RESUMEN
Over the last four decades, chromaffin cells originating from the adrenal medulla have been probably one of the most popular cell models to study neurosecretion at the molecular level. Accordingly, numerous seminal discoveries in the field, including the characterization of role of the cytoskeleton, fusogenic lipids, and soluble N-ethylmaleimide-sensitivefactor attachment protein receptor (SNARE) proteins, have been made using this model. In this chapter, we describe a standard method currently used to isolate and culture bovine chromaffin cells, and we illustrate a catecholamine secretion assay based on the successive transformation of adrenaline into adrenochrome and adrenolutine for fluorescence measurements. We also provide some guidelines for efficient cell recovery and for the use of this assay in the laboratory.
Asunto(s)
Médula Suprarrenal/metabolismo , Secreciones Corporales/metabolismo , Técnicas de Cultivo de Célula/métodos , Células Cromafines/citología , Animales , BovinosRESUMEN
Specific forms of fatty acids are well known to have beneficial health effects, but their precise mechanism of action remains elusive. Phosphatidic acid (PA) produced by phospholipase D1 (PLD1) regulates the sequential stages underlying secretory granule exocytosis in neuroendocrine chromaffin cells, as revealed by pharmacological approaches and genetic mouse models. Lipidomic analysis shows that secretory granule and plasma membranes display distinct and specific composition in PA. Secretagogue-evoked stimulation triggers the selective production of several PA species at the plasma membrane near the sites of active exocytosis. Rescue experiments in cells depleted of PLD1 activity reveal that mono-unsaturated PA restores the number of exocytotic events, possibly by contributing to granule docking, whereas poly-unsaturated PA regulates fusion pore stability and expansion. Altogether, this work provides insight into the roles that subspecies of the same phospholipid may play based on their fatty acyl chain composition.