Lipid Metabolites Enhance Secretion Acting on SNARE Microdomains and Altering the Extent and Kinetics of Single Release Events in Bovine Adrenal Chromaffin Cells

Abstract

Lipid molecules such as arachidonic acid (AA) and sphingolipid metabolites have been implicated in modulation of neuronal and endocrine secretion. Here we compare the effects of these lipids on secretion from cultured bovine chromaffin cells. First, we demonstrate that exogenous sphingosine and AA interact with the secretory apparatus as confirmed by FRET experiments. Examination of plasma membrane SNARE microdomains and chromaffin granule dynamics using total internal reflection fluorescent microscopy (TIRFM) suggests that sphingosine production promotes granule tethering while arachidonic acid promotes full docking. Our analysis of single granule release kinetics by amperometry demonstrated that both sphingomyelinase and AA treatments enhanced drastically the amount of catecholamines released per individual event by either altering the onset phase of or by prolonging the off phase of single granule catecholamine release kinetics. Together these results demonstrate that the kinetics and extent of the exocytotic fusion pore formation can be modulated by specific signalling lipids through related functional mechanisms.

Emerging evidence for the modulation of exocytosis by signalling lipids

“…In this neuroendocrine model, incubation with FTY‐720, increases the frequency of vesicle fusions during the first round of cell stimulation by depolarization but decreases the amount of vesicles recruited in subsequent stimulations, (V. Garcia‐Martinez, J. Villanueva, Y. Gimenez‐Molina & L. M. Gutiérrez, unpublished results). Furthermore, by using FRET, we have observed that FTY‐720 interacts at the molecular level with SNARE clusters in chromaffin cells as was previously described for sphingosine and AA . In addition, FTY720 could also interact with F‐actin governing the motion of the vesicles in the close proximity of secretory sites .…”

“…Simultaneously, our group at the Institute of Neurosciences of Alicante performed experiments in adrenomedullary chromaffin cells, using the amperometry technique to detect the release of catecholamines from individual fusion events by their oxidation at the tip of a carbon fibre . In our study, sphingosine and derivatives were produced by SMase treatment of the cells, and resulted in the increase of the amount of catecholamines released in individual fusions with detection of changes in the kinetics of the process suggesting changes in the mode of fusion of the vesicles.…”

“…In neuroendocrine cells SNAREs proteins are located in the fluid mosaic of the plasma membrane composed of a diversity of lipids including phospholipids, sphingolipids and cholesterol , and stabilized by a matrix of cytoskeletal elements forming the dynamic cytoarchitecture of active sites . In this environment, the activity of phospholipases could release either saturated and also polyunsaturated fatty acids (PUFAs) that normally are present in the sn‐2 position , and often, the released PUFAs could act as a intracellular messengers regulating a diversity of cellular processes including exocytosis . Specifically, phospholipase type A2 (PLA2) acting in the sn‐2 site release lysophospholipids and free unsaturated fatty acids, and the latter ones can diffuse into cytosol where they interact with their targets of action, likely in hydrophobic domains .…”

“…In order to demonstrate that the endogenous sphingosine production could mimic these results, the activity of external sphingomyelinases (SMase) and intracellular ceramidases releasing sphingosine into the cytosol in isolated nerve terminals , or cultured chromaffin cells was tested on potentiation of exocytosis. The obtained results support this mechanism and further implicate synaptobrevin 2 since the treatment of the cells with Botulinum Neurotoxin type D, cleaving vesicular synaptobrevin, prevented the enhancement of neurosecretion due to the production of sphingosine and derivatives.…”

Role of sphingomyelinases in neurological disorders

“…Analysis of single granule release kinetics by amperometry shows that both sphingomyelinase and arachidonic acid treatment markedly enhance the amount of catecholamines released per individual event [88]. In comparison, another study reports that disruption of microdomains by sphingomyelinase inhibits Ca 2+ sensitivity and late kinetics of secretory vesicle fusion [89].…”