Juliana M. Rosa

Sphingosine Facilitates SNARE Complex Assembly and Activates Synaptic Vesicle Exocytosis

Summary Synaptic vesicles loaded with neurotransmitters fuse with the plasma membrane to release their content into the extracellular space, thereby allowing neuronal communication. The membrane fusion process is mediated by a conserved set of SNARE proteins: vesicular synaptobrevin and plasma membrane syntaxin and SNAP-25. Recent data suggest that the fusion process may be subject to regulation by local lipid metabolism. Here, we have performed a screen of lipid compounds to identify positive regulators of vesicular synaptobrevin. We show that sphingosine, a releasable backbone of sphingolipids, activates synaptobrevin in synaptic vesicles to form the SNARE complex implicated in membrane fusion. Consistent with the role of synaptobrevin in vesicle fusion, sphingosine upregulated exocytosis in isolated nerve terminals, neuromuscular junctions, neuroendocrine cells and hippocampal neurons, but not in neurons obtained from synaptobrevin-2 knockout mice. Further mechanistic insights suggest that sphingosine acts on the synaptobrevin/phospholipid interface, defining a novel function for this important lipid regulator.

Calcium entry through slow-inactivating L-type calcium channels preferentially triggers endocytosis rather than exocytosis in bovine chromaffin cells

Calcium (Ca(2+))-dependent endocytosis has been linked to preferential Ca(2+) entry through the L-type (α(1D), Ca(V)1.3) of voltage-dependent Ca(2+) channels (VDCCs). Considering that the Ca(2+)-dependent exocytotic release of neurotransmitters is mostly triggered by Ca(2+) entry through N-(α(1B), Ca(V)2.2) or PQ-VDCCs (α(1A), Ca(V)2.1) and that exocytosis and endocytosis are coupled, the supposition that the different channel subtypes are specialized to control different cell functions is attractive. Here we have explored this hypothesis in primary cultures of bovine adrenal chromaffin cells where PQ channels account for 50% of Ca(2+) current (I(Ca)), 30% for N channels, and 20% for L channels. We used patch-clamp and fluorescence techniques to measure the exo-endocytotic responses triggered by long depolarizing stimuli, in 1, 2, or 10 mM concentrations of extracellular Ca(2+) ([Ca(2+)](e)). Exo-endocytotic responses were little affected by ω-conotoxin GVIA (N channel blocker), whereas ω-agatoxin IVA (PQ channel blocker) caused 80% blockade of exocytosis as well as endocytosis. In contrast, nifedipine (L channel blocker) only caused 20% inhibition of exocytosis but as much as 90% inhibition of endocytosis. Conversely, FPL67146 (an activator of L VDCCs) notably augmented endocytosis. Photoreleased caged Ca(2+) caused substantially smaller endocytotic responses compared with those produced by K(+) depolarization. Using fluorescence antibodies, no colocalization between L, N, or PQ channels with clathrin was found; a 20-30% colocalization was found between dynamin and all three channel antibodies. This is incompatible with the view that L channels are coupled to the endocytotic machine. Data rather support a mechanism implying the different inactivation rates of L (slow-inactivating) and N/PQ channels (fast-inactivating). Thus a slow but more sustained Ca(2+) entry through L channels could be a requirement to trigger endocytosis efficiently, at least in bovine chromaffin cells.

Permissive role of sphingosine on calcium-dependent endocytosis in chromaffin cells

Sphingosine has been shown to modulate neurotransmitter release. Because membrane fusion and fission involve lipid metabolism, we asked here whether sphingosine had a role in regulating endocytosis. To explore this hypothesis, we monitored changes of membrane capacitance (Cm) to study the effects of intracellular sphingosine on membrane retrieval after chromaffin cell stimulation with depolarising pulses (DPs). We found that: (1) sphingosine dialysis through the patch-clamp pipette (SpD) using the whole-cell configuration of the patch-clamp technique (WCC) favours the appearance of a pronounced endocytotic response; (2) SpD-elicited endocytosis was Ca2+-dependent but Ba2+ did not substitute Ca2+; (3) under WCC, such endocytotic response disappeared with repetitive DPs; (4) in cells preincubated with sphingomyelinase to augment endogenous sphingosine synthesis, and then voltage-clamped under the perforated-patch configuration of the patch-clamp technique (PPC), endocytosis decayed little with repeated stimulation; (5) sphingosine-1-phosphate (S1P), a metabolite of sphingosine, had a meagre effect on endocytosis; and (6) neither dynamin inhibitor dynasore nor calmodulin blocker calmidazolium affected the sphingosine elicited endocytosis. We believe this is the first report showing that sphingosine plays a permissive role in activating Ca2+-dependent endocytosis during cell depolarisation. This effect requires high subplasmalemmal cytosolic Ca2+ concentrations and a cytosolic factor(s) that is dialysed with the pipette solution. Independence of dynamin and calmodulin suggests that sphingosine-dependent endocytosis could be a novel, more direct pathway for vesicle recycling under mild depolarisation stimuli.

Paradoxical facilitation of exocytosis by inhibition of L-type calcium channels of bovine chromaffin cells.

Ca(2+) entry through the L-subtype (α(1D), Ca(v)1,3) of voltage-dependent calcium channels (VDCCs) seems to selectively regulate the endocytotic response after the application of a single depolarizing pulse to voltage-clamped bovine chromaffin cells. Here we have found that L channel blockade with nifedipine transformed the exocytotic responses elicited by a double-pulse protocol, from depression to facilitation. This apparent paradoxical effect was mimicked by pharmacological interventions that directly block endocytosis namely, dynasore, calmidazolium, GTP-γS and GDP-βS. This reinforces our view that Ca(2+) entry through PQ channels (α(1A); Ca(v)2.1) regulates fast exocytosis while Ca(2+) entry through L channels preferentially controls rapid endocytosis.

Augmentation of catecholamine release elicited by an Eugenia punicifolia extract in chromaffin cells

Plant extracts of Eugenia punicifolia (Kunth) DC., Myrtaceae, are used in Amazon region of Brazil to treat diarrhea and stomach disturbances, and as hypoglycemic medicine. We have recently shown that an aqueous extract of E. punicifolia augmented cholinergic neurotransmission in a rat phrenic nerve-diaphragm preparation. In this study, we investigated the effects of an E. punicifolia dichloromethane extract (EPEX) in a neuronal model of cholinergic neurotransmission, the bovine adrenal chromaffin cell. EPEX augmented the release of catecholamine triggered by acetylcholine (ACh) pulses but did not enhance ACh-evoked inward currents, which were inhibited by 30%. Since EPEX did not cause a blockade of acetylcholinesterase or butyrylcholinesterase, it seems that EPEX is not directly activating the cholinergic system. EPEX also augmented K+-elicited secretion without enhancing the whole-cell inward calcium current. This novel and potent effect of EPEX in enhancing exocytosis might help to identify the active component responsible for augmenting exocytosis. When elucidated, the molecular structure of this active principle could serve as a template to synthesise novel compounds to regulate the exocytotic release of neurotransmitters.