RESUMEN
Cannabidiol (CBD) has been recently approved as an antiseizure agent in Dravet Syndrome (DS), a pediatric epileptic encephalopathy, but CBD could also be active against associated comorbidities. Such associated comorbidities were also attenuated by the sesquiterpene ß-caryophyllene (BCP). Here, we have compared the efficacy of both compounds and further initiated the analysis of a possible additive effect between both compounds in relation with these comorbidities using two experimental approaches. The first experiment was aimed at comparing the benefits of CBD and BCP, including their combination in conditional knock-in Scn1a-A1783V mice, an experimental model of DS, treated since the postnatal day 10th to 24th. As expected, DS mice showed impairment in limb clasping, delay in the appearance of hindlimb grasp reflex and additional behavioural disturbances (e.g., hyperactivity, cognitive deterioration, social interaction deficits). This behavioural impairment was associated with marked astroglial and microglial reactivities in the prefrontal cortex and the hippocampal dentate gyrus. BCP and CBD administered alone were both able to partially attenuate the behavioural disturbances and the glial reactivities, with apparently greater efficacy against glial reactivities obtained with BCP, whereas superior effects in a few specific parameters were obtained when both compounds were combined. In the second experiment, we investigated this additive effect in cultured BV2 cells treated with BCP and/or CBD and stimulated with LPS. As expected, addition of LPS induced a marked increase in several inflammation-related markers (e.g., TLR4, COX-2, iNOS, catalase, TNF-α, IL-1ß), as well as elevated Iba-1 immunostaining. Treatment with BCP or CBD attenuated these elevations, but, again and in general, superior results were obtained when both cannabinoids were combined. In conclusion, our results support the interest to continue investigating the combination of BCP and CBD to improve the therapeutic management of DS in relation with their disease-modifying properties.
Asunto(s)
Cannabidiol , Epilepsias Mioclónicas , Ratones , Animales , Cannabidiol/farmacología , Cannabidiol/uso terapéutico , Lipopolisacáridos , Epilepsias Mioclónicas/tratamiento farmacológico , Canal de Sodio Activado por Voltaje NAV1.1RESUMEN
Dravet syndrome (DS) is an epileptic encephalopathy caused by mutations in the Scn1a gene encoding the α1 subunit of the Nav1.1 sodium channel, which is associated with recurrent and generalized seizures, even leading to death. In experimental models of DS, histological alterations have been found in the brain; however, the retina is a projection of the brain and there are no studies that analyze the possible histological changes that may occur in the disease. This study analyzes the retinal histological changes in glial cells (microglia and astrocytes), retinal ganglion cells (RGCs) and GABAergic amacrine cells in an experimental model of DS (Syn-Cre/Scn1aWT/A1783V) compared to a control group at postnatal day (PND) 25. Retinal whole-mounts were labeled with anti-GFAP, anti-Iba-1, anti-Brn3a and anti-GAD65/67. Signs of microglial and astroglial activation, and the number of Brn3a+ and GAD65+67+ cells were quantified. We found retinal activation of astroglial and microglial cells but not death of RGCs and GABAergic amacrine cells. These changes are similar to those found at the level of the hippocampus in the same experimental model in PND25, indicating a relationship between brain and retinal changes in DS. This suggests that the retina could serve as a possible biomarker in DS.
Asunto(s)
Epilepsias Mioclónicas , Canal de Sodio Activado por Voltaje NAV1.1 , Ratones , Animales , Canal de Sodio Activado por Voltaje NAV1.1/genética , Epilepsias Mioclónicas/genética , Epilepsias Mioclónicas/patología , Retina/patología , Convulsiones/genética , Microglía/patología , Modelos Animales de EnfermedadRESUMEN
Cannabis plant has been used from ancient times with therapeutic purposes for treating human pathologies, but the identification of the cellular and molecular mechanisms underlying the therapeutic properties of the phytocannabinoids, the active compounds in this plant, occurred in the last years of the past century. In the late 1980s and early 1990s, seminal studies demonstrated the existence of cannabinoid receptors and other elements of the so-called endocannabinoid system. These G protein-coupled receptors (GPCRs) are a key element in the functions assigned to endocannabinoids and appear to serve as promising pharmacological targets. They include CB1, CB2, and GPR55, but also non-GPCRs can be activated by endocannabinoids, like ionotropic receptor TRPV1 and even nuclear receptors of the PPAR family. Their activation, inhibition, or simply modulation have been associated with numerous physiological effects at both central and peripheral levels, which may have therapeutic value in different human pathologies, then providing a solid experimental explanation for both the ancient medicinal uses of Cannabis plant and the recent advances in the development of cannabinoid-based specific therapies. This chapter will review the scientific knowledge generated in the last years around the research on the different endocannabinoid-binding receptors and their signaling mechanisms. Our intention is that this knowledge may help readers to understand the relevance of these receptors in health and disease conditions, as well as it may serve as the theoretical basis for the different experimental protocols to investigate these receptors and their signaling mechanisms that will be described in the following chapters.
Asunto(s)
Cannabinoides , Cannabis , Endocannabinoides , Humanos , Receptores Activados del Proliferador del Peroxisoma , Receptor Cannabinoide CB2 , Receptores de Cannabinoides , Receptores Acoplados a Proteínas GRESUMEN
New strategies for tacrolimus administration that conserve its immunosuppressive effect but avoiding fluctuations in tacrolimus circulating levels are needed. The aim was to analyze if subcutaneous biodegradable tacrolimus-loaded microspheres injection promoted a significant immunosuppressive response in rats. Rats received two subcutaneous tacrolimus-loaded microspheres injections at different days, the first injection was done at day 0 and the second injection was done 12 days after. Plasma circulating levels of tacrolimus, interleukin-2 (IL-2) and calcineurin phosphatase (PP2B) activity in mononuclear cells were measured. Tacrolimus plasma levels were significantly increased from the day after tacrolimus-loaded microspheres injection and remained increased during 10days. Compared to control, plasma IL-2 levels and PP2B activity in mononuclear cells were significantly decreased during ten days. At day 12, a new subcutaneous injection of tacrolimus-loaded microspheres was performed and two days after injection, tacrolimus plasma levels were again increased and both IL-2 plasma levels and PP2B activity decreased. A single subcutaneous tacrolimus-loaded microspheres injection was enough to reduce tacrolimus-related immunosuppressive parameters. These results open the possibility of new therapeutic strategies to administrate calcineurin inhibitors reducing the variability of their circulating levels related to gastrointestinal drug absorption/metabolism modifications.
Asunto(s)
Rechazo de Injerto/prevención & control , Inmunosupresores/uso terapéutico , Microesferas , Trasplante de Órganos , Tacrolimus/uso terapéutico , Animales , Sistemas de Liberación de Medicamentos , Humanos , Terapia de Inmunosupresión , Inyecciones Subcutáneas , Masculino , Modelos Animales , Ratas , Ratas Endogámicas WKY , Resultado del TratamientoRESUMEN
We have demonstrated recently that branched-chain α-keto acid stimulation of insulin secretion is dependent on islet GABA (γ-aminobutyric acid) metabolism: GABA transamination to succinic semialdehyde is increased by 2-oxoglutarate, generated in α-keto acid transamination to its corresponding α-amino acid. The present work was aimed at investigating whether glucose also promotes islet GABA metabolism and whether the latter contributes to the stimulation of insulin secretion. Glucose (20 mM) decreased both the content and release of islet GABA. Gabaculine (1 mM), a GABA transaminase inhibitor, partially suppressed the secretory response of rat perifused islets to 20 mM glucose at different L-glutamine concentrations (0, 1 and 10 mM), as well as the glucose-induced decrease in islet GABA. The drug also reduced islet ATP content and the ATP/ADP ratio at 20 mM glucose. Exogenous succinic semialdehyde induced a dose-dependent increase in islet GABA content by reversal of GABA transamination and a biphasic insulin secretion in the absence of glucose. It depolarized isolated ß-cells and triggered action potential firing, accompanied by a reduction of membrane currents through ATP-sensitive K(+) channels. The gene expression and enzyme activity of GABA transaminase were severalfold higher than that of 2-oxoglutarate dehydrogenase in islet homogenates. We conclude that, at high glucose concentrations, there is an increased diversion of glucose metabolism from the citric acid cycle into the 'GABA shunt'. Semialdehyde succinic acid is a cell-permeant 'GABA-shunt' metabolite that increases ATP and the ATP/ADP ratio, depolarizes ß-cells and stimulates insulin secretion. In summary, an increased islet GABA metabolism may trigger insulin secretion.
Asunto(s)
Glucosa/metabolismo , Células Secretoras de Insulina/metabolismo , Insulina/metabolismo , Ácido gamma-Aminobutírico/metabolismo , Adenosina Trifosfato/metabolismo , Animales , Secreción de Insulina , Masculino , Potenciales de la Membrana , Técnicas de Placa-Clamp , Ratas , Ratas WistarRESUMEN
We have previously shown that oxo-4-methylpentanoate promotes islet GABA (gamma-aminobutyric acid) metabolism and stimulates insulin secretion. The main aim of this work was to explore the participation of the transamination of branched-chain 2-oxoacids in these processes with the aid of several inhibitors of this enzyme activity. No correlation was found between the transamination of branched-chain 2-oxoacids in islet homogenates and insulin secretion. However, in vivo transamination rates correlated better with the secretion capacity of the different branched-chain 2-oxoacids. Gabapentin, a specific inhibitor of the cytosolic isoenzyme, showed greater potential to decrease the in vitro transamination rates of oxo-3-methylbutyrate and oxo-3-methylpentanoate than those of oxo-4-methylpentanoate and oxohexanoate; this correlated with its capacity to decrease insulin secretion. 4-Methylvaleric acid very strongly inhibited the transamination of all the branched-chain 2-oxoacids and blocked their capacity to decrease islet GABA and to stimulate insulin secretion. KCl at 70 mM at stimulated islet GABA release, subsequently decreasing its tissue concentration. This 'non-metabolic' decrease of GABA suppressed the second phase of insulin secretion triggered by oxo-4-methylpentanoate and oxohexanoate. Oxo-4-methylpentanoate and oxo-3-methylpentanoate suppressed dose-dependent 2-oxoglutarate dehydrogenase activity in islet homogenates. In conclusion, the transamination of branchedchain 2-oxoacids is more important to the stimulation of insulin secretion than their catabolism, and transamination decreases islet GABA concentrations by promoting GABA metabolism. Also, inhibition of 2-oxoglutarate dehydrogenase by branched-chain 2-oxoacids may increase metabolic flux in the 'GABA-shunt' at the expense of reduced tricarboxylic acid cycle flux.
Asunto(s)
Insulina/metabolismo , Ácido gamma-Aminobutírico/metabolismo , 4-Aminobutirato Transaminasa/antagonistas & inhibidores , 4-Aminobutirato Transaminasa/metabolismo , Aminas/farmacología , Animales , Ácidos Ciclohexanocarboxílicos/farmacología , Gabapentina , Secreción de Insulina , Islotes Pancreáticos/efectos de los fármacos , Islotes Pancreáticos/metabolismo , Masculino , Modelos Biológicos , Cloruro de Potasio/farmacología , Ratas , Ratas Wistar , Ácido gamma-Aminobutírico/farmacologíaRESUMEN
OMP (oxo-4-methylpentanoic acid) stimulates by itself a biphasic secretion of insulin whereas L-leucine requires the presence of L-glutamine. L-Glutamine is predominantly converted into GABA (gamma-aminobutyric acid) in rat islets and L-leucine seems to promote its metabolism in the 'GABA shunt' [Fernández-Pascual, Mukala-Nsengu-Tshibangu, Martín del Río and Tamarit-Rodríguez (2004) Biochem. J. 379, 721-729]. In the present study, we have investigated how 10 mM OMP affects L-glutamine metabolism to uncover possible differences with L-leucine that might help to elucidate whether they share a common mechanism of stimulation of insulin secretion. In contrast with L-leucine, OMP alone stimulated a biphasic insulin secretion in rat perifused islets and decreased the islet content of GABA without modifying its extracellular release irrespective of the concentration of L-glutamine in the medium. GABA was transaminated to L-leucine whose intracellular concentration did not change because it was efficiently transported out of the islet cells. The L-[U-14C]-Glutamine (at 0.5 and 10.0 mM) conversion to 14CO2 was enhanced by 10 mM OMP within 30% and 70% respectively. Gabaculine (250 microM), a GABA transaminase inhibitor, suppressed OMP-induced oxygen consumption but not L-leucine- or glucose-stimulated respiration. It also suppressed the OMP-induced decrease in islet GABA content and the OMP-induced increase in insulin release. These results support the view that OMP promotes islet metabolism in the 'GABA shunt' generating 2-oxo-glutarate, in the branched-chain alpha-amino acid transaminase reaction, which would in turn trigger GABA deamination by GABA transaminase. OMP, but not L-leucine, suppressed islet semialdehyde succinic acid reductase activity and this might shift the metabolic flux of the 'GABA shunt' from gamma-hydroxybutyrate to succinic acid production.