Acciones de alcaloides de Gelsemium en receptores inhibitorios y en la función sináptica.
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Date
2023
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Universidad de Concepción
Abstract
Los receptores ionotrópicos de GABA del tipo A (GABAARs) y de glicina (GlyRs) son fundamentales para la inhibición sináptica en el sistema nervioso central (SNC). En la sinapsis, los neurotransmisores GABA y glicina activan GlyRs y GABAARs postsinápticos, permitiendo el influjo de iones cloruro y la hiperpolarización del potencial de membrana, controlando así la excitabilidad neuronal. Alteraciones en la neurotransmisión GABAérgica o glicinérgica se han asociado con trastornos prevalentes del SNC tales como epilepsia, depresión, ansiedad, dolor crónico, entre otros. La relevancia de la función GABAérgica ha sido resaltada por la buena eficacia de varios fármacos de uso clínico que poseen como blanco principal a los GABAARs. Por el contrario, actualmente no hay fármacos que tengan como blanco especifico a los GlyRs.
Con relación a la farmacología de estos receptores, es importante mencionar que se han identificado diversos moduladores de GlyRs y GABAARs a partir de plantas y hongos. En este contexto, los alcaloides de las plantas de Gelsemium han demostrado efectos beneficiosos en diversas patologías del SNC. Sin embargo, estos compuestos han mostrado una alta toxicidad intrínseca, con un perfil toxicológico que sugiere posibles efectos a nivel neuronal. Los blancos moleculares de estos alcaloides en el SNC aún no están definidos. No obstante, existen estudios que han sugerido la posible participación de GlyRs y GABAARs en sus acciones. Este trabajo de tesis logró definir y caracterizar la actividad de los cuatro principales alcaloides del Gelsemium (gelsemina, gelsevirina, koumina y humantenmina) en GABAARs y GlyRs, y en la función sináptica de neuronas corticales. Utilizando registros electrofisiológicos de GABAARs y GlyRs recombinantes, se demostró que gelsemina, koumina y gelsevirina inhibieron las corrientes glicinérgicas y GABAérgicas a concentraciones micromolares. El análisis de las concentraciones inhibitorias medias (IC50) y del porcentaje de máxima inhibición mostró que los GlyRs poseen una sensibilidad mayor a estos alcaloides que los GABAARs. Contrariamente, humantenmina no modificó significativamente la actividad de GABAARs y GlyRs. Utilizando receptores quiméricos y mutados en combinación con técnicas bioinformáticas, se logró determinar que dos mutaciones puntuales en el sitio ortostérico fueron suficientes para generar GlyRs insensibles a gelsemina, koumina y gelsevirina, lo que sugiere que las acciones de estos alcaloides dependen únicamente de su unión al sitio ortostérico. Por otra parte, en el contexto de la sinapsis, se logró establecer que gelsemina disminuyó la frecuencia de los eventos sinápticos GABAérgicos y glutamatérgicos en miniatura de neuronas corticales, sin modificar significativamente la amplitud. De modo interesante, se obtuvieron resultados similares utilizando el alcaloide humantenmina, demostrando que la inhibición directa de GlyRs o GABAARs no es necesaria para los efectos sinápticos. Finalmente, estudios de fluorometría de Ca2+ intracelular lograron demostrar que gelsemina disminuyó la frecuencia de las señales espontaneas de Ca2+, sugiriendo que gelsemina, y posiblemente otros alcaloides del Gelsemium, modulan la sinapsis a través de la interacción con blancos moleculares presinápticos.
En conjunto, los resultados de esta tesis proporcionan información novedosa sobre las acciones funcionales y los sitios moleculares de los principales alcaloides del Gelsemium en GABAARs y GlyRs. El perfil de actividad de gelsemina, koumina y gelsevirina en estos canales iónicos sugiere que estas interacciones pudieran estar asociadas a su toxicidad. Por el contrario, los resultados encontrados para humantemina y gelsemina a nivel neuronal sugieren que su actividad biológica estaría relacionada con blancos moleculares presinápticos, diferentes a GABAARs y GlyRs. Estas acciones sinápticas proporcionan una hipótesis novedosa para explicar los diversos efectos beneficiosos de los alcaloides del Gelsemium. En resumen, los resultados de este trabajo contribuyen a dilucidar, al menos en parte, los mecanismos involucrados en las acciones beneficiosas y tóxicas de los alcaloides del Gelsemium a nivel del SNC de mamíferos.
Ionotropic gamma-aminobutyric acid type A receptors (GABA A Rs) and glycine receptors (GlyRs) play a fundamental role in synaptic inhibition within the central nervous system (CNS). At synapses, neurotransmitters GABA and glycine actívate postsynaptic GlyRs and GABA A Rs, f acilitating the entry of chloride ions and inducing membrane hyperpolarization to regulate neuronal excitability . Dysregulation of GABAergic or glycinergic neurotransmission has been associated with prevalent CNS disorders such as epilepsy, depression, anxiety, and chronic pain. The significance of GABAergi c function is underscored by the efficacy of various clinically used drugs targeting GABAARs. In contrast, there are currently no drugs specifically targeting GlyRs. Regarding the pharmacology of these receptors, various modulators of GlyRs and GABA A Rs have been identified from plants and fungi. In this context, alkaloids from Gelsemium plants have demonstrated beneficial effects in various CNS pathologies. However, these compounds exhibit high intrinsic toxicity, with a toxicological profile suggesting potential neuronal effects. The molecular targets of these alkaloids in the CNS are ye t to be defined. Nevertheless, studies have suggested the possible involvement of G lyRs and GABA A Rs in their actions. This tesis successfully defined and characterized the activity of the four main Gelsemium alkaloids ( g elsemine, g elsevirine, k oumine, and h umantenmine) on GABA A Rs, GlyRs, and synaptic function in cortical neurons. Using electrophysiological recordings from recombinant GABA A Rs and GlyRs, it was demonstrated that Gelsemine, Koumine, and Gelsevirine inhibited glycinergic and GABAergic currents at micromolar concentrations. Analysis of the half maximal inhibitory concentrations (IC 50 ) and the percentage of maximum inhibition revealed that GlyRs exhibit greater sensitivity to these alkaloids than GABA A Rs. Conversely, Humantenmine did not significantly alter the activity of GABA A Rs and GlyRs. Using chimeric and mutated receptors in combination with bioinformatics, it was determined that two point mutations in the orthosteric site were sufficient to generate GlyRs insensitive to Ge lsemine, Koumine, and Gelsevirine, suggesting that the functional effects of these alkaloids depend solely on their binding to the orthosteric site. Moreover, in the synaptic context, Gelsemine was found to decrease the frequency of GABAergic and glutamatergic miniature synaptic events in cortical neurons without significantly affecting amplitude. Interestingly, similar results were obtained with the a lkaloid h umantenmine, demonstrating that direct inhibition of GlyRs or GABA A Rs is not necessary for these synaptic effects. Finally, intracelular Ca 2+ fluorimetry studies demonstrated that g elsemine significantly reduced the frequency of spontaneous Ca 2+ signals, suggesting that Gelsemine, and possibly other Gelsemium alkaloids, modulate synapses through interaction with presynaptic molecular targets. Collectively, the results of this thesis provide novel insights into the functional actions and molecular sites of Gelsemium 's main alkaloids on GABA A Rs and GlyRs. The activity profile of g elsemine, k oumine, and g elsevirine on these ion channels suggests a potential association with their toxicity. Conversely, the findings for humantenmine and g elsemine at the neuronal level suggest that their biological activity may be related to presynaptic molecular targets, different from GABA A Rs and GlyRs. These synaptic actions provide a novel hypothesis to explain the diverse beneficial effects of Gelsemium alkaloids. In summary, the results of this work contribute to elucidating, at least in part, the mechanisms involved in the beneficial and toxic actions of Gelsemium alkaloids in the mammalian CNS.
Ionotropic gamma-aminobutyric acid type A receptors (GABA A Rs) and glycine receptors (GlyRs) play a fundamental role in synaptic inhibition within the central nervous system (CNS). At synapses, neurotransmitters GABA and glycine actívate postsynaptic GlyRs and GABA A Rs, f acilitating the entry of chloride ions and inducing membrane hyperpolarization to regulate neuronal excitability . Dysregulation of GABAergic or glycinergic neurotransmission has been associated with prevalent CNS disorders such as epilepsy, depression, anxiety, and chronic pain. The significance of GABAergi c function is underscored by the efficacy of various clinically used drugs targeting GABAARs. In contrast, there are currently no drugs specifically targeting GlyRs. Regarding the pharmacology of these receptors, various modulators of GlyRs and GABA A Rs have been identified from plants and fungi. In this context, alkaloids from Gelsemium plants have demonstrated beneficial effects in various CNS pathologies. However, these compounds exhibit high intrinsic toxicity, with a toxicological profile suggesting potential neuronal effects. The molecular targets of these alkaloids in the CNS are ye t to be defined. Nevertheless, studies have suggested the possible involvement of G lyRs and GABA A Rs in their actions. This tesis successfully defined and characterized the activity of the four main Gelsemium alkaloids ( g elsemine, g elsevirine, k oumine, and h umantenmine) on GABA A Rs, GlyRs, and synaptic function in cortical neurons. Using electrophysiological recordings from recombinant GABA A Rs and GlyRs, it was demonstrated that Gelsemine, Koumine, and Gelsevirine inhibited glycinergic and GABAergic currents at micromolar concentrations. Analysis of the half maximal inhibitory concentrations (IC 50 ) and the percentage of maximum inhibition revealed that GlyRs exhibit greater sensitivity to these alkaloids than GABA A Rs. Conversely, Humantenmine did not significantly alter the activity of GABA A Rs and GlyRs. Using chimeric and mutated receptors in combination with bioinformatics, it was determined that two point mutations in the orthosteric site were sufficient to generate GlyRs insensitive to Ge lsemine, Koumine, and Gelsevirine, suggesting that the functional effects of these alkaloids depend solely on their binding to the orthosteric site. Moreover, in the synaptic context, Gelsemine was found to decrease the frequency of GABAergic and glutamatergic miniature synaptic events in cortical neurons without significantly affecting amplitude. Interestingly, similar results were obtained with the a lkaloid h umantenmine, demonstrating that direct inhibition of GlyRs or GABA A Rs is not necessary for these synaptic effects. Finally, intracelular Ca 2+ fluorimetry studies demonstrated that g elsemine significantly reduced the frequency of spontaneous Ca 2+ signals, suggesting that Gelsemine, and possibly other Gelsemium alkaloids, modulate synapses through interaction with presynaptic molecular targets. Collectively, the results of this thesis provide novel insights into the functional actions and molecular sites of Gelsemium 's main alkaloids on GABA A Rs and GlyRs. The activity profile of g elsemine, k oumine, and g elsevirine on these ion channels suggests a potential association with their toxicity. Conversely, the findings for humantenmine and g elsemine at the neuronal level suggest that their biological activity may be related to presynaptic molecular targets, different from GABA A Rs and GlyRs. These synaptic actions provide a novel hypothesis to explain the diverse beneficial effects of Gelsemium alkaloids. In summary, the results of this work contribute to elucidating, at least in part, the mechanisms involved in the beneficial and toxic actions of Gelsemium alkaloids in the mammalian CNS.
Description
Tesis presentada para optar al grado de Doctor en Ciencias Biológicas.
Keywords
Alcaloides, Sinapsis, Sistema nervioso central