Histamine-induced cytosolic calcium mobilization in human bronchial smooth muscle cells

Abstract

Histamine is a well-known mediator of bronchoconstriction. Despite the widespread use of histamine as a tool to study the bronchial smooth muscle function, the precise mechanism by which it causes calcium mobilization in bronchial smooth muscle cells remains unclear. Therefore, the current study aimed to investigate the mechanism of action of histamine in calcium mobilization in cultured human bronchial smooth muscle cells. A series of in vitro calcium imaging experiments have shown that histamine increases intracellular calcium levels in a concentration-dependent manner. The half maximum concentration of cytosolic Ca²⁺ peak was 3.00 ± 0.25 µM of histamine. Histamine was able to mobilize calcium from intracellular stores, even in the absence of extracellular calcium. These histamine-induced calcium elevations were completely blocked by the H₁ receptor antagonist chlorpheniramine (1 µM). Histamine-induced calcium elevation was also completely inhibited by the phospholipase C (PLC) inhibitor U73122 (1 µM) and inositol 1,4,5-trisphosphate (InsP₃) receptor inhibitor caffeine (20 mM). Cyanide p-(trifluoromethoxy)phenylhydrazone (1 µM) and oligomycin (1 µg/ml) effectively attenuated histamine-induced calcium release from intracellular stores. In the presence of histamine, cytosolic calcium elevation induced by reperfusion of 1.28 mM extracellular calcium after the depletion of stores was significantly inhibited by FCCP and oligomycin, unlike in the presence of thapsigargin. Based on the above results, we can conclude that histamine activates the intracellular PLC/InP₃ pathway through the H₁ receptor, which in turn activates the InP₃ receptor present in intracellular stores to mobilize calcium in human bronchial smooth muscle cells. In addition, the mitochondria appear to be involved in the release of calcium from intracellular stores. These results provide insights into the mechanisms underlying histamine-induced calcium mobilization for bronchoconstriction under pathophysiological conditions.

Introduction

Histamine is a well-known mediator of allergic reactions, and it has been extensively studied to examine its role in the regulation of bronchial smooth muscle tone (1, 2). Bronchoconstriction of smooth muscle mediated by specific histamine receptors is recognized as one of the initial events characterizing the physiological actions of histamine on the respiratory system (3). Bronchoconstriction is a common response to respiratory diseases, such as asthma, which is triggered by allergens, irritants, and infections (4, 5). Despite its well-established role in bronchoconstriction, the exact mechanism by which histamine causes calcium (Ca²⁺) mobilization in bronchial smooth muscle cells remains unclear. It is important to understand the subcellular mechanism of histamine-induced Ca²⁺ mobilization because Ca²⁺ plays a crucial role in regulating bronchial smooth muscle tone and contraction.

Calcium is a key intracellular signaling molecule that regulates a variety of cellular processes, such as smooth muscle contraction, secretion, metabolism, and differentiation. Ca²⁺ signals are typically initiated by specific receptors that stimulate phospholipase C (PLC) and, in turn, the formation of inositol 1,4,5-trisphosphate (InsP₃), which induces the release of Ca²⁺ from the sarcoplasmic reticulum through InsP₃ receptors within the intracellular Ca²⁺ store membrane (6,7,8). In human airway smooth muscle, the major physiological contractile stimulus is acetylcholine, which is released from parasympathetic terminals and stimulates PLC activity and mobilizes cytosolic Ca²⁺. Increased intracellular Ca²⁺ levels have been shown to lead to bronchoconstriction, and histamine has also been shown to trigger this increase (9). A deeper understanding of the precise mechanisms of histamine-induced bronchoconstriction is essential for developing more effective treatments for respiratory diseases, such as asthma, chronic obstructive pulmonary disease, and allergic rhinitis. Therefore, in this study, we aimed to elucidate the subcellular signaling pathways involved in histamine-induced Ca²⁺ mobilization using a series of intracellular Ca²⁺ imaging experiments. We herein report that histamine-induced intracellular Ca²⁺ mobilization occurs through H₁ receptors, leading to the activation of PLC, which effectively mobilizes Ca²⁺ from intracellular stores through InsP₃ receptors. Additionally, mitochondria may play a critical role in the histamine-induced release of Ca²⁺ from intracellular stores in human bronchial smooth muscle cells.

Materials and Methods

Culture of human bronchial smooth muscle cells (hBSMCs)

Human bronchial smooth muscle cells (hBSMCs, passage 3) from three male donors (aged 34, 57, and 83 years) were obtained from Lonza (material number CC-2576; Basel, Switzerland). Bronchial smooth muscle cells were cultured in a growth medium containing Dulbecco’s Modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 100 U/ml penicillin, and 100 µg/ml streptomycin. Cells were maintained in a humidified atmosphere at 37°C and 5% CO₂. After reaching confluence, cells were trypsinized, counted, and seeded into new culture dishes. The cells were passaged every 4–5 days until they reached the desired number of passages. To confirm the identity of the bronchial smooth muscle cells, the cells were subjected to immunofluorescence staining using specific markers, such as alpha-smooth muscle actin (α-SMA) and calponin. There were no significant changes in growth rate or Ca²⁺ responsiveness in passages 4–10. To examine the effect of histamine on Ca²⁺ mobilization in hBSMCs, cells were treated with various concentrations of histamine in the presence or absence of specific inhibitors. Fluorescence-based Ca²⁺ imaging techniques were used to measure the changes in intracellular Ca²⁺ levels.

Intracellular Ca²⁺ measurements

To measure intracellular Ca²⁺, cultured cells were loaded with 5 μM Fura-2/AM, a Ca²⁺-sensitive dye, for 30 min at room temperature. Fura-2/AM-loaded cells were continuously perfused with HEPES-buffered physiological saline solution (HEPES-PSS) containing 5.5 mM glucose, 137 mM NaCl, 0.56 mM MgCl₂, 4.7 mM KCl, 1 mM Na₂HPO₄, 10 mM HEPES (pH 7.4), 1.28 mM CaCl₂, and 1% (w/v) bovine serum albumin until ready for use. All materials were obtained from Sigma Chemical Co. (MO, USA). When necessary, Ca²⁺-free solutions were prepared by removing CaCl₂ from HEPES-PSS and adding 0.5 mM ethylene glycol-bis-(β-aminoethyl ether)-N,N,N′,N′-tetraacetic acid (EGTA). The perfusion rate was maintained at 1 ml/min using an electronically controlled perfusion system (Warner Instruments, CT, USA). The intracellular Ca²⁺ levels were monitored using a TILL Photonics imaging system (CA, USA). Fura-2/AM-loaded cells were excited alternately with light at 340 nm and 380 nm. Fluorescence images were captured at 510 nm through a 40× fluorescence objective lens using a Cool-SNAP HQ₂ camera (Photometrics, AZ, USA) attached to an Olympus IX71 inverted microscope (Olympus, Tokyo, Japan). Changes in intracellular Ca²⁺ levels were calculated using the ratio of fluorescence intensities at the two wavelengths, as previously described (10).

Data analysis

The data were analyzed using specialized software to calculate changes in intracellular Ca²⁺ levels in response to histamine or other agents. Fluorescence intensity was corrected for background fluorescence, cell autofluorescence, and dye photobleaching. Time-course data were used to generate concentration-response curves and to determine EC₅₀ values. Changes in cytosolic Ca²⁺ levels are expressed as representative traces in individual cells. The average of all responding cells at each time point was used for the statistical analysis. Summated results are presented as the mean ± S.E.M. Differences were considered statistically significant when the P-value was less than 0.05, as determined by Student’s t-test.

Results

Histamine-induced Ca²⁺ mobilization in cultured hBSMCs

Initial experiments were performed to investigate whether histamine can generate cytosolic Ca²⁺ signals in bronchial smooth muscle cells. As shown in Fig. 1A, changes in intracellular Ca²⁺ concentration were monitored using various concentrations of histamine (0.3–100 µM) for 100 sec in the presence of 1.28 mM extracellular Ca²⁺ (Fig. 1A). The cells did not respond to histamine concentrations <1 µM, but began to respond at histamine concentrations >3 µM. The maximum effect on cytosolic Ca²⁺ elevation was observed when approximately 10 µM histamine was used. The half maximum concentration of cytosolic Ca²⁺ peak was 3.00 ± 0.25 µM of histamine (Fig. 1B). These results suggested that histamine can generate cytosolic Ca²⁺ signals in hBSMCs in a concentration-dependent manner. The following experiment was performed to determine the types of histamine receptors involved in Ca²⁺ mobilization.

Fig. 1.

Histamine-induced cytosolic Ca²⁺ elevation in hBSMCs. (A) Representative intracellular Ca²⁺ elevation following a stepwise increase in histamine concentration (0.3–100 µM) (B) Concentration-response curve of histamine expressed as the % of the maximum Ca²⁺ peak. Each value represents the mean ± S.E.M (n=8). Histamine significantly stimulated cytosolic Ca²⁺ mobilization in a concentration-dependent manner.

Intracellular Ca²⁺ mobilization via H₁ receptor in hBSMCs

To determine the type of histamine receptor that contributes to histamine-induced intracellular Ca²⁺ elevation, cells were perfused with receptor antagonists 200 sec prior to histamine stimulation. In a control experiment, histamine effectively induced an increase in intracellular Ca²⁺ when stimulated 3 times for 100 sec at 300-sec intervals, although the second peak was slightly reduced to 83.33 ± 6.35% of the first peak (Fig. 2A, 2F). Chlorpheniramine, an H₁ receptor antagonist, completely and irreversibly blocked histamine-induced cytosolic Ca²⁺ elevation at 1 µM (Fig. 2B, 2F). On the other hand, for 100 µM cimetidine, 100 µM thioperamide, and 10 µM JNJ7777120, specific H₂, H₃ and H₄ receptor antagonists, respectively, had no effect on the Ca²⁺ mobilization induced by histamine, despite using higher concentrations in comparison to chlorpheniramine (Fig. 2C–2F). As shown in Fig. 2F, when comparing the percent response to the first Ca²⁺ peak for histamine, chlorpheniramine was completely inhibited at 7.81 ± 5.72%. However, cimetidine (82.56 ± 8.21%), thioperamide (71.78 ± 7.47%), and JNJ7777120 (87.10 ± 10.45%) showed no significant difference from the second peak value of 83.33 ± 6.35% observed in the control group (Fig. 2A). These data clearly indicate that histamine can mobilize Ca²⁺ ions for bronchoconstriction through H₁ receptors present on the cell membranes of bronchial smooth muscle cells.

Fig. 2.

Effects of histamine antagonists on cytosolic Ca²⁺ mobilization in hBSMCs. (A) Representative changes of cytosolic Ca²⁺ following continuous histamine stimulation (n=8). Representative cytosolic Ca²⁺ elevation obtained by pretreatment with chlorpheniramine (1 µM, H₁ receptor antagonist, n=8) (B), cimetidine (100 µM, H₂ receptor antagonist, n=6) (C), thioperamide (100 µM, H₃ receptor antagonist, n=5) (D), and JNJ7777120 (10 µM, H₄ receptor antagonist, n=6) (E) in hBSMCs. (F) Pooled data showing the effects of histamine antagonists expressed as the % of the initial Ca²⁺ peak. Each bar represents the mean ± S.E.M. Asterisk indicates the value is significantly different from the second peak value observed in the control group (P<0.05). Chlorpheniramine, an H₁ receptor antagonist, completely blocked histamine-induced cytosolic Ca²⁺ elevation in hBSMCs.

Histamine mobilizes initial Ca²⁺ from intracellular Ca²⁺ stores

To determine whether histamine mobilizes Ca²⁺ from intracellular stores or from the extracellular space, histamine stimulation was performed in the absence of extracellular Ca²⁺. As shown in Fig. 3A and 3B, in the absence of extracellular Ca²⁺, histamine stimulation for the first 100 sec induced Ca²⁺ elevation by 87.33 ± 8.35% of the initial peak value in the presence of extracellular Ca²⁺. However, this response was significantly reduced to 17.48 ± 5.68% upon the second histamine stimulation in the absence of extracellular Ca²⁺. These responses indicate that histamine releases Ca²⁺ from intracellular stores, and that the second response is reduced because intracellular stores are depleted by the first stimulation. Additionally, histamine-induced Ca²⁺ mobilization was restored to 81.49 ± 7.89% after intracellular stores were refilled by reperfusion with 1.28 mM extracellular Ca²⁺. When thapsigargin, an irreversible Ca²⁺ store-depleting agent produced by sarcoendoplasmic reticulum Ca²⁺-ATPase (SERCA) inhibition, was administered in the absence of extracellular Ca²⁺, intracellular Ca²⁺ gradually increased and then slowly decreased to the baseline level. In this state, histamine failed to mobilize cytosolic Ca²⁺ (Fig. 3C). These results suggest that the histamine-induced Ca²⁺ increase is sufficiently mobilized from intracellular Ca²⁺ stores in the absence of extracellular Ca²⁺ in hBSMCs.

Fig. 3.

Histamine-induced Ca²⁺ mobilization initiated from intracellular stores in hBSMCs. (A) Representative changes of cytosolic Ca²⁺ in the presence and absence of extracellular Ca²⁺. (B) Pooled data showing the effect of extracellular Ca²⁺ removal on histamine-induced Ca²⁺ mobilization (n=6). Each bar represents the mean ± S.E.M. Asterisk indicates that the value is significantly different from the first peak value (control) observed in the presence of extracellular Ca²⁺ (P<0.05). (C) Representative changes of cytosolic Ca²⁺ by histamine after the depletion of intracellular Ca²⁺ stores by thapsigargin (TG) treatment in the absence of extracellular Ca²⁺ (n=5). Histamine mobilized sufficient Ca²⁺ from stores, even in the absence of extracellular calcium, but failed to mobilize Ca²⁺ after the intracellular stores were depleted.

Mitochondria inhibits histamine-induced Ca²⁺ release from intracellular stores

Interestingly, as shown in Fig. 3A, histamine-induced Ca²⁺ mobilization was fully restored but store-operated Ca²⁺ entry (SOCE) was not detected upon reperfusion with 1.28 mM extracellular Ca²⁺ after the depletion of intracellular stores. Therefore, we hypothesized that the entered Ca²⁺ would rapidly refill intracellular stores, thereby preventing global Ca²⁺ elevation within the cytosol. To investigate this hypothesis, SOCE was measured in the presence or absence of histamine while perfusing 1.28 mM extracellular Ca²⁺ after depletion of stores by histamine stimulation for 300 s. After the stores were depleted, when 1.28 mM extracellular Ca²⁺ was perfused in the presence of histamine, the increase in cytosolic Ca²⁺ was 97.50 ± 9.12% relative to the first peak induced by histamine (Fig. 4C, 4G), but there was no increase in the absence of histamine (8.51 ± 1.35%, Fig. 4A, 4G). These results indicate that Ca²⁺ entering through store-operated Ca²⁺ channels can not only be rapidly refilled into intracellular stores, but can also be efficiently released into the cytosol by histamine stimulation, which seems to be detected as global cytosolic Ca²⁺ elevation. After the depletion of intracellular stores with thapsigargin (TG), perfusion of 1.28 mM extracellular Ca²⁺ effectively generated SOCE that was 102.35 ± 5.55% of the initial Ca²⁺ peak induced by TG (Fig. 4E, 4G). These results indicate that Ca²⁺ entering the extracellular fluid is not refilled into intracellular stores due to SERCA blockade, but instead diffuses into the cytosol, resulting in an increase in cytosolic Ca²⁺. The following experiments aimed to investigate the role of the mitochondria in intracellular Ca²⁺ mobilization in the presence of carbonyl cyanide p-(trifluoromethoxy)phenylhydrazone (FCCP), a known mitochondrial oxidative phosphorylation uncoupler. FCCP and oligomycin did not affect the cytosolic Ca²⁺ changes induced by reperfusion with 1.28 mM extracellular Ca²⁺ in the absence of histamine (6.48 ± 2.43% of the initial Ca²⁺ peak induced by histamine, Fig. 4B, 4G) or in the presence of thapsigargin (98.24 ± 8.47% of the initial Ca²⁺ peak induced by TG, Fig. 4F, 4G) after depletion of stores. However, as shown in Fig. 4D and 4G, the cytosolic Ca²⁺ elevation induced by reperfusion of 1.28 mM extracellular Ca²⁺ in the presence of histamine after the depletion of stores was significantly inhibited (44.12 ± 7.32% of the initial Ca²⁺ peak induced by histamine), relative to the control value (97.50 ± 9.12% of the initial Ca²⁺ peak induced by histamine). These results suggest that mitochondria are not involved in the process of refilling intracellular Ca²⁺ stores through SOCE after store depletion but are partially involved in the release of Ca²⁺ from the stores into the cytosol.

Fig. 4.

Mitochondria inhibits histamine-induced Ca²⁺ release from intracellular stores in hBSMCs. (A) Representative Ca²⁺ entry in the absence of histamine (n=6). (B) Effects of FCCP and oligomycin on Ca²⁺ entry in the absence of histamine (n=5). (C) Representative Ca²⁺ entry in the presence of histamine (n=6). (D) Effects of FCCP and oligomycin on Ca²⁺ entry in the presence of histamine (n=7). (E) Representative changes of thapsigargin-induced Ca²⁺ entry (n=6). (F) Effects of FCCP and oligomycin on thapsigargin-induced Ca²⁺ entry (n=5). (G) Pooled data showing Ca²⁺ entry expressed as the % of the initial Ca²⁺ peak. Each bar represents the mean ± S.E.M. Open bars represent control experiments. Filled bars represent the effects of FCCP and oligomycin. Asterisk indicates that the value is significantly different from the corresponding control value obtained without FCCP and oligomycin (P<0.05). Mitochondria may participate in histamine-induced Ca²⁺ release from intracellular stores in hBSMCs.

Histamine activates PLC to stimulate InsP₃ receptors on intracellular Ca²⁺ stores

To investigate the pathway involved in histamine-induced Ca²⁺ mobilization from intracellular Ca²⁺ stores, the following experiments were performed using 20 mM caffeine, a drug known to inhibit InsP₃ receptors while simultaneously activating ryanodine receptors (11,12,13). As shown in Fig. 5A and 5D, pretreatment with caffeine alone had no effect on Ca²⁺ mobilization, but caffeine strongly inhibited histamine-induced Ca²⁺ mobilization by 19.56 ± 3.38% of the first peak induced by histamine relative to the second peak value of 83.33 ± 6.35% observed in the control group (Fig. 2A). These results suggest that ryanodine receptors do not play a significant functional role in hBSMCs and that histamine-induced Ca²⁺ mobilization is mediated by InsP₃ receptors. Since InsP₃ receptor activation generally occurs through the activation of the PLC system, we performed histamine stimulation in the presence of pretreatment with a PLC inhibitor. As a result, 1 µM U73122, a PLC inhibitor, completely inhibited the histamine-induced cytosolic Ca²⁺ increase by 3.15 ± 2.32% relative to the second peak value of the control group (Fig. 5B, 5D). The subsequent experiments were performed in the presence of 1 µM FCCP and 1 µg/ml oligomycin. In the presence of FCCP and oligomycin, the effect of histamine was significantly inhibited by 23.05 ± 8.92% relative to the second peak value of the control group (Fig. 5C, 5D). These results suggested that histamine-induced Ca²⁺ mobilization is closely linked to mitochondrial Ca²⁺ buffering. Interestingly, FCCP and oligomycin induced a transient increase in cytosolic Ca²⁺, but this increase was not observed in the depleted state of intracellular Ca²⁺ as shown in Fig. 4B, 4D, and 4F, suggesting that this may be due to Ca²⁺ release from the mitochondria. Taken together, these results suggest that histamine activates PLC to stimulate InsP₃ receptors present on the membrane of intracellular Ca²⁺ stores, thereby transporting Ca²⁺ from the sarcoplasmic reticulum into the cytoplasm in hBSMCs. In addition, mitochondria are thought to participate in histamine-induced Ca²⁺ release.

Fig. 5.

Histamine activates phospholipase C and InsP₃ receptors to mobilize cytosolic Ca²⁺ from sarcoplasmic reticulum in hBSMCs. (A) The effect of caffeine, an inhibitor of InsP₃ receptor, on histamine-induced cytosolic Ca²⁺ elevation (n=7). (B) The effect of U73122, an inhibitor of phospholipase C, on histamine-induced cytosolic Ca²⁺ elevation (n=7). (C) The effect of FCCP, an uncoupling agent of mitochondrial oxidative phosphorylation, with oligomycin on histamine-induced cytosolic Ca²⁺ elevation (n=6). (D) Pooled data showing the effects of PLC, InsP₃ receptor, and mitochondrial inhibitors expressed as the % of the initial Ca²⁺ peak. Each bar represents the mean ± S.E.M. Asterisks indicate the value is significantly different from the second peak value observed in the control group (P<0.05). Histamine can activate PLC to stimulate InsP₃ receptors, thus transporting Ca²⁺ from the intracellular stores into the cytosol, and mitochondria may be involved in this process in hBSMCs.

Discussion

Bronchial smooth muscle is the contractile tissue of the airway in the respiratory system. Its position and arrangement within the airway wall are optimized to produce changes in the airway diameter through active force generation and cell shortening under normal conditions (14). Therefore, understanding the contractile function of the airway is important for elucidating the mechanisms of diseases characterized by airway obstruction, such as asthma and chronic obstructive pulmonary disease (15). Histamine is a well-known inflammatory mediator that directly affects bronchial smooth muscle cells, causing bronchoconstriction and airway hyper-responsiveness in respiratory diseases (16). When bronchial smooth muscle is stimulated by classical contractile agonists such as histamine or acetylcholine, a contractile response is initiated via a PLC-dependent pathway (17, 18). PLC-activated intracellular Ca²⁺ mobilization is a ubiquitous subcellular signal that is closely linked to the initiation of bronchial smooth muscle contraction (19, 20). These results suggest that histamine may initiate airway constriction in allergic diseases such as asthma by potently stimulating Ca²⁺ mobilization in bronchial smooth muscle cells.

Various effects of histamine are mediated by different histamine membrane receptors. Four different subtypes of G protein-coupled histamine receptors (H₁, H₂, H₃, and H₄) have been described, which exert multiple effects, including vasodilation, increased vascular permeability, and modulation of immune cell activity (21). Immunocytochemistry and western blot analysis revealed that bronchial epithelial cells express histamine H₁, H₂, and H₃ receptors, with the pronounced expression of the H₁ receptor (22). The H₁ receptor coupled with Gαq/11-proteins is known to activate PLC and increase intracellular Ca²⁺ levels. Histamine has been reported to induce smooth muscle contraction in the respiratory tract via the H₁ receptor (9). The Gαs-coupled H₂ receptor is highly expressed in immune cells, smooth muscle cells, and brain tissue. Activation of the H₂ receptor is known to have a profound effect on relaxation of the airways, uterus, and blood vessels (23). The H₃ receptor is coupled to Gαi/o and is exclusively expressed in neurons (24). The histamine H₄ receptor is coupled to Gα/io proteins and is expressed in immune cells, intestinal epithelia, lungs, neurons, and cancer cells (2). In the present study, pretreatment with 1 µM chlorpheniramine (H₁ antagonist) significantly attenuated histamine-induced Ca²⁺ mobilization, whereas pretreatment with 100 µM cimetidine (H₂ antagonist), 100 µM thioperamide (H₃ antagonist), and 10 µM JNJ7777120 (H₄ antagonist) did not alter the histamine-induced Ca²⁺ response, even though the concentrations were used were higher than those of chlorpheniramine. Similar experimental results involving Ca²⁺ mobilization through the H₁ receptor have been reported in human ciliary muscle cells (25) and human lung fibroblast cells (26). In vivo studies have shown that the H₁ receptor blocker chlorpheniramine suppresses bronchial constriction in response to inhaled histamine in non-asthmatic humans (27). From the above results, we suggest that the H₁ receptor is predominantly responsible for mediating Ca²⁺ mobilization in hBSMCs, which provides crucial insight into the mechanisms underlying histamine-induced bronchoconstriction.

Further investigation of the source of histamine-induced Ca²⁺ mobilization revealed that it is initiated by release from intracellular stores rather than by influx from the extracellular fluid. Histamine sufficiently mobilized Ca²⁺ even in Ca²⁺-free medium, suggesting that it mobilizes Ca²⁺ from intracellular stores. Thapsigargin, a drug known to discharge Ca²⁺ from InsP₃-sensitive stores by inhibiting SERCA, was used to deplete intracellular Ca²⁺ stores (28). The finding that histamine failed to mobilize Ca²⁺ after the depletion of intracellular stores by treatment with thapsigargin further supports the hypothesis that histamine releases Ca²⁺ from intracellular stores. To explore the signaling pathway involved in histamine-induced Ca²⁺ release, we focused on the PLC-InsP₃ signaling axis, which is a well-known pathway for mobilizing Ca²⁺ from intracellular stores. In mammalian cells, numerous membrane-bound receptors are coupled to PLC via a regulatory G-protein, which produces two secondary messengers, InsP₃ and diacylglycerol. In smooth muscle, InsP₃ triggers the release of Ca²⁺ from intracellular stores, thus increasing intracellular free Ca²⁺, which activates Ca²⁺-calmodulin-dependent myosin light-chain kinases (29). Subsequent phosphorylation of the myosin light chain activates actin-myosin ATPase, resulting in the initial phase of muscle contraction. In our study, pretreatment with U73122, a PLC inhibitor, markedly reduced the histamine-induced Ca²⁺ mobilization. This finding implicates PLC activation as a critical step in the signaling cascade initiated by histamine. We also confirmed the involvement of InsP₃ receptors using caffeine, an InsP₃ receptor inhibitor, which significantly attenuated the Ca²⁺ response. Caffeine is known to sensitize ryanodine receptors (RyRs) in the sarcoendoplasmic reticulum membrane to release Ca²⁺, but it is also known to inhibit InsP₃ receptor-mediated Ca²⁺ release from intracellular Ca²⁺ stores (11,12,13). Although pretreatment with caffeine alone had no effect on basal Ca²⁺ levels, it markedly attenuated the histamine-induced Ca²⁺ mobilization. These results suggest that RyRs do not play a significant role in the mobilization of Ca²⁺ in hBSMCs, whereas histamine-induced Ca²⁺ mobilization appears to be mediated through InsP₃ receptors. These results indicate that InsP₃ receptors play a critical role in the histamine-induced mobilization of Ca²⁺. In our study, when stores were depleted by histamine perfusion in the absence of extracellular Ca²⁺ and then perfused with 1.28 mM extracellular Ca²⁺, there was a significant increase in cytosolic Ca²⁺ in the presence of histamine; however, no increase was observed in the absence of histamine. These results can be interpreted as histamine-promoting Ca²⁺ influx through receptor-operated Ca²⁺ (ROC) channels (30). However, the histamine-induced increase in calcium was almost completely suppressed by caffeine. It is reasonable to assume that Ca²⁺ influx through store-operated Ca²⁺ (SOC) channels can be rapidly restored to intracellular stores and efficiently released into the cytosol by histamine stimulation, resulting in an overall increase in cytosolic Ca²⁺. However, our results alone do not completely rule out the possibility that histamine can induce calcium influx through ROC channels. Transient receptor potential canonical (TRPC) channels are known to be associated with the PLC signaling pathway (31), are expressed in hBSMCs (32), and can act as ROC channels (20). Further studies are needed to elucidate the relationship between histamine and TRPC channels in hBSMCs.

It is well known that the sarcoplasmic reticulum and mitochondria are two major intracellular Ca²⁺-storing organelles that exhibit close structural and functional interaction with each other (33). The pattern and rate of mitochondrial Ca²⁺ mobilization can vary depending on the cell type, the location of mitochondria relative to the sarcoplasmic reticulum and plasma membrane, and the expression of InsP₃ receptor subtypes (34, 35). Thus, mitochondria can perform their role by buffering the released Ca²⁺ from the sarcoplasmic reticulum (36) or calcium entering from outside (37). Our data demonstrated that histamine-induced Ca²⁺ mobilization was inhibited by FCCP and oligomycin, indicating that mitochondria participate in Ca²⁺ mobilization. In fact, FCCP acts by dissipating ΔΨm and impairing mitochondrial Ca²⁺ uptake through a uniporter driven by an electrical gradient. It has also been reported that FCCP itself may induce a transient increase in cytosolic Ca²⁺ because it induces ΔΨm loss in the mitochondria, thereby triggering the release of Ca²⁺ from the organelle (38). However, FCCP also impairs the ATP-generation process, resulting in massive ATP consumption (39). Therefore, we used FCCP with oligomycin, which inhibits ATP consumption and minimizes the deleterious effects induced by FCCP itself. In our experiments, FCCP and oligomycin significantly inhibited the cytosolic Ca²⁺ elevation induced by reperfusion with 1.28 mM calcium after the depletion of intracellular stores in the presence of histamine, but had no effect on the calcium elevation induced by thapsigargin. Although the results of this study alone cannot provide a detailed mechanism of mitochondrial Ca²⁺ regulation, mitochondria seem to be partially involved in the release of Ca²⁺ from stores rather than the refilling of Ca²⁺ into stores induced by histamine in hBSMC. These findings may increase our understanding of the subcellular mechanisms underlying histamine-induced Ca²⁺ mobilization for bronchoconstriction, and provide a basis for the development of targeted therapies for respiratory diseases.

Conclusion

Histamine acts through the H₁ receptor, a G protein-coupled receptor, to activate PLC. This activation results in efficient mobilization of Ca²⁺ from intracellular stores through InsP₃ receptors. Mitochondria are thought to be more closely involved in histamine-induced Ca²⁺ release from intracellular stores. A schematic illustration of the study findings is shown in Fig. 6.

Fig. 6.

A schematic illustration of the study findings. The subcellular mechanism of histamine-induced cytosolic calcium mobilization in human bronchial smooth muscle cells. PLC: phospholipase C; InsP₃: inositol 1,4,5-trisphosphate; PIP₂: phosphatidylinositol 4,5-bisphosphate; DAG: diacylglycerol; SOC: store-operated calcium channel; SERCA: sarcoendoplasmic reticulum calcium ATPase; MCU: mitochondrial calcium uniporter; mPTP: mitochondrial permeability transition pore.

Data Availability

The data are available from the corresponding author on reasonable request.

Author Contributions

Investigation and Design: MYL, SHK, HSP, Methodology: KJC, WYJ, Data analysis: KJC, WYJ, SHK, Writing-original draft: KJC, HSP, Writing-review & editing: KJC, WYJ, MYL, SHK, HSP.

Funding

This study was supported by the 2022 Konyang University Myunggok Research Fund.

Conflicts of Interest

The authors declare no conflicts of interest in association with the present study.