Non-Nitrogen-Containing Bisphosphonates Prevent Pyrophosphorylation of Exocytosis Proteins.

BACKGROUND: Clodronate, a non-nitrogen-containing bisphosphonate (non-NBP), is intracellularly converted into non-hydrolyzable ATP analogs. Clodronate and its analogs impair normal cell functions, including the exocytosis process. However, how this occurs in mast cells is still not well characterized. OBJECTIVE: To summarize the possible mechanisms of clodronate-mediated exocytosis inhibition in mast cells. RESULTS: Non-NBPs display several possible mechanisms of exocytosis inhibition in various cell types, including vesicular nucleotide transporter (VNUT) and purinergic receptor inhibition. Inhibition of purinergic receptors has been shown in mast cells, but VNUT inhibition remains to be confirmed. Inhibition of protein prenylation by non-NBPs has also been shown; however, direct evidence of non-NBPs in prenylated exocytosis proteins is still contradictory. Finally, non-NBPs may inhibit mast cell exocytosis via impairment of protein pyrophosphorylation. This mechanism is less studied, and direct evidence of the involvement of pyrophosphorylated proteins in exocytosis is still lacking. CONCLUSION: Non-NBPs may affect mast cell exocytosis by interacting with purinergic receptors or VNUT or by preventing post-translational modifications of exocytosis protein(s), i.e., prenylation and pyrophosphorylation. The latter needs further investigation to provide direct evidence of a role for non- NBPs.

Inhibition of Histamine Release from RBL-2H3 Cells by Zoledronate Did Not Affect Rab27a/Doc2a Interaction.

Mast cell (MC) exocytosis is organized by prenylated protein, including Rab families. Among Rab proteins, Rab3a, Rab27a, and Rab11 are responsible for exocytosis arrangement. Rab3a and Rab27a are contributed to exocytosis by interacting with other exocytosis proteins. Zoledronate administration disrupted the Rab prenylation process that affected its interaction with other proteins, and finally, its function. The present study has investigated the effect of zoledronate on the histamine release (HR) from RBL-2H3 cells. The main focus is to answer the question of whether zoledronate affects Rab27a/Doc2a interaction. Histamine release on RBL-2H3 cells after zoledronate or clodronate administration was measured using HPLC-fluorometry. Dinitrophenylated bovine serum albumin (DNP-BSA) (20 ng/mL) or ionomycin (1 µM) are used as secretagogues. Calcium (Ca2+) influx observation was performed using Fura-2A/M. In situ proximity ligation assay (PLA) is used to investigate Rab27a/Doc2a interaction after bisphosphonates (BPs) treatment. Histamine concentration measurement with HPLC-fluorometry showed that zoledronate (30, 100 µM) inhibited HR from antigen-activated RBL-2H3 cells. Zoledronate showed less inhibition in cells activated with ionomycin. Intracellular Ca2+ concentration and Ca2+ flux rate from the extracellular compartment was not changed by zoledronate administration. No changes in Rab27a/Doc2a interaction after zoledronate treatment. Histamine release inhibition by zoledronate in DNP-BSA-activated RBL-2H3 cells is not related to the disruption of Rab27a/Doc2a interaction and is not involve the change in Ca2+ influx.

Radioactive gold nanocluster (198-AuNCs) showed inhibitory effects on cancer cells lines.

Cancer is a global epidemic disease responsible for over ten millions death worldwide. The early diagnosis and the precise treatment with reduced adverse reactions are the main goal worldwide. In this study, we produced, characterized and evaluated (in vitro) in three different cancer cell lines (protaste, breast and melanoma) a radioactive gold nanocluster (R-AuNC) (198Au25(Capt)18). The pharmacokinetics as the influence in the ABC transporter (MRP1 Efflux Transporter Protein) was also evaluated. The results showed that R-AuNC (198Au25(Capt)18) are capable to kill the cancer cells lines of protaste, breast and melanoma. The pharmacokinetics showed a fast clearance and great volume of distribution, confirming the use of R-AuNC as nanomedicine for cancer treatment. Finally, the ABC transporter assay corroborated that the R-AuNC (198Au25(Capt)18) has no risk of being pumped out of cells by this efflux transporter. The results validate the use of gold nanoparticles as therapeutic nanomedicine for cancer treatment.

Mast cell activation markers for in vitro study.

Mast cells (MCs) are well known for their role in allergic conditions. This cell can be activated by various types of secretagogues, ranging from a small chemical to a huge protein. Mast cell activation by secretagogues triggers the increase in intracellular calcium (iCa2+) concentration, granule trafficking, and exocytosis. Activated mast cells release their intra-granular pre-stored mediator or the newly synthesized mediator in the exocytosis process, in the form of degranulation or secretion. There are at least three types of exocytosis in mast cells, which are suggested to contribute to the release of different mediators, i.e.,, piecemeal, kiss-and-run, and compound exocytosis. The status of mast cells, i.e., activated or resting, is often determined by measuring the concentration of the released mediator such as histamine or ß-hexosaminidase. This review summarizes several mast cell components that have been and are generally used as mast cell activation indicator, from the classical histamine and ß-hexosaminidase measurement, to eicosanoid and granule trafficking observation. Basic principle of the component determination is also explained with their specified research application and purpose. The information will help to predict the experiment results with a certain study design.

Tachykinin-1 receptor antagonism suppresses substance-P- and compound 48/80-induced mast cell activation from rat mast cells expressing functional mas-related GPCR B3.

OBJECTIVE: Mice and rats are important animal models for mast cell (MC) study. However, rat Mas-related-GPCR-B3 receptor (MRGPRB3) has been less studied than its mouse counterpart. Therefore, we aimed to characterize rat MRGPRB3. METHODS: Mrgprb3 mRNA expression was assessed in peritoneal cells (RPCs) and peritoneal MCs (RPMCs) of wild-type rats, RPCs of MC-deficient rats, and RBL-2H3 cells by reverse-transcriptase polymerase chain reaction (RT-PCR). RPMCs, MRGPRX2-transfected and non-transfected RBL-2H3 cells were activated by 15-30 min incubation with DNP-BSA, substance-P (SP), or compound-48/80. L732138 or CP96344 was used as a tachykinin/neurokinin-1-receptor antagonist. Histamine release from MCs was measured by HPLC fluorometry. RESULTS: Mrgprb3 mRNA expression was found in all cells, with the highest level in wild-type RPCs. All cells responded to DNP-BSA, but only MRGPRX2-transfected-RBL-2H3 cells and RPMCs responded to all activators. L732138 (0.1-10 µM) and CP96344 (1-100 µM) suppressed SP (10 µM)-induced RPMC activation. L732138 inhibition was dose independent, whereas CP96344 inhibition occurred in a dose-dependent manner. Additionally, only CP96344 suppressed SP (100 µM)- and compound-48/80 (10 µg/mL)-induced RPMC activation. CONCLUSIONS: RPMCs expressing functional MRGPRB3 response upon MRGPRX2 ligands to regulated MC-mediated activities. It`s provide novel insights for future pseudo-allergic studies in rodents.

Morphological and functional analysis of beige (Chèdiak-Higashi syndrome) mouse mast cells with giant granules.

Chèdiak-Higashi syndrome is a rare autosomal recessive disease that causes hypopigmentation, recurrent infections, mild coagulation defects and neurological problems. Beige mice carry a mutation in the lysosome trafficking regulator (LYST) gene and display some of the key characteristics of human Chèdiak-Higashi syndrome, in particular, a high susceptibility to infection due to aberrant natural killer (NK) cell and polymorphonuclear leucocyte function. Morphological analysis of beige mice reveals the presence of enlarged lysosomes in a variety of cell types, including leucocytes, hepatocytes, fibroblasts and renal tubule cells. To examine the process of granule maturation and degranulation in beige mice mast cells, morphological studies have been conducted using a combination of electrophysiological techniques; however, few functional studies have been conducted with mast cells, such as mediator release. The aim of the present study was to determine the morphological and functional characteristics of skin and peritoneal mast cells and bone marrow-derived mast cells of homozygous (bg/bg) and heterozygous (bg/+) beige mice and wild-type (+/+) mice. The histamine concentration was lower in the peritoneal and bone marrow-derived mast cells of bg/bg mice compared with those of bg/+ and +/+ mice, but the histamine release response was potentiated. In vivo studies of passive cutaneous anaphylaxis showed no differences between bg/bg mice and either bg/+ or +/+ mice. Although bg/bg mast cells with enlarged granules display specific exocytotic processes in vitro, the consequences of mast cell activation in beige mice were similar to those of wild-type mice in vivo.

Histamine uptake mediated by plasma membrane monoamine transporter and organic cation transporters in rat mast cell lines.

The resources of released histamine from activated mast cells, as initial effectors of allergic disease, include not only endogenous prepackaged histamine and newly synthesized histamine, but also histamine that is obtained through the de novo reuptake pathway. To investigate the de novo histamine production pathway, a mast cell line, RBL-2H3 Sc98 in which endogenous histamine production is lacking and only the de novo histamine release pathway via transporters is maintained, was used to dissect histamine reuptake in the present study. Histamine content measurements indicated that RBL-2H3 Sc98 cells took up extracellular histamine for storage in granules and subsequent release after stimulation by an antigen. Profiling and inhibition analysis of possible transporters suggested that the plasma membrane monoamine transporter and organic cation transporter 1 may be candidate transporters for histamine uptake from extracellular spaces, and that vesicular monoamine transporter 2 was responsible for intracellular vesicle uptake. These results may provide the foundation to understand the contribution of exogenous histamine to outward histamine release that is mediated by mechanisms other than conventional exocytosis.

High-throughput screening system for dynamic monitoring of exocytotic vesicle trafficking in mast cells.

Mast cells, in addition to endocrine cells and neurons, are typical secretory cells. Their function in allergic inflammation is to secrete inflammatory mediators from secretory vesicles. Intracellular synthesized inflammatory mediators are transported by vesicular monoamine transporters (VMATs) to vesicles where they are stored. After stimulation, the contents of the secretory vesicles are released via exocytosis. This study established a high throughput imaging screening system to monitor the functions of secretory vesicles in mast cells, including molecular uptake via VMAT2 and the exocytotic process, by using a novel fluorescent probe, FFN206, which was developed as a VMAT2 substrate. After loading with FFN206, the rapid uptake of FFN206 was observed and secretory vesicles in mouse bone marrow derived mast cells and a cultured mast cell line were clearly visualized. FFN206 uptake by secretory vesicles was time-dependent and was blocked by reserpine. Furthermore, exocytotic trafficking was monitored dynamically by real-time high-throughput fluorescence quantitation. In the present study, we verified the application of FFN206 for the monitoring of functional vesicles. This high-throughput screening system may benefit instinctive drug evaluation.

Zoledronate modulates intracellular vesicle trafficking in mast cells via disturbing the interaction of myosinVa/Rab3a and sytaxin4/VAMP7.

Nitrogen-containing bisphosphonates (NBPs) have been widely used as bone anti-resorptive drugs for the treatment of osteoclast-dependent bone disorders. Zoledronate is currently the most potent NBP, and has potential as an inhibitor of farnesyl pyrophosphate synthase. The present study was undertaken to elucidate the possible effects of zoledronate on FcεRI-dependent mast cell activity in vitro, which is essential for in maintaining homeostasis of the gastrointestinal mucosa. Treatment with zoledronate significantly diminished exocytosis of mast cells, which was reflected by a decrease of FcεRI-dependent histamine release compared to that in vehicle-treated mast cells. Our single-vesicle monitoring and biochemical results suggested that zoledronate modulates intracellular formation of the myosinVa/Rab3a complex and syntaxin4/VAMP7 complex, which are critical in vesicle motility, and therefore disturbs exocytosis via suppression of the velocity of intracellular vesicles and inhibition of membrane fusion. Our findings imply that oral administration of zoledronate could modulate mucosal immune function by blocking mast cell function, and this risk should be of concern in the clinical usage of NBPs.

Effects of 8-Hydroxyisocapnolactone-2-3-diol and friedelin on mast cell degranulation.

OBJECTIVE: To investigate the effects of friedelin (terpenoid) and 8-hydroxyisocapnolactone-2-3-diol (coumarin) with concentration 10 µM, 30 µM, and 100 µM on inhibiting mast cells (MCs) degranulation. METHODS: The investigation was performed in vitro by administering each compound into rat peritoneal MCs and rat basophilic leukemia-2H3 cells followed by activation with 50 µg/mL of compound 48/80 or 1 µM of ionomycin. The concentration of histamine released from each group was measured by a high-performance liquid chromatography-fluorometry system with post-column derivatization using o-phthalaldehyde. RESULTS: 8-Hydroxyisocapnolactone-2-3-diol inhibited degranulation of compound 48/80 activated-rat peritoneal MCs with the histamine release percentages of 74.57%, 72.21% and 51.79% when the 10 µM, 30 µM and 100 µM concentrations were used, respectively. Where as about 81% histamine was released by the control group. Degranulation inhibition ability was also observed in ionomycin-activated rat basophilic leukemia-2H3 cells. In contrast, friedelin failed to inhibit degranulation in either cell type. The inhibition of 8-hydroxyisocapnolactone-2-3-diol was not related to the depletion of histamine synthesis as implied by the total histamine measurement. CONCLUSIONS: These results exhibit the promising of 8-hydroxyisocapnolactone-2-3-diol is a potential parent structure for developing a MCs stabilizer.

Inhibition of the mevalonate pathway by simvastatin interferes with mast cell degranulation by disrupting the interaction between Rab27a and double C2 alpha proteins.

Statins are well-known inhibitors of 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase, which block the mevalonate pathway. The activity of statins not only decreases cholesterol levels but also ameliorates inflammation and modulates the immune system. In this study, we investigated the effects of simvastatin on histamine release using rat basophilic leukaemia (RBL-2H3) cells, and examined its interaction with proteins involved in the exocytosis process. Treatment with simvastatin for 24h inhibited histamine release in RBL-2H3 cells in a concentration-dependent manner after stimulation with dinitrophenylated bovine serum albumin (DNP-BSA, as an antigen), ionomycin (a calcium ion [Ca2+] ionophore), and thapsigargin (an inhibitor of Ca2+-ATPase in the endoplasmic reticulum). Simvastatin-induced inhibition was counteracted by co-administration of mevalonolactone or geranylgeraniol, but not farnesol. Indeed, several exocytotic proteins were post-translationally modified by isoprenylation, which is required for proper localization in the lipid membrane. RBL-2H3 cells express proteins involved in the fusion of granules and the plasma membrane, such as Ras-like protein in the brain 27a (Rab27a), synaptosome-associated protein 23 (SNAP23), and vesicle-associated membrane protein 7 (VAMP7), as well as Ca2+ binding proteins, such as double C2 alpha (Doc2a), synaptotagmin2, and mammalian uncoordinated13-4 (munc13-4). The interaction of Rab27a and Doc2a proteins was detected using proximity ligation assays. Antigen stimulation caused these proteins to interact, and this interaction could be disrupted by co-administration of simvastatin. In conclusion, simvastatin inhibited the mevalonate pathway, which suppressed the geranylgeranylation of Rab27a by depleting geranylgeranyl pyrophosphate and interfering with the Rab27a-Doc2a interaction. This activity resulted in the inhibition of exocytosis in RBL-2H3 cells.

CRACM3 regulates the stability of non-excitable exocytotic vesicle fusion pores in a Ca(2+)-independent manner via molecular interaction with syntaxin4.

Ca(2+) release-activated calcium channel 3 (CRACM3) is a unique member of the CRAC family of Ca(2+)-selective channels. In a non-excitable exocytosis model, we found that the extracellular L3 domain and the cytoplasmic C-terminus of CRACM3 interacted in an activity-dependent manner with the N-peptide of syntaxin4, a soluble N-ethylmaleimide-sensitive factor attachment receptor protein. Our biochemical, electrophysiological and single-vesicle studies showed that knockdown of CRACM3 suppressed functional exocytosis by decreasing the open time of the vesicle fusion pore without affecting Ca(2+) influx, the activity-dependent membrane capacitance (Cm) change, and the total number of fusion events. Conversely, overexpressing CRACM3 significantly impaired cell exocytosis independent of Ca(2+), led to an impaired Cm change, decreased the number of fusion events, and prolonged the dwell time of the fusion pore. CRACM3 changes the stability of the vesicle fusion pore in a manner consistent with the altered molecular expression. Our findings imply that CRACM3 plays a greater role in exocytosis than simply acting as a compensatory subunit of a Ca(2+) channel.