INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

ASTHMA IS A COMMON DISORDER associated with airway inflammation, bronchial hyperresponsiveness, and bronchospasm. Airway obstruction because of bronchial constriction and airway hyperreactivity is a major cause for acute respiratory incapacity in patients with asthma. Airway smooth muscle can also undergo hypertrophy, which contributes to the development of persistent airway obstruction and increased nonspecific airway hyperresponsiveness in chronic severe asthma (6, 13, 22, 23).

Intracellular Ca²⁺ is a critical signal transduction element in regulating muscle contraction, cell proliferation, and gene expression (4, 9, 26-29). A rise in cytosolic free Ca²⁺ concentration ([Ca²⁺]_cyt) in bronchial smooth muscle cells (BSMC) would trigger bronchial constriction and cause bronchospasm (24), whereas increases in [Ca²⁺]_cyt and nuclear Ca²⁺ concentration ([Ca²⁺]) would stimulate BSMC growth and cause bronchial wall thickening (4, 23, 25). Many bronchoconstrictors, growth factors, cytokines, and inflammatory mediators increase [Ca²⁺]_cyt in BSMC (2). The agonist-induced increases in [Ca²⁺]_cyt usually consist of an initial Ca²⁺ release from intracellular Ca²⁺ stores [mainly sarcoplasmic reticulum (SR)] followed by a sustained Ca²⁺ influx through plasmalemmal Ca²⁺ channels (2-5, 16, 26-29).

In airway smooth muscle cells, there are at least three classes of Ca²⁺ channels in the plasma membrane (18): voltage-dependent Ca²⁺ channels, receptor-operated Ca²⁺ channels, and store-operated Ca²⁺ channels (SOC) (21, 24, 30). By governing Ca²⁺ influx via voltage-dependent Ca²⁺ channels, membrane potential plays a critical role in regulating [Ca²⁺]_cyt (8). Agonist- or mitogen-induced Ca²⁺ influx is mainly caused by receptor-mediated activation of receptor-operated Ca²⁺ channels and store depletion-mediated opening of SOC (2, 21, 28).

Depletion of the SR Ca²⁺ triggers capacitative Ca²⁺ entry (CCE), a mechanism involved in maintaining sustained Ca²⁺ influx and refilling Ca²⁺ in the SR (3-5, 24, 28). The transient receptor potential channel (TRPC) genes have recently been demonstrated essential for agonist-activated CCE. Expression of TRPC genes in mammalian cells results in the formation of Ca²⁺-permeable channels that are activated by Ca²⁺ store depletion. These findings suggest that the TRPC-encoded proteins are the putative SOC responsible for CCE (5, 31, 32).

When BSMCs are exposed to cytokines, growth factors, and inflammatory mediators during acute and chronic allergic responses in patients with asthma, a critical signal transduction pathway on activation of respective receptors is the rise in [Ca²⁺]_cyt due to Ca²⁺ influx through sarcolemmal Ca²⁺ channels and Ca²⁺ release from intracellular Ca²⁺ stores. CCE, potentially through TRPC-related SOC, is critical in maintaining the sustained rise in [Ca²⁺]_cyt and refilling of Ca²⁺ into the SR. Thus the function of SOC and amplitude of CCE may contribute to regulating bronchial constriction and BSMC growth by modulating [Ca²⁺]_cyt and intracellularly stored [Ca²⁺] in the SR ([Ca²⁺]_SR). The intracellular Ca²⁺ may serve as a shared signal transduction element that leads to the bronchial hyperresponsiveness and bronchospasm as well as bronchial wall thickening in asthma. By using combined approaches of patch clamp, molecular biology, contractility, and fluorescence microscopy, this study was designed to test the hypothesis that CCE through TRPC-encoded SOC plays an important role in agonist-mediated bronchial constriction and that upregulation of TRPC gene expression is essential for BSMC proliferation.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Cell preparation and culture. Primary cultures of rat BSMC were prepared from Sprague-Dawley rats. Divisions 6-8 of bronchial branches of the right tracheas were isolated, the connective tissue and adventitia were carefully stripped off, and the epithelium was removed by gently scratching the intimal surface. The remaining smooth muscle was digested with 1.5 mg/ml collagenase and 0.5 mg/ml elastase (Sigma Chemical, St. Louis, MO) for 45 min at 37°C. The cells were plated onto 25-mm coverslips in Petri dishes (for electrophysiological and fluorescent experiments) or 10-cm Petri dishes (for molecular biological experiments) and cultured in DMEM containing 10% fetal bovine serum (FBS) in a 37°C, 5% CO₂, humidified incubator.

Isometric contraction measurement in isolated bronchial rings. The isolated bronchioles from rats were cut into 1-mm-long rings. Two stainless steel hooks (0.1-mm diameter) were inserted through the lumen of the bronchial rings. One hook was mounted into a perfusion chamber (0.75-ml volume), and the other hook was connected to an isometric force transducer (Harvard Apparatus). Isometric tension was continuously monitored and recorded on an IBM-compatible PC using DATAQ data acquisition software (DATAQ Instruments). Rings were equilibrated for ~60 min at resting tension (600-650 mg) before experimentation. Isolated bronchial rings were superfused with modified Krebs solution (at 37°C), which contained (in mM) 138 NaCl, 4.7 KCl, 1.2 MgSO₄, 1.2 NaH₂PO₄, 5 HEPES, 1.8 CaCl₂, and 10 glucose (pH 7.4). In Ca²⁺-free solution, CaCl₂ was replaced by equimolar MgCl₂, and 0.1 mM EGTA was added to chelate residual Ca²⁺. Acetylcholine (ACh) and atropine (Sigma Chemical) were dissolved in the perfusate on the day of use. The pH values of all solutions were measured after addition of drugs and readjusted to 7.4.

Measurement of [Ca²+]_cyt. In single BSMC, [Ca²⁺]_cyt was measured by using the Ca²⁺-sensitive fluorescent indicator, fura 2 (10, 11). Cells were loaded with the acetoxymethylester form of fura 2, fura 2-AM (3 µM for 30 min), in the dark at room temperature (22-24°C) under an atmosphere of 5% CO₂-95% air. The fura 2-loaded cells on coverslips were then transferred to a recording cell chamber on the microscope stage and superfused with modified Krebs solution for 30 min to remove extracellular fura 2 and allow cytosolic esterases to cleave fura 2-AM into active fura 2. Fura 2 fluorescence (510-nm light emission excited by 340- and 380-nm illuminations) from the cell, as well as background fluorescence, was collected at 32°C by using the Nikon ultraviolet-fluor objectives. The fluorescence signals emitted from the cells were monitored continuously by using an intracellular imaging fluorescence microscopy system and recorded in an IBM-compatible computer for later analysis. The 340:380-nm ratios (F₃₄₀/F₃₈₀) were used to indicate the changes of [Ca²⁺]_cyt in BSMC treated with agonists. In some experiments, F₃₄₀/F₃₈₀ was converted to [Ca²⁺]_cyt on the basis of the equation previously described by Grynkiewicz et al. (11).

Electrophysiological measurement of store-operated Ca²+ currents. Whole cell store-operated Ca²⁺ currents (I_SOC) were recorded at 22-24°C with an Axopatch-1D amplifier and a DigiData 1200 interface (Axonpatch) by using patch-clamp techniques (10, 12). Patch pipettes (2-4 M) were made on a Sutter electrode puller by using borosilicate glass tubes and fire polished on a microforge (Narishige). Voltage stimuli lasting 300 ms were delivered from a holding potential of 0 mV (to inactivate voltage-gated Ca²⁺ and Na⁺ channels) by using voltage steps from 80 to +20 mV. Traces recorded before the activation of SOCs were used as a template to subtract leak currents. SOCs were activated by passive depletion of the SR by using 10 µM cyclopiazonic acid (CPA, Sigma Chemical) dissolved in the Ca²⁺-free solution. The bath (extracellular) solution for recording whole cell I_SOC contained (in mM) 120 sodium methane sulfonate, 20 calcium aspartate, 0.5 3,4-diaminopyridine, 10 glucose, and 10 HEPES (pH 7.4 with methane sulfonic acid). The pipette (intracellular) solution contained (in mM) 138 cesium aspartate, 1.15 EGTA, 1 Ca(OH)₂, 2 Na₂-ATP, and 10 HEPES (pH 7.2). These ionic conditions eliminated the currents through K⁺ or Cl channels. In Ca²⁺-free solution, CaCl₂ was replaced by equimolar MgCl₂, and 0.1 mM EGTA was added to chelate residual Ca²⁺. CPA was dissolved into DMSO to make a stock solution of 30 mM. Aliquots of the stock solution were then diluted 1:3,000 into the bath solution or culture medium to make a final concentration of 10 µM CPA (pH 7.4). Ni²⁺ (Sigma Chemical) was directly dissolved in the bath solution on the day of use. The pH values of all solutions were checked after addition of the drugs and readjusted to 7.4.

RT-PCR assay. Total RNA from rat BSMC was reverse-transcribed by using the First-Strand cDNA Synthesis Kit (Pharmacia). The sense (5'-CAAGATTTTGGAAAATTTCTTG-3'; nucleotide 2238-2359) and antisense (5'-TTTGTCTTCATGATTTGCTAT-3'; nucleotide 2689-2709) primers were designed from the coding region of human TRPC1 (U31110). Two microliters of the first-strand cDNA reaction mixture were used in a 50-µl PCR reaction consisting of 0.1 µM of each primer, 10 mM Tris · HCl (pH 8.3), 50 mM KCl, 2 mM MgCl₂, 200 µN each dNTP, and 2 units of Taq DNA polymerase (Perkin-Elmer). The cDNA samples were amplified in a DNA thermal cycler (Perkin-Elmer) under the following conditions: the mixture was annealed at 50-61°C (1 min), extended at 72°C (2.0 min), and denatured at 94°C (1 min) for 25 cycles. This was followed by a final extension at 72°C (10 min) to ensure complete product extension. PCR products were electrophoresed through a 1% agarose gel, and amplified cDNA bands were visualized by ethidium bromide staining. To quantify the PCR products, an invariant mRNA of human -actin was used as an internal control. The optical density values for each band on the gel were measured by a gel documentation system. Optical density values in the channel signals were normalized to the optical density values in the -actin signals; the ratios are expressed as arbitrary units for quantitative comparison (10).

Statistical analysis. The composite data are expressed as means ± SE. Statistical analyses were performed by using unpaired Student's t-test or ANOVA and post hoc tests (Student-Newman-Keuls) where appropriate. Differences were considered to be significant when P < 0.05.

	RESULTS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

CCE-mediated bronchial constriction. Cytosolic Ca²⁺ stemming from extracellular fluids and intracellular organelles (e.g., SR) is important in triggering and maintaining agonist-mediated airway smooth muscle contraction. In isolated rat bronchial rings superfused with solutions containing 1.8 mM Ca²⁺, activation of M receptors by 5 µM ACh caused a rapid increase in isometric tension due to bronchial contraction, which maximized and reached a plateau within a few minutes. The ACh-induced bronchial contraction was completely abolished by removal of extracellular Ca²⁺ (replacing extracellular Ca²⁺ with Mg²⁺; Fig. 1A) or chelation of extracellular Ca²⁺ with 2 mM EGTA (which reduces [Ca²⁺] from 1.8 to ~0.0005 mM; Fig. 1B). The reversible effects of removal/chelation of extracellular Ca²⁺ on ACh-induced tension suggest that Ca²⁺ influx is required for sustained bronchial contraction.

View larger version (28K): [in this window] [in a new window]   Fig. 1.   Role of Ca2+ and capacitative Ca2+ entry (CCE) in rat bronchial constriction. A: representative record showing that removal of extracellular Ca2+ abolished bronchial contraction induced by 10 µM ACh (left). Ca2+-free solution (0Ca) was applied to the bronchial ring when ACh-mediated contraction reached plateau. The vertical and horizontal bars denote 100 mg and 2 min, respectively. Right: summarized data (means ± SE) of basal tension and active tension induced by ACh (n = 8) before (1.8 Ca), during (0 Ca), and after (1.8 Ca) application of Ca2+-free solution. ** P < 0.01 vs. closed bars. B: representative record showing that chelation of extracellular Ca2+ with 2 mM EGTA abolished bronchial constriction induced by 10 µM ACh (left). EGTA-containing solution was applied to the bronchial ring when ACh-mediated contraction reached plateau. Vertical and horizontal bars denote 100 mg and 2 min, respectively. Right: summarized data (means ± SE) of basal tension and active tension induced by ACh (n = 8) before (EG), during (+EG), and after (EG) application of EGTA. ** P < 0.01 vs. closed bars. C: representative record showing CCE-mediated bronchial constriction. ACh (5 µM) was first applied to the bronchial ring in the absence of extracellular Ca2+. M-receptor blocker atropine (10 µM) was applied to the ring when ACh-induced transient contraction declined back to baseline. In the presence of atropine, restoration of extracellular Ca2+ induced a contraction that was most likely due to CCE. Vertical and horizontal bars denote 150 mg/mg and 2 min, respectively. Right: summarized data (means ± SE) of ACh-induced initial transient constriction in the absence (Ca release) of extracellular Ca2+ and the active tension induced by restoration of extracellular Ca2+ in the presence of atropine (CCE). *** P < 0.001 vs. open bars.

Extracellular Ca²+ and intracellularly stored Ca²⁺ are required for BSMC growth. In cell cycle, there are multiple Ca²⁺/calmodulin-sensitive steps. Increases in [Ca²⁺]_cyt and nuclear [Ca²⁺] would thus propel transitions from the quiescent phase to DNA synthesis phase, accelerate mitosis, and stimulate cell proliferation (4). Indeed, chelation of extracellular free [Ca²⁺] in culture media (from 1.6 mM to 523 nM) with the use of 2 mM EGTA (7) significantly inhibited rat (Fig. 2) and human (data not shown) BSMC growth in the presence of serum and growth factors. Furthermore, passive depletion of intracellular Ca²⁺ stores with 10 µM CPA also markedly inhibited BSMC proliferation in the presence of extracellular Ca²⁺ (Fig. 2). Chelation of extracellular Ca²⁺ with 2 mM EDTA or depletion of the SR Ca²⁺ with thapsigargin, an irreversible inhibitor of the SR Ca²⁺-Mg²⁺ ATPase, caused similar inhibition on rat and human BSMC growth in media containing serum and growth factors, as did EGTA and CPA. These results suggest that continuous Ca²⁺ influx as well as maintaining sufficient Ca²⁺ in the SR are both essential for BSMC growth. Because CCE is involved in maintaining sustained increase in [Ca²⁺]_cyt and refilling Ca²⁺ in the SR, it is likely that mitogen/agonist-mediated CCE contributes to the sustained Ca²⁺ influx and preservation of Ca²⁺ in the SR, which are required for BSMC proliferation.

View larger version (25K): [in this window] [in a new window]   Fig. 2.   Chelation of extracellular Ca2+ and depletion of intracellularly stored Ca2+ in the sarcoplasmic reticulum (SR) inhibit rat bronchial smooth muscle cell (BSMC) growth. Cell numbers were determined before (basal) and after 2-day incubation of cells in medium without serum [0% fetal bovine serum (FBS)] or in media with 10% FBS containing vehicle (DMSO; cont), EGTA (2 mM), or cyclopiazonic acid (CPA; 10 µM). Data are means ± SE (n = 6). *** P < 0.001 vs. solid bar.

Blockade of the SR Ca²+ pump with CPA depletes Ca²⁺ from the SR. CPA is a specific blocker of the Ca²⁺-Mg²⁺-ATPase in the SR membrane, which reversibly blocks Ca²⁺ sequestration in the SR and thus induces a transient increase in [Ca²⁺]_cyt due to leakage of Ca²⁺ from the SR to the cytosol. In human BSMC superfused with Ca²⁺-free solution, extracellular application of ATP induced a huge transient increase in [Ca²⁺]_cyt due to Ca²⁺ mobilization from the SR (Fig. 3, A, left, and B). Pretreatment of the cells with 10 µM CPA for ~5 min completely abolished the ATP-induced transient increase in [Ca²⁺]_cyt due to Ca²⁺ release (Fig. 3, A, right, and B). These results suggest that blockade of the SR Ca²⁺ pump with CPA is able to deplete the receptor-coupled and inositol 1,4,5-trisphosphate (IP₃)-sensitive intracellular Ca²⁺ stores (3, 5).

View larger version (21K): [in this window] [in a new window]   Fig. 3.   CPA mediates store depletion in rat BSMC. A: representative records showing time course of ATP-induced changes in cytosolic free [Ca2+] ([Ca2+]cyt) in the absence of extracellular Ca2+ (0 Ca). ATP (10 µM) was superfused to the cells with (right) or without (left) pretreatment with 10 µM CPA. B: summarized data (means ± SE) showing [Ca2+]cyt measured before (control) and during (ATP) application of ATP in rat BSMC pretreated with vehicle (DMSO, open bars) or CPA (solid bars). *** P < 0.001.

Increased resting [Ca²+]_cyt and enhanced CCE in normal BSMC during proliferation. [Ca²⁺]_cyt can be increased by Ca²⁺ release from intracellular Ca²⁺ stores (e.g., the SR) and/or Ca²⁺ influx through sarcolemmal Ca²⁺ channels. The inhibitory effects of removal of extracellular Ca²⁺ or depletion of the [Ca²⁺]_SR on BSMC proliferation suggest that both [Ca²⁺]_cyt and [Ca²⁺]_SR are important in stimulating BSMC growth.

View larger version (22K): [in this window] [in a new window]   Fig. 4.   Enhancement of the CCE induced by CPA-mediated store depletion in proliferating BSMC. A: representative records showing time course of [Ca2+]cyt changes in growth-arrested (0% FBS) and proliferating (10% FBS) rat BSMC. CPA (10 µM) was continuously applied to the cells in the absence and presence of extracellular Ca2+. Ni2+ (0.5 mM) was applied to the cells when CCE reached a plateau. Bottom: summarized data (means ± SE) of the increases in [Ca2+]cyt due to CPA-induced Ca2+ release or CCE (Ca2+ influx). *** P < 0.001 vs. 0% FBS. B: representative records showing the time course of [Ca2+]cyt changes in growth-arrested (SMBM) and proliferating (SMGM) human BSMC. Bottom: summarized data (means ± SE) of the resting (basal) [Ca2+]cyt (left) and the increase in [Ca2+]cyt due to CCE (right). *** P < 0.001 vs. SMBM.

Enhanced I_SOC and TRPC1 expression in proliferating BSMC. It has been demonstrated that CCE is caused by Ca²⁺ influx through TRPC-encoded SOC, which are opened by depletion of the SR Ca²⁺ (5, 24, 31-33). TRPC1 is a member of the TRPC gene family, which is highly expressed in lung tissues and smooth muscle cells. The next set of experiments was designed to determine whether TRPC1-encoded SOC is responsible for the increased [Ca²⁺]_cyt and enhanced CCE during proliferation by comparing I_SOC and TRPC1 mRNA expression in proliferating and growth-arrested BSMC.

View larger version (19K): [in this window] [in a new window]   Fig. 5.   Whole cell store depletion-activated current (ISOC) is enhanced in proliferating BSMC. A: representative currents (a) elicited by test potentials from 80 to +20 mV in 20-mV increments (holding potential = 0 mV) before (cont) and after 10-min application of CPA (10 µM) in growth arrested (0% FBS) and proliferating (10% FBS) rat BSMC. Right shows ISOC between the currents recorded before and after application of CPA. b: Current-voltage relationships (I-V curves; means ± SE) for ISOC recorded in cells cultured in media with (10% FBS) or without (0% FBS) serum. P < 0.001 between the two I-V curves. B: representative currents (a) elicited by test potentials from 80 to +20 mV in 20-mV increments (holding potential = 0 mV), before (cont) and after 10-min application of CPA (10 µM) in growth arrested (SMBM) and proliferating (SMGM) human BSMC. Right shows ISOC between currents recorded before and after application of CPA. b: I-V curves (means ± SE) for ISOC recorded in cells cultured in media with (SMGM) or without (SMBM) serum and growth factors. P < 0.001 between the two I-V curves.

View larger version (36K): [in this window] [in a new window]   Fig. 6.   TRPC1 mRNA expression when rat BSMC proliferate. A: PCR-amplified products displayed in agarose gel stained with ethidium bromide for TRPC1 (372 bp) and -actin (244 bp) in proliferating (10% FBS) and growth-arrested (0% FBS) rat BSMC. M, molecular weight marker (100-bp DNA ladder). B: summarized data (means ± SE) of TRPC1 mRNA levels that were normalized to the amount of -actin (n = 4). ** P < 0.01 vs. 10% FBS.

Inhibition of SOC with Ni²+ attenuates BSMC proliferation. The TRPC1-encoded protein has been demonstrated to form heterotetrameric or homotetrameric SOC in many cell types (5, 31, 32). Extracellular application of Ni²⁺, a relative selective cationic blocker of SOC, inhibits Ca²⁺ influx through SOC in vascular smooth muscle cells (10) and lymphocytes (33). Pharmacological blockade of SOC with Ni²⁺ (0.5 mM) significantly decreased I_SOC (Fig. 7, A and B) and CCE (Fig. 4A) in proliferating rat and human BSMC cultured in media containing serum and growth factors and markedly inhibited rat BSMC growth (Fig. 7C). Ni²⁺ also attenuated human pulmonary artery smooth muscle cell proliferation in media containing 5% FBS and growth factors (data not shown). These results suggest that an increase in SOC activity is indeed an important mechanism involved in the elevated [Ca²⁺]_cyt and [Ca²⁺]_SR during BSMC proliferation.

View larger version (17K): [in this window] [in a new window]   Fig. 7.   Inhibitory effects of Ni2+ on CPA-induced whole cell ISOC and BSMC proliferation. A: representative currents (a) elicited by test potentials from 80 to +20 mV in 20-mV increments (holding potential = 0 mV) before (cont) and after 10-min application of CPA (10 µM) in the absence (CPA) or presence (CPA+Ni) of 0.5 mM Ni2+ in rat BSMC. b: Summarized data (means ± SE) of the current amplitude of ISOC at 80 and +20 mV before (CPA) and during (CPA+Ni) application of 0.5 mM Ni2+. *** P < 0.001 vs. solid bars. B: representative currents (a) elicited by test potentials from 80 to +20 mV in 20-mV increments (holding potential = 0 mV) before (cont) and after 10-min application of CPA (10 µM) in the absence (CPA) or presence (CPA+Ni) of 0.5 mM Ni2+ in human BSMC. b: Summarized data (means ± SE) of the current amplitude of ISOC at 80 and +20 mV before (CPA) and during (CPA+Ni) application of 0.5 mM Ni2+. *** P < 0.001 vs. solid bars. C: cell numbers were determined before (basal) and after 2-day incubation of rat BSMC in 10% FBS-containing culture media with (+Ni) or without (-Ni) 0.5 mM Ni2+. Data are means ± SE (n = 6). ** P < 0.01 vs. -Ni.

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

The contractile abnormalities of airway or bronchial smooth muscle and structural changes in airway wall greatly contribute to the development of persistent airway obstruction and increased airway hyperresponsiveness in asthma. Hypertrophy and hyperplasia of airway smooth muscle cells are believed to play a role in airway or bronchial narrowing observed in asthmatics (6, 13, 14). In human and animal BSMC, intracellular Ca²⁺ is an important signal transduction element in regulating smooth muscle contractility, responsiveness of bronchioles to inflammatory mediators and allergic factors, and expression of genes that are required for cell proliferation (6, 13, 14).

Intracellular Ca²⁺ diffuses quickly between the cytosol and nucleus because of a high permeability of the nuclear membrane to Ca²⁺ (1). Under normal conditions, most cytosolic free Ca²⁺ is sequestered by the SR Ca²⁺-Mg²⁺-ATPase into the SR, a cytoplasmic organelle involved in protein sorting and lipid synthesis, to keep [Ca²⁺]_cyt as low as ~100 nM. When BSMC is stimulated by agonists or mitogens, [Ca²⁺]_cyt is increased by Ca²⁺ influx due to opening of Ca²⁺ channels in the plasma membrane and Ca²⁺ mobilization due to opening of Ca²⁺ release channels (e.g., IP₃ receptors) in the SR membrane. When [Ca²⁺]_cyt rises in airway or BSMCs, Ca²⁺ binds to calmodulin and then activates myosin light chain kinase, which activates contractile apparatus (actomyosin), causes BSMC contraction, and induces bronchial constriction (13, 26). In contrast, removal or chelation of extracellular Ca²⁺ significantly inhibited ACh-mediated bronchial contraction (Fig. 1).

In isolated bronchioles from rats, we observed that, in the absence of extracellular Ca²⁺, ACh-induced a transient contraction (lasted for ~3-5 min) by activating the M₂ receptor and its downstream signal transduction molecule IP₃, which induces mobilization of Ca²⁺ from the SR to the cytosol (3, 16). After the SR Ca²⁺ was depleted (i.e., when the ACh-induced transient contraction declined to the baseline level), the M-receptor blocker atropine was applied to the bronchioles to terminate production of IP₃ and diacylglycerol and thus to eliminate diacylglycerol/protein kinase C-mediated activation of receptor-operated Ca²⁺ channels. Under these conditions, addition of extracellular Ca²⁺ to the perfusate induced a large contraction. This contraction was apparently due to an increase in [Ca²⁺]_cyt from CCE because pharmacological blockade of the M receptor with atropine did not interfere with the bronchial contraction induced by restoration of extracellular Ca²⁺. These results suggest that increases in [Ca²⁺]_cyt due to Ca²⁺ release and CCE were both involved in the ACh-mediated constriction in isolated bronchial rings.

A rise in [Ca²⁺]_cyt is also involved in stimulating cell proliferation because of the Ca²⁺-sensitive transitions in the cell cycle: 1) the transition from G₀ to G₁ phase, 2) the transition from G₁ to S phase, 3) the transition from G₂ to M phase, and 4) mitosis itself (4). In rat and human BSMC, removal of extracellular free Ca²⁺ with the use of EGTA, a potent Ca²⁺ chelator, and depletion of the intracellularly stored Ca²⁺ with the use of CPA, a specific inhibitor of Ca²⁺-Mg²⁺-ATPase in the SR, significantly attenuated BSMC proliferation in the presence of serum and growth factors. These results indicate that a sustained increase in [Ca²⁺]_cyt due to Ca²⁺ influx through sarcolemmal Ca²⁺ channels and maintenance of sufficient Ca²⁺ within the SR are both required for BSMC growth.

Among the sarcolemmal Ca²⁺ channels expressed in airway smooth muscle cells (18), the store depletion-activated SOC is an important Ca²⁺ influx pathway on activation of respective cell surface receptors (5, 24). CCE through SOC is a critical mechanism in maintaining sustained Ca²⁺ influx and in refilling Ca²⁺ in the SR (3-5, 24, 28). In rat and human BSMC, passive depletion of intracellular stores with CPA induced inward Ca²⁺ currents (I_SOC) by opening SOC and increased [Ca²⁺]_cyt by mediating CCE. The CPA- (or store depletion-) induced I_SOC and CCE were significantly enhanced in proliferating BSMC compared with growth-arrested cells. The increases in [Ca²⁺]_cyt induced by Ca²⁺ leakage from the SR and by CCE were both higher in proliferating BSMC than in growth-arrested cells. These results suggest that the increased I_SOC and CCE are an essential mechanism to maintain the high levels of [Ca²⁺]_cyt and [Ca²⁺]_SR required for BSMC proliferation.

The amplitude of CCE is positively proportional to the current amplitude and density of whole cell I_SOC, which is a function of the total number of SOC and the single-channel activity of SOC. Therefore, an increase in the channel expression and/or in the channel function can lead to an increase in whole cell I_SOC or CCE. Electrophysiological studies on the store depletion-activated I_SOC indicate that there are multiple SOC on the basis of the single-channel conductance (0.2-110 pS) (15, 17, 19, 20). Because of the diversity and variability of the biophysical and pharmacological properties of I_SOC, SOC are believed to be complex and heterogeneous in molecular composition and in cellular regulation. It has been recently demonstrated that native SOC in smooth muscle cells may be composed of subunits encoded by transient receptor potential (TRP) genes. Indeed, overexpression of TRPs in mammalian cells and Xenopus oocytes enhances I_SOC and CCE induced by store depletion by extracellular application of CPA or thapsigargin and by intracellular application of Ca²⁺ chelators (5, 24, 31-33). These results suggest that TRP-encoded proteins are involved in forming native SOC responsible for the store depletion-induced I_SOC and CCE.

Our observations from this study indicate that the mRNA expression of TRPC1, a member of the TRP gene family that is highly expressed in lung tissues and smooth muscle cells, was upregulated in proliferating BSMC compared with growth-arrested cells. Taken together with the electrophysiological and fluorescent microscopy results, we suggest that the upregulated TRPC1 is, at least in part, responsible for the increased I_SOC and CCE during BSMC proliferation. The precise cellular mechanism involved in the upregulation of TRPC1 gene when BSMC proliferate is unclear and needs further study. On the basis of sequence analysis, the promoter of TRPC1 gene contains binding sequences for many transcription factors (such as AP-1, signal transducer and activator of transcription, Smad, and c-myc) that are involved in progression of the cell cycle and regulation of cell proliferation, differentiation, and apoptosis. It would be interesting to investigate whether inflammation mediators (such as cytokines, chemokines, and histamine) and growth factors, which are upregulated in asthmatics, affect TRP expression in normal BSMC by regulating these transcription factors and their DNA binding activity with the TRP genes.

Airway obstruction in asthma is mainly due to airway inflammation, bronchospasm, bronchial hyperresponsiveness, and bronchial wall thickening. A high level of inflammatory mediators, bronchoconstrictors, and growth factors has been implicated in asthma. A critical signal transduction pathway on activation of respective receptors in the plasma membrane is the rise in [Ca²⁺]_cyt due to Ca²⁺ release and influx. CCE, potentially through TRPC1-encoded SOC, is a critical mechanism in maintaining a sustained rise in [Ca²⁺]_cyt and refilling Ca²⁺ into the SR in BSMC. Thus expression and function of TRPC1-encoded Ca²⁺ channels as well as amplitude of CCE may all contribute to regulating bronchial constriction and BSMC growth by modulating [Ca²⁺]_cyt and [Ca²⁺]_SR. Development of drugs that target TRP gene expression and SOC function may be an additional strategy to develop novel therapeutic approaches for patients with asthma.