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In epithelial layers in culture, immediately after an injury a fast calcium wave (FCW) propagates from the wound borders toward the rest of the monolayer. We show here that similarly to other tissues, during the FCW in bovine corneal endothelial (BCE) cells in culture many cells exhibit calcium oscillations mediated by IP3 signaling. In this study we perform a detailed characterization of this oscillatory behavior and explore its possible role in the process of wound healing. In previous work we showed that, in BCE cells in culture, the healing cells undergo two stages of caspase-dependent apoptosis, at approximately two and eight hours after wounding. We determined that inhibition of the FCW greatly increases the apoptotic rate of the two stages, suggesting that the wave prevents excessive apoptosis of the healing cells. Taking this into account, we investigated the possible participation of the calcium oscillations during the FCW in apoptosis of the healing cells. For this, we employed ARL-67156 (ARL), a weak competitive inhibitor of ecto-ATPases, and the calcium chelator EGTA. We show here that, in healing BCE cells, ARL enhances cellular calcium oscillations during the FCW, while EGTA decreases oscillations. We found that ARL produces a significant decrease (to about half the control value) in the apoptotic index of the first stage of apoptosis, while EGTA increases it. Neither drug noticeably affects the second stage. We have interpreted the effect of ARL on apoptosis as due to the maintenance of moderately risen ATP levels during the FCW, which is in turn the cause for the enhancement of ATP-dependent calcium oscillations. Correspondingly, EGTA would increase the apoptotic index of the first stage by promoting a decrease in the calcium oscillatory rate. The fact that the second stage of apoptosis is not affected by the drugs suggests that the two stages are at least partially subject to different signaling pathways.

The epithelial sodium channel (ENaC) mediates passive sodium transport across the apical membranes of sodium absorbing epithelia, like the distal nephron, the intestine, and the lung airways. Additionally, the channel has been involved in the transduction of mechanical stimuli, such as hydrostatic pressure, membrane stretch, and shear stress from fluid flow. Thus, in vascular endothelium, it participates in the control of the vascular tone via its activity both as a sodium channel and as a shear stress transducer. Rather recently, ENaC has been shown to participate in the processes of wound healing, a role that may also involve its activities as sodium transporter and as mechanotransducer. Its presence as the sole channel mediating sodium transport in many tissues and the diversity of its functions probably underlie the complexity of its regulation. This brief review describes some aspects of ENaC regulation, comments on evidence about ENaC participation in wound healing, and suggests possible regulatory mechanisms involved in this participation.

This paper studies the electrical phenomena in biological processes, specially through a laboratory studio that reveals that epithelial cells can employ modifications of the electric field to produce structural and functional changes in various physiological circumstances., La comprensión de los fenómenos eléctricos y el diseño de máquinas generadoras y utilizadoras de electricidad han ocurrido en forma relativamente reciente, y han permitido el gran desarrollo científico y tecnológico de los últimos doscientos años. Sin embargo, los fenómenos eléctricos ocurren naturalmente en los procesos biológicos. Se acepta que ya las células primitivas poseían membranas a través de las cuales se generaron diferencias de potencial eléctrico, que constituyeron baterías eléctricas primitivas que las células emplearon con fines energéticos. El gradual incremento de complejidad de las células y el desarrollo de organismos pluricelulares que ocurrieron en el curso de la evolución implicaron el empleo de los fenómenos eléctricos celulares con fines fisiológicos adicionales. Así, la aparición de las células excitables permitió la propagación de señales eléctricas entre células distantes, lo que constituyó la base para diversos procesos biológicos, tales como la cognición y la interacción entre organismos. Recientemente se ha reconocido que las células no excitables también emplean los fenómenos eléctricos de las membranas plasmáticas con fines de señalización. Aquí mostramos algunos resultados de nuestro laboratorio que revelan que las células epiteliales pueden emplear modificaciones del campo eléctrico de dichas membranas para producir cambios estructurales y funcionales en diferentes circunstancias fisiológicas.

The establishment and maintenance of the polarized epithelial phenotype require a characteristic organization of the cytoskeletal components. There are many cellular effectors involved in the regulation of the cytoskeleton of epithelial cells. Recently, modifications in the plasma membrane potential (PMP) have been suggested to participate in the modulation of the cytoskeletal organization of epithelia. Here, we review evidence showing that changes in the PMP of diverse epithelial cells promote characteristic modifications in the cytoskeletal organization, with a focus on the actin cytoskeleton. The molecular paths mediating these effects may include voltage-sensitive integral membrane proteins and/or peripheral proteins sensitive to surface potentials. The voltage dependence of the cytoskeletal organization seems to have implications in several physiological processes, including epithelial wound healing and apoptosis.

Gramicidin is a thoroughly studied cation ionophore widely used to experimentally manipulate the plasma membrane potential (PMP). In addition, it has been established that the drug, due to its hydrophobic nature, is capable of affecting the organization of membrane lipids. We have previously shown that modifications in the plasma membrane potential of epithelial cells in culture determine reorganizations of the cytoskeleton. To elucidate the molecular mechanisms involved, we explored the effects of PMP depolarization on some putative signaling intermediates. In the course of these studies, we came across some results that could not be interpreted in terms of the properties of gramicidin as an ionic channel. The purpose of the present work is to communicate these results and, in general, to draw attention to the fact that gramicidin effects can be misleadingly attributed to its ionic or electrical properties. In addition, this work also contributes with some novel findings of the modifications provoked on the signaling intermediates by PMP depolarization and hyperpolarization. ANII: PR_FCE_2009_1_2474 CSIC: I+D 2014

The epithelial sodium channel (ENaC) mediates passive sodium transport across the apical membranes of sodium absorbing epithelia, like the distal nephron, the intestine, and the lung airways. Additionally, the channel has been involved in the transduction of mechanical stimuli, such as hydrostatic pressure, membrane stretch, and shear stress from fluid flow. Thus, in vascular endothelium, it participates in the control of the vascular tone via its activity both as a sodium channel and as a shear stress transducer. Rather recently, ENaC has been shown to participate in the processes of wound healing, a role that may also involve its activities as sodium transporter and as mechanotransducer. Its presence as the sole channel mediating sodium transport in many tissues and the diversity of its functions probably underlie the complexity of its regulation. This brief review describes some aspects of ENaC regulation, comments on evidence about ENaC participation in wound healing, and suggests possible regulatory mechanisms involved in this participation.

Dionisio Díaz: Estudiante de Medicina, Ciclo de Metodología Científica II, Facultad de Medicina, Universidad de la República. Uruguay. La contribución en la realización del trabajo fue equivalente a la de los demás estudiantes.-- Rodrigo Dorelo: Estudiante de Medicina, Ciclo de Metodología Científica II, Facultad de Medicina, Universidad de la República. Uruguay. La contribución en la realización del trabajo fue equivalente a la de los demás estudiantes.-- Diego Fleitas: Estudiante de Medicina, Ciclo de Metodología Científica II, Facultad de Medicina, Universidad de la República. Uruguay. La contribución en la realización del trabajo fue equivalente a la de los demás estudiantes.-- Silvia Chifflet: Docente supervisor. Departamento de Bioquímica de la Facultad de Medicina, Universidad de la República, Montevideo, Uruguay. Contacto: Silvia Chifflet. Departamento de Bioquímica, Facultad de Medicina, Gral Flores 2125, 11800 Montevideo, Uruguay. Tel: (5982) 924 3414 ext. 3403. E-mail: schiffle@mednet.org.uy La enfermedad de Crohn, un tipo de enfermedad inflamatoria intestinal, afecta la integridad estructural y funcional de la barrera intestinal. Esta barrera está constituida por una capa de células epiteliales que forman una monocapa continua y polarizada, estrechamente conectada por varios tipos de uniones celulares, entre las que se destacan las uniones estrechas. Las uniones estrechas forman una estructura continua entre los dominios apical y basolateral en las células epiteliales y endoteliales, creando una barrera paracelular selectiva fundamental para la homeostasis del organismo. La disfunción de la barrera epitelial juega un papel central en la fisiopatología de la enfermedad de Crohn. Los pacientes con enfermedad de Crohn presentan una pérdida de la función de barrera de las uniones estrechas y un aumento de la producción de citoquinas pro-inflamatorias, así como una desregulación del sistema inmune. En esta revisión se describe brevemente la composición molecular, estructura, interacciones y función de las uniones estrechas, además de mostrar evidencia reciente acerca de la relación entre la disfunción de las mismas y la enfermedad de Crohn. También se detalla el rol de la microbiota en la regulación de las uniones estrechas, así como el papel de la desregulación de estas uniones en el desarrollo del cáncer asociado a la enfermedad de Crohn. Finalmente, se describen hallazgos de la literatura actual respecto a la posible utilidad de las uniones estrechas como blanco de nuevos medicamentos. Crohn’s disease is an inflammatory bowel disease that affects the intestinal wall. This wall consists of an epithelial cell layer that forms a continuous polarized monolayer, closely connected by various types of cellular junctions. Among these, tight junctions play a relevant role. They form a continuous structure delimiting the apical and basolateral domains in epithelial and endothelial cells, creating in this way a selective paracellular barrier, fundamental for the homeostasis of the organism. There is increasing evidence that the dysfunction of the epithelial barrier plays a central role in the physio pathology of Crohn’s disease. Crohn’s disease patients exhibit a loss of the barrier function of the tight junctions, an increased production of pro-inflammatory cytokines and dysregulation of the immune system. In his review the molecular composition, structure, interactions and function of the tight junctions are briefly described, and recent evidence on the relationship between the dysfunction of the tight junctions and Crohn’s disease is commented. The role of microbiota in the regulation of tight junctions and the participation of tight junctions deregulation in the development of cancer associated to Crohn’s disease are also detailed. Finally, recent findings about the employment of tight junctions as targets for the development of new drugs are described.