Olopatadine Inhibits Exocytosis in Rat Peritoneal Mast Cells by Counteracting Membrane Surface Deformation

Abstract

Backgroud/Aims: Besides its anti-allergic properties as a histamine receptor antagonist, olopatadine stabilizes mast cells by inhibiting the release of chemokines. Since olopatadine bears amphiphilic features and is preferentially partitioned into the lipid bilayers of the plasma membrane, it would induce some morphological changes in mast cells and thus affect the process of exocytosis. Methods: Employing the standard patch-clamp whole-cell recording technique, we examined the effects of olopatadine and other anti-allergic drugs on the membrane capacitance (Cm) in rat peritoneal mast cells during exocytosis. Using confocal imaging of a water-soluble fluorescent dye, lucifer yellow, we also examined their effects on the deformation of the plasma membrane. Results: Low concentrations of olopatadine (1 or 10 µM) did not significantly affect the GTP-γ-S-induced increase in the Cm. However, 100 µM and 1 mM olopatadine almost totally suppressed the increase in the Cm. Additionally, these doses completely washed out the trapping of the dye on the cell surface, indicating that olopatadine counteracted the membrane surface deformation induced by exocytosis. As shown by electron microscopy, olopatadine generated inward membrane bending in mast cells. Conclusion: This study provides electrophysiological evidence for the first time that olopatadine dose-dependently inhibits the process of exocytosis in rat peritoneal mast cells. Such mast cell stabilizing properties of olopatadine may be attributed to its counteracting effects on the plasma membrane deformation in degranulating mast cells.

Clarithromycin Dose-Dependently Stabilizes Rat Peritoneal Mast Cells

“…The protocol for animal use was approved by the Animal Care and Use Committee of the Tohoku University Graduate School of Medicine. Peritoneal mast cells were obtained by washing the peritoneal cavity with a standard external (bathing) solution containing: 145 m M NaCl, 4.0 m M KCl, 1.0 m M CaCl 2 , 2.0 m M MgCl 2 , 5.0 m M HEPES, and 0.01% bovine serum albumin (pH 7.2 adjusted with NaOH) as described in our previous studies [17,18,19]. They were maintained at room temperature (22-24°C) for use within 8 h. The mast cell suspension was scattered in a chamber placed on the head stage of an inverted microscope (Nikon, Tokyo, Japan).…”

“…They were maintained at room temperature (22-24°C) for use within 8 h. The mast cell suspension was scattered in a chamber placed on the head stage of an inverted microscope (Nikon, Tokyo, Japan). Single mast cells were easily distinguishable from other cells by their intracellular inclusion of secretory granules [17,18,19]. …”

“…Erythromycin and roxithromycin (Sigma-Aldrich Co., St. Louis, Mo., USA) and azithromycin (LKT Laboratories Inc., St. Paul, Minn., USA) were also dissolved in the external solution at a final concentration of 100 μ M . After incubation of mast cells in these solutions or a solution containing no drug for 10 min, exocytosis was externally induced by compound 48/80 (final concentration 10 μg/ml; Sigma-Aldrich) [17,18,19]. Bright-field images were acquired from randomly selected 0.1-mm 2 fields of view (10 views from 6 of each condition) as described in our previous studies [17,18,19].…”

“…However, due to the limitations of the methods used in molecular biology [16], the effects of these drugs on the exocytotic process could not directly been examined in mast cells. Recently, by monitoring the changes in whole-cell membrane capacitance (Cm) in mast cells, we provided for the first time electrophysiological evidence that antiallergic drugs, such as olopatadine, ketotifen, and tranilast, inhibit the process of exocytosis and thus have mast cell-stabilizing properties [17,18]. In the present study, employing this standard patch clamp whole-cell recording technique in rat peritoneal mast cells, we examined the effects of macrolide antibiotics, such as clarithromycin, erythromycin, roxithromycin, and azithromycin, on changes in Cm in order to determine the mechanism by which they stabilize mast cells.…”

Antihistamines in Atopic Dermatitis Therapy

“…The study provided electrophysiological evidence for the first time that olopatadine dose-dependently inhibited the process of exocytosis in rat peritoneal mast cells. Such mast cell stabilizing properties of olopatadine might be attributed to its counteracting effects on the plasma membrane deformation in degranulating mast cells [17].…”