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

Olopatadine is one of the most potent anti-allergic drugs that are widely used in the treatment of allergic disorders, such as seasonal pollinosis, chronic rhinitis, urticaria and allergic conjunctivitis. Olopatadine was originally developed as a second-generation antihistamine drug [1], which was to thought to exert its anti-allergic effects primarily by antagonizing histamine H1 receptors. Additionally, studies using human conjunctival mast cells further demonstrated that this drug also exerts inhibitory effects on the release of chemokines [2,3,4]. Concerning the mechanisms by which drugs exert such mast cell stabilizing properties, previous studies indicated that the drug-induced changes in the intracellular Ca²⁺ concentration ([Ca²⁺]_i) are primarily responsible [5,6]. However, later studies also revealed the involvement of G proteins [7] or mechanical stimuli to the plasma membranes [8] in modulating the process of exocytosis in mast cells. In our recent study, chlorpromazine, a positively charged membrane amphipath, changed the plasma membrane curvature in rat peritoneal mast cells [9], and the induced mechanical stretch of the membranes significantly inhibited the process of exocytosis. Since olopatadine, a relatively cationic and lipophilic antihistamine, shares common pharmacological features with those of the membrane amphipaths [10], this drug should also induce some morphological changes in mast cell plasma membranes, and thus may affect their exocytotic process. To test this, employing the standard patch-clamp whole-cell recording technique in rat peritoneal mast cells [9,11,12], we examined the effects of olopatadine on the membrane capacitance. By confocal imaging of a hydrophilic fluorescent dye that is trapped on the cell surface [9,13], we also examined the effects of this drug on the plasma membrane deformation. Here, using rat peritoneal mast cells, we provide electrophysiological evidence for the first time that olopatadine dose-dependently inhibits the process of exocytosis. We also show that such mast cell stabilizing properties of olopatadine can be ascribed to its counteracting effects on the plasma membrane deformation in degranulating mast cells.

Male Wistar rats more than 25 weeks old, supplied by Japan SLC Inc. (Shizuoka, Japan), were deeply anaesthetized with isoflurane and then killed by cervical dislocation. The protocol for animal use was approved by the Animal Care and Use Committee of Tohoku University Graduate School of Medicine. Peritoneal mast cells were obtained by washing the peritoneal cavity with standard external (bathing) solution containing (in mM): NaCl, 145; KCl, 4.0; CaCl₂, 1.0; MgCl₂, 2.0; HEPES, 5.0; bovine serum albumin, 0.01 % (pH 7.2 adjusted with NaOH) as described in our previous study [9]. They were maintained at room temperature (22-24ºC) for use within 8 hours. The mast cell suspension was scattered in a chamber placed on the headstage of an inverted microscope (Nikon, Tokyo, Japan). Single mast cells were easily distinguishable from other cells by their intracellular inclusion of secretory granules [9].

Olopatadine hydrochloride, kindly provided by Kyowa Hakko Kirin Co., Ltd. (Tokyo, Japan), was separately dissolved in the external solution at final concentrations of 1,10,100 µM and 1 mM, respectively Fexofenadine hydrochloride, purchased from LKT Laboratories, Inc. (St. Paul, Min., USA), and loratadine, from Tokyo Chemical Industry Co., Ltd. (Tokyo, Japan), were also separately dissolved in the external solution at the final concentrations of 100 µM. After mast cells were incubated in these solutions or a solution containing no drug, exocytosis was externally induced by compound 48/80 (Sigma-Aldrich, St. Louis, MO, USA; final concentration 10 µg/ml) [9]. Bright-field images were acquired from randomly selected 0.1-mm² fields of view (10 views from 6 of each condition), as described in our previous studies [9,14]. Degranulated mast cells were defined as cells associated with ≥ 8 granules outside the cell membrane, as described previously [15]. These mast cells were then counted and their ratio to all mast cells was calculated.

We conducted standard whole-cell patch-clamp recordings using an EPC-9 patch-clamp amplifier system (HEKA Electronics, Lambrecht, Germany) as described previously [9,16]. The patch pipette resistance was 4-6 MΩ when filled with internal (patch pipette) solution containing (in mM): K-glutamate, 145; MgCl₂, 2.0; Hepes, 5.0 (pH 7.2 adjusted with KOH). One-hundred µM guanosine 5'-o-(3-thiotriphosphate) (GTP-γ-S) (EMD Bioscience Inc., La Jolla, CA, USA) was additionally included in the internal solution to induce exocytosis in the mast cells [9,11,12]. After a giga-seal formation on mast cells scattered in the external solutions containing no drug, the different concentrations of olopatadine (1, 10, 100 µM and 1 mM), 100 µM fexofenadine or loratadine, we applied suction briefly to the pipette to rupture the patch membrane and to dialyze the cells with GTP-γ-S. The series resistance of the whole-cell recordings was maintained below 10 MΩ during the experiments. To measure the membrane capacitance of the mast cells, we employed a sine plus DC protocol using the Lock-in amplifier of an EPC-9 Pulse program. An 800-Hz sinusoidal command voltage was superimposed on the holding potential of -80 mV. The membrane capacitance (Cm), as well as membrane conductance (Gm) and series conductance (Gs), was continuously recorded during the whole-cell recording configuration. All experiments were carried out at room temperature.

After the mast cells were incubated in the external solutions containing no drug, the different concentrations of olopatadine (1, 10, 100 µM and 1 mM), 100 µM fexofenadine or loratadine, exocytosis was externally induced by compound 48/80 (10 µg/ml). Then, the cells were incubated for 5 min at room temperature in the external solution containing a hydrophilic fluorescent dye, lucifer yellow [9,13] (Wako, Osaka, Japan; final concentration 10 µM), and were washed 2 to 3 times thoroughly with dye-free external solutions. Fluorescent images were taken using a TE 2000-E Nikon Eclipse confocal microscope (Nikon, Tokyo, Japan).

The mast cells, incubated in the external solutions containing no drug or 1mM olopatadine for 10 min were fixed with 4% paraformaldeyde and 3% glutaraldehyde in 0.1 M cacodylate buffer, pH 7.4 for 2 h at room temperature. After being trimmed into small pieces, the above specimen containing the cells was postfixed in 1% osmium tetroxide for 1 h at 4°C, rinsed in PBS, dehydrated in a graded series of alcohol and propylene oxide, and finally embedded in epoxy resin. Ultrathin (80 nm) sections were prepared on an ultramicrotome (Ultracut R, Leica, Heerbrugg, Switzerland) with a diamond knife and then were stained with uranyl acetate and lead citrate, and viewed using an electron microscope (JEM-1200, JOEL, Tokyo, Japan).

Data were analyzed using PulseFit software (HEKA Electronics, Lambrecht, Germany) and Microsoft Excel (Microsoft Corporation, Redmond, Wash., USA) and reported as means ± SEM. Statistical significance was assessed by two-way ANOVA followed by Dunnett's or Student's t test. A value of p < 0.05 was considered significant.

In the absence of olopatadine, compound 48/80 (10 µg/ml) induced degranulation in 80.5 ± 1.1 % of the mast cells observed (n=6). In human, the physiological plasma concentration of olopatadine ranges from 1 to 10 µM when orally administered for the treatment of systemic allergic reactions [17,18,19]. On the other hand, when topically administered for allergic conjunctivitis, doses as high as 559 to 653 µM olopatadine were required to effectively elicit its inhibitory propeties on histamine release from human conjunctival mast cells [2,3,4]. Since doses as high as 10 mM did not cause any cytotoxic effects in a previous in vitro study [20], we tried doses starting from 1 µM up to 1 mM in the present study. Relatively lower concentrations of olopatadine, such as 1 and 10 µM, did not significantly affect the ratio of degranulating mast cells (Fig. 1A). However, doses as high as 100 µM and 1 mM markedly decreased the numbers of degranulating mast cells (100 µM, 17.1 ± 1.8 %; 1 mM, 10.6 ± 0.55 %; n=6, P<0.05; Fig. 1A). From our results, olopatadine, which is also known as a mast cell stabilizer [21], actually inhibited the degranulation from rat peritoneal mast cells in a concentration-dependent manner. These results were consistent with the previous findings demonstrated in human conjunctival mast cells that olopatadine dose-dependently inhibited the release of histamine [2,3,4].

Besides olopatadine, some anti-allergic drugs are also known to exert mast cell stabilizing properties in addition to their antihistaminic properties [22,23]. Among them, fexofenadine exerts relatively lower [23,24], whereas loratadine exerts higher mast cell stabilizing properties than olopatadine does [22,25]. Therefore, we also examined the effects of these drugs on the degranulation of mast cells (Fig. 1B). As expected, fexofenadine did not significantly affect the ratio of degranulating mast cells (76.1 ± 0.85 % vs. external solution alone: 81.4 ± 1.4 %, n=6, Fig. 1B), but loratadine markedly decreased the number of degranulating mast cells (11.3 ± 0.26 %, n=6, P<0.05).

In megakaryocytes or thymus lymphocytes, microscopic changes in the cell surface area were induced by membrane invagination during thrombopoiesis [26] or by the accumulation of lipophilic drugs into the lipid bilayers [27]. In our previous studies, such microscopic changes in these cells were most precisely monitored by measurement of the whole-cell membrane capacitance (Cm) [16,28,29,30,31]. In mast cells, it was also established that the increase in the Cm well indicates the degranulating process during exocytosis [9,32,33]. Therefore, in the present study, to determine the effects of olopatadine on mast cell exocytosis, we incubated the cells in olopatadine-containing external solutions and examined the changes in the Cm (Fig. 2). The effects of different concentrations of olopatadine on the Cm, Gs and Gm are shown in the figure and the numerical changes in the parameters are summarized in Table 1. Consistent with previous findings [9,11,12], internal application of GTP-γ-S to mast cells incubated in the external solution induced a more than 3-fold increase in the Cm (from 9.78 ± 1.26 to 30.5 ± 4.15 pF, n=6, P<0.05; Table 1), which occurred within 30 to 100 s after the establishment of the whole-cell recording configuration (Fig. 2A).

Incubation of mast cells with relatively lower concentrations of olopatadine (1 or 10 µM) did not significantly affect this increase in the Cm (Fig. 2B, Table 1). However, incubation with 100 µM or 1 mM olopatadine almost completely suppressed the incremental effect of GTP-γ-S on the Cm (Fig. 2C and D, Table 1). As previously shown in degranulating mast cells [9,11,33], the Gs tended to decrease after the establishment of the whole-cell recording configuration (Fig. 2A-D), reflecting the gradual increase in the series resistance between the pipette electrodes and the cells interior. These results provide electrophysiological evidence for the first time that olopatadine inhibits the process of exocytosis in a concentration-dependent manner, which strongly supported our findings obtained from Fig. 1.

We also examined the effects of loratadine and fexofenadine on the Cm (Table 1), since these drugs inhibited or failed to inhibit the degranulation of mast cells (Fig. 1B). In mast cells incubated with 100 µM fexofenadine, the Cm also significantly increased (from 9.37 ± 0.61 to 24.4 ± 1.79 pF, n=6, P<0.05; Table 1). However, in mast cells incubated with 100 µM loratadine, such increase in the Cm was almost totally suppressed (from 9.36 ± 1.32 to 11.8 ± 1.61 pF, n=6; Table 1).

In our recent study, amphiphilic drugs, such as salicylate and chlorpromazine, changed the plasma membrane curvature in rat peritoneal mast cells [9], which modulated the process of exocytosis. In the present study, since olopatadine inhibited the exocytotic process in mast cells (Fig. 1, 2), the drug-induced morphological changes in the cells would also affect this process. Therefore, after incubating mast cells in fexofenadine, loratadine or olopatadine-containing solutions, we externally induced exocytosis in the cells and examined the effects of this drug on the plasma membranes (Fig. 3A). By the external addition of compound 48/80, mast cells incubated in the external solution showed more wrinkles on their cell surface and released secretory granules as a result of exocytosis (Fig. 3 Ab vs. a). Mast cells incubated in 100 µM fexofenadine- (Fig. 3 Ac) or 1 and 10 µM olopatadine-containing solutions (Fig. 3 Ae, f) were not different from those incubated in the external solution alone (Fig. 3 Ab). However, in mast cells incubated in 100 µM loratadine- (Fig. 3Ad) or 100 µM and 1 mM olopatadine-containing solutions (Fig. 3Ag,h), the findings suggestive of exocytosis, such as the wrinkles on their cell surface and the release of secretory granules, were totally absent.

To determine whether the wrinkles observed in the degranulating mast cells (Fig. Ab,c,e,f) represented membrane surface deformation as a result of exocytosis, we then used lucifer yellow (Fig. 4B), a water-soluble fluorescent dye, that is retained in the invaginated folds created in the plasma membranes [13]. In mast cells that were treated with external solution alone (Fig. 3Bb), fexofenadine (Fig. 3Bc) or relatively lower concentrations of olopatadine solution (1 and 10 µM; Fig. 3Be and f), lucifer yellow was trapped partially on the cell surface area. Since the dye, which is normally membrane-impermeable [34], was completely absent in the cells before exocytosis was induced (Fig. 3Ba), the staining indicated its retention in the opened pores formed by exocytosis [35]. However, after incubating mast cells in loratadine- (Fig. 3Bd) or 100 µM and 1 mM olopatadine-containing solutions (Fig. 3Bg,h), the dye was completely washed out. These results indicated that olopatadine inhibited the formation of the invaginated folds, suggesting that this drug counteracted the membrane surface deformation induced by exocytosis.

To examine the direct effect of olopatadine on the membrane surface deformation in mast cells (Fig. 3B), we took the electron microscopic images of mast cells after incubating them in the external solutions containing no drug or 1 mM olopatadine (Fig. 4). Mast cells incubated in the external solution alone showed the flat surface of the cellular membranes which consisted of the lipid bilayers (Fig. 4A). However, in mast cells incubated in the olopatadine-containing external solution, there were some inward invaginations in the cellular membranes (Fig. 4B, arrows), suggesting that the drug induced the inward membrane bending in mast cells.

In previous in vitro studies using human conjunctival mast cells, olopatadine was demonstrated to exert inhibitory effects on the release of histamine [2,3,4], in addition to its primary properties as a histamine receptor antagonist [1]. Mast cells that are derived from mucosal tissues, including conjunctiva, are known to produce larger amounts of chemical mediators than those from serosal tissues, such as the peritoneal cavity [36]. Therefore, in previous studies, mast cells derived from human conjunctiva required extremely high doses of olopatadine to effectively elicit their inhibitory properties on exocytosis [2,3,4]. In the present study, using rat peritoneal mast cells, we provide electrophysiological evidence for the first time for the mast cell-stabilizing properties of olopatadine (Fig. 2). Additionally, from our results, mast cells derived from peritoneal serosa required relatively lower doses of olopatadine than those derived from conjunctival mucosa [2,3,4]. According to previous in vivo studies, serosal-type mast cells are frequently involved in the development of fibrosis in many organs, including kidney, skin, lung and liver [37,38,39]. From our results, since olopatadine exerted inhibitory effects on exocytosis more effectively in mast cells derived from serosal tissues, this drug may also be used as an anti-fibrotic agent for mast cell-triggered organ sclerosis.

In the present study, as we recently demonstrated with chlorpromazine [9], olopatadine prevented plasma membrane deformation in rat peritoneal mast cells (Fig. 3). In many types of secretory cells, the exocytotic process can be modulated by mechanical stimuli, such as changes in the membrane tension, shear stress, hydrostatic pressure and compression [8]. Therefore, the counteracting effect of olopatadine on the plasma membrane deformation in degranulating mast cells was thought to contribute to its inhibitory effect on the exocytotic process. Structurally, since olopatadine has both a carboxylic functional group and a tertiary amino group, this drug exists in different ionic forms depending upon the pH of the solution [19]. Since the pKa of the groups are 4.18 and 9.79, respectively, olopatadine usually exists as a zwitterion in the pH under a physiological condition [40]. However, since olopatadine was conjugated with hydrochloride in the present study, the external solution containing higher concentrations of olopatadine tended to be more acidic. Therefore, as is usually the case with the other anti-allergic reagents [10], olopatadine was thought to be relatively cationic under the condition applied in the present study. Additionally, in previous studies using erythrocyte membranes, lipophilic olopatadine actually interacted directly with lipid layers of the plasma membranes [20,41]. Therefore, this drug was thought to share similar pharmacological features with those of membrane amphipaths, and thus easily partitions into cellular membranes [20]. As previously demonstrated with chlorpromazine in both mast cells [9] and erythrocytes [42], a positively charged membrane amphipath, such as cationic olopatadine, would preferentially partition into the inner leaflet of the plasma membranes and generate inward membrane bending (Fig. 5A), which was actually demonstrated by electron microscopy in the present study (Fig. 4). On the “curved regions” of the membranes, where secretory granules started to dock or fuse into the cellular membranes (Fig. 5B top), such induced inward stretch would mechanically counteract the further docking or fusion forces of the secretory granules, as we recently demonstrated with membrane amphipaths [9]. Consequently, this closes the opened pores of the mast cell membrane surface and thereby inhibits the further process of exocytosis (Fig. 5B bottom). In contrast, on the “flat part” of the membranes, where the secretory granules are about to dock or fuse (Fig. 5B top), the inward stretch of the membranes may rather facilitate the soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE)-induced fusion process of the granules (Fig. 5B bottom), as previously demonstrated at neuronal synapses [43]. However, since anti-allergic drugs, including olopatadine, are known to exert membrane stabilizing effects by decreasing the fluidity of the membranes [44], it would halt the further progression of exocytosis at this stage (Fig. 5B bottom).

According to previous patch-clamp studies, a rise in the [Ca²⁺]_i was one of the main triggers of exocytosis in mast cells [7,11]. Recently, Cruse et al. further demonstrated in human lung mast cells that the rise in the [Ca²⁺]_i was dependent on the activity of Ca²⁺ activated K⁺ channels (Kc_a 3.1), which provide the driving force for Ca²⁺ influx through store-operated calcium channels (SOCs) [45]. Previous studies revealed that the activity of SOCs, such as canonical transient receptor potential 1 (TRPC1), and Kc_a -channels was mechanically modified by the stretch of the plasma membranes [46,47]. From our results, olopatadine actually generated inward membrane stretch in mast cells (Fig. 4), and thus counteracted the exocytosis-induced deformation of the membrane surface (Fig. 5). Therefore, this drug was thought to influence the activity of these channels through such mechanical stimuli to the membranes. As a result, such induced changes in the [Ca²⁺]_i may also have additional effects on the olopatadine-induced inhibition of exocytosis.

In summary, this study provided 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 could be ascribed to its counteracting effects on the plasma membrane deformation in degranulating mast cells.

We thank Ms. Chika Tazawa and her colleagues at Biomedical Research Core of Tohoku University Graduate School of Medicine for their technical support.

The authors declare no conflicts of interest.