrosis Pulmonar/metabolismo , Pericitos/metabolismo , Miofibroblastos/metabolismo , Guanilato Ciclasa/metabolismo , Fibrosis , Colágeno/metabolismoRESUMEN
Gastrointestinal dysfunctions in individuals with autism spectrum disorder are poorly understood, although they are common among this group of patients. FOXP1 haploinsufficiency is characterized by autistic behavior, language impairment, and intellectual disability, but feeding difficulties and gastrointestinal problems have also been reported. Whether these are primary impairments, the result of altered eating behavior, or side effects of psychotropic medication remains unclear. To address this question, we investigated Foxp1+/- mice reflecting FOXP1 haploinsufficiency. These animals show decreased body weight and altered feeding behavior with reduced food and water intake. A pronounced muscular atrophy was detected in the esophagus and colon, caused by reduced muscle cell proliferation. Nitric oxide-induced relaxation of the lower esophageal sphincter was impaired and achalasia was confirmed in vivo by manometry. Foxp1 targets (Nexn, Rbms3, and Wls) identified in the brain were dysregulated in the adult Foxp1+/- esophagus. Total gastrointestinal transit was significantly prolonged due to impaired colonic contractility. Our results have uncovered a previously unknown dysfunction (achalasia and impaired gut motility) that explains the gastrointestinal disturbances in patients with FOXP1 syndrome, with potential wider relevance for autism.
Asunto(s)
Trastorno Autístico/genética , Acalasia del Esófago/genética , Factores de Transcripción Forkhead/genética , Tránsito Gastrointestinal , Proteínas Represoras/genética , Animales , Trastorno Autístico/fisiopatología , Encéfalo/metabolismo , Proliferación Celular , Colon/metabolismo , Colon/patología , Colon/fisiopatología , Acalasia del Esófago/fisiopatología , Esófago/metabolismo , Esófago/patología , Esófago/fisiopatología , Conducta Alimentaria , Femenino , Factores de Transcripción Forkhead/metabolismo , Heterocigoto , Masculino , Ratones , Proteínas de Microfilamentos/genética , Proteínas de Microfilamentos/metabolismo , Músculo Liso/metabolismo , Músculo Liso/patología , Músculo Liso/fisiopatología , Proteínas de Unión al ARN/genética , Proteínas de Unión al ARN/metabolismo , Proteínas Represoras/metabolismo , Síndrome , Transactivadores/genética , Transactivadores/metabolismoRESUMEN
Nitric oxide-sensitive guanylyl cyclase (NO-GC) has been shown to regulate a plethora of different functions in the body. These include, among many others, the fine-tuning of vascular tone, platelet reactivity and gastrointestinal motility. Evidence for the participation of NO-GC in these functions has been obtained from various species including humans, rodents, as well as insects. Clearly, individual cell types that express NO-GC contribute differentially to organ-specific NO/cGMP signaling in the body. Hence, identification of NO-GC-expressing cells and their individual involvement in NO/cGMP signaling constituted the focus of many studies over the last 40 years. Probably most information has been obtained from vascular smooth muscle cells and platelets, in which NO-GC is known to induce relaxation and inhibition of aggregation, respectively. Many other cell types that express the enzyme have been linked to certain functions, e.g. cardiomyocyte/inotropy or gastrointestinal smooth muscle cells/motility. However, in some cell types, e.g. myofibroblasts or pericytes, NO-GC expression is evident but individual functions of NO/cGMP signaling have yet to be assigned, whereas in other cell types, e.g. in erythrocytes, expression and role of NO-GC is still a matter of debate. This review discusses the current knowledge on 'less popular' cell types that express NO-GC (pericytes, myofibroblasts, cardiomyocytes, adipocytes, interstitial cells of Cajal, fibroblast-like cells and blood cells) and outlines possible further functions in cell types that have not gained strong attention so far.
Asunto(s)
Guanilil Ciclasa Soluble/metabolismo , Adipocitos/citología , Adipocitos/metabolismo , Animales , Fibroblastos/citología , Fibroblastos/metabolismo , Humanos , Células Intersticiales de Cajal/citología , Células Intersticiales de Cajal/metabolismo , Miocitos Cardíacos/citología , Miocitos Cardíacos/metabolismo , Miocitos del Músculo Liso/citología , Miocitos del Músculo Liso/metabolismo , Miofibroblastos/citología , Miofibroblastos/metabolismo , Pericitos/citología , Pericitos/metabolismo , Transducción de SeñalRESUMEN
Aims: It has been suggested that the nitric oxide-sensitive guanylyl cyclase (NO-GC)/cyclic guanosine monophosphate (cGMP)-dependent signalling pathway affords protection against cardiac damage during acute myocardial infarction (AMI). It is, however, not clear whether the NO-GC/cGMP system confers its favourable effects through a mechanism located in cardiomyocytes (CMs). The aim of this study was to evaluate the infarct-limiting effects of the endogenous NO-GC in CMs in vivo. Methods and results: Ischemia/reperfusion (I/R) injury was evaluated in mice with a CM-specific deletion of NO-GC (CM NO-GC KO) and in control siblings (CM NO-GC CTR) subjected to an in vivo model of AMI. Lack of CM NO-GC resulted in a mild increase in blood pressure but did not affect basal infarct sizes after I/R. Ischemic postconditioning (iPost), administration of the phosphodiesterase-5 inhibitors sildenafil and tadalafil as well as the NO-GC activator cinaciguat significantly reduced the amount of infarction in control mice but not in CM NO-GC KO littermates. Interestingly, NS11021, an opener of the large-conductance and Ca2+-activated potassium channel (BK), an important downstream effector of cGMP/cGKI in the cardiovascular system, protects I/R-exposed hearts of CM NO-GC proficient and deficient mice. Conclusions: These findings demonstrate an important role of CM NO-GC for the cardioprotective signalling following AMI in vivo. CM NO-GC function is essential for the beneficial effects on infarct size elicited by iPost and pharmacological elevation of cGMP; however, lack of CM NO-GC does not seem to disrupt the cardioprotection mediated by the BK opener NS11021.
Asunto(s)
Benzoatos/farmacología , GMP Cíclico/metabolismo , Activadores de Enzimas/farmacología , Poscondicionamiento Isquémico/métodos , Infarto del Miocardio/prevención & control , Daño por Reperfusión Miocárdica/prevención & control , Miocitos Cardíacos/efectos de los fármacos , Óxido Nítrico/metabolismo , Inhibidores de Fosfodiesterasa 5/farmacología , Citrato de Sildenafil/farmacología , Guanilil Ciclasa Soluble/metabolismo , Tadalafilo/farmacología , Animales , Modelos Animales de Enfermedad , Femenino , Canales de Potasio de Gran Conductancia Activados por el Calcio/metabolismo , Masculino , Ratones Noqueados , Infarto del Miocardio/enzimología , Infarto del Miocardio/genética , Infarto del Miocardio/patología , Daño por Reperfusión Miocárdica/enzimología , Daño por Reperfusión Miocárdica/genética , Daño por Reperfusión Miocárdica/patología , Miocitos Cardíacos/enzimología , Miocitos Cardíacos/patología , Transducción de Señal/efectos de los fármacos , Guanilil Ciclasa Soluble/deficiencia , Guanilil Ciclasa Soluble/genética , Tetrazoles/farmacología , Tiourea/análogos & derivados , Tiourea/farmacología , Factores de Tiempo , Regulación hacia ArribaRESUMEN
CAR-like membrane protein (CLMP), an immunoglobulin cell adhesion molecule (IgCAM), has been implicated in congenital short-bowel syndrome in humans, a condition with high mortality for which there is currently no cure. We therefore studied the function of CLMP in a Clmp-deficient mouse model. Although we found that the levels of mRNAs encoding Connexin43 or Connexin45 were not or were only marginally affected, respectively, by Clmp deficiency, the absence of CLMP caused a severe reduction of both proteins in smooth muscle cells of the intestine and of Connexin43 in the ureter. Analysis of calcium signaling revealed a disordered cell-cell communication between smooth muscle cells, which in turn induced an impaired and uncoordinated motility of the intestine and the ureter. Consequently, insufficient transport of chyme and urine caused a fatal delay to thrive, a high rate of mortality, and provoked a severe hydronephrosis in CLMP knockouts. Neurotransmission and the capability of smooth muscle cells to contract in ring preparations of the intestine were not altered. Physical obstructions were not detectable and an overall normal histology in the intestine as well as in the ureter was observed, except for a slight hypertrophy of smooth muscle layers. Deletion of Clmp did not lead to a reduced length of the intestine as shown for the human CLMP gene but resulted in gut malrotations. In sum, the absence of CLMP caused functional obstructions in the intestinal tract and ureter by impaired peristaltic contractions most likely due to a lack of gap-junctional communication between smooth muscle cells.
Asunto(s)
Conexina 43/metabolismo , Conexinas/metabolismo , Proteína de la Membrana Similar al Receptor de Coxsackie y Adenovirus/metabolismo , Intestinos/fisiología , Contracción Muscular , Músculo Liso/fisiología , Uréter/fisiología , Animales , Peso Corporal , Señalización del Calcio , Comunicación Celular , Proteína de la Membrana Similar al Receptor de Coxsackie y Adenovirus/deficiencia , Femenino , Humanos , Hidronefrosis/patología , Intestinos/citología , Intestinos/ultraestructura , Ratones Endogámicos C57BL , Miocitos del Músculo Liso/metabolismo , Peristaltismo , Análisis de Supervivencia , Transmisión SinápticaRESUMEN
Phosphodiesterase 3 (PDE3) exists in two isoforms (PDE3A and PDE3B) and is known to act as cGMP-inhibited cAMP-degrading PDE. Therefore, PDE3 may likely be involved in the interaction between the two second messenger pathways. NO-sensitive guanylyl cyclase (NO-GC) is the most important cytosolic generator of cGMP. Here, we investigated the effect of NO-GC deletion on PDE3A-mediated signaling in animals lacking NO-GC either globally (GCKO) or specifically in smooth muscle cells (SMC-GCKO). PDE3A expression is detected in murine aortic smooth muscle, platelets, and heart tissue. Expression and activity of PDE3A in aortae from GCKO and SMC-GCKO mice was reduced by approx. 50% compared to that in control animals. PDE3A downregulation can be linked to the reduction in NO-GC and is not an effect of the increased blood pressure levels resulting from NO-GC deletion. Despite the different PDE3A expression levels, smooth muscle relaxation induced by forskolin to stimulate cAMP signaling was similar in all genotypes. Basal and forskolin-stimulated cAMP levels in aortic tissue were not different between KO and control strains. However, the potency of milrinone, a selective inhibitor of PDE3A, to induce relaxation was higher in aortae from GCKO and SMC-GCKO than that in aorta from control animals. These data were corroborated by the effect of milrinone in vivo, which led to an increase in systolic blood pressure in both KO strains but not in control mice. We conclude that NO-GC modulates PDE3A expression and activity in SMC in vivo conceivably to preserve functional cAMP signaling.
Asunto(s)
Aorta/metabolismo , Fosfodiesterasas de Nucleótidos Cíclicos Tipo 3/metabolismo , Guanilato Ciclasa/metabolismo , Óxido Nítrico/metabolismo , Animales , Aorta/efectos de los fármacos , Plaquetas/efectos de los fármacos , Plaquetas/metabolismo , Presión Sanguínea/efectos de los fármacos , Presión Sanguínea/fisiología , Colforsina/farmacología , AMP Cíclico/metabolismo , GMP Cíclico/metabolismo , Regulación hacia Abajo/efectos de los fármacos , Regulación hacia Abajo/fisiología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Milrinona/farmacología , Relajación Muscular/efectos de los fármacos , Miocitos del Músculo Liso/efectos de los fármacos , Miocitos del Músculo Liso/metabolismo , Transducción de Señal/efectos de los fármacos , Transducción de Señal/fisiologíaRESUMEN
The optical control over biological function with small photoswitchable molecules has gathered significant attention in the last decade. Herein, we describe the design and synthesis of a small library of photoswitchable peptidomimetics based upon human atrial natriuretic peptide (ANP), in which the photochromic amino acid [3-(3-aminomethyl)phenylazo]phenylacetic acid (AMPP) is incorporated into the peptide backbone. The endogeneous hormone ANP signals via the natriuretic peptide receptor A (NPR-A) through raising intracellular cGMP concentrations, and is involved in blood pressure regulation and sodium homeostasis, as well as lipid metabolism and pancreatic function. The cis- and trans-isomers of one of our peptidomimetics, termed TOP271, exhibit a four-fold difference in NPR-A mediated cGMP synthesis in vitro. Despite this seemingly small difference, TOP271 enables large, optically-induced conformational changes ex vivo and transforms the NPR-A into an endogenous photoswitch. Thus, application of TOP271 allows the reversible generation of cGMP using light and remote control can be afforded over vasoactivity in explanted murine aortic rings, as well as pancreatic beta cell function in islets of Langerhans. This study demonstrates the broad applicability of TOP271 to enzyme-dependent signalling processes, extends the toolbox of photoswitchable molecules to all classes of transmembrane receptors and utilizes photopharmacology to deduce receptor activation on a molecular level.
RESUMEN
In the gastrointestinal (GI) tract, nitric oxide (NO) has been shown over the last 25 years to exert a prominent function as inhibitory neurotransmitter. Apart from the regulation of secretion and resorption, NO from nitrergic neurons has been demonstrated to be crucial for GI smooth muscle relaxation and motility. In fact, several human diseases such as achalasia, gastroparesis, slow transit constipation or Hirschsprung's disease may involve dysfunctional nitrergic signaling. Most of NO's effects as neurotransmitter are mediated by NO-sensitive guanylyl cyclase (NO-GC) and further transduced by cGMP-dependent mechanisms. In contrast to the vascular system where NO from the endothelium induces relaxation by acting on NO-GC solely in smooth muscle cells, GI tissues contain several different NO-GCexpressing cell types that include smooth muscle cells, interstitial cells of Cajal and fibroblast-like cells. Based on this diverse localization of the NO receptor, the exact pathway(s) leading to NO-induced relaxation are still unknown. Global and cell-specific knockout mouse strains have been generated that lack enzymes participating in nitrergic signaling. These animals have been helpful in examining the role of NO in smooth muscle of the GI tract. Here, we discuss the current knowledge on NO-mediated mechanisms in the relaxation of GI smooth muscle in stomach, small and large intestine including sphincters. Special focus is placed on the integration of nitrergic signals by specialized cell types within the gut smooth muscle layers.
Asunto(s)
Motilidad Gastrointestinal/fisiología , Tracto Gastrointestinal/metabolismo , Óxido Nítrico/metabolismo , Animales , GMP Cíclico/metabolismo , Humanos , Células Intersticiales de Cajal/metabolismo , Contracción Muscular , Músculo Liso/metabolismo , Transducción de Señal , Guanilil Ciclasa Soluble/metabolismoRESUMEN
Asthma is defined by airway inflammation and hyperresponsiveness, and contributes to morbidity and mortality worldwide. Although bronchodilation is a cornerstone of treatment, current bronchodilators become ineffective with worsening asthma severity. We investigated an alternative pathway that involves activating the airway smooth muscle enzyme, soluble guanylate cyclase (sGC). Activating sGC by its natural stimulant nitric oxide (NO), or by pharmacologic sGC agonists BAY 41-2272 and BAY 60-2770, triggered bronchodilation in normal human lung slices and in mouse airways. Both BAY 41-2272 and BAY 60-2770 reversed airway hyperresponsiveness in mice with allergic asthma and restored normal lung function. The sGC from mouse asthmatic lungs displayed three hallmarks of oxidative damage that render it NO-insensitive, and identical changes to sGC occurred in human lung slices or in human airway smooth muscle cells when given chronic NO exposure to mimic the high NO in asthmatic lung. Our findings show how allergic inflammation in asthma may impede NO-based bronchodilation, and reveal that pharmacologic sGC agonists can achieve bronchodilation despite this loss.
Asunto(s)
Antiasmáticos/farmacología , Asma/tratamiento farmacológico , Benzoatos/farmacología , Compuestos de Bifenilo/farmacología , Broncodilatadores/farmacología , Guanilato Ciclasa/efectos de los fármacos , Hidrocarburos Fluorados/farmacología , Pirazoles/farmacología , Piridinas/farmacología , Animales , Antiasmáticos/uso terapéutico , Asma/enzimología , Asma/fisiopatología , Benzoatos/uso terapéutico , Compuestos de Bifenilo/uso terapéutico , Hiperreactividad Bronquial/tratamiento farmacológico , Hiperreactividad Bronquial/enzimología , Broncodilatadores/uso terapéutico , Técnicas de Cocultivo , GMP Cíclico/metabolismo , Evaluación Preclínica de Medicamentos , Activación Enzimática/efectos de los fármacos , Femenino , Humanos , Hidrocarburos Fluorados/uso terapéutico , Pulmón/enzimología , Ratones , Ratones Endogámicos BALB C , Músculo Liso/efectos de los fármacos , Músculo Liso/enzimología , Óxido Nítrico/farmacología , Pirazoles/uso terapéutico , Piridinas/uso terapéutico , Solubilidad , Tráquea/efectos de los fármacosRESUMEN
In the enteric nervous systems, NO is released from nitrergic neurons as a major inhibitory neurotransmitter. NO acts via NO-sensitive guanylyl cyclase (NO-GC), which is found in different gastrointestinal (GI) cell types including smooth muscle cells (SMCs) and interstitial cells of Cajal (ICC). The precise mechanism of nitrergic signalling through these two cell types to regulate colonic spontaneous contractions is not fully understood yet. In the present study we investigated the impact of endogenous and exogenous NO on colonic contractile motor activity using mice lacking nitric oxide-sensitive guanylyl cyclase (NO-GC) globally and specifically in SMCs and ICC. Longitudinal smooth muscle of proximal colon from wild-type (WT) and knockout (KO) mouse strains exhibited spontaneous contractile activity ex vivo. WT and smooth muscle-specific guanylyl cyclase knockout (SMC-GCKO) colon showed an arrhythmic contractile activity with varying amplitudes and frequencies. In contrast, colon from global and ICC-specific guanylyl cyclase knockout (ICC-GCKO) animals showed a regular contractile rhythm with constant duration and amplitude of the rhythmic contractions. Nerve blockade (tetrodotoxin) or specific blockade of NO signalling (L-NAME, ODQ) did not significantly affect contractions of GCKO and ICC-GCKO colon whereas the arrhythmic contractile patterns of WT and SMC-GCKO colon were transformed into uniform motor patterns. In contrast, the response to electric field-stimulated neuronal NO release was similar in SMC-GCKO and global GCKO. In conclusion, our results indicate that basal enteric NO release acts via myenteric ICC to influence the generation of spontaneous contractions whereas the effects of elevated endogenous NO are mediated by SMCs in the murine proximal colon.
Asunto(s)
Colon/fisiología , Células Intersticiales de Cajal/fisiología , Contracción Muscular/fisiología , Músculo Liso/fisiología , Óxido Nítrico/fisiología , Animales , Guanilato Ciclasa/genética , Guanilato Ciclasa/fisiología , Masculino , Ratones Endogámicos C57BL , Ratones Noqueados , Miocitos del Músculo Liso/fisiología , Receptores Citoplasmáticos y Nucleares/genética , Receptores Citoplasmáticos y Nucleares/fisiología , Transducción de Señal , Guanilil Ciclasa SolubleRESUMEN
Nitric oxide (NO) acts as essential regulator of vasculogenesis and angiogenesis and is critical for arteriogenesis. Whether NO's effects in vivo are mediated through NO-sensitive guanylyl cyclase (NO-GC) and thus by cGMP-dependent mechanisms has been only poorly addressed. Mice lacking NO-GC globally or specifically in smooth muscle cells (SMC) or endothelial cells (EC) were subjected to two established models for arteriogenesis and angiogenesis, namely hindlimb ischemia and oxygen-induced retinopathy. Our data clearly show the involvement of NO-GC in the recovery of blood flow after hindlimb ischemia, and this effect could be attributed to NO-GC in SMC. In the retina, global deletion of NO-GC led to reduced oxygen-induced vessel loss and hypoxia-induced capillary regrowth, whereas pathological neovascularization was increased. These effects were also seen in mice with SMC-specific NO-GC deletion but not in animals lacking NO-GC in EC. Intriguingly, NO-GC was found to be strongly expressed in retinal pericytes. Our data prove the involvement of NO-GC in growth and plasticity of hindlimb and retinal vasculature after ischemic/hypoxic insult.
Asunto(s)
Guanilato Ciclasa/metabolismo , Neovascularización Patológica , Óxido Nítrico/metabolismo , Receptores Citoplasmáticos y Nucleares/metabolismo , Animales , GMP Cíclico/metabolismo , Células Endoteliales/metabolismo , Exones , Guanilato Ciclasa/genética , Miembro Posterior/irrigación sanguínea , Hipoxia/patología , Procesamiento de Imagen Asistido por Computador , Inmunohistoquímica , Isquemia/patología , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Miocitos del Músculo Liso/metabolismo , Oxígeno/química , Pericitos/metabolismo , Receptores Citoplasmáticos y Nucleares/genética , Retina/metabolismo , Retina/patología , Enfermedades de la Retina/patología , Transducción de Señal , Guanilil Ciclasa Soluble , Factores de TiempoRESUMEN
Oesophageal achalasia is a disease known to result from reduced relaxation of the lower oesophageal sphincter (LES). Nitric oxide (NO) is one of the main inhibitory transmitters. NO-sensitive guanylyl cyclase (NO-GC) acts as the key target of NO and, by the generation of cGMP, mediates nitrergic relaxation in the LES. To date, the exact mechanism of nitrergic LES relaxation is still insufficiently elucidated. To clarify the role of NO-GC in LES relaxation, we used cell-specific knockout (KO) mouse lines for NO-GC. These include mice lacking NO-GC in smooth muscle cells (SMC-GCKO), in interstitial cells of Cajal (ICC-GCKO) and in both SMC/ICC (SMC/ICC-GCKO). We applied oesophageal manometry to study the functionality of LES in vivo. Isometric force studies were performed to monitor LES responsiveness to exogenous NO and electric field stimulation of intrinsic nerves in vitro. Cell-specific expression/deletion of NO-GC was monitored by immunohistochemistry. Swallowing-induced LES relaxation is strongly reduced by deletion of NO-GC in ICC. Basal LES tone is affected by NO-GC deletion in either SMC or ICC. Lack of NO-GC in both cells leads to a complete interruption of NO-induced relaxation and, therefore, to an achalasia-like phenotype similar to that seen in global GCKO mice. Our data indicate that regulation of basal LES tone is based on a dual mechanism mediated by NO-GC in SMC and ICC whereas swallow-induced LES relaxation is mainly regulated by nitrergic mechanisms in ICC.
Asunto(s)
Esfínter Esofágico Inferior/metabolismo , Guanilato Ciclasa/metabolismo , Células Intersticiales de Cajal/metabolismo , Relajación Muscular , Óxido Nítrico/metabolismo , Receptores Citoplasmáticos y Nucleares/metabolismo , Animales , Esfínter Esofágico Inferior/citología , Esfínter Esofágico Inferior/fisiología , Guanilato Ciclasa/genética , Células Intersticiales de Cajal/fisiología , Contracción Isométrica , Ratones , Ratones Endogámicos C57BL , Miocitos del Músculo Liso/metabolismo , Miocitos del Músculo Liso/fisiología , Receptores Citoplasmáticos y Nucleares/genética , Guanilil Ciclasa SolubleRESUMEN
Nitroxyl (HNO), the reduced and protonated form of nitric oxide (NO·), confers unique physiological effects including vasorelaxation and enhanced cardiac contractility. These features have spawned current pharmaceutical development of HNO donors as heart failure therapeutics. HNO interacts with selective redox sensitive cysteines to effect signaling but is also proposed to activate soluble guanylate cyclase (sGC) in vitro to induce vasodilation and potentially enhance contractility. Here, we tested whether sGC stimulation is required for these HNO effects in vivo and if HNO also modifies a redox-sensitive cysteine (C42) in protein kinase G-1α to control vasorelaxation. Intact mice and isolated arteries lacking the sGC-ß subunit (sGCKO, results in full sGC deficiency) or expressing solely a redox-dead C42S mutant protein kinase G-1α were exposed to the pure HNO donor, CXL-1020. CXL-1020 induced dose-dependent systemic vasodilation while increasing contractility in controls; however, vasodilator effects were absent in sGCKO mice whereas contractility response remained. The CXL-1020 dose reversing 50% of preconstricted force in aortic rings was ≈400-fold greater in sGCKO than controls. Cyclic-GMP and cAMP levels were unaltered in myocardium exposed to CXL-1020, despite its inotropic-vasodilator activity. In protein kinase G-1α(C42S) mice, CXL-1020 induced identical vasorelaxation in vivo and in isolated aortic and mesenteric vessels as in littermate controls. In both groups, dilation was near fully blocked by pharmacologically inhibiting sGC. Thus, sGC and cGMP-dependent signaling are necessary and sufficient for HNO-induced vasodilation in vivo but are not required for positive inotropic action. Redox modulation of protein kinase G-1α is not a mechanism for HNO-mediated vasodilation.
Asunto(s)
Cardiotónicos/farmacología , Guanilato Ciclasa/fisiología , Óxidos de Nitrógeno/farmacología , Receptores Citoplasmáticos y Nucleares/fisiología , Vasodilatación/fisiología , Animales , Aorta/efectos de los fármacos , GMP Cíclico/fisiología , Proteína Quinasa Dependiente de GMP Cíclico Tipo I/química , Proteína Quinasa Dependiente de GMP Cíclico Tipo I/deficiencia , Proteína Quinasa Dependiente de GMP Cíclico Tipo I/genética , Cisteína/química , Guanilato Ciclasa/deficiencia , Guanilato Ciclasa/genética , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Músculo Liso Vascular/fisiopatología , Contracción Miocárdica/efectos de los fármacos , Contracción Miocárdica/fisiología , Miocardio/metabolismo , Óxido Nítrico/fisiología , Donantes de Óxido Nítrico/farmacología , Oxidación-Reducción , Receptores Citoplasmáticos y Nucleares/deficiencia , Receptores Citoplasmáticos y Nucleares/genética , Sistemas de Mensajero Secundario/fisiología , Guanilil Ciclasa Soluble , Sulfonamidas/farmacología , Vasodilatación/efectos de los fármacosRESUMEN
Nitric oxide (NO) is a major inhibitory neurotransmitter in the gastrointestinal (GI) tract. Its main effector, NO-sensitive guanylyl cyclase (NO-GC), is expressed in several GI cell types, including smooth muscle cells (SMC), interstitial cells of Cajal (ICC), and fibroblast-like cells. Up to date, the interplay between neurons and these cells to initiate a nitrergic inhibitory junction potential (IJP) is unclear. Here, we investigate the origin of the nitrergic IJP in murine fundus and colon. IJPs were determined in fundus and colon SMC of mice lacking NO-GC globally (GCKO) and specifically in SMC (SM-GCKO), ICC (ICC-GCKO), and both SMC/ICC (SM/ICC-GCKO). Nitrergic IJP was abolished in ICC-GCKO fundus and reduced in SM-GCKO fundus. In the colon, the amplitude of nitrergic IJP was reduced in ICC-GCKO, whereas nitrergic IJP in SM-GCKO was reduced in duration. These results were corroborated by loss of the nitrergic IJP in global GCKO. In conclusion, our results prove the obligatory role of NO-GC in ICC for the initiation of an IJP. NO-GC in SMC appears to enhance the nitrergic IJP, resulting in a stronger and prolonged hyperpolarization in fundus and colon SMC, respectively. Thus NO-GC in both cell types is mandatory to induce a full nitrergic IJP. Our data from the colon clearly reveal the nitrergic IJP to be biphasic, resulting from individual inputs of ICC and SMC.
Asunto(s)
Colon/inervación , Fundus Gástrico/inervación , Células Intersticiales de Cajal/metabolismo , Inhibición Neural , Neuronas Nitrérgicas/metabolismo , Óxido Nítrico/metabolismo , Transmisión Sináptica , Animales , Guanilato Ciclasa/genética , Guanilato Ciclasa/metabolismo , Potenciales Postsinápticos Inhibidores , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Miocitos del Músculo Liso/metabolismo , Factores de TiempoRESUMEN
The action of nitric oxide (NO) to stimulate NO-sensitive guanylyl cyclase (NO-GC), followed by production of cGMP, and eventually to cause smooth muscle relaxation is well known. In the lower urinary tract (LUT), in contrast to the cardiovascular system and the gastrointestinal tract, specific localization in combination with function of NO-GC has not been investigated to date. Consequently, little is known about the mechanisms regulating relaxation of the lower urinary tract in general and the role of NO-GC-expressing cells in particular. To study the distribution and function of NO-GC in the murine lower urinary tract, we used internal urethral sphincter and bladder detrusor from global (GCKO) and smooth muscle cell-specific (SM-GCKO) NO-GC knock-out mice for immunohistochemical analyses and organ bath experiments. In urethral sphincter, NO-GC-positive immunofluorescence was confined to smooth muscle cells (SMCs). Deletion of NO-GC in SMCs abolished NO-induced relaxation. In bladder detrusor, exposure to NO did not cause relaxation although immunohistochemistry uncovered the existence of NO-GC in the tissue. In contrast to the urethral sphincter, expression of NO-GC in bladder detrusor was limited to platelet-derived growth factor receptor α (PDGFRα)-positive interstitial cells. In conclusion, NO-GC found in SMCs of the urethral sphincter mediates NO-induced relaxation; bladder detrusor is unique as NO-GC is not expressed in SMCs and, thus, NO does not induce relaxation. Nevertheless, NO-GC expression was found in PDGFRα-positive interstitial cells of the murine bladder with an as yet unknown function. Further investigation is needed to clarify the role of NO-GC in the detrusor.
Asunto(s)
Guanilato Ciclasa/metabolismo , Receptores Citoplasmáticos y Nucleares/metabolismo , Uretra/metabolismo , Vejiga Urinaria/metabolismo , Animales , Guanilato Ciclasa/genética , Ratones , Ratones Endogámicos C57BL , Relajación Muscular , Miocitos del Músculo Liso/enzimología , Receptores Citoplasmáticos y Nucleares/genética , Guanilil Ciclasa Soluble , Uretra/citología , Vejiga Urinaria/citologíaRESUMEN
BACKGROUND AND PURPOSE: Nitric oxide (NO) is known to activate NO-sensitive guanylyl cyclase (NO-GC) and to elicit cGMP production. However, NO has also been proposed to induce cGMP-independent effects. It is accepted practice to use specific NO-GC inhibitors, such as ODQ or NS2028, to assess cGMP-dependent NO effects. Consequently, NO-induced reactions seen in the presence of these inhibitors commonly serve as an affirmation of cGMP independence. EXPERIMENTAL APPROACH: We evaluated the use of ODQ to discriminate between cGMP-dependent and cGMP-independent NO effects. NO-GC-expressing HEK cells, platelets and tissues from wild type (WT) and NO-GC-deficient mice (GCKO) were used. KEY RESULTS: NO donors led to accumulation of cGMP in platelets and GC-expressing HEK cells and induced phosphorylation of the vasodilator-stimulated phosphoprotein in platelets; both effects were reduced by ODQ. High concentrations of NO donors, however, overrode the inhibitory effect of ODQ. Correspondingly, ODQ inhibited but did not fully eliminate NO-induced relaxation of aorta and fundus from WT mice. Relaxation induced by endogenously released NO was fully or partially inhibited by ODQ in fundus and aorta, respectively. In aorta and fundus of GCKO mice NO-induced relaxation was absent and served as standard for complete NO-GC inhibition. CONCLUSIONS AND IMPLICATIONS: High NO concentrations can overcome the inhibitory effect of ODQ on NO-GC. Smooth muscle relaxation induced by NO donors/endogenously released NO in the presence of ODQ in WT was absent in GCKO animals indicating involvement of NO-GC. Accordingly, NO-induced effects in the presence of ODQ do not necessarily prove cGMP independence.