Efectos conductuales y modulación de la síntesis de monoaminas y de la vía de quinasas mitogénicas en cerebro de rata tras tratamientos con cannabinoides y etanol

A pesar de que el uso del cannabis y el alcohol como sustancias psicoactivas es muy antiguo y que su uso como drogas recreacionales está muy extendido, los mecanismos a través de los cuales producen sus efectos psicoactivos se han empezado a conocer hace relativamente poco tiempo. En la presente tesis se estudia como afectan distintos tratamientos sistémicos, tanto con etanol como con distintos compuestos cannabinoides, simultáneamente sobre la síntesis de las distintas monoaminas en diferentes regiones cerebrales de rata. Además, se analizó como afectaban estos tratamientos a los distintos componentes de la vía de señalización intracelular de las MAPK en la corteza frontal de rata. Se realizaron distintos tratamientos agudos con estos compuestos así como tratamientos a más largo plazo (crónicos) y además se estudiaron estos parámetros durante el llamado síndrome de abstinencia que aparece una vez que se detiene esta administración crónica

Casos prácticos como herramienta educativa en las asignaturas de fisiología humana

[EN] The education plans of the universities represent a coincidence in the subject of Human Physiology (Physiotherapy, Biology, Biochemistry, Nursing and Medicine degrees), in which teaching for small groups are a good strategy to achieve certain skills that could not be achieved during large groups time. The main methodology followed is the realization of monographic works. During the 2018-2019 academic year, the University of the Balearic Islands awarded a teaching innovation project in which the design and coordination of seminars was proposed through an active learning methodology through the resolution of practical cases that would help students to acquire skills in critical thinking, leadership, emotional intelligence, adaptability and management of new technologies. The students had to solve a supposed clinical case, exposing the normal physiological process and the physiological alteration associated with the proposed case. The results obtained (n = 174 students) indicated an improvement in the acquisition of knowledge (average of 8.2) and in the satisfaction (average of 8.1) obtained by the students, who preferred this methodology to monographic expositions or the use of simulators by computer. In conclusion, the methodology contributed was received with a high degree of satisfaction for its innovative application in the small groups.[ES] Los planes docentes de diversas titulaciones suponen una coincidencia en la asignatura de Fisiología Humana (grados: Fisioterapia, Biología, Bioquímica y Medicina), en los cuales las horas docentes destinadas a grupos pequeños suponen una buena estrategia para alcanzar determinadas competencias que no se podrían alcanzar con grupos grandes. La realización de trabajos monográficos es la principal metodología seguida. Durante el curso 2018/2019, la Universidad de las Islas Baleares concedió un proyecto de innovación docente donde se planteó el diseño y la coordinación de la realización de seminarios mediante una metodología activa de aprendizaje a través de resolución de casos prácticos con el objetivo de adquirir destrezas en pensamiento crítico, liderazgo, inteligencia emocional, adaptabilidad y manejo de las nuevas tecnología. Los alumnos debían resolver un supuesto caso clínico, exponiendo el proceso fisiológico normal y la alteración fisiológica asociada al caso propuesto. Los resultados obtenidos (n=174 alumnos) indicaron una mejora en la adquisición de conocimientos (media de 8,2) y en la satisfacción (media de 8,1)obtenida por parte del alumnado, que prefirió esta metodología a las exposiciones monográficas o el uso de simuladores por ordenador. En conclusión, la metodología aportada fue recibida con alto grado de satisfacción por su aplicación innovadora en los grupos pequeños.Tejada Gavela, S.; Bibiloni Esteva, MDM.; Moranta Mesquida, D.; Esteban Valdés, SC.; Sureda Gomila, A. (2019). Casos prácticos como herramienta educativa en las asignaturas de fisiología humana. En IN-RED 2019. V Congreso de Innovación Educativa y Docencia en Red. Editorial Universitat Politècnica de València. 675-683. https://doi.org/10.4995/INRED2019.2019.10439OCS67568

Efectos conductuales y modulación de la síntesis de monoaminas y de la vía de quinasas mitogénicas en cerebro de rata tras tratamientos con cannabinoides y etanol

A pesar de que el uso del cannabis y el alcohol como sustancias psicoactivas es muy antiguo y que su uso como drogas recreacionales está muy extendido, los mecanismos a través de los cuales producen sus efectos psicoactivos se han empezado a conocer hace relativamente poco tiempo. En la presente tesis se estudia como afectan distintos tratamientos sistémicos, tanto con etanol como con distintos compuestos cannabinoides, simultáneamente sobre la síntesis de las distintas monoaminas en diferentes regiones cerebrales de rata. Además, se analizó como afectaban estos tratamientos a los distintos componentes de la vía de señalización intracelular de las MAPK en la corteza frontal de rata. Se realizaron distintos tratamientos agudos con estos compuestos así como tratamientos a más largo plazo (crónicos) y además se estudiaron estos parámetros durante el llamado síndrome de abstinencia que aparece una vez que se detiene esta administración crónica

Ethanol desensitizes cannabinoid CB1 receptors modulating monoamines synthesis in the rat brain in vivo

[eng] The endocannabinoid system and the cannabinoid CB1 receptors are involved in the development of ethanol tolerance and dependence. This study aimed to investigate the in vivo sensitivity of a CB1 receptor agonist (WIN 55,212-2) modulating the synthesis of 3,4-dihydroxyphenylalanine/dopamine/noradrenaline (DOPA/DA/NA) and that of 5-hydroxy-tryptophan/serotonin (5-HTP/5-HT) in rat brain after ethanol treatment and withdrawal. In control rats, WIN 55,212-2 (4 mg/kg, i.p., for 1 h), through a mechanism sensible to the CB1 antagonist SR 141716A, increased the synthesis of DOPA/NA in a slice of brainstem containing the locus ceruleus (250%) and in the hippocampus (64%), and it reduced DOPA/DA synthesis in the striatum (47%). WIN 55,212-2 also decreased the synthesis of 5-HTP/5-HT in the locus ceruleus (43%), hippocampus (35%) and striatum (35%). In the locus ceruleus of ethanol-treated rats, the stimulatory effect of WIN 55,212-2 on DOPA/NA synthesis was abolished (acute treatment) or markedly attenuated (53-55%, chronic treatment and withdrawal), whereas in the hippocampus this effect was reduced only in chronic ethanol-withdrawn rats (33%). In the striatum of ethanol-treated rats (acute, chronic and withdrawal), the inhibitory effect of WIN 55,212-2 on DOPA/DA synthesis was completely blunted or markedly reduced. Similarly, the inhibitory effect of WIN 55,212-2 on 5-HTP/5-HT synthesis was reduced or abolished in the three brain regions after chronic ethanol and during withdrawal. These results indicate that treatment with ethanol in rats induces a functional desensitization of CB1 receptors modulating the synthesis of brain monoamines

Acute, chronic and withdrawal effects of the cannabinoid receptor agonist WIN55212-2 on the sequential activation of MAPK/Raf-MEK-ERK signaling in the rat cerebral frontal cortex: short-term regulation by intrinsic and extrinsic pathways.

[eng] The cannabinoids (CB) modulate the extracellular signalregulated kinase (ERK), leading to various forms of plasticity in the brain. Little is known, however, on the in vivo short- and long-term activation and regulation of the components of mitogen-activated protein kinase (MAPK)/ERK signaling by CB. The CB agonist WIN55212-2 (8 mg/kg) increased the immunodensities of phosphorylated c-Raf-1 (42%), MEK1/2 (63%), ERK1 (24%), and ERK2 (28%) in the rat cerebral frontal cortex. These effects were antagonized by SR141716A (rimonabant, 10 mg/kg), a selective CB1 receptor antagonist. Repeated WIN55212-2 treatment (2-8 mg/kg for 5 days) resulted in tachyphylaxis to the acute activation of Raf-MEK-ERK signaling. Acute WIN55212-2 also induced a hypothermic effect in rats, which was reduced after repeated administration (tolerance). Treatment with SR141716A after chronic WIN55212-2 resulted in the expected cannabinoid withdrawal syndrome, without concomitant alterations in the phosphorylation state of c-Raf-1, MEK1/2, or ERK1/2. Pretreatment with SL327 (20 mg/kg, a MEK1/2 inhibitor) increased the basal phosphorylation of c-Raf-1 (40%) and MEK1/2 (74%; feedback regulation) and fully prevented the up-regulation of ERK1/2 (23-31%) induced by WIN55212-2. Pretreatment with MK801 (1 mg/kg, a NMDA receptor antagonist) effectively blocked the up-regulation c-Raf-1 (41%), MEK1/2 (57%) and ERK1/2 (25-30%) induced by the CB agonist. The main findings demonstrate that the acute stimulation of CB1 receptors in the frontal cortex results in the sequential phosphorylation of Raf-MEKERK cascade, in which c-Raf-1 activation (rate-limiting process) plays a crucial role. Moreover, the in vivo stimulating effect of WIN55212-2 on Raf-MEK-ERK signaling is under the extrinsic regulation of an excitatory glutamatergic mechanism

Klebsiella pneumoniae increases the levels of Toll-like receptors 2 and 4 in human airway epithelial cells.

[eng] Airway epithelial cells act as the first barrier against pathogens. These cells recognize conserved structural motifs expressed by microbial pathogens via Toll-like receptors (TLRs) expressed on the surface. In contrast to the level of expression in lymphoid cells, the level of expression of TLR2 and TLR4 in airway epithelial cells is low under physiological conditions. Here we explored whether Klebsiella pneumoniae upregulates the expression of TLRs in human airway epithelial cells. We found that the expression of TLR2 and TLR4 by A549 cells and human primary airway cells was upregulated upon infection with K. pneumoniae. The increased expression of TLRs resulted in enhancement of the cellular response upon stimulation with Pam3CSK4 and lipopolysaccharide, which are TLR2 and TLR4 agonists, respectively. Klebsiella-dependent upregulation of TLR expression occurred via a positive I B -dependent NF- pathway and via negative p38 and p44/42 mitogen-activated protein kinase-dependent pathways. We showed that Klebsiella-induced TLR2 and TLR4 upregulation was dependent on TLR activation. An isogenic capsule polysaccharide (CPS) mutant did not increase TLR2 and TLR4 expression. Purified CPS upregulated TLR2 and TLR4 expression, and polymyxin B did not abrogate CPS-induced TLR upregulation. Although no proteins were detected in the CPS preparation by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and colloidal gold staining, we could not rule out the possibility that traces of protein in our CPS preparation could have been responsible, at least in part, for the TLR upregulation

Klebsiella pneumoniae outer membrane protein A is required to prevent the activation of airway epithelial cells.

[eng] Outer membrane protein A (OmpA) is a class of proteins highly conserved among the Enterobacteriaceae family and throughout evolution. Klebsiella pneumoniae is a capsulated Gram-negative pathogen. It is an important cause of community- acquired and nosocomial pneumonia. Evidence indicates that K. pneumoniae infections are characterized by a lack of an early inflammatory response. Data from our laboratory indicate that K. pneumoniae CPS helps to suppress the host inflammatory response. However, it is unknown whether K. pneumoniae employs additional factors to modulate host inflammatory responses. Here, we report that K. pneumoniaeOmpAis important for immune evasion in vitro and in vivo. Infection of A549 and normal human bronchial cells with 52OmpA2, an ompA mutant, increased the levels of IL-8. 52145- wcaK2ompA, which does not express CPS and ompA, induced the highest levels of IL-8. Both mutants could be complemented. In vivo, 52OmpA2 induced higher levels of tnf , kc, and il6 than the wild type. ompA mutants activated NF- B, and the phosphorylation of p38, p44/42, and JNK MAPKs and IL-8 induction was via NF- B-dependent and p38- and p44/42-dependent pathways. 52OmpA2 engaged TLR2 and -4 to activate NF- B, whereas 52145- wcaK2ompA activated not only TLR2 and TLR4 but also NOD1. Finally, we demonstrate that the ompA mutant is attenuated in the pneumonia mouse model. The results of this study indicate that K. pneumoniae OmpA contributes to attenuate airway cell responses. This may facilitate pathogen survival in the hostile environment of the lung

Withdrawal from chronic ethanol increases the sensitivity of presynaptic 5-HT(1A) receptors modulating serotonin and dopamine synthesis in rat brain in vivo

[eng] The in vivo sensitivity of presynaptic 5-HT1A receptors (autoreceptors and heteroreceptors) modulating the synthesis of 5-hydroxytryptophan/serotonin (5-HTP/5-HT) and 3,4-dihydroxyphenylalanine/dopamine (DOPA/DA) in rat brain was investigated after ethanol treatment and withdrawal. In saline-treated rats as well as in acute ethanol (2 g/kg, intraperitoneally (i.p.), 2 h)- and chronic ethanol (2 g/kg for 7 days)-treated rats, a low dose of the 5-HT1A receptor agonist 8-hydroxy-2-di-n-propylamino-tetralin (8-OH-DPAT; 0.1 mg/kg, i.p., 1 h) did not decrease the synthesis of 5-HTP in brain (except modestly in striatum; 20% after the chronic treatment) or that of DOPA in striatum. In contrast, in chronic ethanolwithdrawn rats (24 h), 8-OH-DPAT significantly decreased the synthesis of 5-HTP in the hippocampus (29%), cerebral cortex (41%) and striatum (33%) and that of DOPA in the striatum (28%). Similar effects were induced by the mixed 5-HT1A agonist/D2 antagonist buspirone (1 mg/kg, i.p., 1 h) which also decreased 5-HTP synthesis in the hippocampus (24%), cerebral cortex (36%) and striatum (35%) of chronic ethanol-withdrawn rats. These results indicate that chronic ethanol and more clearly the spontaneous withdrawal from chronic ethanol induce supersensitivity of 5-HT1A-auto/heteroreceptors modulating the synthesis of 5-HT and DA in rat brain

Improving effect of chronic resveratrol treatment on central monoamines synthesis and cognition in aged rats

[eng] Resveratrol is a polyphenol exhibiting antioxidant and neuroprotective effects in neurodegenerative diseases. However, neuroprotective properties during normal aging have not been clearly demonstrated. We analyzed the in vivo effects of chronic administration of resveratrol (20 mg/kg/day for 4 weeks) in old male rats (Wistar, 20 months), on tryptophan hydroxylase (TPH) and tyrosine hydroxylase (TH) activities which mediate central monoaminergic neurotransmitters synthesis, and besides, on hippocampal-dependent working memory test (radial maze). Our results show an age-related decline in neurochemical parameters that were reversed by resveratrol administration. The resveratrol treatment enhances serotonin (5-HT) levels in pineal gland, in hippocampus, and in striatum, and those of noradrenaline (NA) in hippocampus and also dopamine (DA) in striatum. These changes were largely due to an increased activity of TPH-1 (463 % in pineal gland), TPH-2 (70-51 % in hippocampus and striatum), and TH (150-36 % in hippocampus and striatum). Additionally, the observed hippocampal effects correlate with a resveratrol-induced restorative effect on working memory (radial maze). In conclusion, this study suggests resveratrol treatment as a restoring therapy for the impaired cognitive functions occurring along normal aging process, by preventing 5-HT, DA, and NA neurotransmission decline

High affinity binding of β-carbolines to imidazoline I2B receptors and MAO-A in rat tissues: Norharman blocks the effect of morphine withdrawal on DOPA/noradrenaline synthesis in the brain

[eng] This study was designed to determine the affinity and binding profile of h-carbolines for imidazoline I2 receptors and catalytic sites of monoamine oxidase (MAO)-A/B in rat brain and liver. The aim was also directed to assess the in vivo effects of norharman (h-carboline) and LSL 60101 (I2 ligand) on brain 3,4-dihydroxyphenylalanine (DOPA) synthesis in morphine-dependent rats. Competition experiments against [3H]2-BFI revealed that h-carbolines recognize the high- and low-affinity components of the brain imidazoline I2 receptor with the rank order of potency (KiH in nM): noreleagnine (12)>norharman (20)>harmalol (82)>harmaline (177)>harmine (630)>harman (700)HFG-7142 (>100,000). In liver, this rank was different: harmine (51)>harmaline (103)=noreleagnine (103)Hharmalol (1290)>harman (2000)Hnorharman (12,382)HFG-7142 (>100,000). In brain and liver, competition curves for h-carbolines against [3H]Ro41-1049 (MAO-A) and [3H]Ro19-6327 (MAO-B) were monophasic and resulted in different drug potencies for the two MAO isozymes (higher affinities for MAO-A) and in similar pharmacological profiles in both tissues. In morphine-dependent rats, naloxone (2 mg/kg, 2 h)-precipitated withdrawal increased the synthesis of DOPA in the cerebral cortex and hippocampus (50%). Pretreatment with norharman (20 mg/kg) or LSL 60101 (20 mg/kg) (30 min before naloxone) fully prevented the stimulatory effect of opiate withdrawal on DOPA synthesis. Norharman and LSL 60101 also attenuated the severity of the withdrawal syndrome. The results indicate that h-carbolines bind with high affinity to imidazoline I2B receptors, and similarly to I2 ligands (LSL 60101) can block the behavioural and biochemical effects of opiate withdrawal