Large Dense-core Vesicles Research Articles

The granin VGF promotes genesis of secretory vesicles, and regulates circulating catecholamine levels and blood pressure

Secretion of proteins and neurotransmitters from large dense core vesicles (LDCVs) is a highly regulated process. Adrenal LDCV formation involves the granin proteins chromogranin A (CgA) and chromogranin B (CgB); CgA- and CgB-derived peptides regulate catecholamine levels and blood pressure. We investigated function of the granin VGF (nonacronymic) in LDCV formation and the regulation of catecholamine levels and blood pressure. Expression of exogenous VGF in nonendocrine NIH 3T3 fibroblasts resulted in the formation of LDCV-like structures and depolarization-induced VGF secretion. Analysis of germline VGF-knockout mouse adrenal medulla revealed decreased LDCV size in noradrenergic chromaffin cells, increased adrenal norepinephrine and epinephrine content and circulating plasma epinephrine, and decreased adrenal CgB. These neurochemical changes in VGF-knockout mice were associated with hypertension. Germline knock-in of human VGF1-615 into the mouse Vgf locus rescued the hypertensive knockout phenotype, while knock-in of a truncated human VGF1-524 that lacks several C-terminal peptides, including TLQP-21, resulted in a small but significant increase in systolic blood pressure compared to hVGF1-615 mice. Finally, acute and chronic administration of the VGF-derived peptide TLQP-21 to rodents decreased blood pressure. Our studies establish a role for VGF in adrenal LDCV formation and the regulation of catecholamine levels and blood pressure.

The Role of Dense Core Nanoparticles in Regulation of Neuronal Communication

The goal of this project is to introduce a novel bottom-up approach to probe the functionality of the dense core nanoparticles by creating artificial dense core nanoparticles from the native proteins.Communication of neurons is happening through the exocytosis process, which is based on vesicular release of neurotransmitters like dopamine. A portion of these vesicles are large dense core vesicles and have the critical function of storing and excreting neurotransmitters or neuropeptides. The so-called dense core is composed of charged polypeptides from the chromogranin family of proteins. This protein matrix accumulates and stores neurotransmitters in high concentrations but the mechanisms of accumulation and release are unknown and highly debated.So far in this work we have developed a model system for the first time as artificial dense core nanoparticles. The synthesized dense core protein nanoparticles are made of chromogranin A, which is the main protein, located in large dense core vesicles. This protein is proposed to involve in the storage of neurotransmitters, Ca2+, and ATP within secretory vesicle. To explore the interactions between dense core proteins and neurotransmitters like dopamine, we are using isothermal titration calorimetry (ITC). By this study we could determine the binding affinity and stoichiometry of neurotransmitter storage under various physiological conditions, such as the pH gradient between the interior and exterior of the secretory vesicle, and the presence of other small physiological relevant molecules such as calcium and ATP.

Protein kinase C gamma interneurons in the rat medullary dorsal horn: Distribution and synaptic inputs to these neurons, and subcellular localization of the enzyme

The γ isoform of protein kinase C (PKCγ), which is concentrated in interneurons in the inner part of lamina II (IIi ) of the dorsal horn, has been implicated in the expression of tactile allodynia. Lamina IIi PKCγ interneurons were shown to be activated by tactile inputs and to participate in local circuits through which these inputs can reach lamina I, nociceptive output neurons. That such local circuits are gated by glycinergic inhibition and that A- and C-fibers low threshold mechanoreceptors (LTMRs) terminate in lamina IIi raise the general issue of synaptic inputs to lamina IIi PKCγ interneurons. Combining light and electron microscopic immunochemistry in the rat spinal trigeminal nucleus, we show that PKCγ-immunoreactivity is mostly restricted to interneurons in lamina IIi of the medullary dorsal horn, where they constitute 1/3 of total neurons. The majority of synapses on PKCγ-immunoreactive interneurons are asymmetric (likely excitatory). PKCγ-immunoreactive interneurons appear to receive exclusively myelinated primary afferents in type II synaptic glomeruli. Neither large dense core vesicle terminals nor type I synaptic glomeruli, assumed to be the endings of unmyelinated nociceptive terminals, were found on these interneurons. Moreover, there is no vesicular glutamate transporter 3-immunoreactive bouton, specific to C-LTMRs, on PKCγ-immunoreactive interneurons. PKCγ-immunoreactive interneurons contain GABAA ergic and glycinergic receptors. At the subcellular level, PKCγ-immunoreactivity is mostly concentrated on plasma membranes, close to, but not within, postsynaptic densities. That only myelinated primary afferents were found to contact PKCγ-immunoreactive interneurons suggests that myelinated, but not unmyelinated, LTMRs play a critical role in the expression of mechanical allodynia.

Self-Assembly of VPS41 Promotes Sorting Required for Biogenesis of the Regulated Secretory Pathway

The regulated release of polypeptides has a central role in physiology, behavior, and development, but the mechanisms responsible for production of the large dense core vesicles (LDCVs) capable of regulated release have remained poorly understood. Recent work has implicated cytosolic adaptor protein AP-3 in the recruitment of LDCV membrane proteins that confer regulated release. However, AP-3 inmammals has been considered to function in theendolysosomal pathway and in the biosynthetic pathway only in yeast. We now find that the mammalian homolog of yeast VPS41, a member of the homotypic fusion and vacuole protein sorting (HOPS) complex that delivers biosynthetic cargo to the endocytic pathway in yeast, promotes LDCV formation through a common mechanism with AP-3, indicating a conserved role for these proteins in the biosynthetic pathway. VPS41 also self-assembles into a lattice, suggesting that it acts as a coat protein for AP-3 in formation of the regulated secretory pathway.

Deciphering Dead-End Docking of Large Dense Core Vesicles in Bovine Chromaffin Cells

Large dense core vesicle (LDCV) exocytosis in chromaffin cells follows a well characterized process consisting of docking, priming, and fusion. Total internal reflection fluorescence microscopy (TIRFM) studies suggest that some LDCVs, although being able to dock, are resistant to calcium-triggered release. This phenomenon termed dead-end docking has not been investigated until now. We characterized dead-end vesicles using a combination of membrane capacitance measurement and visualization of LDCVs with TIRFM. Stimulation of bovine chromaffin cells for 5 min with 6 μm free intracellular Ca2+ induced strong secretion and a large reduction of the LDCV density at the plasma membrane. Approximately 15% of the LDCVs were visible at the plasma membrane throughout experiments, indicating they were permanently docked dead-end vesicles. Overexpression of Munc18-2 or SNAP-25 reduced the fraction of dead-end vesicles. Conversely, expressing open-syntaxin increased the fraction of dead-end vesicles. These results indicate the existence of the unproductive target soluble N-ethylmaleimide-sensitive factor attachment protein receptor acceptor complex composed of 2:1 syntaxin-SNAP-25 in vivo. More importantly, they define a novel function for this acceptor complex in mediating dead-end docking.

The role of chromogranins in the secretory pathway

Chromogranins (Cgs) are acidic proteins implicated in several physiological processes, including the biogenesis and sorting of secretory vesicles, the generation of bioactive peptides, and the accumulation of soluble species inside large dense core vesicles (LDCV). Indeed, Cgs are the main protein component of the vesicular matrix in LDCV, and they are involved in the concentration of soluble species like neurotransmitters and calcium. Experiments using electrochemical techniques such amperometry, patch amperometry, and intracellular electrochemistry have clarified the functional roles of Cgs in the accumulation and release of catecholamines. We have focused this review at a single event of exocytosis of chromaffin cells from three mouse strains lacking Cgs. Accordingly, in this brief review, we will focus on the role of Cgs in maintaining the intravesicular environment of secretory vesicles and in exocytosis, bringing together the most recent findings from studies on adrenal chromaffin cells.

Widespread Dysregulation of Peptide Hormone Release in Mice Lacking Adaptor Protein AP-3

The regulated secretion of peptide hormones, neural peptides and many growth factors depends on their sorting into large dense core vesicles (LDCVs) capable of regulated exocytosis. LDCVs form at the trans-Golgi network, but the mechanisms that sort proteins to this regulated secretory pathway and the cytosolic machinery that produces LDCVs remain poorly understood. Recently, we used an RNAi screen to identify a role for heterotetrameric adaptor protein AP-3 in regulated secretion and in particular, LDCV formation. Indeed, mocha mice lacking AP-3 have a severe neurological and behavioral phenotype, but this has been attributed to a role for AP-3 in the endolysosomal rather than biosynthetic pathway. We therefore used mocha mice to determine whether loss of AP-3 also dysregulates peptide release in vivo. We find that adrenal chromaffin cells from mocha animals show increased constitutive exocytosis of both soluble cargo and LDCV membrane proteins, reducing the response to stimulation. We also observe increased basal release of both insulin and glucagon from pancreatic islet cells of mocha mice, suggesting a global disturbance in the release of peptide hormones. AP-3 exists as both ubiquitous and neuronal isoforms, but the analysis of mice lacking each of these isoforms individually and together shows that loss of both is required to reproduce the effect of the mocha mutation on the regulated pathway. In addition, we show that loss of the related adaptor protein AP-1 has a similar effect on regulated secretion but exacerbates the effect of AP-3 RNAi, suggesting distinct roles for the two adaptors in the regulated secretory pathway.

Estradiol regulates large dense core vesicles in the hippocampus of adult female rats

Previous work has shown that the steroid hormone estradiol facilitates the release of anticonvulsant neuropeptides from inhibitory neurons in the hippocampus to suppress seizures. Because neuropeptides are packaged in large dense core vesicles, estradiol may facilitate neuropeptide release through regulation of dense core vesicles. In the current study, we used serial section electron microscopy in the hippocampal CA1 region of adult female rats to test three hypotheses about estradiol regulation of dense core vesicles: (1) Estradiol increases the number of dense core vesicles in axonal boutons, (2) Estradiol increases the size of dense core vesicles in axonal boutons, (3) Estradiol shifts the location of dense core vesicles toward the periphery of axonal boutons, potentially lowering the threshold for neuropeptide release during seizures. We found that estradiol increases the number and size of dense core vesicles in inhibitory axonal boutons, consistent with increased neuropeptide content, but does not shift the location of dense core vesicles closer to the bouton periphery. These effects were specific to large dense core vesicles (>80nm) in inhibitory boutons. Estradiol had no effects on small dense core vesicles or dense core vesicles in excitatory boutons. Our results indicate that estradiol suppresses seizures at least in part by increasing the potentially releasable pool of neuropeptides in the hippocampus, and that estradiol facilitation of neuropeptide release involves a mechanism other than mobilization of dense core vesicles toward sites of release.

Ultrastructure and synaptic connectivity of main and accessory olfactory bulb efferent projections terminating in the rat anterior piriform cortex and medial amygdala

Neurons in the main olfactory bulb relay peripheral odorant signals to the anterior piriform cortex (aPir), whereas neurons of the accessory olfactory bulb relay pheromone signals to the medial amygdala (MeA), suggesting that they belong to two functionally distinct systems. To help understand how odorant and pheromone signals are further processed in the brain, we investigated the synaptic connectivity of identified axon terminals of these neurons in layer Ia of the aPir and posterodorsal part of the MeA, using anterograde tracing with horseradish peroxidase, quantitative ultrastructural analysis of serial thin sections, and immunogold staining. All identified boutons contained round vesicles and some also contained many large dense core vesicles. The number of postsynaptic dendrites per labeled bouton was significantly higher in the aPir than in the MeA, suggesting higher synaptic divergence at a single bouton level. While a large fraction of identified boutons (29%) in the aPir contacted 2-4 postsynaptic dendrites, only 7% of the identified boutons in the MeA contacted multiple postsynaptic dendrites. In addition, the majority of the identified boutons in the aPir (95%) contacted dendritic spines, whereas most identified boutons in the MeA (64%) contacted dendritic shafts. Identified boutons and many of the postsynaptic dendrites showed glutamate immunoreactivity. These findings suggest that odorant and pheromone signals are processed differently in the brain centers of the main and accessory olfactory systems.

Long-term potentiation in mammalian autonomic ganglia: An inclusive proposal of a calcium-dependent, trans-synaptic process

Ganglionic synapses have the capability to express long-term potentiation (gLTP) after application of a brief high-frequency stimulus. It has been suggested a possible role of gLTP in some cardiovascular diseases. Although a number of characteristics of gLTP have been described, the precise locations and mechanisms underlying gLTP are not completely known. Current findings support two major conflicting presynaptic and postsynaptic hypotheses. The presynaptic hypothesis posits a presynaptic increase in acetylcholine (ACh) release, whereas the postsynaptic hypothesis proposes a long-lasting enhancement of the nicotinic response on the postsynaptic membrane. An alternative trans-synaptic hypothesis proposes the presynaptic release of a cotransmitter from large dense core vesicles, which postsynaptically enhances synaptic efficacy and accounts for gLTP. Here, we review the studies of LTP, with emphasis on gLTP in mammals, and we examine the findings that support the presynaptic, the postsynaptic and the trans-synaptic hypotheses. We then review our data on the contribution of calcium to gLTP as an approach to elucidate the mechanisms of gLTP. Data on the contribution of calcium to gLTP and on prolonged high-frequency stimulus-dependent fading of LTP have led us to support the trans-synaptic process as responsible for gLTP. Finally, we present a formal working model for the mechanisms of gLTP.

Sodium/hydrogen exchanger NHA2 is critical for insulin secretion in β-cells

NHA2 is a sodium/hydrogen exchanger with unknown physiological function. Here we show that NHA2 is present in rodent and human β-cells, as well as β-cell lines. In vivo, two different strains of NHA2-deficient mice displayed a pathological glucose tolerance with impaired insulin secretion but normal peripheral insulin sensitivity. In vitro, islets of NHA2-deficient and heterozygous mice, NHA2-depleted Min6 cells, or islets treated with an NHA2 inhibitor exhibited reduced sulfonylurea- and secretagogue-induced insulin secretion. The secretory deficit could be rescued by overexpression of a wild-type, but not a functionally dead, NHA2 transporter. NHA2 deficiency did not affect insulin synthesis or maturation and had no impact on basal or glucose-induced intracellular Ca(2+) homeostasis in islets. Subcellular fractionation and imaging studies demonstrated that NHA2 resides in transferrin-positive endosomes and synaptic-like microvesicles but not in insulin-containing large dense core vesicles in β-cells. Loss of NHA2 inhibited clathrin-dependent, but not clathrin-independent, endocytosis in Min6 and primary β-cells, suggesting defective endo-exocytosis coupling as the underlying mechanism for the secretory deficit. Collectively, our in vitro and in vivo studies reveal the sodium/proton exchanger NHA2 as a critical player for insulin secretion in the β-cell. In addition, our study sheds light on the biological function of a member of this recently cloned family of transporters.

Molecular components required for resting and stimulated endocytosis of botulinum neurotoxins by glutamatergic and peptidergic neurons

Proteins responsible for basal and stimulated endocytosis in nerves containing small clear synaptic vesicles (SCSVs) or large dense-core vesicles (LDCVs) are revealed herein, using probes that exploit surface-exposed vesicle proteins as acceptors for internalization. Basal uptake of botulinum neurotoxins (BoNTs) by both SCSV-releasing cerebellar granule neurons (CGNs) and LDCV-enriched trigeminal ganglionic neurons (TGNs) was found to require protein acceptors and acidic compartments. In addition, dynamin, clathrin, adaptor protein complex-2 (AP2), and amphiphysin contribute to the depolarization-evoked entry. For fast recycling of SCSVs, knockdown and knockout strategies demonstrated that CGNs use predominantly dynamin 1, whereas isoform 2 and, to a smaller extent, isoform 3 support a less rapid mode of stimulated endocytosis. Accordingly, proximity ligation assay confirmed that dynamin 1 and 2 colocalize with amphiphysin 1 in CGNs, and the latter copurified with both dynamins from cell extracts. In contrast, LDCV-releasing TGNs preferentially employ dynamins 2 and 3 and amphiphysin 1 for evoked endocytosis and lack the fast phase. Hence, stimulation recruits dynamin, clathrin, AP2, and amphiphysin to augment BoNT internalization, and neurons match endocytosis mediators to the different demands for locally recycling SCSVs or replenishing distally synthesized LDCVs.

Understanding the Direct Synaptic Effects of Estradiol

More than 35 years ago, Kelly and coworkers (1) demonstrated that iontophoretic application of an estradiol ester altered the firing rates of a subset of hypothalamic neurons in a matter of milliseconds, an effect that appeared to be stereospecific in that responses were observed to esters of 17 -estradiol, but not its 17 isomer. These observations accompanied the almost simultaneous demonstration by Pietras and Szego (2) that endometrial cells had estradiol recognition sites on their plasma membranes. Kelly et al (1) proposed that neurons might also express stereospecific membrane receptor sites, capable of mediating rapid electrophysiological responses to changes in ovarian estradiol secretion. Progress in characterizing these putative membrane receptors was initially slow, because most work continued to focus on estrogen receptor (ER) actions in the cell nucleus and the regulation of transcriptional response mechanisms. This situation changed dramatically in the early 1990s with the demonstration of rapid effects of estradiol on a number of intracellular signaling pathways (3, 4) as well as direct evidence of ER-like immunoreactivity in dendrites, axon terminals, and postsynaptic densities in the brain (5–7) (reviewed in Blaustein, Ref. 8). Over the ensuing 20 years, multiple potential receptor systems for estradiol have been identified in the central nervous system, signaling via a number of different intracellular pathways (reviewed in Micevych and Christensen, Ref. 9). Studies in cells transfected with vectors expressing ER or ER have shown that whereas newly synthesized receptors concentrate in the cell nucleus, they also incorporate into the plasma membrane (10) where they can initiate estradiol-dependent changes in ERK phosphorylation (11), suggesting that plasma membrane and nuclear ER or ER may be derived from a common source. The demonstration that ER immunoreactivity is present in multiple neuronal cellular compartments raises the question of how the distribution of extranuclear ER is controlled. Are ERs associated with specific cellular organelles or do synaptic and dendritic ERs represent receptors en passant to the plasma membrane? Studies in nonneuronal systems have demonstrated that that ER is trafficked to the plasma membrane via a caveolin-dependent process (12) where it can be anchored in place by palmitoylation (13). Is the same true for ER in neurons? Is the density of ER in the plasma membrane regulated independently of nuclear receptor concentrations? Is ER distributed evenly throughout neuronal cell membranes or is it concentrated specifically in target sites where it can both respond to locally synthesized steroids and exert rapid effects on neurotransmission? The paper by Tabatadze et al (14) in this issue of Endocrinology begins to answer some of these questions. Using tissue fractionationtechniques, theauthorsdemonstrate that ER is concentrated in synaptosomes, particularly in association with synaptic vesicles and neuropeptide-containing large dense-core vesicles. Immunoisolation experiments showthatER is associatedwithbothgammaaminobutryic acid (GABA)and glutamate-containing vesicles. On Western blots, synaptosomal ER immunoreactivity migrates as two bands at approximately 66 and 70 kDa, the ratio of the higher to lower molecular mass forms being greatly decreased by phosphatase treatment, suggesting that the 70kDa band represents a phosphorylated form of the receptor. Finally, treatment of hippocampal slices in vitro was found to decrease the quantity of ER associated with synaptosomal membranes, via a depalmitoylation-dependent mechanism. Overall, the results suggest that ER associates with synaptosomal membranes via a palmitoyla-

Distinct Modes of Dopamine and GABA Release in a Dual Transmitter Neuron

We now know of a surprising number of cases where single neurons contain multiple neurotransmitters. Neurons that contain a fast-acting neurotransmitter, such as glutamate or GABA, and a modulatory transmitter, such as dopamine, are a particularly interesting case because they presumably serve dual signaling functions. The olfactory bulb contains a large population of GABA- and dopamine-containing neurons that have been implicated in normal olfaction as well as in Parkinson's disease. Yet, they have been classified as nonexocytotic catecholamine neurons because of the apparent lack of vesicular monoamine transporters. Thus, we examined how dopamine is stored and released from tyrosine hydroxylase-positive GFP (TH(+)-GFP) mouse periglomerular neurons in vitro. TH(+) cells expressed both VMAT2 (vesicular monoamine transporter 2) and VGAT (vesicular GABA transporter), consistent with vesicular storage of both dopamine and GABA. Carbon fiber amperometry revealed that release of dopamine was quantal and calcium-dependent, but quantal size was much less than expected for large dense core vesicles, suggesting that release originated from small clear vesicles identified by electron microscopy. A single action potential in a TH(+) neuron evoked a brief GABA-mediated synaptic current, whereas evoked dopamine release was asynchronous, lasting for tens of seconds. Our data suggest that dopamine and GABA serve temporally distinct roles in these dual transmitter neurons.

Complexin activates exocytosis of distinct secretory vesicles controlled by different synaptotagmins.

Complexins are SNARE-complex binding proteins essential for the Ca(2+)-triggered exocytosis mediated by synaptotagmin-1, -2, -7, or -9, but the possible role of complexins in other types of exocytosis controlled by other synaptotagmin isoforms remains unclear. Here we show that, in mouse olfactory bulb neurons, synaptotagmin-1 localizes to synaptic vesicles and to large dense-core secretory vesicles as reported previously, whereas synaptotagmin-10 localizes to a distinct class of peptidergic secretory vesicles containing IGF-1. Both synaptotagmin-1-dependent synaptic vesicle exocytosis and synaptotagmin-10-dependent IGF-1 exocytosis were severely impaired by knockdown of complexins, demonstrating that complexin acts as a cofactor for both synaptotagmin-1 and synaptotagmin-10 despite the functional differences between these synaptotagmins. Rescue experiments revealed that only the activating but not the clamping function of complexins was required for IGF-1 exocytosis controlled by synaptotagmin-10. Thus, our data indicate that complexins are essential for activation of multiple types of Ca(2+)-induced exocytosis that are regulated by different synaptotagmin isoforms. These results suggest that different types of regulated exocytosis are mediated by similar synaptotagmin-dependent fusion mechanisms, that particular synaptotagmin isoforms confer specificity onto different types of regulated exocytosis, and that complexins serve as universal synaptotagmin adaptors for all of these types of exocytosis independent of which synaptotagmin isoform is involved.

Diabetes Insipidus: A Review

th position. Oxytocin is also produced in the hypothalamic nuclei, with a structure similar to ADH, but with leucine in the 8 th position. Both ADH and oxytocin are produced in the magnocellular neurosecretory cells (MNC) of the hypothalamus, mainly in the supraoptic (SON) and paraventricular (PVN) nuclei. While each MNC was initially thought to produce either ADH or oxytocin alone, more recent evidence indicates that there can be some overlap of production [5]. ADH is created as a composite precursor molecule composed of ADH, neurophysin-II (NPII), which is ADH’s carrier protein, and copeptin- a glycopeptides [5,6]. Both NPII and ADH are produced from the same precursor mRNA. After passage through the golgi apparatus the prepropeptide complex is packaged into large dense core vesicles (LDCV) which exit from the trans golgi network. Inside the LDCV enzymatic processing of the precursor molecules takes place. This is facilitated by the LDCV’s mildly acidic (pH 5-6) internal environment. Additionally the acidic environment keeps the fully processed ADH nonapeptide bound to the NPII. After their production, the LDCVs are transported in an anterograde fashion, down the neuronal axon along microtubules at a rate near 140 mm/day. LDCV’s containing ADH is stored in the nerve terminals of the posterior pituitary, awaiting neurosecretion. When an action potential causes an influx of Ca 2+

Role of Intracellular Calcium in Release from Nerve Terminals

Oxytocin (OT) and vasopressin (AVP) are released from terminals in the neurohypophysis (NH). These neuropeptides (NP) are contained in large dense core vesicles (LDCV). Ryanodine receptors (RyR) in NH terminals can induce spontaneous focal Ca2+ transients (DeCrescenzo, et al., 2004 J. Neurosci.24:1226-1235), making them ideal for studying the role of intraterminal Ca2+ in NP release.Fluorescent immunolabeling and immunogold micrographs of NH terminals show that RyR are localized specifically to LDCV. Furthermore, a large conductance non-specific cation channel, previously identified in the LDCV membrane with properties similar to that of a RyR (Lee, et al., 1992 Neuron 8:335-342), is pharmacologically affected in the same characteristic manner as a RyR.We found that individual Ca2+ release events alone are not enough to drive peptide release (McNally, et al., 2009 J. Neurosci. 29:14120-14126). However, these RyR sensitive events could potentially play a role in modulating NP release. To test this hypothesis, the association of LDCV within an area 0.45 µm of the plasma membrane was assessed using immunolabeling of Neurophysins I (OT) and II (AVP) in isolated NH terminals. We found that the amount of membrane associated NP-immunoreactivity varies significantly between terminal types and that this distribution pattern can be enhanced by agonist concentrations of ryanodine, but only in OT terminals.Importantly, Ca2+-evoked NP release from permeabilized-terminals was increased by agonist concentrations of ryanodine and decreased by antagonist concentrations of this drug. Amperometric recording of spontaneous release events from artificial transmitter-loaded terminals corroborated these ryanodine effects. Agonist concentrations of ryanodine were also able to increase the asynchronous phase of low frequency electrically-stimulated capacitance increases in NH terminals. Thus, the ryanodine-sensitive mobilization of LDCV seems to have a functional role in modulating secretion of NP from NH terminals. [Support: NS29470, NS40966, P60037094900000].

Copper modulates the large dense core vesicle secretory pathway in PC12 cells

Copper (Cu) is an essential biometal involved in a number of cell functions. Abnormal Cu homeostasis has been identified as a major factor in a number of neurodegenerative disorders. However, little is known about how cells of brain origin maintain Cu homeostasis and in particular, how they respond to an elevated Cu environment. Understanding these processes is essential to obtaining a greater insight into the pathological changes in neurodegeneration and ageing. Although previous studies have shown that Cu in neurons can be associated with synaptic function, there is little understanding of how Cu modulates the regulated secretory vesicle pathways in these cells. In this study, we examined the effect of elevated intracellular Cu on proteins associated with the regulated secretory vesicle pathway in NGF-differentiated PC12 cells that exhibit neuronal-like properties. Increasing intracellular Cu with a cell-permeable Cu-complex (Cu(II)(gtsm)) resulted in increased expression of synaptophysin and robust translocation of this and additional vesicular proteins from synaptic-like microvesicle (SLMV) fractions to chromogranin-containing putative large dense core vesicle (LDCV) fractions in density gradient preparations. The LDCV fractions also contained substantially elevated Cu levels upon treatment of cells with Cu(II)(gtsm). Expression of the H(+) pump, V-ATPase, which is essential for vesicle maturation, was increased in Cu-treated cells while inhibition of V-ATPase prevented translocation of synaptophysin to LDCV fractions. Cu treatment was found to inhibit release of LDCVs in chromaffin cells due to reduced Ca(2+)-mediated vesicle exocytosis. Our findings demonstrate that elevated Cu can modulate LDCV metabolism potentially resulting in sequestration of Cu in this vesicle pool.

Secretion-related structures of hypothalamo-hypophysial terminals in the rat posterior pituitary

Hypothalamic terminals were investigated in the rat posterior pituitary (PP). Injection of wheat germ agglutinin conjugated horseradish peroxidase (WGA-HRP) and co-injection of WGA-HRP with Rab3A-siRNA were made into the hypothalamus, respectively. Additional injection of WGA-HRP was made into the hypothalamus in the animals exposed to ethanol. These injections resulted in heavy labeling of fibers exclusively confined to the PP. Ultrastructural observations showed terminals, fibers, pituicytes, capillaries and vascular spaces in the PP. Although the majority of terminals were observed to contain large dense core vesicles (LDCVs) and HRP-reaction products (HRP-RPs), exocytosis of LDCVs in close proximity to cell membrane was not found. Interestingly, a few terminals showed alteration of cell membrane called "apocrine-like structure" containing LDCV and RP. The narrow neck portion of the structure gave the appearance that it may have been in some stage of separating from terminals. Other remarkable feature was that terminals occasionally reveal the structure of "leakage" of RP discharged into vascular spaces crossing cell membrane. Such hormone-releasing mechanism might be involved in one of "diacrine-like secretion". In the present study secretion-related structures of hypothalamic terminals in the PP are quite different from normal vesicular exocytosis.

Distribution and Posttranslational Modification of Synaptic ERα in the Adult Female Rat Hippocampus

Acute 17β-estradiol (E2) signaling in the brain is mediated by extranuclear estrogen receptors. Here we used biochemical methods to investigate the distribution, posttranslational modification, and E2 regulation of estrogen receptor-α (ERα) in synaptosomal fractions isolated by differential centrifugation from the adult female rat hippocampus. We find that ERα is concentrated presynaptically and is highly enriched with synaptic vesicles. Immunoisolation of vesicles using vesicle subtype-specific markers showed that ERα is associated with both glutamate and γ-aminobutyric acid-containing neurotransmitter vesicles as well as with some large dense core vesicles. Experiments using broad spectrum and residue-specific phosphatases indicated that a portion of ERα in synaptosomal fractions is phosphorylated at serine/threonine residues leading to a mobility shift in SDS-PAGE and creating a double band on Western blots. The phosphorylated form of ERα runs in the upper of the two bands and is particularly concentrated with synaptic vesicles. Finally, we used E2 with or without the acyl protein thioesterase 1 inhibitor, Palmostatin B, to show that 20 min of E2 treatment of hippocampal slices depletes ERα from the synaptosomal membrane by depalmitoylation. We found no evidence that E2 regulates phosphorylation of synaptosomal ERα on this time scale. These studies begin to fill the gap between detailed molecular characterization of extranuclear ERα in previous in vitro studies and acute E2 modulation of hippocampal synapses in the adult brain.