RESUMEN
Myocardial infarction (MI) is one of the leading causes of death worldwide. Prognosis and mortality rate are directly related to infarct size and post-infarction pathological heart remodeling, which can lead to heart failure. Hypoxic MI-affected areas increase the expression of hypoxia-inducible factor (HIF-1), inducing infarct size reduction and improving cardiac function. Hypoxia translocates HIF-1 to the nucleus, activating carbonic anhydrase IX (CAIX) transcription. CAIX regulates myocardial intracellular pH, critical for heart performance. Our objective was to investigate CAIX participation and relation with sodium bicarbonate transporters 1 (NBC1) and HIF-1 in cardiac remodeling after MI. We analyzed this pathway in an "in vivo" rat coronary artery ligation model and isolated cardiomyocytes maintained under hypoxia. Immunohistochemical studies revealed an increase in HIF-1 levels after 2 h of infarction. Similar results were observed in 2-h infarcted cardiac tissue (immunoblotting) and in hypoxic cardiomyocytes with a nuclear distribution (confocal microscopy). Immunohistochemical studies showed an increase CAIX in the infarcted area at 2 h, mainly distributed throughout the cell and localized in the plasma membrane at 24 h. Similar results were observed in 2 h in infarcted cardiac tissue (immunoblotting) and in hypoxic cardiomyocytes (confocal microscopy). NBC1 expression increased in cardiac tissue after 2 h of infarction (immunoblotting). CAIX and NBC1 interaction increases in cardiac tissue subjected to MI for 2h when CAIX is present (immunoprecipitation). These results suggest that CAIX interacts with NBC1 in our infarct model as a mechanism to prevent acidic damage in hypoxic tissue, making it a promising therapeutic target.
Asunto(s)
Anhidrasa Carbónica IX/metabolismo , Factor 1 Inducible por Hipoxia/metabolismo , Hipoxia/enzimología , Infarto del Miocardio/enzimología , Simportadores de Sodio-Bicarbonato/metabolismo , Animales , Masculino , Cultivo Primario de Células , Ratas Wistar , Remodelación VentricularRESUMEN
Rab GTPases define the identity and destiny of vesicles. Some of these small GTPases present isoforms that are expressed differentially along developmental stages or in a tissue-specific manner, hence comparative analysis is difficult to achieve. Here, we describe the intracellular distribution and function in lipid transport of the poorly characterized Rab39 isoforms using typical cell biology experimental tools and new ones developed in our laboratory. We show that, despite their amino acid sequence similarity, Rab39a and Rab39b display non-overlapping intracellular distribution. Rab39a localizes in the late endocytic pathway, mainly at multivesicular bodies. In contrast, Rab39b distributes in the secretory network, at the endoplasmic reticulum/cis-Golgi interface. Therefore, Rab39a controls trafficking of lipids (sphingomyelin and phospholipids) segregated at multivesicular bodies, whereas Rab39b transports sphingolipids biosynthesized at the endoplasmic reticulum-Golgi factory. Interestingly, lyso bis-phosphatidic acid is exclusively transported by Rab39a, indicating that both isoforms do not exert identical functions in lipid transport. Conveniently, the requirement of eukaryotic lipids by the intracellular pathogen Chlamydia trachomatis rendered useful for dissecting and distinguishing Rab39a- and Rab39b-controlled trafficking pathways. Our findings provide comparative insights about the different subcellular distribution and function in lipid transport of the two Rab39 isoforms.
Asunto(s)
Fosfolípidos/metabolismo , Esfingolípidos/metabolismo , Proteínas de Unión al GTP rab/metabolismo , Transporte Biológico , Chlamydia trachomatis/metabolismo , Retículo Endoplásmico/metabolismo , Aparato de Golgi/metabolismo , Células HeLa , Humanos , Imagen de Lapso de TiempoRESUMEN
Given their obligate intracellular lifestyle, Chlamydia trachomatis ensure that they have access to multiple host sources of essential lipids by interfering with vesicular transport. These bacteria hijack Rab6-, Rab11- and Rab14-controlled trafficking pathways to acquire sphingomyelin from the Golgi complex. Another important source of sphingolipids, phospholipids and cholesterol are multivesicular bodies (MVBs). Despite their participation in chlamydial inclusion development and bacterial replication, the molecular mechanisms mediating the interaction between MVBs and chlamydial inclusions remain unknown. In the present study, we demonstrate that Rab39a labels a subset of late endocytic vesicles - mainly MVBs - that move along microtubules. Moreover, Rab39a is actively recruited to chlamydial inclusions throughout the pathogen life cycle by a bacterial-driven process that depends on the Rab39a GTP- or GDP-binding state. Interestingly, Rab39a participates in the delivery of MVBs and host sphingolipids to maturing chlamydial inclusions, thereby promoting inclusion growth and bacterial development. Taken together, our findings indicate that Rab39a favours chlamydial replication and infectivity. This is the first report showing that a late endocytic Rab GTPase is involved in chlamydial infection development.
Asunto(s)
Chlamydia trachomatis/metabolismo , Cuerpos Multivesiculares/metabolismo , Esfingolípidos/metabolismo , Proteínas de Unión al GTP rab/metabolismo , Chlamydia trachomatis/patogenicidad , Colesterol/metabolismo , Aparato de Golgi/química , Células HeLa , Interacciones Huésped-Patógeno/genética , Humanos , Cuerpos Multivesiculares/microbiología , Fosfolípidos/metabolismo , Esfingomielinas/metabolismo , Vesículas Transportadoras/metabolismoRESUMEN
Pseudomonasaeruginosa uses choline as a source of carbon and nitrogen, and also for the synthesis of glycine betaine, an osmoprotectant under stress conditions such as drought and salinity. The transcription factor GbdR is the specific regulator of choline metabolism and it belongs to the Arac/XylS family of transcriptional regulators. Despite the link between choline catabolism and bacterial pathogenicity, gbdR regulation has not been explored in detail. In the present work, we describe how gbdR transcription can be initiated from a σ54-dependent promoter. gbdR transcription can be activated by NtrC in the absence of a preferential nitrogen source, by CbrB in the absence of a preferential carbon source, and by the integration host factor favouring DNA bending. In addition, we found that BetI negatively regulates gbdR expression in the absence of choline. We identified two overlapping BetI binding sites in the gbdR promoter sequence, providing an additional example of σ54-promoter down-regulation. Based on our findings, we propose a model for gdbR regulation and its impact on choline metabolism.
Asunto(s)
Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Regulación Bacteriana de la Expresión Génica , Regiones Promotoras Genéticas , Pseudomonas aeruginosa/genética , Pseudomonas aeruginosa/metabolismo , Transcripción Genética , Factores de Integración del Huésped/metabolismo , Mutación , Unión ProteicaRESUMEN
Chlamydia trachomatis is the most commonly reported agent of sexually transmitted bacterial infections worldwide. This pathogen frequently leads to persistent, long-term, subclinical infections, which in turn may cause severe pathology in susceptible hosts. This is in part due to the strategies that Chlamydia trachomatis uses to survive within epithelial cells and to evade the host immune response, such as subverting intracellular trafficking, interfering signaling pathways and preventing apoptosis. Innate immune receptors such as toll-like receptors expressed on epithelial and immune cells in the genital tract mediate the recognition of chlamydial molecular patterns. After bacterial recognition, a subset of pro-inflammatory cytokines and chemokines are continuously released by epithelial cells. The innate immune response is followed by the initiation of the adaptive response against Chlamydia trachomatis, which in turn may result in T helper 1-mediated protection or in T helper 2-mediated immunopathology. Understanding the molecular mechanisms developed by Chlamydia trachomatis to avoid killing and host immune response would be crucial for designing new therapeutic approaches and developing protective vaccines. In this review, we focus on chlamydial survival strategies and the elicited immune responses in male genital tract infections.
Asunto(s)
Antígenos Bacterianos/inmunología , Infecciones por Chlamydia/inmunología , Infecciones por Chlamydia/microbiología , Chlamydia trachomatis/inmunología , Genitales Masculinos/inmunología , Inmunidad Innata , Animales , Interacciones Huésped-Patógeno , Humanos , Masculino , Viabilidad MicrobianaRESUMEN
Chlamydia, an obligate intracellular bacterium which passes its entire lifecycle within a membrane-bound vacuole called the inclusion, has evolved a variety of unique strategies to establish an advantageous intracellular niche for survival. This review highlights the mechanisms by which Chlamydia subverts vesicular transport in host cells, particularly by hijacking the master controllers of eukaryotic trafficking, the Rab proteins. A subset of Rabs and Rab interacting proteins that control the recycling pathway or the biosynthetic route are selectively recruited to the chlamydial inclusion membrane. By interfering with Rab-controlled transport steps, this intracellular pathogen not only prevents its own degradation in the phagocytic pathway, but also creates a favourable intracellular environment for growth and replication. Chlamydia, a highly adapted and successful intracellular pathogen, has several redundant strategies to re-direct vesicles emerging from biosynthetic compartments that carry host molecules essential for bacterial development. Although current knowledge is limited, the latest findings have shed light on the role of Rab proteins in the course of chlamydial infections and could open novel opportunities for anti-chlamydial therapy.
Asunto(s)
Chlamydia/fisiología , Proteínas Bacterianas/metabolismo , Chlamydia/crecimiento & desarrollo , Infecciones por Chlamydia/metabolismo , Infecciones por Chlamydia/microbiologíaRESUMEN
Chlamydia trachomatis, an obligate intracellular pathogen, survives within host cells in a special compartment named 'inclusion' and takes advantage of host vesicular transport pathways for its growth and replication. Rab GTPases are key regulatory proteins of intracellular trafficking. Several Rabs, among them Rab11 and Rab14, are implicated in chlamydial development. FIP2, a member of the Rab11-Family of Interacting Proteins, presents at the C-terminus a Rab-binding domain that interacts with both Rab11 and Rab14. In this study, we determined and characterized the recruitment of endogenous and GFP-tagged FIP2 to the chlamydial inclusions. The recruitment of FIP2 is specific since other members of the Rab11-Family of Interacting Proteins do not associate with the chlamydial inclusions. The Rab-binding domain of FIP2 is essential for its association. Our results indicate that FIP2 binds to Rab11 at the chlamydial inclusion membrane through its Rab-binding domain. The presence of FIP2 at the chlamydial inclusion favours the recruitment of Rab14. Furthermore, our results show that FIP2 promotes inclusion development and bacterial replication. In agreement, the silencing of FIP2 decreases the bacterial progeny. C. trachomatis likely recruits FIP2 to hijack host intracellular trafficking to redirect vesicles full of nutrients towards the inclusion.
Asunto(s)
Proteínas Portadoras/metabolismo , Chlamydia trachomatis/crecimiento & desarrollo , Chlamydia trachomatis/patogenicidad , Interacciones Huésped-Patógeno , Cuerpos de Inclusión/microbiología , Proteínas de la Membrana/metabolismo , Células HeLa , Humanos , Unión Proteica , Mapeo de Interacción de Proteínas , Proteínas de Unión al GTP rab/metabolismoRESUMEN
BACKGROUND INFORMATION: Rab11 is a small GTPase that controls diverse intracellular trafficking pathways. However, the molecular machinery that regulates the participation of Rab11 in those different transport events is poorly understood. In resting cells, Rab11 localizes at the endocytic recycling compartment (ERC), whereas the different protein kinase C (PKC) isoforms display a cytosolic distribution. RESULTS: Sustained phorbol ester stimulation induces the translocation of the classical PKCα and PKCßII isoenzymes to the ERC enriched in Rab11, and results in transferrin recycling inhibition. In contrast, novel PKCε and atypical PKCζ isoenzymes neither redistribute to the perinucleus nor modify transferrin recycling transport after phorbol ester stimulation. Although several Rabs have been shown to be phosphorylated, there is to date no evidence indicating Rab11 as a kinase substrate. In this report, we show that Rab11 appears phosphorylated in vivo in phorbol ester-stimulated cells. A bioinformatic analysis of Rab11 allowed us to identify several high-probability Ser/Thr kinase phosphorylation sites. Our results demonstrate that classical PKC (PKCα and PKCßII but not PKCßI) directly phosphorylate Rab11 in vitro. In addition, novel PKCε and PKCη but not PKCδ isoenzymes also phosphorylate Rab11. Mass spectrometry analysis revealed that Ser 177 is the Rab11 residue to be phosphorylated in vitro by either PKCßII or PKCε. In agreement, the phosphomimetic mutant, Rab11 S177D, retains transferrin at the ERC in the absence of phorbol-12-myristate-13-acetate stimulus. CONCLUSIONS: This report shows for the first time that Rab11 is differentially phosphorylated by distinct PKC isoenzymes and that this post-translational modification might be a regulatory mechanism of intracellular trafficking.
Asunto(s)
Endosomas/enzimología , Proteína Quinasa C/metabolismo , Proteínas de Unión al GTP rab/metabolismo , Biología Computacional , Citosol/efectos de los fármacos , Citosol/metabolismo , Endosomas/efectos de los fármacos , Células HeLa , Humanos , Isoenzimas/metabolismo , Espectrometría de Masas , Fosforilación , Plásmidos , Procesamiento Proteico-Postraduccional , Transporte de Proteínas/efectos de los fármacos , Transporte de Proteínas/fisiología , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Serina/metabolismo , Especificidad por Sustrato , Acetato de Tetradecanoilforbol/farmacología , Transfección , Transferrina/antagonistas & inhibidores , Transferrina/metabolismo , Proteínas de Unión al GTP rab/genéticaRESUMEN
The significant impact of Chlamydia trachomatis(Ct) infections worldwide highlights the need to develop a prophylactic vaccine that elicits effective immunity and protects the host from the immunopathological effects of Ct infection. The aim of this study was to evaluate a vaccine based on a fragment of the Polymorphic membrane protein D (FPmpD) of C. trachomatis as an immunogen using a heterologous DNA prime-protein boost strategy in female mice Three different formulations were evaluated as protein boost: free recombinant FPmpD (rFPmpD) or rFPmpD formulated with a liposomal adjuvant alternatively supplemented with CpG or a cationic gemini lipopeptide as immunostimulants. The three candidates induced an increase in the cervicovaginal and systemic titers of anti-rFPmpD antibodies in two strains of mice (BALB/c and C57BL/6), with no evidence of fertility alterations. The three formulations induced a rapid and robust humoral immune response upon the Ct challenge. However, the booster with free rFPmpD more efficiently reduced the shedding of infective Ct and prevented the development of immunopathology. The formulations containing adjuvant induced a strong inflammatory reaction in the uterine tissue. Hence, the prime-boost strategy with the adjuvant-free FPmpD vaccine formulation might constitute a promissory candidate to prevent C. trachomatis intravaginal infection.
Asunto(s)
Infecciones por Chlamydia , Vacunas , Femenino , Animales , Ratones , Chlamydia trachomatis , Proteínas de la Membrana , Infecciones por Chlamydia/prevención & control , Ratones Endogámicos C57BL , Adyuvantes Inmunológicos , Proteínas RecombinantesRESUMEN
The control of the worldwide spread of sexually transmitted Chlamydia trachomatis (Ct) infection urgently demands the development of a preventive vaccine. In this work, we designed a vaccine based on a fragment of polymorphic protein D (FPmpD) that proved to be immunogenic enough to generate a robust systemic and mucosal IgG humoral immune response in two strains of mice. We used a heterologous prime-boost strategy, including simultaneous systemic and mucosal administration routes. The high titers of anti-PmpD antibodies elicited by this immunization scheme did not affect murine fertility. We tested the vaccine in a mouse model of Ct intravaginal infection. Anti-PmpD antibodies displayed potent neutralizing activity in vitro and protective effects in uterine tissues in vivo. Notably, the humoral immune response of PmpD-vaccinated mice was faster and stronger than the primary immune response of non-vaccinated mice when exposed to Ct. FPmpD-based vaccine effectively reduced Ct shedding into cervicovaginal fluids, bacterial burden at the genitourinary tract, and overall infectivity. Hence, the FPmpD-based vaccine might constitute an efficient tool to protect against Ct intravaginal infection and decrease the infection spreading.
Asunto(s)
Infecciones por Chlamydia , Chlamydia trachomatis , Animales , Anticuerpos Antibacterianos , Vacunas Bacterianas , Infecciones por Chlamydia/prevención & control , Ratones , VacunaciónRESUMEN
During cross-presentation, exogenous antigens (i.e. intracellular pathogens or tumor cells) are internalized and processed within the endocytic system and also by the proteasome in the cytosol. Then, antigenic peptides are associated with Major Histocompatibility Complex (MHC) class I molecules and these complexes transit to the plasma membrane in order to trigger cytotoxic immune responses through the activation of CD8+ T lymphocytes. Dendritic cells (DCs) are particularly adapted to achieve efficient antigen cross-presentation and their endocytic network displays important roles during this process, including a sophisticated MHC-I transport dependent on recycling compartments. In this study, we show that C. trachomatis, an obligate intracellular pathogen that exhibits multiple strategies to evade the immune system, is able to induce productive infections in the murine DC line JAWS-II. Our results show that when C. trachomatis infects these cells, the bacteria-containing vacuole strongly recruits host cell recycling vesicles, but no other endosomal compartments. Furthermore, we found that chlamydial infection causes significant alterations of MHC-I trafficking in JAWS-II DCs: reduced levels of MHC-I expression at the cell surface, disruption of the perinuclear MHC-I intracellular pool, and impairment of MHC-I endocytic recycling to the plasma membrane. We observed that all these modifications lead to a hampered cross-presentation ability of soluble and particulate antigens by JAWS-II DCs and primary bone marrow-derived DCs. In summary, our findings provide substantial evidence that C. trachomatis hijacks the DC endocytic recycling system, causing detrimental changes on MHC-I intracellular transport, which are relevant for competent antigen cross-presentation.
Asunto(s)
Presentación de Antígeno/inmunología , Chlamydia trachomatis/inmunología , Reactividad Cruzada/inmunología , Células Dendríticas/inmunología , Células Dendríticas/microbiología , Antígenos de Histocompatibilidad Clase I/inmunología , Animales , Células de la Médula Ósea/inmunología , Línea Celular , Chlamydia trachomatis/patogenicidad , Endocitosis , Ratones , Ratones Endogámicos C57BL , Transporte de ProteínasRESUMEN
Rab proteins belong to the Ras superfamily of small monomeric GTPases. These G proteins are the main controllers of vesicular transport in every tissue, among them, the endometrium. They are in charge of to the functional subcellular compartmentalization and cargo transport between organelles and the plasma membrane. In turn, intracellular trafficking contributes to endometrial changes during the menstrual cycle, secretion to the uterine fluid, and trophoblast implantation; however, few reports analyze the role of Rab proteins in the uterus. In general, Rab proteins control the release of cytokines, growth factors, enzymes, hormones, cell adhesion molecules, and mucus. Further, the secretion of multiple compounds into the uterine cavity is required for successful implantation. Therefore, alterations in Rab-controlled intracellular transport likely impair secretory processes to the uterine fluid that may correlate with abnormal endometrial development and failed reproductive outcomes. Overall, they could explain recurrent miscarriages, female infertility, and/or assisted reproductive failure. Interestingly, estrogen (E2) and progesterone (P) regulate gene expression of Rab proteins involved in secretory pathways. This review aims to gather information regarding the role of Rab proteins and intracellular trafficking in the endometrium during the different menstrual phases, and in the generation of a receptive stage for embryo implantation, modulated by E2 and P. This knowledge might be useful for the development of novel reproductive therapies that overcome low implantation rates of assisted reproductive procedures.
Asunto(s)
Endometrio/metabolismo , Ciclo Menstrual/metabolismo , Proteínas de Unión al GTP rab/metabolismo , Animales , Endometrio/microbiología , Endometrio/virología , Estradiol/metabolismo , Femenino , Interacciones Huésped-Patógeno , Humanos , Progesterona/metabolismo , Transporte de Proteínas , Enfermedades Bacterianas de Transmisión Sexual/metabolismo , Enfermedades Bacterianas de Transmisión Sexual/microbiología , Enfermedades Virales de Transmisión Sexual/metabolismo , Enfermedades Virales de Transmisión Sexual/virologíaRESUMEN
Chlamydia trachomatis is the causing agent of the most frequent bacterial sexually-transmitted diseases worldwide and is an underlying cause of chronic pelvic inflammatory diseases and cervical cancer. It is an obligate intracellular bacterium that establishes a close relationship with the Golgi complex and parasites the biosynthetic machinery of host cells. In a recent study, we have demonstrated that Rab14, a newly-described Golgi-associated Rab, is involved in the delivery of sphingolipids to the growing bacteria-containing vacuole. The interference with Rab14-controlled trafficking pathways delays chlamydial inclusion enlargement, decreases bacterial lipid uptake, negatively impact on bacterial differentiation, and reduces bacterial progeny and infectivity. C. trachomatis manipulation of host trafficking pathways for the acquisition of endogenously-biosynthesized nutrients arises as one of the characteristics of this highly evolved pathogen. The development of therapeutic strategies targeted to interfere with bacterium-host cell interaction is a new challenge for pharmacological approaches to control chlamydial infections.
RESUMEN
Chlamydia trachomatis are obligate intracellular bacteria that survive and replicate in a bacterial-modified phagosome called inclusion. As other intracellular parasites, these bacteria subvert the phagocytic pathway to avoid degradation in phagolysosomes and exploit trafficking pathways to acquire both energy and nutrients essential for their survival. Rabs are host proteins that control intracellular vesicular trafficking. Rab14, a Golgi-related Rab, controls Golgi to endosomes transport. Since Chlamydia establish a close relationship with the Golgi apparatus, the recruitment and participation of Rab14 on inclusion development and bacteria growth were analyzed. Time course analysis revealed that Rab14 associated with inclusions by 10 h post infection and was maintained throughout the entire developmental cycle. The recruitment was bacterial protein synthesis-dependent but independent of microtubules and Golgi integrity. Overexpression of Rab14 dominant negative mutants delayed inclusion enlargement, and impaired bacteria replication as determined by IFU. Silencing of Rab14 by siRNA also decreased bacteria multiplication and infectivity. By electron microscopy, aberrant bacteria were observed in cells overexpressing the cytosolic negative Rab14 mutant. Our results showed that Rab14 facilitates the delivery of sphingolipids required for bacterial development and replication from the Golgi to chlamydial inclusions. Novel anti-chlamydial therapies could be developed based on the knowledge of how bacteria subvert host vesicular transport events through Rabs manipulation.
Asunto(s)
Chlamydia trachomatis/crecimiento & desarrollo , Aparato de Golgi/metabolismo , Esfingolípidos/metabolismo , Proteínas de Unión al GTP rab/metabolismo , Proteínas Bacterianas/metabolismo , Transporte Biológico , Chlamydia trachomatis/metabolismo , Chlamydia trachomatis/fisiología , Aparato de Golgi/ultraestructura , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Células HeLa , Interacciones Huésped-Patógeno , Humanos , Cuerpos de Inclusión/metabolismo , Cuerpos de Inclusión/ultraestructura , Microscopía Confocal , Microscopía Electrónica de Transmisión , Interferencia de ARN , Proteínas de Unión al GTP rab/genéticaRESUMEN
ANTECEDENTES: Las kininas son oligopéptidos que generados localmente en corazón y vasos sanguíneos estimulan la producción endotelial de factores relajantes (prostaciclina, óxido nítrico y factor endotelial hiperpolarizante). El nivel de kininas depende de: a) la tasa de producción por kalikreínas y b) la tasa de destrucción por kininasas, entre las que se encuentra la enzima de conversión de angiotensina (CE). Los inhibidores de la enzima de conversión (CEI) empleados con notable éxito en el tratamiento de la hipertensión arterial y la insuficiencia cardíaca actuarían en parte prolongando la vida media del péptido. El objetivo de este trabajo es examinar: a) si los componentes del sistema kalikreína-kininógeno-kininas (SKKK) se liberan al perfusado en corazones aislados y perfundidos, b) la influencia de ramiprilat sobre la tasa de secreción de kininas y c) la acción biológica de las kininas en una preparación de vasos coronarios aislados. MATERIAL Y METODO: Los corazones de Ratas Wistar (300 ñ 30 gr) se aislaron y perfundieron en forma retrógrada con buffer Krebs-Henseleit equilibrado con 95 por ciento de oxígeno y 5 por ciento de anhídrido carbónico a un flujo constante (5 ml/min) mediante una bomba de perfusión Gilson. Kalikreína se midió por su capacidad de generar kininas; kininógeno, por una modificación de un método previamente descripto; y las kininas extraídas con un Set-Pack Cartridge, se dosaron por RIA. RESULTADOS: Los componentes del SKKK se forman y liberan continuamente en la preparación de corazón aislado (hasta 120 minutos). El pretratamiento con puromicina, un inhibidor de la síntesis proteica, descendió significativamente el release de kalikreína y kininógeno, confirmando la síntesis ex novo en el propio tejido cardíaco. El agregado del CEI, ramiprilat, incrementó significativamente el nivel de kininas en el perfusado. Kalikreína, per se, actuando sobre su sustrato en la propia pared del vaso libera kininas que relajan los vasos coronarios. Las kininas generadas permanentemente en la pared vascular en pequeñas cantidades se ponen de manifiesto en presencia de ramiprilat, relajando los vasos coronarios. El agregado de HOE 140, un inhibidor selectivo B2 de las kininas, bloquea el efecto relajante, confirmando que tanto la acción de las kalikreínas liberadas localmente como el efecto de los CEIs es mediado por kininas endógenas...
Asunto(s)
Animales , Ratas , Inhibidores de la Enzima Convertidora de Angiotensina/administración & dosificación , Inhibidores de la Enzima Convertidora de Angiotensina/uso terapéutico , Calicreínas , Sistema Calicreína-Quinina/fisiología , Vasos CoronariosRESUMEN
ANTECEDENTES: La hipertensión arterial y la hiperlipemia dañan el endotelio vascular y predisponen al desarrollo de lesiones ateromatosas y enfermedades coronarias. Las células endoteliales y musculares son capaces de modular la función cardiovascular mediante la producción de factores autocrino/paracrinos. Se ha descripto la presencia de un sistema generador de kininas en vasos y corazón. El propósito de este estudio es examinar si existe modificación de la actividad kininogenásica en tejido cardiovascular de ratas dislipidémicas e hipertensas por inhibición de la producción del factor relajante derivado del endotelio. MATERIAL Y METODO: Se utilizaron ratas Wistar macho (430 ñ 50 g peso), las que se sometieron a los siguientes tratamientos: control (n= 10): dieta normal; lípidos (n= 10): dieta con sobrecarga de 16 por ciento de ácidos grasos saturados y 2 por ciento de colesterol durante 8 semanas; L-NAME (n= 10): administración oral de un inhibidor de la síntesis de factor relajante endotelial, L-nitro arginina metil éster, 100 mg/kg/ día durante las 2 últimas semanas del período experimental; lípidos + L-NAME (n= 10): administración de dieta con sobrecarga grasa durante 8 semanas y de L-NAME durante las 2 últimas semanas. Se midió la presión arterial, la concentración de lípidos plasmáticos y la actividad kininogenásica en homogenados de corazón y vasos sanguíneos (pgBK/mg/h). Resultados: La manipulación dietética de los animales no produjo cambios en su presión arterial, pero fue efectiva para instaurar un perfil lipoproteico aterogénico (p< 0,05). La administración de L-NAME produjo un aumento significativo de los niveles tensionales (p< 0,05), sin modificar las concentraciones de los metabolitos plasmáticos dosados. La actividad kininogenásica presente en homogenados de corazón aumentó significativamente en los animales sometidos a sobrecarga dietética de grasa y ese incremento fue aún mayor cuando se les adicionó al agua de bebida el inhibidor de la síntesis de factor relajante endotelial (p< 0,05). Un patrón semejante se observa en homogenados de aorta, arterias caudales y venas caudales (p< 0,05). CONCLUSIONES: El incrmento local de la capacidad generadora de kininas en tejido cardiovascular podría representar un esfuerzo tisular por contraponerse a la agresión endotelial causada por el aumento de los lípidos plasmáticos y el aumento de la presión arterial. Las kininas liberadas in situ tendrían efectos hemodinámicos y metabólicos ...