Multiple sclerosis drug FTY-720 toxicity is mediated by the heterotypic fusion of organelles in neuroendocrine cells.

FTY-720 (Fingolimod) was one of the first compounds authorized for the treatment of multiple sclerosis. Among its other activities, this sphingosine analogue enhances exocytosis in neuroendocrine chromaffin cells, altering the quantal release of catecholamines. Surprisingly, the size of chromaffin granules is reduced within few minutes of treatment, a process that is paralleled by the homotypic fusion of granules and their heterotypic fusion with mitochondria, as witnessed by dynamic confocal and TIRF microscopy. Electron microscopy studies support these observations, revealing the fusion of several vesicles with individual mitochondria to form large, round mixed organelles. This cross-fusion is SNARE-dependent, being partially prevented by the expression of an inactive form of SNAP-25. Fused mitochondria exhibit an altered redox potential, which dramatically enhances cell death. Therefore, the cross-fusion of intracellular organelles appears to be a new mechanism to be borne in mind when considering the effect of FTY-720 on the survival of neuroendocrine cells.

Impact of Chromogranin A deficiency on catecholamine storage, catecholamine granule morphology and chromaffin cell energy metabolism in vivo.

Chromogranin A (CgA) is a prohormone and granulogenic factor in neuroendocrine tissues with a regulated secretory pathway. The impact of CgA depletion on secretory granule formation has been previously demonstrated in cell culture. However, studies linking the structural effects of CgA deficiency with secretory performance and cell metabolism in the adrenomedullary chromaffin cells in vivo have not previously been reported. Adrenomedullary content of the secreted adrenal catecholamines norepinephrine (NE) and epinephrine (EPI) was decreased 30-40 % in Chga-KO mice. Quantification of NE and EPI-storing dense core (DC) vesicles (DCV) revealed decreased DCV numbers in chromaffin cells in Chga-KO mice. For both cell types, the DCV diameter in Chga-KO mice was less (100-200 nm) than in WT mice (200-350 nm). The volume density of the vesicle and vesicle number was also lower in Chga-KO mice. Chga-KO mice showed an ~47 % increase in DCV/DC ratio, implying vesicle swelling due to increased osmotically active free catecholamines. Upon challenge with 2 U/kg insulin, there was a diminution in adrenomedullary EPI, no change in NE and a very large increase in the EPI and NE precursor dopamine (DA), consistent with increased catecholamine biosynthesis during prolonged secretion. We found dilated mitochondrial cristae, endoplasmic reticulum and Golgi complex, as well as increased synaptic mitochondria, synaptic vesicles and glycogen granules in Chga-KO mice compared to WT mice, suggesting that decreased granulogenesis and catecholamine storage in CgA-deficient mouse adrenal medulla is compensated by increased VMAT-dependent catecholamine update into storage vesicles, at the expense of enhanced energy expenditure by the chromaffin cell.

Sphingomyelin derivatives increase the frequency of microvesicle and granule fusion in chromaffin cells.

Sphingomyelin derivatives like sphingosine have been shown to enhance secretion in a variety of systems, including neuroendocrine and neuronal cells. By studying the mechanisms underlying this effect, we demonstrate here that sphingomyelin rafts co-localize strongly with synaptosomal-associated protein of 25Kda (SNAP-25) clusters in cultured bovine chromaffin cells and that they appear to be linked in a dynamic manner. In functional terms, when cultured rat chromaffin cells are treated with sphingomyelinase (SMase), producing sphingomyelin derivatives, the secretion elicited by repetitive depolarizations is enhanced. This increase was independent of cell size and it was significant 15min after initiating stimulation. Interestingly, by evaluating the membrane capacitance we found that the events in control untreated cells corresponded to two populations of microvesicles and granules, and the fusion of both these populations is clearly enhanced after treatment with SMase. Furthermore, SMase does not increase the size of chromaffin granules. Together, these results strongly suggest that SNARE-mediated exocytosis is enhanced by the generation of SMase derivatives, reflecting an increase in the frequency of fusion of both microvesicles and chromaffin granules rather than an increase in the size of these vesicles.

Controlling synaptotagmin activity by electrostatic screening.

Exocytosis of neurosecretory vesicles is mediated by the SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) proteins syntaxin-1, synaptobrevin and SNAP-25, with synaptotagmin functioning as the major Ca(2+) sensor for triggering membrane fusion. Here we show that bovine chromaffin granules readily fuse with large unilamellar liposomes in a SNARE-dependent manner. Fusion is enhanced by Ca(2+), but only when the target liposomes contain phosphatidylinositol-4,5-bisphosphate and when polyphosphate anions, such as nucleotides or pyrophosphate, are present. Ca(2+)-dependent enhancement is mediated by endogenous synaptotagmin-1. Polyphosphates operate by an electrostatic mechanism that reverses an inactivating cis association of synaptotagmin-1 with its own membrane without affecting trans binding. Hence, the balancing of trans- and cis-membrane interactions of synaptotagmin-1 could be a crucial element in the pathway of Ca(2+)-dependent exocytosis.

The association of dynamin with synaptophysin regulates quantal size and duration of exocytotic events in chromaffin cells.

Although synaptophysin is one of the most abundant integral proteins of synaptic vesicle membranes, its contribution to neurotransmitter release remains unclear. One possibility is that through its association with dynamin it controls the fine tuning of transmitter release. To test this hypothesis, we took advantage of amperometric measurements of quantal catecholamine release from chromaffin cells. First, we showed that synaptophysin and dynamin interact in chromaffin granule-rich fractions and that this interaction relies on the C terminal of synaptophysin. Experimental maneuvers that are predicted to disrupt the association between these two proteins, such as injection of antibodies against dynamin or synaptophysin, or peptides homologous to the C terminal of synaptophysin, increased the quantal size and duration of amperometric spikes. In contrast, the amperometric current that precedes the spike remained unchanged, indicating that synaptophysin/dynamin association does not regulate the initial fusion pore, but it appears to target a later step of exocytosis to control the amount of catecholamines released during a single vesicle fusion event.

Design, synthesis, and biological evaluation of fluorinated analogues of salicylihalamide.

Salicylihalamide A (SA), a benzolactone enamide compound, possesses potent cytotoxicity against human tumor cell lines. SA is a selective inhibitor of mammalian vacuolar type H(+)-ATPase (V-ATPase), and is distinct from previously known V-ATPase inhibitors such as bafilomycins and concanamycins that do not discriminate between mammalian and nonmammalian V-ATPases. Because of its potent antitumor activity and structural simplicity, SA is a promising candidate for an anticancer drug. Although a number of structure-activity relation studies using synthetic analogues have been reported, no fluorinated derivative of SA has been evaluated even though selective addition of a fluorine atom into a therapeutic small molecule candidate often enhances pharmacokinetic and physicochemical properties. We designed and synthesized fluorinated analogues of SA and evaluated their V-ATPase inhibitory activities. Compared to the natural product, the synthetic analogues were potent V-ATPase inhibitors, suggesting that these analogues are potential drug candidates and potential molecular probes for mode-of-action studies using fluorine-based analytical methods such as (19)F-NMR spectroscopy.

F-actin and myosin II accelerate catecholamine release from chromaffin granules.

The roles of nonmuscle myosin II and cortical actin filaments in chromaffin granule exocytosis were studied by confocal fluorescence microscopy, amperometry, and cell-attached capacitance measurements. Fluorescence imaging indicated decreased mobility of granules near the plasma membrane following inhibition of myosin II function with blebbistatin. Slower fusion pore expansion rates and longer fusion pore lifetimes were observed after inhibition of actin polymerization using cytochalasin D. Amperometric recordings revealed increased amperometric spike half-widths without change in quantal size after either myosin II inhibition or actin disruption. These results suggest that actin and myosin II facilitate release from individual chromaffin granules by accelerating dissociation of catecholamines from the intragranular matrix possibly through generation of mechanical forces.

Vesicular monoamine transporter substrate/inhibitor activity of MPTP/MPP+ derivatives: a structure-activity study.

The active metabolite of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), N-methyl-4-phenylpyridinium (MPP(+)), selectively destroys the dopaminergic neurons and induces the symptoms of Parkinson's disease. Inhibition of mitochondrial complex I and/or the perturbation of dopamine metabolism through cellular and granular accumulation have been proposed as some of the major causes of neurotoxicity. In the present study we have synthesized and characterized a number of MPTP and MPP(+) derivatives that are suitable for the comparative neurotoxicity and complex I inhibition versus dopamine metabolism perturbation studies. Structure-activity studies with bovine chromaffin granule ghosts show that 3'-hydroxy-MPP(+) is one of the best known substrates for the vesicular monoamine transporter (VMAT). A series of compounds that combine the structural features of MPP(+) and a previously characterized VMAT inhibitor, 3-amino-2-phenyl-propene, have been identified as the most effective VMAT inhibitors. These derivatives have been used to define the structural requirements of the VMAT substrate and inhibitor activities.

Copper ions disrupt dopamine metabolism via inhibition of V-H+-ATPase: a possible contributing factor to neurotoxicity.

The involvement of copper in the pathophysiology of neurodegeneration has been well documented but is not fully understood. Commonly, the effects are attributed to increased reactive oxygen species (ROS) production due to inherent redox properties of copper ions. Here we show copper can have physiological effects distinct from direct ROS production. First, we show that extragranular free copper inhibits the vesicular H(+)-ATPase of resealed chromaffin granule ghosts. Extragranular ascorbate potentiates this inhibition. The inhibition is mixed type with K(is) = 6.8 +/- 2.8 micromol/L and K(ii) = 3.8 +/- 0.6 micromol/L, with respect to ATP. Second, extracellular copper causes an inhibition of the generation of a pH-gradient and rapid dissipation of pre-generated pH and catecholamine gradients. Copper chelators and the ss-amyloid peptide 1-42 were found to effectively prevent the inhibition. The inhibition is reversible and time-independent suggesting the effects of extracellular copper on H(+)-ATPase is direct, and not due to ROS. The physiological significance of these observations was shown by the demonstration that extracellular copper causes a dramatic perturbation of dopamine metabolism in SH-SY5Y cells. Thus, we propose that the direct inhibition of the vesicular H(+)-ATPase may also contribute to the neurotoxic effects of copper.

One-electron oxidation of catecholamines generates free radicals with an in vitro toxicity correlating with their lifetime.

One-electron oxidation of dopamine by ferricyanide generates a highly reactive free radical intermediate that inactivates the V-type H(+)-ATPase proton pump in catecholamine storage vesicles, i.e., the driving force in both the vesicular uptake and the storage of catecholamines, in a cell-free in vitro model system at pH 7.0. Electron paramagnetic resonance spectroscopy revealed that a radical with g=2.0045, formed by this oxidation, was relatively long-lived (t(1/2) obs=79 s at pH 6.5 and 25 degrees C). Experimental evidence is presented that the observed radical most likely represents dopamine semiquinone free radical, although an o-quinone free radical cannot be ruled out. Oxidation of noradrenaline and adrenaline by ferricyanide generated similar isotropic radicals, but of shorter half-lives (i.e., 43 and 5.3 s, respectively), and the efficacy of inactivation of the H(+)-ATPase correlated with the half-life of the respective catecholamine free radical (i.e., dopamine >noradrenaline>>adrenaline). Thus, the generation of relatively long-lived semiquinone free radicals, although at low concentrations, in dopaminergic and noradrenergic neurons may represent a common mechanism of cytotoxicity linked to neurodegeneration of the respective neurons related to Parkinson disease.

Adrenal chromaffin cells exhibit impaired granule trafficking in NCAM knockout mice.

Neural cell adhesion molecule (NCAM) plays several critical roles in neuron path-finding and intercellular communication during development. In the clinical setting, serum NCAM levels are altered in both schizophrenic and autistic patients. NCAM knockout mice have been shown to exhibit deficits in neuronal functions including impaired hippocampal long term potentiation and motor coordination. Recent studies in NCAM null mice have indicated that synaptic vesicle trafficking and active zone targeting are impaired, resulting in periodic synaptic transmission failure under repetitive physiological stimulation. In this study, we tested whether NCAM plays a role in vesicle trafficking that is limited to the neuromuscular junction or whether it may also play a more general role in transmitter release from other cell systems. We tested catecholamine release from neuroendocrine chromaffin cells in the mouse adrenal tissue slice preparation. We utilize electrophysiological and electrochemical measures to assay granule recruitment and targeting in wild-type and NCAM -/- mice. Our data show that NCAM -/- mice exhibit deficits in normal granule trafficking between the readily releasable pool and the highly release-competent immediately releasable pool. This defect results in a decreased rate of granule fusion and thus catecholamine release under physiological stimulation. Our data indicate that NCAM plays a basic role in the transmitter release mechanism in neuroendocrine cells through mediation of granule recruitment and is not limited to the neuromuscular junction and central synapse active zones.

Differential regulation of multiple populations of granules in rat adrenal chromaffin cells by culture duration and cyclic AMP.

We employed carbon fiber amperometry to measure the amount of catecholamine released from individual granules (i.e. the quantal size, Q) of rat chromaffin cells. The distribution of Q1/3 of amperometric events could be reasonably described by the summation of at least three Gaussians, suggesting that rat chromaffin cells contained at least three distinct populations of granules, with a small, medium or large modal Q. After 3 days of culture, the mean cellular Q reduced by approximately 14%, which did not arise from a uniform percentage decrease in the Q of every granule. Instead, the rundown involved a > 11% decrease in the proportional release from large Q granules and a > 19% decrease in the modal Q-value of small Q granules. In contrast, when cells were cultured with dibutyryl-cAMP (dBcAMP) for 3 days, their mean cellular Q increased by approximately 38% (relative to time-matched controls). This increase in Q was not associated with any shift in the proportional release from the three populations of granules. Instead, cAMP increased the average amount of catecholamines released from all three populations of granules. Our data raise the possibility that distinct populations of granules in rat chromaffin cells can be regulated either differentially or uniformly.

Secretory vesicles membrane area is regulated in tandem with quantal size in chromaffin cells.

The number of transmitter molecules released in a quantal event can be regulated, and recent studies suggest that the modulation of quantal size is associated with corresponding changes in vesicle volume (Colliver et al., 2000; Pothos et al., 2002). If so, this could occur either by distension of the vesicle membrane or by incorporation and removal of vesicle membrane. We performed simultaneous measurements of vesicle membrane area and catecholamine release in individual quantal events from chromaffin cells using cell-attached patch amperometry. Cells were treated with reserpine, a vesicular monoamine transport blocker that decreases quantal size, or l-dopa, a catecholamine precursor that increases quantal size. We show that decrease and increase in quantal size are associated with a respective decrease and increase in vesicle membrane area. These results point to a novel mechanism of vesicle membrane dynamics by which vesicles physically change their membrane area in response to changes in transmitter content such that the intravesicular concentration of transmitter is maintained.

Exocytosis of single chromaffin granules in cell-free inside-out membrane patches.

In chromaffin cells, exocytosis of single granules and properties of the fusion pore--the first connection between vesicular lumen and extracellular space --can be studied by cell-attached patch amperometry, which couples patch-clamp capacitance measurements with simultaneous amperometric recordings of transmitter release. Here we have studied exocytosis of single chromaffin granules and endocytosis of single vesicles in cell-free inside-out membrane patches by patch capacitance measurements and patch amperometry. We excised patches from chromaffin cells by using methods developed for studying properties of single ion channels. With low calcium concentrations in the pipette and bath, the patches showed no spontaneous exocytosis, but exocytosis could be induced in some patches by applying calcium to the cytoplasmic side of the patch. Exocytosis was also stimulated by calcium entry through the patch membrane. Initial conductances of the fusion pore were undistinguishable in cell-attached and excised patch recordings, but the subsequent pore expansion was slower in excised patches. The properties of exocytotic fusion pores in chromaffin cells are very similar to those observed in mast cells and granulocytes. Excised patches provide a tool with which to study the mechanisms of fusion pore formation and endocytosis in vitro.

Hydralazine reduces the quantal size of secretory events by displacement of catecholamines from adrenomedullary chromaffin secretory vesicles.

The effects of the antihypertensive agent hydralazine (1 to 100 nmol/L) on the exocytotic process of single adrenal chromaffin cells have been studied using amperometry. Hydralazine does not reduce the frequency of exocytotic spikes but rapidly slows the rate of catecholamine release from individual exocytotic events by reducing the quantal size of catecholamine exocytosis. Confocal and standard epifluorescence microscopy studies show that hydralazine rapidly accumulates within secretory vesicles. The blockade of the vesicular H+ pump with bafilomycin A1 inhibits hydralazine uptake. Experiments with permeabilized cells show that hydralazine displaces catecholamines from secretory vesicles. The drug also displaces vesicular Ca2+, as shown by fura-2 microfluorimetry. These data suggest that hydralazine acts, at least partially, by interfering with the storage of catecholamines. These effects of hydralazine occurred within seconds, and at the tissue concentrations presumably reached in antihypertensive therapy; these concentrations are a thousand times lower than those described for relaxing vascular tissues in vitro. We proposed that these novel effects could explain many of the therapeutic and side effects of this drug that are likely exerted in sympathetic nerve terminals.

Stereospecific inhibition of monoamine uptake transporters by meta-hydroxyephedrine isomers.

Meta-hydroxyephedrine (HED) comprises four stereoisomers consisting of two enantiomeric pairs related to ephedrine and pseudoephedrine. HED is transported into adrenergic neurons and radiolabeled HED has been employed in positron emission tomography (PET) to image adrenergic neurons in vivo. To extend structure-activity analyses of binding sites within monoamine transporters and to determine which stereoisomer displayed the best selectivity for PET imaging applications, we tested the HED compounds for their abilities to inhibit [(3)H]neurotransmitter uptake into platelets, transfected cells, and chromaffin vesicles. We hypothesized that the HED compounds would be most potent at the norepinephrine transporter (NET) compared to the serotonin or dopamine transporters and that the 1R diastereomers would be more effective than 1S diastereomers. Supporting the hypotheses, all stereoisomers were most potent at the NET and the 1R,2S stereoisomer was the most potent inhibitor overall. However, the 1S,2R isomer may be preferred for PET applications because of better selectivity among the transporters and reduced neuronal recycling.

cAmp modulates exocytotic kinetics and increases quantal size in chromaffin cells.

The role of cAMP/cAMP-dependent protein kinase (PKA) on the late phase of exocytosis has been studied by amperometry on Ba(2+)-stimulated single bovine chromaffin cells. Forskolin (FSK) increases the intracellular cAMP levels in a concentration-dependent manner. Forskolin (100 nM) does not increase the number of exocytotic events, although it significantly increases the net granule content of catecholamines (CA), which is accompanied by a slowing of the process of degranulation. These effects are reversible, occur within 15 to 60 s, and are not due to newly synthesized CA. Isoprenaline, pituitary adenylate cyclase-activating polypeptide-38 or dB-cAMP reproduce FSK effects as does cholera toxin. The inhibition of phosphodiesterases with 3-isobutyl-1-methylxanthine mimics and potentiates the effect of FSK and isoprenaline. Rolipram and okadaic acid also produce a drastic increase in net granule content of CA, whereas H-89 attenuates the FSK response. These data indicate that cyclic AMP/PKA might favor the granule aggregation before its fusion with cell membrane and slow the late step of the exocytotic process.

ATP-sensitive K+ transport in adrenal chromaffin granules.

In the present study the influx of 86Rb+, a K+ analogue, was studied in mitochondria, microsomes and chromaffin granules prepared from adrenal gland medulla. The most active electrogenic 86Rb+ transport was found in the membrane fraction identified as chromaffin granules by marker enzyme estimation. The transport was found to be sensitive to ATP, ATP gamma S, ADP and to the triazine dyes, but not to AMP and cAMP. The inhibition induced by ATP was observed in the absence of externally added Mg2+, suggesting that a free nucleotide, rather than the ATP-Mg complex, was required for inhibition. Furthermore, the 86Rb+ influx was found to be inhibited by Mg2+ alone, but not by Ca2+ and antidiabetic sulfonylureas. The 86Rb+ influx was not stimulated by potassium channel openers. In conclusion, our results indicate that an electrogenic, ATP-sensitive potassium transport system operates in the chromaffin granule membrane.

Energy-dependent accumulation of calcium antagonists in catecholamine storage vesicles.

The calcium antagonists verapamil, nitrendipine, mibefradil, and amlodipine accumulate in chromaffin granule ghosts with apparent equilibrium partition coefficients [(mol/mg membrane lipid)/(mol/mg solvent water)] of 246 +/- 105 (N = 8), 2700 +/- 600 (N = 4), 7400 +/- 2200 (N = 4), and 8100 +/- 1100 (N = 5), respectively. In the presence of 1.2 mM MgATP, the partition coefficients were 854 +/- 206 (N = 10), 2300 +/- 600 (N = 4), 32,700 +/- 8,900 (N = 7), and 20,300 +/- 5,000 (N = 11) for verapamil, nitrendipine, mibefradil, and amlodipine, respectively. Except for nitrendipine, the apparent partition coefficients in the presence of MgATP were significantly different from the control (P < 0.001). For amlodipine and verapamil, the vacuolar H(+)-ATPase inhibitors bafilomycin A1 (30 nM) and N-ethylmaleimide (2 mM) and the protonophore (uncoupler) carbonyl cyanide m-chlorophenylhydrazone (CCCP, 10 microM) completely blocked the increase in partition coefficients in response to MgATP. The extra amlodipine, mibefradil, and verapamil that accumulated in response to MgATP were released into the medium by CCCP (10 microM) by 18% (N = 5), 30% (N = 5), and 88% (N = 5) for amlodipine, mibefradil, and verapamil, respectively. Thus, amlodipine, mibefradil, and verapamil, but not nitrendipine, accumulate in catecholamine storage vesicles in response to delta mu H+ generated by the endogenous V-type H(+)-ATPase, and are partially released by de-energetisation. Hence, these calcium antagonists can reach unexpectedly high concentrations in certain target cells, and give pharmacodynamic properties not shared by nitrendipine.

Nitric oxide modulates a late step of exocytosis.

The effects of nitric oxide (NO) on the late phase of exocytosis have been studied, by amperometry, on Ba(2+)-stimulated chromaffin cells. Acute incubation with NO or NO donors (sodium nitroprusside, spermine-NO, S-nitrosoglutathione) produced a drastic slowdown of the granule emptying. Conversely, cell treatment with N(omega)-nitro-l-arginine methyl ester (a NO synthase inhibitor) or with NO scavengers (methylene blue, 2-(4-carboxyphenyl)-4,4,5, 5-tetramethyl-imidazoline-1-oxyl-3-oxide potassium) accelerated the extrusion of catecholamines from chromaffin granules, suggesting the presence of a NO modulatory tone. The incubation with phosphodiesterase inhibitors (3-isobutyl-1-methylxanthine or zaprinast) or with the cell-permeant cGMP analog 8-bromo-cGMP, mimicked the effects of NO, suggesting the involvement of the guanylate cyclase cascade. NO effects were not related to changes in intracellular Ba(2+). NO did not modify the duration of feet. Effects were evident even on pre-fusioned granules, observed under hypertonic conditions, suggesting that the fusion pore is not the target for NO, which probably acts by modifying the affinity of catecholamines for the intragranular matrix. NO could modify the synaptic transmitter efficacy through a novel mechanism, which involves the regulation of the emptying of secretory vesicles.