RESUMEN
Chromosome Y (chrY) variation has been associated with many complex diseases ranging from cancer to cardiovascular disorders. Functional roles of chrY genes outside of testes are suggested by the fact that they are broadly expressed in many other tissues and correspond to regulators of basic cellular functions (such as transcription, translation, and protein stability). However, the unique genetic properties of chrY (including the lack of meiotic crossover and the presence of numerous highly repetitive sequences) have made the identification of causal variants very difficult. Despite the prior lack of reliable sequences and/or data on genetic polymorphisms, earlier studies with animal chrY consomic strains have made it possible to narrow down the phenotypic contributions of chrY. Some of the evidence so far indicates that chrY gene variants associate with regulatory changes in the expression of other autosomal genes, in part via epigenetic effects. In humans, a limited number of studies have shown associations between chrY haplotypes and disease traits. However, recent sequencing efforts have made it possible to greatly increase the identification of genetic variants on chrY, which promises that future association of chrY with disease traits will be further refined. Continuing studies (both in humans and in animal models) will be critical to help explain the many sex-biased disease states in human that are contributed to not only by the classical sex steroid hormones, but also by chrY genetics.
Asunto(s)
Variación Genética , Modelos Animales , Cromosoma Y , Animales , Ensamble y Desensamble de Cromatina , Epigénesis Genética , Femenino , Perfilación de la Expresión Génica , Hormonas Esteroides Gonadales/fisiología , Haplotipos , Humanos , Masculino , Ratones , FenotipoRESUMEN
We recently demonstrated early metabolic alterations in the dystrophin-deficient mdx heart that precede overt cardiomyopathy and may represent an early "subclinical" signature of a defective nitric oxide (NO)/cGMP pathway. In this study, we used genetic and pharmacological approaches to test the hypothesis that enhancing cGMP, downstream of NO formation, improves the contractile function, energy metabolism, and sarcolemmal integrity of the mdx heart. We first generated mdx mice overexpressing, in a cardiomyocyte-specific manner, guanylyl cyclase (GC) (mdx/GC(+/0)). When perfused ex vivo in the working mode, 12- and 20-week-old hearts maintained their contractile performance, as opposed to the severe deterioration observed in age-matched mdx hearts, which also displayed two to three times more lactate dehydrogenase release than mdx/GC(+/0). At the metabolic level, mdx/GC(+/0) displayed a pattern of substrate selection for energy production that was similar to that of their mdx counterparts, but levels of citric acid cycle intermediates were significantly higher (36 +/- 8%), suggesting improved mitochondrial function. Finally, the ability of dystrophin-deficient hearts to resist sarcolemmal damage induced in vivo by increasing the cardiac workload acutely with isoproterenol was enhanced by the presence of the transgene and even more so by inhibiting cGMP breakdown using the phosphodiesterase inhibitor sildenafil (44.4 +/- 1.0% reduction in cardiomyocyte damage). Overall, these findings demonstrate that enhancing cGMP signaling, specifically downstream and independent of NO formation, in the dystrophin-deficient heart improves contractile performance, myocardial metabolic status, and sarcolemmal integrity and thus constitutes a potential clinical avenue for the treatment of the dystrophin-related cardiomyopathies.
Asunto(s)
Cardiomiopatías/prevención & control , GMP Cíclico/metabolismo , Distrofina/deficiencia , Miocitos Cardíacos/efectos de los fármacos , Miocitos Cardíacos/metabolismo , Piperazinas/farmacología , Transducción de Señal/efectos de los fármacos , Sulfonas/farmacología , Animales , Factor Natriurético Atrial/genética , Factor Natriurético Atrial/metabolismo , Cardiomiopatías/enzimología , Cardiomiopatías/fisiopatología , Activación Enzimática/efectos de los fármacos , Regulación de la Expresión Génica/efectos de los fármacos , Guanilato Ciclasa/genética , Guanilato Ciclasa/metabolismo , Frecuencia Cardíaca/efectos de los fármacos , Técnicas In Vitro , Ratones , Ratones Transgénicos , Mitocondrias/efectos de los fármacos , Mitocondrias/metabolismo , Contracción Miocárdica/efectos de los fármacos , Miocitos Cardíacos/enzimología , Miocitos Cardíacos/patología , Especificidad de Órganos/efectos de los fármacos , Purinas/farmacología , Sarcolema/efectos de los fármacos , Sarcolema/metabolismo , Citrato de SildenafilRESUMEN
To advance the spontaneous hypertensive rat (SHR) model of attention deficit/hyperactivity disorder (ADHD), experiments examined the SHR in tasks recognized to assess functioning of the prefrontal cortex or dorsal striatal. Tasks included odor-delayed win-shift (nonspatial working and reference memory), win-stay (habit learning), and attentional set-shifting (attention and behavioral flexibility). In Experiment 1, the SHR strain was compared with Wistar-Kyoto (WKY) and Wistar-Kyoto Hypertensive (WKHT) strains on the first 2 tasks. In Experiment 2, oral methylphenidate (1.5 mg/kg) and vehicle (water) were evaluated on all 3 tasks in SHR and WKY strains. Results demonstrated that the SHR made significantly more errors in the odor-delayed win-shift, win-stay, and attentional set-shifting tasks compared with the WKY. Similar performances in the WKY and WKHT indicated that deficits observed in the SHR were not related solely to hypertension. Treating the SHR with methylphenidate eliminated strain differences in all 3 tasks. These findings provide evidence that the SHR is a valid model for studying ADHD-associated neurocognitive deficits. Moreover, the current behavioral approach is appropriate to assess novel medications developed to target ADHD-associated neurocognitive deficits.
Asunto(s)
Aprendizaje por Asociación/fisiología , Trastorno por Déficit de Atención con Hiperactividad/fisiopatología , Atención/fisiología , Aprendizaje Discriminativo/fisiología , Modelos Animales de Enfermedad , Corteza Prefrontal/fisiología , Animales , Trastorno por Déficit de Atención con Hiperactividad/complicaciones , Trastorno por Déficit de Atención con Hiperactividad/tratamiento farmacológico , Estimulantes del Sistema Nervioso Central/uso terapéutico , Conducta Impulsiva/complicaciones , Conducta Impulsiva/tratamiento farmacológico , Conducta Impulsiva/fisiopatología , Memoria a Corto Plazo/fisiología , Metilfenidato/uso terapéutico , Neostriado/fisiología , Ratas , Ratas Endogámicas SHR , Ratas Endogámicas WKY , Ratas Mutantes , Disposición en PsicologíaRESUMEN
Wistar-Kyoto (WKY) and WKY-derived hyperactive (WKHA) rats are two genetically-related inbred strains of rats that are both normotensive yet exhibit differences in left ventricular mass (LVM). We had shown previously that cardiomyocytes from male WKHA are wider than that of male WKY, and that there was genetic linkage between LVM and a locus on chromosome 5 (RNO5) in the male progeny of a F2 WKHA/WKY cross. We show here that cardiomyocyte width is linked to the same RNO5 locus in male reciprocal congenic rats derived from WKHA and WKY. Contrary to males, we found no genetic linkage between LVM and the RNO5 locus in female rats. However, ventricular hypertrophy in females might be of a different nature, because cardiomyocytes from female WKHA were shorter than their WKY counterparts (with no difference in width). The RNO5 locus contains that of the natriuretic peptide precursor A (Nppa) gene. In male congenic rats, changes in cardiomyocyte width always correlated with reciprocal changes in the LV concentration of atrial natriuretic factor (ANF, i.e., the peptide product of Nppa). Taken together with other functional data, the small size of the RNO5 locus (approximately 63 cR) increased the likelihood that both cardiomyocyte width and LV ANF concentration could be linked to only one gene (possibly Nppa) in male rats. Moreover, our results support the notion that genes and sex interact to regulate cardiomyocyte width and length independently from one another.
Asunto(s)
Ventrículos Cardíacos/anatomía & histología , Sitios de Carácter Cuantitativo , Animales , Animales Congénicos , Factor Natriurético Atrial , Mapeo Cromosómico , Femenino , Ligamiento Genético , Ventrículos Cardíacos/química , Hipertrofia Ventricular Izquierda/etiología , Hipertrofia Ventricular Izquierda/genética , Masculino , Péptido Natriurético Tipo-C/análisis , Péptido Natriurético Tipo-C/genética , Precursores de Proteínas/análisis , Precursores de Proteínas/genética , Ratas , Ratas Endogámicas SHR , Ratas Endogámicas WKY , Reproducibilidad de los Resultados , Factores Sexuales , Especificidad de la EspecieRESUMEN
In vivo, the normal heart obtains at least 60% of its energy from lipids and the remainder from glucose. Several lines of evidence indicate that an increase in the utilization of glucose [at the expense of fatty acids (FA)] may play a role in the genesis of hypertrophy. Primary cultures of neonatal cardiomyocytes have been used extensively to study the phenotype of these cells as well as their responses to hormonal hypertrophic agents. Unfortunately, such cultures are most typically cultured in glucose-rich FA-free media, and thus might be hypertrophied to start with. We therefore tested the effects of FA-albumin complexes on three different surrogate end points of hypertrophy of cardiomyocytes. Oleate-albumin complexes decreased the baseline values of all three variables, and increased the relative response of these variables to administration of norepinephrine. Oleate:palmitate-albumin complexes also affected all three variables and their responses to norepinephrine, but the effects differed somewhat from that of oleate-albumin complexes. Our results suggest that addition of long-chain FA, by providing conditions that more closely resemble physiological situations, may optimize the expression of hypertrophic responses in such cells. However, the differences between the effects of oleate and oleate:palmitate also suggest that the precise composition of FA may affect the phenotype of cardiomyocytes and how these cells respond to hypertrophic agents.
Asunto(s)
Animales Recién Nacidos , Cardiomegalia/patología , Ácidos Grasos/farmacología , Corazón/efectos de los fármacos , Miocardio/patología , Animales , Factor Natriurético Atrial/metabolismo , Cardiomegalia/metabolismo , Tamaño de la Célula/efectos de los fármacos , Células Cultivadas , Medios de Cultivo , Miocardio/metabolismo , Norepinefrina/farmacología , Ácido Oléico/farmacología , Ácido Palmítico/farmacología , Biosíntesis de Proteínas , Ratas , Ratas Sprague-Dawley , Albúmina Sérica Bovina/farmacologíaRESUMEN
Cardiac left ventricular hypertrophy (LVH) is commonly associated with hypertension, but its variance is determined for more than 50% by blood pressure-independent genetic factors. Because it constitutes one of the most important risk factors for cardiovascular mortality, we have performed a genome-wide scan of the F2 progeny of crosses between inbred WKY and WKHA rats to detect quantitative trait loci (QTL) linked to cardiac mass. In addition to left ventricular mass (LVM), we also measured left ventricle (LV) concentration of atrial natriuretic factor (ANF), because we have previously established that there was a genetic link between these 2 traits in the same animal cross. We found 2 contiguous QTL on chromosome 5 that were linked to either LVM (logarithm of odds [LOD]=3.5) or log(n) (LV ANF) (LOD=12). The 1-LOD support intervals of both QTL shared a region overlapping the locus of natriuretic peptide precursor A (NPPA:) (ie, the ANF-coding gene). We found by sequencing 2 single nucleotide polymorphisms (SNPs) within the first 650 bp of the NPPA: minimal promoters of the genes from both strains. One of these SNPs increased the transcriptional activity of the NPPA: minimal promoter in transfected neonatal cardiomyocytes in keeping with the higher LV concentration of ANF observed in WKY versus WKHA rats. Taken together with the previous reports showing that ANF may protect cardiomyocytes against hypertrophy, our genetic data single out NPPA: as a strong candidate gene for the determination of LVM.
Asunto(s)
Cruzamientos Genéticos , Ligamiento Genético , Guanilato Ciclasa/genética , Hipertrofia Ventricular Izquierda/genética , Regiones Promotoras Genéticas , Receptores del Factor Natriurético Atrial/genética , Animales , Factor Natriurético Atrial/metabolismo , Células Cultivadas , Perfilación de la Expresión Génica , Escala de Lod , Masculino , Miocardio/citología , Miocardio/metabolismo , Tamaño de los Órganos/genética , Mapeo Físico de Cromosoma , Polimorfismo de Nucleótido Simple , Carácter Cuantitativo Heredable , Ratas , Ratas Endogámicas WKY , TransfecciónRESUMEN
In most rat models studied to date, increased ventricular mass is associated with high ventricular expression of the atrial natriuretic factor (ANF) gene. However, it is unknown whether ANF plays a beneficial or detrimental role in the course of left ventricular hypertrophy or whether ANF gene expression could be genetically linked to cardiac mass. To address such questions, we performed a cosegregation analysis in genetic crosses of inbred strains of rats. To select strains with the appropriate phenotypic characteristics, we first compared the ventricular abundance of ANF mRNA to ventricular mass (corrected for body weight) in 2 recombinant inbred strains derived from Wistar-Kyoto (WKY)/spontaneously hypertensive rat (SHR) hybrid crosses, ie, WKY-derived hyperactive (WKHA) and WKY-derived hypertensive (WKHT) rats, as well as in their parental inbred strains. In the 2 such strains that were normotensive, we observed that ventricular mass was higher in WKHA than in WKY rats, yet ventricular ANF mRNA was less abundant in WKHA than in WKY rats. Within a segregating population of F2 animals generated from a cross between WKY and WKHA genitors, the abundance of ventricular ANF mRNA and peptide correlated inversely with left ventricular mass, in contrast to the positive correlation observed with beta-myosin heavy chain mRNA. Finally, in the equally hypertensive SHR and WKHT strains, we found that ventricular mass was higher in SHR than in WKHT, yet ventricular ANF mRNA was less abundant in SHR than in WKHT. These results demonstrate for the first time that low ventricular ANF gene expression can be linked genetically to high cardiac mass independently of blood pressure and are consistent with a protective role for ANF against left ventricular hypertrophy.
Asunto(s)
Factor Natriurético Atrial/sangre , Factor Natriurético Atrial/genética , Hipertrofia Ventricular Izquierda/sangre , Hipertrofia Ventricular Izquierda/genética , Actinas/sangre , Animales , Biomarcadores , Presión Sanguínea , Cruzamientos Genéticos , Modelos Animales de Enfermedad , Expresión Génica/fisiología , Antígenos de Histocompatibilidad Clase II/sangre , Hipertrofia Ventricular Izquierda/fisiopatología , Masculino , Sondas de Oligonucleótidos , Fenotipo , ARN Mensajero/análisis , Radioinmunoensayo , Ratas , Ratas Endogámicas SHR , Ratas Endogámicas WKYRESUMEN
Functional angiotensin IV (Ang IV) receptors (denoted AT4) are localized to the outer stripe of the medulla in the rat kidney, and may play a critical role in salt homeostasis. The purpose of this study was to determine if AT4 receptor binding in the kidney is differently regulated in the salt-sensitive spontaneously hypertensive (SH) rat compared to Wistar Kyoto (WKY) controls. AT4 receptor binding was determined using in vitro receptor autoradiography. AT4 receptor binding in the outer stripe of the medulla was similar in WKY and SH rats maintained on a 1% salt diet. A high salt diet (8%) resulted in a statistically significant increase (28%) in AT4 receptor binding in kidneys from WKY rats. However, there was no change in AT4 receptor binding in the kidneys of SH rats fed the same diet. The present data indicate that AT4 binding sites are regulated by salt intake. In addition, regulation of this receptor may be impaired in the kidneys of SH rats, explaining in part the salt-sensitivity of this strain.
Asunto(s)
Hipertensión/metabolismo , Médula Renal/metabolismo , Ratas Endogámicas SHR/fisiología , Ratas Endogámicas WKY/fisiología , Receptores de Angiotensina/metabolismo , Cloruro de Sodio Dietético/administración & dosificación , Animales , Autorradiografía , Presión Sanguínea , Peso Corporal , RatasRESUMEN
Angiotensin II (Ang II) increases the level of tyrosine phosphorylation of several proteins in nondifferentiated NG108-15 cells, a hybrid derived from the fusion of mouse neuroblastoma and rat glioma cells. Conversely, incubation of NG108-15 cells with an angiotensin-converting enzyme (ACE) inhibitor decreased the basal level of tyrosine phosphorylation of proteins, suggesting that locally secreted Ang II may act as an autocrine regulator. By RT-PCR, we found that nondifferentiated NG108-15 cells contained the mRNA transcript of the rat angiotensinogen, mouse renin and rat ACE genes, thus confirming that NG108-15 cells contain all the elements of a local renin-angiotensin system.
Asunto(s)
Angiotensinógeno/genética , Glioma/genética , Neuroblastoma/genética , Peptidil-Dipeptidasa A/genética , Sistema Renina-Angiotensina/genética , Angiotensina II/farmacología , Inhibidores de la Enzima Convertidora de Angiotensina/farmacología , Animales , Línea Celular , Cartilla de ADN/genética , Células Híbridas/metabolismo , Lisinopril/farmacología , Ratones , Fosforilación/efectos de los fármacos , Reacción en Cadena de la Polimerasa , ARN Mensajero/genética , RatasRESUMEN
The natriuretic peptide receptor (NPR) family consists of three receptor subtypes: two transmembrane forms that contain a guanylyl cyclase intracellular domain (NPR-A and NPR-B), and one truncated form (NPR-C). Because of the lack of specific agonists and antagonists for each receptor subtype and to the difficulty to detect the presence of small quantities of NPR-B by ligand binding studies, polyclonal antibodies against a peptide whose sequence was chosen from a region of the extracellular domain of rat NPR-B that is not homologous to sequences in NPR-A and NPR-C were developed. Western blotting with affinity-purified anti-NPR-B (413-426)-Tyr revealed a polypeptide of approximately 120 kD on COS-1 cell membranes transfected with rat NPR-B cDNA. The antibody recognized a second polypeptide, approximately 5 to 10 kD smaller, which probably represents the unglycosylated receptor. Anti-NPR-B (413-426)-Tyr did not show crossreactivity to any other NPR. Western blotting analysis with anti-NPR-B (413-426)-Tyr also identified a protein of appropriate size in renal vascular membranes. These results were supported by immunohistochemistry findings that demonstrated staining for NPR-B on papillary and medullary capillaries, glomeruli, and renal arteries. This study concludes that NPR-B is present in the rat kidney, although it was only detected in vascular structures.
Asunto(s)
Guanilato Ciclasa/análisis , Riñón/metabolismo , Receptores del Factor Natriurético Atrial/análisis , Animales , Membrana Celular/metabolismo , Células Cultivadas , Immunoblotting , Inmunohistoquímica , Riñón/citología , Ratas , Ratas Sprague-Dawley , Sensibilidad y EspecificidadRESUMEN
The formation of the vasoactive peptide angiotensin II (AII) is dependent on the sequential action of two enzymes, renin and angiotensin converting enzyme (ACE), on the substrate angiotensinogen. Although the renin-producing cells of the kidney do not express angiotensinogen, they contain large amounts of AII in the same storage granules that contain renin. When renin expression is suppressed in these cells, AII also disappears. In the current study, we have tested whether the renin-associated disappearance of AII in renal juxtaglomerular (JG) cells is due to a renin-dependent down-regulation of granule biosynthesis and whether receptor-mediated internalization of AII could account for its concentration in these cells. Our results support a model whereby AII peptides are generated within JG cells, presumably by a mechanism which involves the action of endogenous renin on internalized, exogenous angiotensinogen.
Asunto(s)
Angiotensina II/biosíntesis , Aparato Yuxtaglomerular/metabolismo , Antagonistas de Receptores de Angiotensina , Animales , Aparato Yuxtaglomerular/citología , Aparato Yuxtaglomerular/ultraestructura , Masculino , Ratas , Ratas Sprague-Dawley , Receptores de Angiotensina/metabolismo , Renina/antagonistas & inhibidores , Renina/metabolismoRESUMEN
In recent years, it has become apparent that astrocytes (at least in vitro) harbor functional receptors to almost all possible neurotransmitters (with the potential noticeable exception of acetylcholine nicotinic receptors). Peptides are no exception, since receptors to all neuropeptides known to be produced in the CNS have been found on cultured astrocytes, and the presence of many of these has been confirmed on astrocytes in vivo. A variety of methodologies have been used to detect peptide receptors on astrocytes, as summarized in the current review. Special emphasis is also put on the possible roles that peptides may play in the regulation of astrocyte functions. These include proliferation, morphology, release of eicosanoids and arachidonic acid, induction of calcium transients and calcium waves, and control of internal pH, glucose uptake, glycogen metabolism, and gap junctional conductance. Recent data concerning the effects of natriuretic peptides on astrocytes are reviewed, and why these peptides may constitute priviledged tools to test the effects of peptides on astrocyte-neuron interactions is also discussed.
Asunto(s)
Astrocitos/fisiología , Neuropéptidos/fisiología , Receptores de Péptidos/fisiología , Animales , HumanosRESUMEN
Neonatal rats (3-14 days old) exhibit a period of adrenal hyporesponsiveness characterized by blunted corticosterone (B) responses to stress and reduced adrenal sensitivity to adrenocorticotropic hormone (ACTH). Several adrenomedullary peptidergic systems like vasoactive intestinal peptide (VIP) are postulated to influence cortical function. VIP is known to stimulate corticosterone secretion in vitro and to be released from the adrenal medulla following splanchnic nerve stimulation. Here, we tested whether 1) accelerated sympathetic innervation of the adrenal gland by daily L-thyroxine (T4) treatment modified the ontogeny of adrenal VIP and 2) an increase in VIP synthesis could prematurely increase adrenal sensitivity and corticosteroid output during neonatal life. Immunohistochemical VIP staining revealed a different ontogenetic pattern between adrenal regions from days 2-18 and different sensitivities to T4 treatment. Capsular staining was most abundant at all ages and increased with T4 treatment, whereas medullary staining was seen by day 18 and was not affected by T4. Throughout development, VIP receptors were detected mostly in the capsular region, but not in the adrenal cortex. Although receptor levels were not modified by T4 injections, T4 significantly enhanced VIP mRNA levels in the whole adrenal at all ages. In vivo administration of VIP (0.1-2.0 mg/kg body wt ip) to 9- to 12-day-old neonates increased pituitary ACTH, adrenal B, and aldosterone secretion significantly. Corticotropin-releasing factor immunoneutralization before VIP injection diminished VIP-induced ACTH release but still produced small but significant B and aldosterone secretion. Our results show that 1) VIP innervation of the adrenal capsule is present soon after birth and is increased by sympathetic activity whereas VIP appears only much later in the medulla and does not coincide with the onset of splanchnic innervation and 2) exogenous VIP stimulates ACTH, B, and aldosterone release during development and the effect of VIP on steroidogenic secretion is occurring through ACTH secretion, but also, at least in part, directly at the level of the adrenal gland.
Asunto(s)
Corticoesteroides/biosíntesis , Corteza Suprarrenal/fisiología , Médula Suprarrenal/fisiología , Envejecimiento/fisiología , Regulación del Desarrollo de la Expresión Génica , Hipófisis/fisiología , Nervios Esplácnicos/fisiología , Tiroxina/farmacología , Péptido Intestinal Vasoactivo/biosíntesis , Corteza Suprarrenal/crecimiento & desarrollo , Médula Suprarrenal/crecimiento & desarrollo , Médula Suprarrenal/inervación , Hormona Adrenocorticotrópica/sangre , Aldosterona/sangre , Animales , Animales Recién Nacidos , Corticosterona/sangre , Fibras Nerviosas/fisiología , Fibras Nerviosas/ultraestructura , Hipófisis/crecimiento & desarrollo , Reacción en Cadena de la Polimerasa , Ratas , Ratas Sprague-Dawley , Nervios Esplácnicos/efectos de los fármacos , Péptido Intestinal Vasoactivo/farmacologíaRESUMEN
Evidence suggests that astrocytes might play an important role in cerebral energy metabolism. A recently developed cell line, called DI TNC1, displays several characteristic features of astrocytes. Thus, we have investigated in these cells a number of parameters related to energy metabolism. First, glycogen, the major energy reserve in the brain, is present in these cells and its levels are influenced by the glucose content of the growth medium and the presence of serum. Second, several neurotransmitters including noradrenaline and vasoactive intestinal peptide (VIP) induce a glycogenolytic response. Their effect on glycogen is paralleled by a similar effect on the formation of cyclic AMP, which is presumably the second messenger involved. Third, noradrenaline stimulates glucose utilization (as reflected by 2-deoxyglucose uptake) in DI TNC1 cells, an effect which is mimicked by the second messenger arachidonate. Interestingly, two actions of neurotransmitters, which are well characterized in primary astrocytes, are absent in DI TNC1 cells. These are the noradrenaline- and VIP-induced resynthesis of glycogen and the glutamate-stimulated glycolysis. In summary, the observations reported here lend further support to the concept that astrocytes are important for the control of brain energy metabolism. In addition, DI TNC1 cells might represent an interesting preparation to help decipher some of the astrocytic functions related to energy metabolism.
Asunto(s)
Astrocitos/metabolismo , Encéfalo/metabolismo , Metabolismo Energético/efectos de los fármacos , Neurotransmisores/farmacología , Animales , Ácido Araquidónico/metabolismo , Astrocitos/efectos de los fármacos , Línea Celular , Línea Celular Transformada , Desoxiglucosa/metabolismo , Glucosa/metabolismo , Ácido Glutámico/farmacología , Glucógeno/metabolismo , Glucólisis/efectos de los fármacos , Norepinefrina/farmacología , Ratas , Sistemas de Mensajero Secundario , Péptido Intestinal Vasoactivo/farmacologíaRESUMEN
Angiotensin II (Ang II) is an octapeptide generated by the sequential proteolytic action of renin and angiotensin converting enzyme on the glycoprotein angiotensinogen. While numerous mammalian tissues have been shown to express some or all of the components of the renin-angiotensin system (RAS), the function of most of these tissue RAS remains a matter of conjecture. To test for tissue-specific functions of Ang II and as an alternative to co-expressing all the components of RAS, we have engineered a fusion protein that leads to direct Ang II release within specific tissues. The angiotensin peptide is cleaved from the fusion protein within the secretory pathway by the ubiquitous endoprotease furin and is released from the cell by constitutive secretion. Direct injection of an expression vector encoding such a fusion protein into rat cardiac ventricles results in a highly localized expression of atrial natriuretic peptide mRNA (an angiotensin responsive marker of cardiac hypertrophy), demonstrating the utility of this approach for local targeting of mature peptides to tissues in animal models.
Asunto(s)
Angiotensina II/genética , Regulación de la Expresión Génica , Angiotensina II/administración & dosificación , Angiotensina II/metabolismo , Animales , Secuencia de Bases , Vectores Genéticos , Ventrículos Cardíacos/metabolismo , Datos de Secuencia Molecular , Especificidad de Órganos , Ratas , Proteínas Recombinantes de Fusión/administración & dosificación , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismoRESUMEN
There is growing evidence that cyclic GMP (cGMP) plays important roles in the brain. In cultured rat astrocytes, we observed that the cGMP-inducing C-type natriuretic peptide (CNP) and cGMP analogues caused a decrease in intracellular pH (pHi). To examine whether this effect was due to inhibition of an Na+/H+ exchanger (NHE), we acidified cells by replacing extracellular Na+ by choline and examined the kinetics of the pHi recovery that occurred on reintroduction of Na+ in the extracellular medium. Both CNP and amiloride analogues inhibited the Na(+)-dependent pHi recovery, even in the nominal absence of CO2/HCO3-. This indicated that CNP inhibited the activity of an exchanger that was Na(+)-dependent, HCO3(-)-independent, and sensitive to known inhibitors of NHE. However, comparison of the potencies of four distinct amiloride analogues revealed a pharmacological profile that was different from that of any other NHE characterized to date. cGMP mimicked the effect of CNP on sodium-dependent pHi recovery, but the native nucleotide was as potent as membrane-permeant analogues. Intracellularly produced cGMP was very rapidly exported out of astrocytes. Probenecid and niflumic acid slowed down the rate of cGMP egression and inhibited the effect of CNP on Na(+)-dependent recovery, but not that of extracellular cGMP. Altogether, our data indicate that cGMP inhibits a novel type of NHE in astrocytes via an extracellular site of action. If these results with primary cultures transfer to brain, this phenomenon may constitute a mechanism by which natriuretic peptides exert some of their actions in the brain, as pHi transients have been shown to modulate several important astrocytic functions.
Asunto(s)
Astrocitos/efectos de los fármacos , GMP Cíclico/farmacología , Intercambiadores de Sodio-Hidrógeno/antagonistas & inhibidores , Amilorida/análogos & derivados , Amilorida/farmacología , Animales , Antiinflamatorios no Esteroideos/farmacología , Astrocitos/citología , Astrocitos/enzimología , Factor Natriurético Atrial/farmacología , Células Cultivadas/efectos de los fármacos , Células Cultivadas/enzimología , Colina/farmacología , GMP Cíclico/análogos & derivados , Diuréticos/farmacología , Espacio Extracelular/metabolismo , Concentración de Iones de Hidrógeno , Péptido Natriurético Tipo-C , Ácido Niflúmico/farmacología , Nootrópicos/farmacología , Probenecid/farmacología , Proteínas/farmacología , Ratas , Fármacos Renales/farmacología , Sodio/metabolismo , Sodio/farmacología , Intercambiadores de Sodio-Hidrógeno/química , Intercambiadores de Sodio-Hidrógeno/metabolismoRESUMEN
We compared the abundance and sensitivity of atrial natriuretic peptides (ANP) receptors in the brains of spontaneously hypertensive (SHR) and Wistar-Kyoto (WKY) rats and examined the effect of blood pressure on the abundance of brain ANP receptors in several other experimental rat models. Brain slices from SHR generated more guanosine 3',5'-cyclic monophosphate in response to ANP than brain slices from WKY rats. No differences were found in brain particulate guanylate cyclase activity in both strains of rats. In rat brain homogenates, we observed that ANP bound in a specific and saturable fashion to samples from WKY rats, but not in samples from SHR. In vitro receptor autoradiography revealed that ANP binding was reduced in the subfornical organ, the choroid plexus, and the paraventricular nucleus of SHR compared with WKY rat brains. Correction of hypertension in SHR or induction of hypertension in other strains did not affect ANP binding in any of these brain regions. Altogether, our data suggest that the increased sensitivity of SHR brains to the action of ANP may be a consequence of factors other than the abundance of receptors and that it is not secondary to the elevation of blood pressure.
Asunto(s)
Factor Natriurético Atrial/metabolismo , Factor Natriurético Atrial/farmacología , Encéfalo/metabolismo , Hipertensión/metabolismo , Hipotálamo/metabolismo , Receptores del Factor Natriurético Atrial/metabolismo , 1-Metil-3-Isobutilxantina/farmacología , Animales , GMP Cíclico/metabolismo , Guanilato Ciclasa/metabolismo , Hipotálamo/efectos de los fármacos , Técnicas In Vitro , Cinética , Masculino , Péptido Natriurético Tipo-C , Especificidad de Órganos , Proteínas/farmacología , Ratas , Ratas Endogámicas SHR , Ratas Endogámicas WKY , Ratas Sprague-Dawley , Valores de Referencia , Análisis de RegresiónRESUMEN
The precise identification of prorenin-processing enzymes has been hampered by the very low abundance of juxtaglomerular cells in the kidney. Recently, an immortalized renin-producing renal tumor cell line (As4.1) has been proposed as a model to carry out such studies. Despite the fact that they contain secretory granules, we found no evidence (on the basis of enzymatic assays of renin activity in the supernatant of the cells and of immunoprecipitations experiments) that the As4.1 cells can secrete active renin through the regulated pathway. As4.1 cells produce only renin-1, as they derive from a strain of mice expressing only one renin gene. However, stable transfection of these cells with a renin-2 expression plasmid increased the capacity of this cell line to secrete active renin in the regulated pathway. Northern blot and reverse transcriptase-polymerase chain reaction amplification (RT-PCR) assays revealed that furin, PACE4 and PC5 were the only members of the proprotein convertase (PC) family to be present in these cells. As PC5 is the only such enzyme with the demonstrated ability to process mouse prorenin 2, it may constitute a candidate enzyme for the processing of prorenin-2 in mouse juxtaglomerular cells. However, it is not likely to be involved in the processing of mouse prorenin 1.
Asunto(s)
Precursores Enzimáticos/metabolismo , Riñón/citología , Renina/metabolismo , Serina Endopeptidasas/metabolismo , Animales , Northern Blotting , Línea Celular/efectos de los fármacos , Línea Celular/enzimología , Línea Celular/ultraestructura , Frío , Gránulos Citoplasmáticos/ultraestructura , Precursores Enzimáticos/genética , Precursores Enzimáticos/aislamiento & purificación , Regulación Enzimológica de la Expresión Génica/fisiología , Ratones , Microscopía Electrónica , Plásmidos , Pruebas de Precipitina , Proproteína Convertasa 2 , Proproteína Convertasa 5 , Proproteína Convertasas , ARN Mensajero/análisis , Renina/genética , Renina/aislamiento & purificación , Serina Endopeptidasas/genética , Subtilisinas/genética , Subtilisinas/metabolismo , Transfección , Transgenes/fisiologíaRESUMEN
Two novel strains of rats have recently been generated from hybrid crosses of spontaneously hypertensive rats (SHR) and Wistar-Kyoto (WKY) rats. The breeding pairs chosen to create these strains were initially selected on the basis of two mutually exclusive phenotypic traits, so that the rats called WKHT are hypertensive but not hyperactive, whereas the rats called WKHA are hyperactive but not hypertensive. These strains have been refined by inbreeding on a strict brother-sister basis for more than 25 generations. To increase usefulness of these strains, we characterized them by use of genetic methods, including DNA finger-printing and simple sequence length polymorphism (SSLP) analyses. We found that these two novel strains are more closely related genetically to either SHR or WKY rats than the degree to which the latter two strains are related to one another; heterozygosities of SSLP marker alleles were extremely rare in WKHA and WKHT, indicating that these strains can be considered as truly inbred (in contrast to WKY rats from two sources); and it was almost always possible to match the SSLP marker alleles found in WKHA and WKHT rats with similarly sized alleles in the parental SHR and WKY alleles, indicating that the WKHA and WKHT strains constitute true mixes of the genomes of SHR and WKY rats. Furthermore, immunogenetic analyses indicated that WKY and WKHT rats belonged to the RT1 l haplotype, whereas SHR and WKHA rats belonged to the k haplotype. These results extend the usefulness of WKHA and WKHT rats for further genetic and physiologic studies.