Caracterización y rol funcional de canales iónicos en células de músculo liso vascular humano

La investigación en fisiología vascular tiene como objetivo relevante la identificación de los mecanismos que median las respuestas contráctiles del músculo liso en condiciones fisiológicas, patológicas o ante la administración de fármacos. Distintos tipos de canales iónicos, permitiendo y modulando el pasaje de iones a través de la membrana celular, participan en la regulación de la función contráctil de las células de músculo liso que forman parte de la pared de los vasos sanguíneos. En particular, el mantenimiento del potencial de membrana celular, la regulación del grado de polarización del mismo y la modulación del influjo de Ca2+ y Na+, constituyen algunos de los mecanismos donde dichos canales cumplen un rol fundamental. Este trabajo de Tesis Doctoral tuvo como objetivo central identificar y caracterizar las propiedades electrofisiológicas de distintos tipos de canales iónicos en células aisladas de músculo liso humano e indagar su participación en el mecanismo de contracción y relajación del tejido intacto, con la finalidad de producir conocimiento acerca de los mecanismos fisiológicos que sustentan la función contráctil del músculo liso vascular humano. En particular, contribuir a la caracterización de las propiedades de los canales iónicos de K+ y de los mecanismos reguladores de los mismos, en un tipo celular muy poco estudiado en este campo, ya sea aportando datos obtenidos en células frescas aisladas como a partir de segmentos intactos donde se pueden poner en juego mecanismos reguladores intrínsecos de la pared vascular (endotelio, subendotelio, uniones “gap”, etc.). De la correlación de los mismos pueden surgir indicaciones sobre distintos roles fisiológicos de dichos canales en estos vasos humanos. Las arterias umbilicales tienen gran relevancia fisiológica ya que estos vasos son responsables de mantener una resistencia al flujo baja para lograr recibir alrededor del 40% del volumen minuto cardíaco fetal que se oxigenará circulando por la placenta. Con respecto a la arteria mamaria interna humana, el objetivo central fue indagar las propiedades del canal de K+ activado por Ca2+ de alta conductancia, su regulación por cambios de pH y la influencia sobre el estado contráctil de este vaso que pueda tener este factor. Estos resultados permitirán conocer mejor la fisiología de este vaso humano ampliamente utilizado en cirugías de revascularización miocárdica.Tesis digitalizada en SEDICI gracias a la Biblioteca Central de la Facultad de Ciencias Exactas (UNLP).Facultad de Ciencias Exacta

Caracterización y rol funcional de canales iónicos en células de músculo liso vascular humano

La investigación en fisiología vascular tiene como objetivo relevante la identificación de los mecanismos que median las respuestas contráctiles del músculo liso en condiciones fisiológicas, patológicas o ante la administración de fármacos. Distintos tipos de canales iónicos, permitiendo y modulando el pasaje de iones a través de la membrana celular, participan en la regulación de la función contráctil de las células de músculo liso que forman parte de la pared de los vasos sanguíneos. En particular, el mantenimiento del potencial de membrana celular, la regulación del grado de polarización del mismo y la modulación del influjo de Ca2+ y Na+, constituyen algunos de los mecanismos donde dichos canales cumplen un rol fundamental. Este trabajo de Tesis Doctoral tuvo como objetivo central identificar y caracterizar las propiedades electrofisiológicas de distintos tipos de canales iónicos en células aisladas de músculo liso humano e indagar su participación en el mecanismo de contracción y relajación del tejido intacto, con la finalidad de producir conocimiento acerca de los mecanismos fisiológicos que sustentan la función contráctil del músculo liso vascular humano. En particular, contribuir a la caracterización de las propiedades de los canales iónicos de K+ y de los mecanismos reguladores de los mismos, en un tipo celular muy poco estudiado en este campo, ya sea aportando datos obtenidos en células frescas aisladas como a partir de segmentos intactos donde se pueden poner en juego mecanismos reguladores intrínsecos de la pared vascular (endotelio, subendotelio, uniones “gap”, etc.). De la correlación de los mismos pueden surgir indicaciones sobre distintos roles fisiológicos de dichos canales en estos vasos humanos. Las arterias umbilicales tienen gran relevancia fisiológica ya que estos vasos son responsables de mantener una resistencia al flujo baja para lograr recibir alrededor del 40% del volumen minuto cardíaco fetal que se oxigenará circulando por la placenta. Con respecto a la arteria mamaria interna humana, el objetivo central fue indagar las propiedades del canal de K+ activado por Ca2+ de alta conductancia, su regulación por cambios de pH y la influencia sobre el estado contráctil de este vaso que pueda tener este factor. Estos resultados permitirán conocer mejor la fisiología de este vaso humano ampliamente utilizado en cirugías de revascularización miocárdica.Tesis digitalizada en SEDICI gracias a la Biblioteca Central de la Facultad de Ciencias Exactas (UNLP).Doctor en Ciencias Exactas, área Ciencias BiológicasUniversidad Nacional de La PlataFacultad de Ciencias Exacta

Considerations about rodent models of binge eating episodes

A binge eating episode is defined as an uncontrolled event of hyperphagia, in which people quickly eat a large amount of food while feeling a sense of loss of control over eating (Wolfe et al., 2009). Binge eating episodes are observed in a variety of human disorders including bulimia nervosa (BN), binge eating disorder (BED), and the binge/purge subtype of anorexia nervosa (AN) (Berger and Tanofsky-Kraff, 2012). Binge eating episodes are also present in overweight and obese people, as well as non-clinical populations under specific circumstances such as stress. The etiology of this behavior is currently unknown. The use of rodent models has been essential for understanding the pathogenesis of many human diseases; however, it is challenging to mimic all features of human binge eating in rodent models (Corwin and Buda-Levin, 2004; Perello et al., 2010a). In particular, these models should not only display the objective characteristics of a binge eating episode, namely the consumption of a large amount of food in a short period of time, but also the subjective characteristics of the feeling of loss of control. Recently, we examined the neuronal circuitries activated in naïve mice allowed to spontaneously eat a high fat diet (HFD) pellet for 2 h (Valdivia et al., 2014). We found that satiated mice with free access to regular chow rapidly consume a significant amount of HFD when exposed to it, and that HFD intake recruits centers of the mesolimbic pathway, which are known to be activated in human beings displaying binge eating behavior (see below). Experts in the field agreed that our simple model of HFD overconsumption could be relevant for studying neuronal aspects of binge eating behaviors. However, some reviewers argued that it was misleading to describe our model as a model of binge eating. Some criticisms were that our model lacked indications of feelings of loss of control, repeated feeding episodes, escalation of intake over time, a significant level of hyperphagia, and evidence that bingeing occurred in the face of aversive consequences. The notable divergence in the opinion of the journal´s reviewers made evident that a comprehensive debate about rodent binge eating models is needed. Here, we briefly present our opinion about the features that a rodent model should fulfill in order to be considered a reasonable model of binge eating episodes and its implications in terms of the neuronal circuits involved.Fil: Perello, Mario. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto Multidisciplinario de Biología Celular. Provincia de Buenos Aires. Gobernación. Comisión de Investigaciones Científicas. Instituto Multidisciplinario de Biología Celular. Universidad Nacional de La Plata. Instituto Multidisciplinario de Biología Celular; ArgentinaFil: Valdivia Torres, Lesly Spring. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto Multidisciplinario de Biología Celular. Provincia de Buenos Aires. Gobernación. Comisión de Investigaciones Científicas. Instituto Multidisciplinario de Biología Celular. Universidad Nacional de La Plata. Instituto Multidisciplinario de Biología Celular; ArgentinaFil: Garcia Romero, Guadalupe. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto Multidisciplinario de Biología Celular. Provincia de Buenos Aires. Gobernación. Comisión de Investigaciones Científicas. Instituto Multidisciplinario de Biología Celular. Universidad Nacional de La Plata. Instituto Multidisciplinario de Biología Celular; ArgentinaFil: Raingo, Jesica. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto Multidisciplinario de Biología Celular. Provincia de Buenos Aires. Gobernación. Comisión de Investigaciones Científicas. Instituto Multidisciplinario de Biología Celular. Universidad Nacional de La Plata. Instituto Multidisciplinario de Biología Celular; Argentin

La actividad constitutiva del receptor de ghrelina reduce la expresión en superficie de los canales de Ca2+ (CaV) dependientes de voltaje de manera dependiente de CaVβ

Los canales de calcio (Ca+2) dependientes del voltaje (CaV) acoplan la despolarización de la membrana al influjo de Ca+2, lo que desencadena una serie de procesos celulares dependientes de Ca+2. Los CaV son, por lo tanto, cruciales en la configuración de la actividad y función neuronal, dependiendo de sus propiedades temporales y espaciales individuales. Además, muchos neurotransmisores y fármacos que actúan a través de los receptores acoplados a la proteína G (GPCR), modulan la actividad neuronal al alterar la expresión, el tráfico o la función de los CaV. El objetivo del actual trabajo es entender el mecanismo a través del cual el receptor de la hormona orexigénica ghrelina (GHSR1a, por sus siglas en Inglés growth hormone secretagogue receptor type 1a) regula la densidad de CaV en la superficie celular. Nuestros resultados indican que GHSR puede inhibir el tráfico hacia la membrana plasmática de varios subtipos de CaV, incluso en ausencia de su agonista natural, a través de su actividad constitutiva. Esta forma crónica de inhibición del GPCR sobre los CaV depende de la presencia de la subunidad auxiliar CaVβ. Esto se demostró tanto en cultivos neuronales primarios hipotalámicos como en sistemas de expresión heterólogos, utilizando electrofisiología de patch clamp y microscopía confocal para examinar las ubicaciones sub-celulares de proteínas marcadas. El efecto de la actividad constitutiva de GHSR en el tráfico de los CaV sugiere un papel para esta vía de señalización en áreas del cerebro que controlan la ingesta de alimentos, la recompensa, el aprendizaje y la memoria donde el mismo se expresa.Eje: BiofísicaInstituto Multidisciplinario de Biología Celula

Mouse models for V103I and I251L gain of function variants of the human MC4R display decreased adiposity but are not protected against a hypercaloric diet

Objective: The melanocortin 4 receptor (MC4R) is a G protein-coupled receptor that plays major roles in the central control of energy balance. Loss-of-function mutations of MC4R constitute the most common monogenic cause of early-onset extreme obesity in humans, whereas gain-offunction mutations appear to be protective. In particular, two relatively frequent alleles carrying the non-synonymous coding mutations V103I or I251L are associated with lower risks of obesity and type-2 diabetes. Although V103I and I251L MC4Rs showed more efficient signalling in transfected cells, their specific effects in live animals remain unexplored. Here, we investigated whether the introduction of V103I and I251L mutations into the mouse MC4R leads to a lean phenotype and provides protection against an obesogenic diet. Methods: Using CRISPR/Cas9, we generated two novel strains of mice carrying single-nucleotide mutations into the mouse Mc4r which are identical to those present in V103I and I251L MCR4 human alleles, and studied their phenotypic outcomes in mice fed with normal chow or a high-fat diet. In particular, we measured body weight progression, food intake and adiposity. In addition, we analysed glucose homeostasis through glucose and insulin tolerance tests. Results: We found that homozygous V103I females displayed shorter longitudinal length and decreased abdominal white fat, whereas homozygous I251L females were also shorter and leaner due to decreased weight in all white fat pads examined. Homozygous Mc4rV103I/V103I and Mc4rI251L/I251L mice of both sexes showed improved glucose homeostasis when challenged in a glucose tolerance test, whereas Mc4rI251L/I251L females showed improved responses to insulin. Despite being leaner and metabolically more efficient, V103I and I251L mutants fed with a hypercaloric diet increased their fasting glucose levels and adiposity similar to their wild-type littermates. Conclusions: Our results demonstrate that mice carrying V103I and I251L MC4R mutations displayed gain-of-function phenotypes that were more evident in females. However, hypermorphic MC4R mutants were as susceptible as their control littermates to the obesogenic and diabetogenic effects elicited by a long-term hypercaloric diet, highlighting the importance of healthy feeding habits even under favourable genetic conditions.Fil: Rojo, Daniela. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular "Dr. Héctor N. Torres"; ArgentinaFil: Mccarthy, Clara Inés. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto Multidisciplinario de Biología Celular. Provincia de Buenos Aires. Gobernación. Comisión de Investigaciones Científicas. Instituto Multidisciplinario de Biología Celular. Universidad Nacional de La Plata. Instituto Multidisciplinario de Biología Celular; ArgentinaFil: Raingo, Jesica. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto Multidisciplinario de Biología Celular. Provincia de Buenos Aires. Gobernación. Comisión de Investigaciones Científicas. Instituto Multidisciplinario de Biología Celular. Universidad Nacional de La Plata. Instituto Multidisciplinario de Biología Celular; ArgentinaFil: Rubinstein, Marcelo. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular "Dr. Héctor N. Torres"; Argentina. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Departamento de Fisiología, Biología Molecular y Celular; Argentina. University of Michigan; Estados Unido

Impact of A118G polymorphism on the Mu opioid receptor function in pain

Mu Opioid Receptor (MOR) activation by exogenous or endogenous agonists causes reduction of pain threshold after a noxious stimulus, relieving pain sensation.MOR is encoded by OPRM1 gene and its messenger RNA suffers extensible modifications by alternative splicing and single nucleotide polymorphisms (SNPs). A118G (N40D) is the most frequent encoding MOR SNP in humans. In this review we discuss the impact of this polymorphism at molecular, cellular and clinical levels. Since some SNPs are unequally distributed among human populations, we also discuss the utility of A118G as an ethnicity marker among worldwide human populations. As an example, we evaluate A118G frequency in an Argentinean humanpopulation and compare it with worldwide frequencies extracted from HapMap database.Instituto Multidisciplinario de Biología Celula

Considerations about rodent models of binge eating episodes

A binge eating episode is defined as an uncontrolled event of hyperphagia, in which people quickly eat a large amount of food while feeling a sense of loss of control over eating (Wolfe et al., 2009). Binge eating episodes are observed in a variety of human disorders including bulimia nervosa (BN), binge eating disorder (BED), and the binge/purge subtype of anorexia nervosa (AN) (Berger and Tanofsky-Kraff, 2012). Binge eating episodes are also present in overweight and obese people, as well as non-clinical populations under specific circumstances such as stress. The etiology of this behavior is currently unknown. The use of rodent models has been essential for understanding the pathogenesis of many human diseases; however, it is challenging to mimic all features of human binge eating in rodent models (Corwin and Buda-Levin, 2004; Perello et al., 2010a). In particular, these models should not only display the objective characteristics of a binge eating episode, namely the consumption of a large amount of food in a short period of time, but also the subjective characteristics of the feeling of loss of control. Recently, we examined the neuronal circuitries activated in naïve mice allowed to spontaneously eat a high fat diet (HFD) pellet for 2 h (Valdivia et al., 2014). We found that satiated mice with free access to regular chow rapidly consume a significant amount of HFD when exposed to it, and that HFD intake recruits centers of the mesolimbic pathway, which are known to be activated in human beings displaying binge eating behavior (see below). Experts in the field agreed that our simple model of HFD overconsumption could be relevant for studying neuronal aspects of binge eating behaviors. However, some reviewers argued that it was misleading to describe our model as a model of binge eating. Some criticisms were that our model lacked indications of feelings of loss of control, repeated feeding episodes, escalation of intake over time, a significant level of hyperphagia, and evidence that bingeing occurred in the face of aversive consequences. The notable divergence in the opinion of the journal´s reviewers made evident that a comprehensive debate about rodent binge eating models is needed. Here, we briefly present our opinion about the features that a rodent model should fulfill in order to be considered a reasonable model of binge eating episodes and its implications in terms of the neuronal circuits involved.Instituto Multidisciplinario de Biología Celula

Impact of A118G polymorphism on the Mu opioid receptor function in pain

Mu Opioid Receptor (MOR) activation by exogenous or endogenous agonists causes reduction of pain threshold after a noxious stimulus, relieving pain sensation.MOR is encoded by OPRM1 gene and its messenger RNA suffers extensible modifications by alternative splicing and single nucleotide polymorphisms (SNPs). A118G (N40D) is the most frequent encoding MOR SNP in humans. In this review we discuss the impact of this polymorphism at molecular, cellular and clinical levels. Since some SNPs are unequally distributed among human populations, we also discuss the utility of A118G as an ethnicity marker among worldwide human populations. As an example, we evaluate A118G frequency in an Argentinean humanpopulation and compare it with worldwide frequencies extracted from HapMap database.Instituto Multidisciplinario de Biología Celula

Potassium channels in human umbilical artery cells

To identify K+ channels of smooth muscle of human umbilical artery using the patch-clamp technique and to study their effect on resting tone of umbilical artery rings. Whole-cell and single-channel patch-clamp recordings in enzymatically isolated smooth muscle cells were made. Measurements of developed isometric force were performed on intact tissue. Delayed rectifier K+ channels (KDR) and large-conductance Ca2+-activated K+ channels (BKCa) contribute to the whole-cell voltage-and time-dependent outward K+ current, as it was specifically inhibited by 5 mM 4-aminopyridine (4-AP; KDR blocker) (92 ± 4% at 0 mV, n = 7), by 1 mM tetraethylammonium (TEA; BKCa blocker) (71 ± 4% at +60 mV, n = 4), and by 200 nM iberiotoxin (BKCa blocker) (64 ± 7% at +60 mV, n = 4). In outside-out patches, BKCa channels had ā single-channel conductance of 132 ± 4 pS (n = 24) in asymmetric K+ conditions and 216 ± 4 pS (n = 4) in a symmetric K+ gradient. The activity of the BKCa channels was significantly augmented by 1 μM Ca2+ in the inside-out configuration. 4-AP had no effect on resting tone of intact arterial rings. TEA produced contraction of arterial rings whereas phloretin, an activator of BKCa, relaxed them, which means that BKCa channels are functional in intact tissue and are involved in the maintenance of resting tone in this human vessel. The identities of K+ channels in the human umbilical artery were shown using the patch-clamp technique, and the physiologic effect of K+ channels on resting tone was documented.Facultad de Ciencias Exacta

Ghrelin Selectively Inhibits CaV3.3 Subtype of Low-Voltage-Gated Calcium Channels

The mechanisms by which ghrelin controls electrical activity in the hypothalamus are not fully understood. One unexplored target of ghrelin is CaV3, responsible for transient calcium currents (T-currents) that control neuronal firing. We investigated the effect of ghrelin on CaV3 subtypes and how this modulation impacts on neuronal activity. We performed whole-cell patch-clamp recordings in primary mouse hypothalamic cultures to explore the effect of ghrelin on T-currents. We also recorded calcium currents from transiently transfected tsA201 cells to study the sensitivity of each CaV3 subtype to GHSR activation. Finally, we ran a computational model combining the well-known reduction of potassium current by ghrelin with the CaV3 biophysical parameter modifications induced by ghrelin to predict the impact on neuronal electrical behavior. We found that ghrelin inhibits native NiCl₂ sensitive current currents in hypothalamic neurons. We determined that CaV3.3 is the only CaV3 subtype sensitive to ghrelin. The modulation of CaV3.3 by ghrelin comprises a reduction in maximum conductance, a shift to hyperpolarized voltages of the I–V and steady-state inactivation curves, and an acceleration of activation and inactivation kinetics. Our model-based prediction indicates that the inhibition of CaV3.3 would attenuate the stimulation of firing originating from the inhibition of potassium currents by ghrelin. In summary, we discovered a new target of ghrelin in neurons: the CaV3.3. This mechanism would imply a negative feed-forward regulation of the neuronal activation exerted by ghrelin. Our work expands the knowledge of the wide range of actions of GHSR, a receptor potentially targeted by therapeutics for several diseases.Facultad de Ciencias ExactasInstituto Multidisciplinario de Biología Celula