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Resistant starch (RS) is present in various natural and processed foods as well as medications. It has garnered significant attention from both scientists and consumers due to its notable health benefits. However, there is a limited understanding of how RS particles are absorbed at the cellular level and their metabolic behavior, resulting in a lack of clarity regarding the intestinal safety implications of prolonged RS exposure. Here, we demonstrate that rice-derived RS nanoparticles (RSNs) can lead to colitis in mice by triggering lysosomal exocytosis. The research shows that RSNs enter the cells through macropinocytosis and clathrin- and caveolin-mediated endocytosis and activate TRPML1 thereafter, causing the release of lysosomal calcium ions. This, in turn, triggered the TFEB signaling pathway and thus upregulated the lysosomal exocytosis level, leading to lysosomal enzymes to be released to the intestinal lumen. As a result, a decreased number of intestinal goblet cells, diminished tight junction protein expression, and imbalanced intestinal flora in mice were observed. These damages to the intestinal barrier ultimately led to the occurrence of colitis. Our study offers important insights into the cellular bioeffects and detrimental effects on intestinal health caused by RS particles and emphasizes the need to re-evaluate the safety of long-term RS consumption.

Munc18-1 is an SM (sec1/munc-like) family protein involved in vesicle fusion and neuronal exocytosis. Munc18-1 is known to regulate the exocytosis process by binding with closed- and open-state conformations of Syntaxin1, a protein belonging to the SNARE family established to be central to the exocytosis process. Our previous work studied peptide p5 as a promising drug candidate for CDK5-p25 complex, an Alzheimer's disease (AD) pathological target. Experimental in vivo and in vitro studies suggest that Munc18-1 promotes p5 to selectively inhibit the CDK5-p25 complex without affecting the endogenous CDK5 activity, a characteristic of remarkable therapeutic implications. In this paper, we identify several binding modes of p5 with Munc18-1 that could potentially affect the Munc18-1 binding with SNARE proteins and lead to off-target effects on neuronal communication using molecular dynamics simulations. Recent studies indicate that disruption of Munc18-1 function not only disrupts neurotransmitter release but also results in neurodegeneration, exhibiting clinical resemblance to other neurodegenerative conditions such as AD, causing diagnostic and treatment challenges. We characterize such interactions between p5 and Munc18-1, define the corresponding pharmacophores, and provide guidance for the in vitro validation of our findings to improve therapeutic efficacy and safety of p5.

Unveiling molecular mechanisms that dominate protein phase dynamics has been a pressing need for deciphering the intricate intracellular modulation machinery. While ions and biomacromolecules have been widely recognized for modulating protein phase separations, effects of small molecules that essentially constitute the cytosolic chemical atmosphere on the protein phase behaviors are rarely understood. Herein, we report that vitamin C (V C ), a key small molecule for maintaining a reductive intracellular atmosphere, drives reentrant phase transitions of myosin II/F-actin (actomyosin) cytoskeletons. The actomyosin bundle condensates dissemble in the low-V C regime and assemble in the high-V C regime in vitro or inside neuronal cells, through a concurrent myosin II protein aggregation-dissociation process with monotonic V C concentration increase. Based on this finding, we employ in situ single-cell and single-vesicle electrochemistry to demonstrate the quantitative modulation of catecholamine transmitter vesicle exocytosis by intracellular V C atmosphere, i.e., exocytotic release amount increases in the low-V C regime and decreases in the high-V C regime. Furthermore, we show how V C regulates cytomembrane-vesicle fusion pore dynamics through counteractive or synergistic effects of actomyosin phase transitions and the intracellular free calcium level on membrane tensions. Our work uncovers the small molecule-based reversive protein phase regulatory mechanism, paving a new way to chemical neuromodulation and therapeutic repertoire expansion.

Designing nanoparticles (NPs) with desirable cell type-specific exocytosis properties, say promoting their exocytosis from scavenging cell types (e.g., macrophages and endothelial cells) or suppressing their exocytosis from target disease cell types (e.g., cancer cells), improves the application of nanomedicines. However, the design parameters available for tuning the exocytosis of NPs remain scarce in the "nano-cell" literature. Here, we demonstrate that surface modification of NPs with hydrocarbyl functional groups, commonly found in biomolecules and NP-based drug carriers, is a critical parameter for tuning the exocytosis of NPs from RAW264.7 macrophages, C166 endothelial cells, and HeLa epithelial cancer cells. To exclude the effect of hydrophobicity, we prepare a collection of hydrophilic NPs that bear a gold NP (AuNP) core, a dense polyethylene glycol (PEG) shell, and different types of hydrocarbyl groups (X) that are attached to the distal end of the PEG strands (termed "Au@PEG-X NPs"). For all three cell types tested, modification of NPs with straight-chain dodecane leads to a >10-fold increase in the level of cellular uptake, drastically higher than those of all other types of X tested. However, the probability of exocytosis of NPs significantly depends on the types of cell and X. Notably, NPs modified with cyclododecanes are most likely to be exocytosed by RAW264.7 and C166 cells (but not HeLa cells), accompanied by the release of intralumenal vesicles to the extracellular milieu. These data suggest a reductionist approach for rationally assembling bionanomaterials for nanomedicine applications by using hydrocarbyl functional groups as building blocks.

The energy carrying molecule adenosine triphosphate (ATP) has been implicated for its role in modulation of chemical signaling for some time. Despite this, the precise effects and mechanisms of action of ATP on secretory cells are not well-known. Here, bovine chromaffin cells have been used as a model system to study the effects of extracellular ATP in combination with the catecholamine transmitter norepinephrine (NE). Both transmitter storage and exocytotic release were quantified using complementary amperometric techniques. Although incubation with NE alone did not cause any changes to either transmitter storage or release, coincubation with NE and ATP resulted in a significant increase that was concentration dependent. To probe the potential mechanisms of action, a slowly hydrolyzable version of ATP, ATP-γ-S, was used either alone or together with NE. The result implicates two different behaviors of ATP acting on both the purinergic autoreceptors and as a source of the energy needed to load chromaffin cell vesicles.

Metallic gold (Au) nanoparticles have great potential for a wide variety of biomedical applications. Yet, slow clearance of Au nanoparticles significantly hinders their clinical translation. Herein, we describe a strategy of utilizing the endogenous copper (Cu) clearance to improve the elimination of Au nanoparticles. Our mechanistic study reveals that a Cu-transporting P-type ATPase, ATP7B, mediates the exocytosis of CuS nanoparticles into bile canaliculi for their rapid hepatobiliary excretion. The efflux of CuS nanoparticles is adopted to facilitate the hepatobiliary clearance of Au nanoparticles through CuS-Au conjugation. Using two different CuS-Au nanoconjugates, we demonstrate that CuS increases the biliary Au excretion of CuS-Au nanospheres or CuS-Au nanorods in mice or rats in comparison to that of their respective unconjugated Au nanoparticles postintravenous injection. The current CuS-Au conjugation approach provides a feasible strategy to enhance the hepatobiliary clearance of Au nanoparticles that may be applicable to various structures.

Electrochemical measurements of exocytosis combined with intracellular vesicle impact electrochemical cytometry have been used to evaluate the effect of an anticancer drug, tamoxifen, on catecholamine release at the single-cell level. Tamoxifen has been used for over 40 years to treat estrogen receptor-positive breast cancers during both early stages of the disease and in the adjuvant setting. Tamoxifen causes memory and cognitive dysfunction, but the reasons for the cognitive impairment and memory problems induced by this anticancer drug are not well-known. We show that tamoxifen, through a nongenomic mechanism, can modulate both exocytosis and vesicle catecholamine storage in a model cell line. The results indicate that exocytosis is inhibited at high concentrations of tamoxifen and is stimulated at low levels. Tamoxifen also elicits a significant concentration-dependent change in total catecholamine content of single vesicles, while sub-nanomolar concentrations of the drug have stimulatory activity on the catecholamine content of vesicles. In addition, it has profound effects on storage at higher concentrations. Tamoxifen also reduces the intracellular free Ca 2+ but only at micromolar concentration, by acting on voltage-gated Ca 2+ channels, which likely affects neurotransmitter secretion.

Neuronal communication relies on vesicular neurotransmitter release from signaling neurons and detection of these molecules by neighboring neurons. Glutamate, the main excitatory neurotransmitter in the mammalian brain, is involved in nearly all brain functions. However, glutamate has suffered from detection schemes that lack temporal and spatial resolution allowed by electrochemistry. Here we show an amperometric, novel, ultrafast enzyme-based nanoparticle modified sensor, measuring random bursts of hundreds to thousands of rapid spontaneous glutamate exocytotic release events at approximately 30 Hz frequency in the nucleus accumbens of rodent brain slices. Characterizing these single submillisecond exocytosis events revealed a great diversity in spike shape characteristics and size of quantal release, suggesting variability in fusion pore dynamics controlling the glutamate release by cells in this brain region. Hence, this novel biosensor allows recording of rapid single glutamate exocytosis events in the brain tissue and offers insight on regulatory aspects of exocytotic glutamate release, which is critical to understanding of brain glutamate function and dysfunction.

Single cell amperometry and intracellular vesicle impact electrochemical cytometry were used to examine whether lidocaine can regulate neurotransmitter release or storage for PC12 cells to explain the biphasic effects whereby it can protect neurons and improve cognitive outcome at low concentration, but can cause neurotoxicity at high concentration. We show that lidocaine affects the behavior of PC12 cell exocytosis in a concentration dependent way, which exactly corresponds to its biphasic effects. At a relatively high concentration, it shows a much narrower pore size and a longer-duration fusion pore with less monoamine released than control cells. However, at a relatively low concentration, the fusion pore is open even longer than at high concentration, and with more monoamine released than control cells. Furthermore, intracellular vesicle impact electrochemical cytometry was used to confirm that lidocaine did not change the catecholamine content of the vesicles. These data provide a mechanism for the observed biphasic effects of the drug and suggest that lidocaine influences exocytosis through multiple mechanisms.

The fruit fly, Drosophila melanogaster, is a popular model organism for studying neurological processes and diseases due to the availability of sophisticated genetic tools. While endogenous neurotransmitter release has been characterized in Drosophila larvae, here, we measured endogenous dopamine release in isolated adult Drosophila brains for the first time. Dopamine was measured with fast-scan cyclic voltammetry (FSCV), and acetylcholine or nicotine were used as the stimulus, as both interact with nicotinic acetylcholine receptors (nAChRs) to evoke endogenous dopamine release. Stimulations with 10 pmol of acetylcholine elicited 0.26 ± 0.05 μM dopamine, while 70 fmol nicotine stimulations evoked 0.29 ± 0.03 μM in the central complex. Nicotine-stimulated dopamine release lasted much longer than acetylcholine-stimulated release. Dopamine release is reduced in the presence of nAChR antagonist α-bungarotoxin and the sodium channel blocker tetrodotoxin, indicating release is mediated by nAChRs and exocytosis. The identity of dopamine was confirmed by using 3-iodotyrosine, a dopamine synthesis inhibitor, and by confirming that release was not changed in octopamine synthesis mutant flies, Tdc2 RO54 . Additionally, the half-decay time ( t 50 ) in fumin (67 ± 15 s), dopamine transporter mutant flies, was larger than in wild-type flies (16 ± 3.7 s) further proving that acetylcholine stimulation evokes dopamine release. This study demonstrates that stimulation of nAChRs can be used to elicit endogenous dopamine release in adult fly brains, which will be a useful technique for future studies probing dopamine changes during aging or in neurodegenerative diseases.

We used time-of-flight secondary ion mass spectrometry (TOF-SIMS) imaging to investigate the effect of cisplatin, the first member of the platinum-based anticancer drugs, on the membrane lipid composition of model cells to see if lipid changes might be involved in the changes in exocytosis observed. Platinum-based anticancer drugs have been reported to affect neurotransmitter release resulting in what is called the "chemobrain"; however, the mechanism for the influence is not yet understood. TOF-SIMS imaging was carried out using a high energy 40 keV (CO 2 ) 6000 + gas cluster ion beam with improved sensitivity for intact lipids in biological samples. Principal components analysis showed that cisplatin treatment of PC12 cells significantly affects the abundance of different lipids and their derivatives, particularly phosphatidylcholine and cholesterol, which are diminished. Treatment of cells with 2 μM and 100 μM cisplatin showed similar effects on induced lipid changes. Lipid content alterations caused by cisplatin treatment at the cell surface are associated with the molecular and bimolecular signaling pathways of cisplatin-induced apoptosis of cells. We suggest that lipid alterations measured by TOF-SIMS are involved, at least in part, in the regulation of exocytosis by cisplatin.

Emerging two-dimensional (2D) nanomaterials, such as transition-metal dichalcogenide (TMD) nanosheets (NSs), have shown tremendous potential for use in a wide variety of fields including cancer nanomedicine. The interaction of nanomaterials with biosystems is of critical importance for their safe and efficient application. However, a cellular-level understanding of the nano-bio interactions of these emerging 2D nanomaterials ( i. e., intracellular mechanisms) remains elusive. Here we chose molybdenum disulfide (MoS 2 ) NSs as representative 2D nanomaterials to gain a better understanding of their intracellular mechanisms of action in cancer cells, which play a significant role in both their fate and efficacy. MoS 2 NSs were found to be internalized through three pathways: clathrin → early endosomes → lysosomes, caveolae → early endosomes → lysosomes, and macropinocytosis → late endosomes → lysosomes. We also observed autophagy-mediated accumulation in the lysosomes and exocytosis-induced efflux of MoS 2 NSs. Based on these findings, we developed a strategy to achieve effective and synergistic in vivo cancer therapy with MoS 2 NSs loaded with low doses of drug through inhibiting exocytosis pathway-induced loss. To the best of our knowledge, this is the first systematic experimental report on the nano-bio interaction of 2D nanomaterials in cells and their application for anti-exocytosis-enhanced synergistic cancer therapy.

Cationic colloidal gold nanorods (GNRs) have a great potential as a theranostic tool for diverse medical applications. GNRs' properties such as cellular internalization and stability are determined by physicochemical characteristics of their surface coating. GNRs modified by (16-mercaptohexadecyl)trimethylammonium bromide (MTAB), MTAB GNRs, show excellent cellular uptake. Despite their promise for biomedicine, however, relatively little is known about the cellular pathways that facilitate the uptake of GNRs, their subcellular fate and intracellular persistence. Here we studied the mechanism of cellular internalization and long-term fate of GNRs coated with MTAB, for which the synthesis was optimized to give higher yield, in various human cell types including normal diploid versus cancerous, and dividing versus nondividing (senescent) cells. The process of MTAB GNRs internalization into their final destination in lysosomes proceeds in two steps: (1) fast passive adhesion to cell membrane mediated by sulfated proteoglycans occurring within minutes and (2) slower active transmembrane and intracellular transport of individual nanorods via clathrin-mediated endocytosis and of aggregated nanorods via macropinocytosis. The expression of sulfated proteoglycans was the major factor determining the extent of uptake by the respective cell types. Upon uptake into proliferating cells, MTAB GNRs were diluted equally and relatively rapidly into daughter cells; however, in nondividing/senescent cells the loss of MTAB GNRs was gradual and very modest, attributable mainly to exocytosis. Exocytosed MTAB GNRs can again be internalized. These findings broaden our knowledge about cellular uptake of gold nanorods, a crucial prerequisite for future successful engineering of nanoparticles for biomedical applications such as photothermal cancer therapy or elimination of senescent cells as part of the emerging rejuvenation approach.

Polymeric micelles were formed from poly(poly(ethylene glycol) methyl ether methacrylate)-block-poly(styrene) (P(PEGMEMA)-b-PS) block copolymer of two different chain lengths. The micelles formed were approximately 16 and 46 nm in diameter and used to encapsulate curcumin. Upon loading of the curcumin into the micelles, their size increased to approximately 34 and 80 nm in diameter, respectively, with a loading efficiency of 58%. The unloaded micelles were not cytotoxic to human colon carcinoma cells, whereas only the smaller loaded micelles were cytotoxic after 72 h of exposure. The micelles were rapidly internalized by the cells within minutes of exposure, with the loaded micelles internalized to a greater extent owing to their enhanced stability compared to that of the unloaded micelles. The larger micelles were more rapidly internalized and exocytosed than the smaller micelles, demonstrating the effect of micelle size and drug loading on drug delivery and cytotoxicity.

α-Synuclein (α-Syn), a major component of Lewy body that is considered as the hallmark of Parkinson's disease (PD), has been implicated in neuroexocytosis. Overexpression of α-Syn decreases the neurotransmitter release. However, the mechanism by which α-Syn buildup inhibits the neurotransmitter release is still unclear. Here, we investigated the effect of nonaggregated α-Syn on SNARE-dependent liposome fusion using fluorescence methods. In ensemble in vitro assays, α-Syn reduces lipid mixing mediated by SNAREs. Furthermore, with the more advanced single-vesicle assay that can distinguish vesicle docking from fusion, we found that α-Syn specifically inhibits vesicle docking, without interfering with the fusion. The inhibition in vesicle docking requires α-Syn binding to acidic lipid containing membranes. Thus, these results imply the existence of at least two mechanisms of inhibition of SNARE-dependent membrane fusion: at high concentrations, nonaggregated α-Syn inhibits docking by binding acidic lipids but not v-SNARE; on the other hand, at much lower concentrations, large α-Syn oligomers inhibit via a mechanism that requires v-SNARE interaction [ Choi et al. Proc. Natl. Acad. Sci. U. S. A. 2013 , 110 ( 10 ), 4087 - 4092 ].

The effect of latrunculin A, an inhibitor of actin cross-linking, on exocytosis in PC12 cells was investigated with single cell amperometry. This analysis strongly suggests that the actin cytoskeleton might be involved in regulating exocytosis, especially by mediating the constriction of the pore. In an extended kiss-and-run release mode, actin could actually control the fraction of neurotransmitters released by the vesicle. This scaffold appears to contribute, with the lipid membrane and the protein machinery, to the closing dynamics of the pore, in competition with other forces mediating the opening of the exocytotic channel.

Gold nanorods (AuNRs) have been largely investigated driven by their promising potentials in drug delivery, imaging, and photodynamic therapy because of their distinctive physicochemical properties. It is widely known that AuNRs can be taken up by different cells, however, the trafficking of the nanorods in cells are less known. In this work, the behaviors and fate of AuNRs in the human breast cancer cell line MDA-MB-231 were intensively probed by transmission electron microscopy (TEM) with detailed time resolution, together with induced couple plasmon mass spectroscopy (ICP-MS), confocal microscopy, Western blot, and cell viability assay. We reveal that AuNRs enter the classic lysosome maturation through endocytosis and are sequestered in the vesicular system even during cell division. AuNRs can escape from the lysosomes occasionally and the escaped AuNRs are recycled back into the lysosomal system through cytoprotective autophagy. The dilution of AuNRs in cells is mainly attributed to the cell division rather than exocytosis, because expelled AuNRs can be re-endocytosed by the cells. The feature of vesicular restriction guarantees other organelles such as mitochondria and nucleus are exempted from the direct exposure to AuNRs.

Real-time investigations of neurotransmitter release provide a direct insight on the mechanisms involved in synaptic communication. Carbon fiber microelectrodes are state-of-the-art tools for electrochemical measurements of single vesicle neurotransmitter release. Yet, they lack high-throughput capabilities that are required for collecting robust statistically significant data across multiple samples. Here, we present a chip-based recording system enabling parallel in vitro measurements of individual neurotransmitter release events from cells, cultured directly on planar multielectrode arrays. The applicability of this cell-based platform to pharmacological screening is demonstrated by resolving minute concentration-dependent effects of the dopamine reuptake inhibitor nomifensine on recorded single-vesicle release events from PC12 cells. The experimental results, showing an increased half-time of the recorded events, are complemented by an analytical model for the verification of drug action.

Hybrid atomic force microscopy (AFM)-fluorescence microscopy (FM) investigation of exocytosis in lung epithelial cells (ATII cells) allows the detection of individual exocytic events by FM, which can be simultaneously correlated to structural changes in individual cells by AFM. Exocytosis of lamellar bodies (LBs) represents a slow form of exocytosis found in many non-neuronal cells. Exocytosis of LBs, following stimulation with adenosine-5'-triphosphate (ATP) and phorbol 12-myristate 13-acetate (PMA), results in a cation influx via P2X(4) receptors at the site of LB fusion with the plasma membrane (PM), which should induce a temporary increase in cell height/volume. AFM measurements were performed in single-line scans across the cell surface. Five minutes after stimulation, ATII cells revealed a cell height and volume increase of 13.7% ± 4.1% and 15.9 ± 4.8% (N = 9), respectively. These transient changes depend on exocytic LB-PM fusion. Nonstimulated cells and cells lacking LB fusions did not show a significant change in cell height/volume (N = 8). In addition, a cell height decrease was observed in ATII cells stimulated by uridine-5'-triphosphate (UTP) and PMA, agonists inducing LB fusion with the PM, but not activation of P2X(4) receptors. The cell height and volume decreased by -8.6 ± 3.6% and -11.2 ± 3.9% (N = 5), respectively. Additionally, low force contact and dynamic mode AFM imaging of cell areas around the nucleus after stimulation with ATP/PMA was performed. Fused LBs are more pronounced in AFM topography images compared to nonfused LBs, concluding that different "dynamic states" of LBs or locations from the PM are captured during imaging.

Poly(N-isopropyl acrylamide) or pNIPAM is a thermoresponsive polymer that is widely studied for use in bioengineering applications. The interest in this polymer lies in the polymer's unique capability to undergo a sharp property change near physiological temperature, which aids in the spontaneous release of biological cells from substrates. Currently, there are many methods for depositing pNIPAM onto substrates, including atom-transfer radical polymerization (ATRP) and electron beam ionization. Each method yields pNIPAM-coated substrates with different surface characteristics that can influence cell behavior. In this work, we compare two methods of pNIPAM deposition: plasma deposition and codeposition with a sol-gel. The resulting pNIPAM films were analyzed for use as substrates for mammalian cell culture based on surface characterization (XPS, ToF-SIMS, AFM, contact angles), cell attachment/detachment studies, and an analysis of exocytosis function using carbon-fiber microelectrode amperometry (CFMA). We find that although both methods are useful for the deposition of functional pNIPAM films, plasma deposition is much preferred for cell-sheet engineering applications because of the films' thermoresponse, minimal change in cell density, and maintenance of supported cell exocytosis function., (© 2011 American Chemical Society)

Botulinum neurotoxins cleave synaptic SNAREs and block exocytosis, demonstrating that these proteins function in neurosecretion. However, the function of the SNARE syntaxin remains less clear because no neurotoxin cleaves it selectively. Starting with a botulinum neurotoxin that cleaves both syntaxin and SNAP-25, we engineered a version that retains activity against syntaxin but spares SNAP-25. These mutants block synaptic release in neurons and norepinephrine release in neuroendocrine cells, thus establishing an essential role for syntaxin in Ca2+-triggered exocytosis. These mutants can generate syntaxin-free cells as a useful experimental system for research and may lead to pharmaceuticals that target syntaxin selectively.

Exocytosis is a crucial process of secreting various signaling molecules such as neurotransmitters, hormones, and other chemical mediators into the extracellular space. Exocytotic release is caused by membrane fusion of intracellular vesicles with the plasma membrane triggered by an increase in intracellular Ca(2+). In the present study, we developed an artificial system of exocytosis that secretes intravesicular contents upon Ca(2+) influx. We prepared artificial secretory cells using cell-sized giant unilamellar liposomal vesicles (GUVs) that contain small liposomes (SUVs) that correspond to secretory vesicles. To observe exocytosis-like secretion in an artificial system, we labeled both an intra-SUV solution and an SUV membrane with a soluble fluorescent dye and a rhodamine-labeled phospholipid, respectively. To induce membrane fusion between SUVs and a GUV as observed in exocytosis, the Ca(2+) concentration of intra-GUV was elevated by incorporating ionomycin (a Ca(2+) ionophore) into the GUV membrane. We succeeded in inducing exocytosis-like secretion by Ca(2+) elevation in a GUV together with the osmolarity difference between the intra-GUV and extra-GUV solutions.

Extracellular UDP-glucose is a natural purinergic receptor agonist, but its mechanisms of cellular release remain unclear. We studied these mechanisms in Saccharomyces cerevisiae, a simple model organism that releases ATP, another purinergic agonist. Similar to ATP, UDP-glucose was released by S. cerevisiae at a rate that was linear over time. However, unlike ATP release, UDP-glucose release was not dependent on glucose stimulation. This discrepancy was resolved by demonstrating the apparent glucose stimulation of ATP release reflected glucose-dependent changes in the intracellular pattern of adenine nucleotides, with AMP release dominating in the absence of glucose. Indeed, total adenine nucleotide release, like UDP-glucose release, did not vary with glucose concentration over the short term. The genetic basis of UDP-glucose release was explored through analysis of deletion mutants, aided by development of a novel bioassay for UDP-glucose based on signaling through heterologously expressed human P2Y 14 receptors. Using this assay, an elevated rate of UDP-glucose release was demonstrated in mutants lacking the putative Golgi nucleotide sugar transporter YMD8. An increased rate of UDP-glucose release in ymd8Delta was reduced by deletion of the YEA4 UDP- N-acetylglucosamine or the HUT1 UDP-galactose transporters, and overexpression of YEA4 or HUT1 increased the rate of UDP-glucose release. These findings suggest an exocytotic release mechanism similar to that of ATP, a conclusion supported by decreased rates of ATP, AMP, and UDP-glucose release in response to the secretory inhibitor Brefeldin A. These studies demonstrate the involvement of the secretory pathway in nucleotide and nucleotide sugar efflux in yeast and offer a powerful model system for further investigation.

A variety of polarized epithelial cells, such as human breast cancer (MCF-7), have mechanistically evolved the ability to adapt to the dynamic cellular environment and maintain homeostasis of an array of micronutrients which display conditional requirements. Active absorption mechanisms, including endocytosis, are able to control cell surface recognition and protein expression which are associated with a substance's intracellular processing and kinetics. Riboflavin (RF), or vitamin B2, has been recognized as an important factor in a multitude of terminal disease states, most notably in breast cancer, where its cellular absorption is significantly enhanced. In order to delineate the regulatory mechanisms and kinetics associated with RF control in human breast cancer tissue, this study aimed to model its absorption profile and identify its intracellular regulatory components. Using both the Michaelis-Menten equation and a modified version of it, incorporating both active internalization and passive diffusion, RF absorption displayed better correlation ( r (2) > 0.998) with the mixed, active and passive, model exhibiting kinetic parameters characteristic of a receptor-mediated uptake mechanism ( J max = 2.58 pmol/5 min, K m = 106 nM) at extracellular RF concentrations under 5 muM and a passive component existing at RF concentrations greater than 5 muM. Following internalization, RF was able to recycle back to the membrane with a half-life of 13.7 min at 37 degrees C, which occurred more rapidly with increasing extracellular RF concentrations ( t 1/2 = 5.4 min at 1 muM) and decreasing temperatures ( t 1/2 = 6.4 min at 4 degrees C). Furthermore, modification to endosomal pH using the lysomotropic agents monensin (25 muM) and primaquine (300 muM) significantly inhibited the exocytosis of RF (61 and 30% of control), whereas biochemical modification of endocytic trafficking with okadaic acid (1 muM) led to a significant increase in RF exocytosis (208%). In conclusion, RF homeostasis in MCF-7 cells is a well regulated process which is dependent upon RF concentration, temperature, and endosomal acidification.

In this work, carbon-fiber microelectrode amperometry is used to characterize serotonin exocytosis from murine peritoneal mast cells cocultured with fibroblasts in the presence of Au nanoparticles. In the case of mast cell exposure to 1 nM 28 nm diameter spherical Au nanoparticles, there is a decrease of greater than 30% in the number of successful granule transport and fusion events, greater than 30% increase in the rate of intragranular matrix expansion, and greater than 20% increase in the number of secreted serotonin molecules per granule. These results suggest that nanoparticles interrupt the dense-core biopolymer intragranular matrix and present the potential for systematic studies showing how exocytotic function is influenced by nanoparticle size, shape, and composition.

Dopaminergic receptors are found on bovine adrenal chromaffin cells and have been implicated in the facilitation of an inward calcium current [Artalejo et al., (1990) Nature 348, 239-242] that could enhance release. However, previous studies using incubations of long duration (minutes) with dopaminergic receptor antagonists have found instead an inhibition of catecholamine release. In this work we used brief (subsecond) chemical depolarizing stimuli to reexamine the role of dopaminergic receptors on exocytosis from bovine adrenal chromaffin cells. Responses to consecutive depolarizing stimuli were compared using amperometry to monitor vesicular release events and intracellular fura-2 to examine Ca2+ dynamics within individual cells. Restoration of intracellular Ca2+ levels to their initial values following exposure to 60 mM K+ was found to be prolonged unless the exposure was brief (0.5 s) and the cells were maintained at 37 degrees C. However, with these optimum conditions, a second stimulation evoked more exocytotic events than the first. This effect was blocked by SCH-23390, a D1 antagonist, in a dose dependent fashion, but not by raclopride, a D2 antagonist. The D1 agonist, SKF-38393, enhanced the number of exocytotic events as did prior exposure of the cell to epinephrine. Taken together, the data indicate that released catecholamines can enhance their own release by interaction with a D1-like receptor on bovine adrenal chromaffin cells.

Split-and-pool synthesis of a 10,000-membered library of molecules resembling the natural product carpanone has been achieved. The synthesis features development of solid-phase multicomponent reactions between nitrogen nucleophiles, enones, and hydroxylamines, and a solid-phase application of the Huisgen cycloaddition affording substituted triazoles. The synthesis was performed in high-capacity (500 microm) polystyrene beads using a one bead-one stock solution strategy that enabled phenotypic screens of the resulting library. Using whole-cell fluorescence imaging, we discovered a series of molecules from the carpanone-based library that inhibit exocytosis from the Golgi apparatus. The most potent member of this series has an IC(50) of 14 microM. We also report structure-activity relationships for the molecules exhibiting this interesting phenotype. These inhibitors of exocytosis may be useful reagents for the study of vesicular traffic.

The effects of cholesterol depletion from the plasma membrane with methyl-beta-cyclodextrin (MbetaCD) on exocytotic processes were investigated in rat basophil leukemia cells (RBL-2H3 cells). Pretreatment of the cells with MbetaCD inhibited antigen-evoked exocytotic release dose-dependently. To elucidate the mechanism of this inhibition, we performed experiments on the effects of MbetaCD on exocytotic membrane fusion and mobilization of Ca(2+) and on the localization of the tyrosine kinase Lyn. Inhibition of degranulation by MbetaCD was observed even under stimulation with the phorbol ester and calcium ionophore. Therefore, MbetaCD affected a process downstream of Ca(2+) influx, or membrane fusion between the granule and the plasma membrane. Intracellular calcium measurements revealed that MbetaCD inhibited the Ca(2+) increase induced by antigen. Furthermore, we found that MbetaCD significantly inhibited Ca(2+) influx from the extracellular medium through the store-operated calcium channel (SOC) but did not affect Ca(2+) release from the intracellular Ca(2+) store. Fluorescent image analysis of cells expressing Lyn-YFP showed that treatment with MbetaCD scarcely affected the localization and lateral mobility of Lyn in the plasma membrane. These results suggest that cholesterol depletion by MbetaCD decreases degranulation mainly by inhibiting the SOC and membrane fusion between the secretory granules and the plasma membrane in mast cells.

In rat adipocytes, insulin-induced GLUT4 recruitment to the plasma membrane (PM) is associated with characteristic changes in the GLUT4 contents of three distinct endosomal fractions, T, H, and L. The organelle-specific marker distribution pattern suggests that these endosomal GLUT4 compartments are sorting endosomes (SR), GLUT4-storage endosomes (ST), and GLUT4 exocytotic vesicules (EV), respectively, prompting us to analyze GLUT4 recycling based upon a four-compartment kinetic model. Our analysis revealed that insulin modulates GLUT4 trafficking at multiple steps, including not only the endocytotic and exocytotic rates, but also the two rate coefficients coupling the three intracellular compartments. This analysis assumes that GLUT4 cycles through PM T, H,L, and back to PM, in that order, with transitions characterized by four first-order coefficients. Values assigned to these coefficients are based upon the four steady-state GLUT4 pool sizes assessed under both basal and insulin stimulated states and the transition time courses observed in the plasma membrane GLUT4 pool. Here we present the first reported experimental measurements of transient changes in each of the four GLUT4 compartments during the insulin-stimulated to basal transition in rat adipocytes and compare these experimental results with the corresponding model simulations. The close correlation of these results offers clear support for the general validity of the assumed model structure and the assignment of the T compartment to the sorting endosome GLUT4 pool. Variations in the recycling pathway from that of an unbranched cyclic topography are also considered in the light of these experimental observations. The possibility that H is a coupled GLUT4 storage compartment lying outside the direct cyclic pathway is contraindicated by the data. Okadaic acid-induced GLUT4 recruitment is accompanied by modulation of the rate coefficients linking individual endosomal GLUT4 compartments, further demonstrating a significant role of the endosomal pathways in GLUT4 exocytosis.

Although rat pheochromocytoma (PC12) neurotransmitter storage vesicles are known to contain a variety of neurotransmitters including catecholamines, there is little evidence that the molecular species detected during amperometric monitoring of exocytosis is a catecholamine. Rather, as these are catecholamine-containing cells, one assumes catecholamines are released. Additionally, although the total amount of transmitter released can be quantified, it has been extremely difficult to evaluate the concentration at the point of release for each exocytosis event. Interpreting voltammograms obtained in the attoliter volume affected between the electrode and the cell and defined by the size of the exocytosis pore during exocytosis is an extreme analytical challenge. Here we use voltammetry of approximately 10(-19) mol released from individual exocytosis events to identify, along with pharmacological evidence, the released compound at PC12 cells as a catecholamine, most likely dopamine. The area of the electrode at which oxidation occurs following an exocytosis event is proportional to the temporal delay prior to acquisition of a voltammogram. This model allows determination of relative concentrations from individual release events and has been used to examine events at control cells and cells incubated with the dopamine precursor, L-3,4-dihydroxyphenylalanine (L-DOPA). Exposure to L-DOPA (100 microM for 1 h) results in 145 detectable events for 11 cells compared to 77 events for 29 control cells, clearly indicating that vesicles can be "loaded" with dopamine. However, the concentrations measured at the electrode surface provide similar distributions for both L-DOPA-treated and control cells. Cyclic voltammetric measurements of relative concentration for zeptomole levels of transmitter in attoliter volumes provide evidence that loading vesicles by increased transmitter synthesis does not lead to elevated concentrations at individual release sites.

The formation and role of arachidonic acid (AA) and its metabolites during gonadotropin releasing hormone- (GnRH-) induced gonadotropin secretion were investigated in primary cultures of rat pituitary cells. Prelabeled cells ([3H]AA) responded to GnRH challenge with increased formation (about 2-fold) of the leukotrienes LTC4, LTD4, and LTE4 as well as 5- and 15-eicosatetraenoic acids (5- and 15-HETE) as identified by HPLC. Formation of leukotrienes and 15-HETE was further verified by specific radioimmunoassays. No significant increase in the formation of 12-HETE or of the cyclooxygenase products prostaglandin E (PGE) and thromboxane A2 by GnRH was noticed. Addition of physiological concentrations of LTC4 enhanced basal LH release, while subphysiological concentrations of LTC4 (10(-15)-10(-12) M) inhibited GnRH-induced LH release by about 35% (p less than 0.02). Using specific lipoxygenase inhibitors L-656,224 and MK 886, we found inhibition of GnRH-induced LH release by about 40% at concentrations known to specifically inhibit the 5-lipoxygenase pathway. The peptidoleukotriene receptor antagonist ICI 198,615 inhibited LTC4- and LTE4-induced LH release and surprisingly also the effect of GnRH on LH release by 40%. The data strongly suggest a role for AA and its lipoxygenase metabolites in the on/off reactions of GnRH upon LH release. The data also present a novel amplification cycle in which newly formed leukotrienes become first messengers and establish an autocrine/paracrine loop.

Structure-function relationships for the lipid component of chromaffin granules isolated from the bovine adrenal medulla have been investigated by employing 31P nuclear magnetic resonance (NMR), freeze-fracture, and spectrophotometric techniques. Two aspects have been studied in detail, namely, the structural preferences of lipids in the isolated granule membrane and derived liposomal model membrane systems as well as the influence of exogenous lipid (in the form of sonicated vesicle systems) on the Ca2+-stimulated release of granule contents. It is shown that at least 90% of endogenous granule membrane phospholipids assume a liquid-crystalline bilayer configuration at physiological temperatures. Liposomal dispersions of total granule lipid also exhibit bilayer structure, consistent with a structural role of phospholipids in vivo. Incubation of intact isolated granules in the presence of up to 10 mM Ca2+ does not induce significant release of contents above background levels. However, it is shown that incubation of granules in the presence of sonicated phospholipid systems which undergo structural transitions in the presence of Ca2+ can cause immediate and total release of granule contents at Ca2+ levels of 2 mM or more. This behavior is attributed to disruption of granule membrane integrity due to fusion of the vesicle systems with the chromaffin granules. Direct evidence for such fusion is obtained by freeze-fracture electron microscopy. On the basis of this information and with the assumption that the inner leaflet of the adrenal cell plasma membrane is composed predominantly of phosphatidylethanolamine and phosphatidylserine, a mechanism of Ca2+-stimulated exocytotic release of catecholamines in vivo is proposed.

A number of substituted 2-[(2,2,2-trifluoroethyl)sulfonyl]-1H-benzimidazoles (4) have demonstrated antiinflammatory activity that appears to have a mechanism distinct from typical cyclooxygenase inhibiting nonsteroidal antiinflammatory drugs. Several of these compounds inhibit adjuvant-induced arthritis in rats at 25 mg/kg while showing no activity in the carrageenan paw edema model at up to 100 mg/kg. Two compounds, 4a and 4b, showed no significant inhibition of cyclooxygenase in vitro at concentrations as high as 5 X 10(-5) M. All compounds 4 active in adjuvant-induced arthritis were also found to inhibit release of lysosomal enzymes from neutrophils, raising the possibility that their antiinflammatory effect is at least partially mediated by an effect on neutrophil function.