Efecto de la Carbamilcolina y el veneno de escorpión Tityus zulianus en la secreción de proteínas de los acinos pancreáticos aislados de ratón NIH

El envenenamiento accidental con el escorpión Tityus zulianus (Tz) produce manifestaciones clínicas principalmente cardiopulmonares. Sin embargo, en modelos experimentales con ratones BALB/c se ha demostrado que el veneno de Tz (TzV) produce daño pancreático y aumento de las concentraciones séricas de catecolaminas. El objetivo de este estudio es determinar los mecanismos fisiopatológicos asociados a la hiperestimulación autonómica que conduce al desarrollo de la pancreatitis en un modelo experimental de acinos pancreáticos aislados. Los acinos pancreáticos se obtuvieron a partir de pancreata de ratón sometida a la digestión con colagenasa, con lo cual se separan los acinos que posteriormente se mantienen en baño térmico. Una vez estabilizados los acinos, se aplicaron tratamientos con carbamilcolina (Cch) y/o TzV a diferentes concentraciones para evaluar la secreción de proteínas en una curva concentración respuesta. Los niveles de proteínas y tripsina se determinaron por espectrofotometría. Los resultados muestran que tanto la Cch (5-30μM) como el TzV (2,5-128,5μg/ml) incrementan significativamente de forma dependiente de la concentración la liberación de proteínas al medio extracelular. Adicionalmente, se observó que al administrar en forma conjunta, Cch 10 μM y TzV 128,5 μg/ml se produce un efecto sinérgico (180±2%), el cual consiste en un incremento superior a lo observado cuando se aplican de manera individual. En conclusión, el TzV incrementa significativamente la secreción de proteínas ya sea de forma individual o en combinación con la Cch, con lo cual se mimetizan los efectos del neurotransmisor endógeno acetilcolina (Ach). Abstract Accidental poisoning with the scorpion Tityus zulianus (Tz) produces mainly cardiopulmonary clinical manifestations. However, in experimental models with BALB/c mice it has been shown that the venom of Tz (TzV) causes pancreatic damage and increased serum catecholamines values. The objective of this study is to determine the pathophysiological mechanisms associated with autonomic hyperstimulation leading to the development of pancreatitis in an experimental model of isolated pancreatic acini. The pancreatic acini were obtained from mouse pancreata subjected with collagenase digestion, whereby the acini are separated and subsequently kept in a thermic bath. The stabilized acini were treated treatments with different concentration carbamylcholine (Cch) and / or TzV to evaluate the secretion of proteins in a concentration response curve. Protein levels were determined by spectrophotometry. The results show that both the Cch (5-30μM) and the TzV (2.5-128.5μg / ml) significantly increase the release of proteins to the extracellular medium of concentration-dependent of Cch and TzV. Synergistic effect were observed with the combinations Cch 10 μM and TzV 128.5 μg/ml in this order with any increment of 180 ± 2%proteins secretion. In conclusion, TzV significantly increases protein secretion either or in combination with Cch, which mimics the effects of the endogenous neurotransmitter acetylcholine (Ach)

Pathobiology of airway smooth muscle remodeling

El mecanismo primario de asma y EBPOC involucra una excesiva contracción de las vías aéreas (VA), cuya severidad se encuentra en relación a inflamación crónica. Evidencia reciente sugiere que las células de músculo liso de las vías aéreas (MLVA) poseen elevada plasticidad celular que puede contribuir en inflamación, resultando en su engrosamiento mediante hiperplasia y/o hipertrofia. La interacción MLVA-microambiente tisular es la base para hiperreactividad y remodelado tisular, con contribuciones importantes de virus y mediadores químicos, especialmente acetilcolina. Esta revisión abarca la fisiopatología del remodelado del MLVA en relación a fenotipos graves de enfermedades inflamatorias bronquiales. Un análisis in silico de hibridación entre secuencias de ARN humano y virales fue realizado, obteniendo datos para apoyar una hipótesis de ‘hit and run’. Como una propuesta de integración, se resumen los últimos hallazgos moleculares con una perspectiva que ayude al establecimiento de fundamentos para investigaciones futuras y la comprensión de las vías de señalización que regulan la biología del MLVA.   Abstract The primary mechanism of morbidity and mortality in asthma and COPD is excessive airway narrowing, which severity is based on chronic inflammation. New evidence suggests airway smooth muscle (ASM) cells show extraordinary cellular plasticity that may contribute to airway inflammation, ensuing ASM thickening by either hyperplasia and/or hypertrophy. Tissue microenvironment-ASM interaction is a complex crosstalk that supports hyperresponsiveness and tissue remodeling, with major contributions of viruses and chemical mediators, especially acetylcholine. This review addresses the ASM pathology in relation with severe phenotypes of airway inflammatory diseases. An in silico analysis of hybridization between human and viral RNA strands was performed, obtaining data to support a ‘hit and run’ hypothesis. As an integrative proposal, we summarized the last molecular findings in this field with a perspective that helps to set the stage for future research toward understanding the signaling pathways regulating ASM biology

M2 Muscarinic acetylcholine receptor modulates rat airway smooth muscle cell proliferation

Airways chronic inflammatory conditions in asthma and COPD are characterized by tissue remodeling, being smooth muscle hyperplasia, the most important feature. Non-neuronal and neuronal Acetylcholine acting on muscarinic receptors (MAChRs) has been postulated as determinant of tissue remodeling in asthma and COPD by promoting proliferation and phenotypic changes of airway smooth muscle cells (ASMC). The objective was to evaluate proliferative responses to muscarinic agonist as carbamylcholine (Cch) and to identify the MAchR subtype involved. ASMC were isolated from tracheal fragments of Sprague–Dawley rats by enzymatic digestion. Proliferation assays were performed by MTS-PMS method. Viability was confirmed by trypan blue exclusion method. Mitogens as, epidermal growth factor (EGF), Tumor necrosis factor-alpha (TNF-α) and fetal bovine serum (FBS) increased ASMC proliferation (p < 0.05, n = 5). Cch alone increased ASMC proliferation at 24 and 48 hrs. However, combination of Cch with other mitogens exhibited a dual effect, synergistic proliferation effect in the presence of EGF (5 ng/mL) and 5% FBS and inhibiting the proliferation induced by 10% FBS, EGF (10 ng/mL) and TNF-α (10 ng/mL). To determine the MAChR subtype involved in these biological responses, a titration curve of selective muscarinic antagonists were performed. The Cch stimulatory and inhibitory effects on ASCM proliferation was blocked by AF-DX-116 (M2AChR selective antagonist), in greater proportion than 4-DAMP (M3AChR selective antagonist), suggesting that the modulation of muscarinic agonist-induced proliferation is M2AChR mediated responses. Thus, M2AChR can activate multiple signal transduction systems and mediate both effects on ASMC proliferation depending on the plethora and variable airway microenvironments existing in asthma and COPD. Keywords: Airway smooth muscle, Muscarinic receptors, Carbamylcholine, ASMC inhibitio