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.

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.

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.