The Effect Of Scoparone On Relaxing Tracheal Smooth Muscle And Regulating Intracellular Calcium Concentration

PrefaceAsthma is a chronic inflammatory disease of airway with a obscure etiology that seriously damages the health of human being. Recently, it has been believed that the elevation of cytosolic calcium concentration ( [ Ca2 +]i) is the main reason for the contraction and increased sensitivity to sensitizer of bronchi. Thus, It is a important subject in the medicine field to study pathological mechanism asthma and develop effective drugs. Scoparone (Sco) , a coumarin derivative extracted from Artemisia Scoparoneparia Waldst. Et Kit growing in Shenyang ( yinchen) , possesses the pharmacological actions including cholagogue, hepatic protection, anti - inflammation, analgesia, hypolipimia, immune suppression, hypotension and so on. The mechanism underlying these actions has been reported to be related to calcium antagonism. Sco possesses anti - inflammatory and calcium antagonistic effects, and we found it could significantly relax tracheal smooth muscle (TSM) in our study. It can be postulated that Sco may find its use in asthmatic therapy though the mechanisms of the anti - asthmatic effects of Sco have not been reported so far. In this study, the possible mechanism of the tracheaectasy effects of Sco was investigated.Materials and MethodsDrug treatment of animalsHealthy guinea - pigs ( male) weighting 150g - 200g were separated into three groups at random: control, asthma and Sco groups. Guinea - pigs from Sco group were treated by Sco (10-3M in 0. 9% NaCl) for two minutes every , day from beginning of experiment to the 28th day. The others were treated by 0. 9% NaCl in the same way.Producing asthma guinea - pig modelThe guinea - pigs from asthma and Sco groups were sensitized by injecting ovalbumin ( OA) 1ml (10% w/v in 0. 9% NaCl, intraperitoneal). After two weeks, they were challenged by exposure to aerosolized OA (1% w/v in 0.9% NaCl) for two minutes one time per day to induce asthma, by total two weeks. The responsiveness of animals was observed when they were challenged. Take deep and fast breath, ventral twitch, etc. as positive responsiveness which suggests successful model. Dead and unsuccessful animals were fell in disuse. The guinea - pigs from control were given 0.9% NaCl in the same way. The animals were killed for experiments within 24 hours after the last challenge.Tracheal ring preparation and tension measurementThe animals were killed by heating head, the trachea were excised immediately and placed in cold Krebs - Henseleit ( K - H) solution ( Composition in mM: NaCl 118.4, KC14.7, CaCl22.5, NaHCO325, KH2PO4 1.2, MgSO4. 7H2O 0.8, Glucose 10.1, pH 7.4) supplemented with mixed gases (95%O2, 5% C02) at 4℃. The main trachea was cleaned of all connective tissue and the epithelium was then mechanically removed by rubbing the lumen of the rings with a cotton - tipped applicator. The trachea was cut into rings of similar 3 mm diameter and 3 to 5mm in length. Tracheal rings were suspended in organ chambers (10ml) maintained at 37 ±0.5℃. and perfuse with normal gas mixture (95% O2, 5% CO2; pH =7.4). The rings were mounted on two tungsten triangles suspended between stainless steel wire hooks, one of which was anchored to the organ bath, and the other was connected to a forces transducer. Tension was continuously recorded with a pen recorder using a force displacement transducer. Before the beginning of each experiment, tracheal rings were allowed to equilibrate for 2 hours during which the tissues were washed with fresh standard K - H solution at 15 min intervals. Also during the equilibration period, a resting ten-sion of 2g was applied to each ring. The rings were then repetitively challenged with 60mM KCl until stable contractile responses were obtained. Histamine and KCl were used as agonist for receptor - operated calcium channel ( ROCC) and voltage - depended calcium channel (VDCC) respectively.Cultured guinea pig tracheal smooth muscle cellsAfter dissection as described in Tracheal ring preparation and tension measurement , The smooth muscle layer of the trachea was dissected free of cartilage, mucosa, connective tissue, and fat and cut into strips (1 mm wide and 1 mm long) with scissors, then the strips were transferred to a digestion solution (Composition in mM: NaCl 137, NaHCO34.2, glucose 10, Na-,HPO4, 3, KCl 5.4, KH2PO4 0.4, CaCl2 1.3, MgCl2 0.5, MgSO4 0.8, and HEPES 5, pH 7. 4) containing 200U/mlof collagenase (type I) , The tissues were incubated 2 times with gentle shaking for 40min at 37℃ respectively, the digestion solution containing the enzymes was changed between 2 times incubation. After second incubation, the digestion solution containing the enzymes was removed, and the preparations were washed three times in digestion solution without enzyme. Digested tissue was then gently pushed and sucked through a pipette to liberate individual myocytes. At the end of each step, isolated myocytes in suspension were collected. The solution was then centrifuged at 1,000 rpm for 5min, and the cells were resuspended at a density of 1 × 10~5 cells/ml in Dulbecco's modified Eagle s medium ( DMEM) containing 20% fetal bovine serum. The cells were then cultured for 3 to 5 days on glass cover slips in 35 mm culture dish in a 95% air -5% CO2 atmosphere at 37℃.Immunofluorescent stainingImmunofluorescent staining with a monoclonal mouse anti - a - smooth muscle actin IgG ( anti - asm - 1) recognizing a - smooth muscle actin. The cultured cells were fixed for 10 min in 4% paraform at 4℃,. anti - asm -1(0. 5% V/V in HBSS) was added to the cells, after which they were kept for 1 hr at room temperature. The muscle cells were washed three times with HBSS and treated with FITC - labeled goat anti - mouse IgG (diluted 1:100) for 30 min at room temperature. Then, the cells were washed three times with HBSS.Measurement of [Cα2+]iPreparation of fluo -3/AM: The concentration of free cytosolic Ca2+ in TSM was determined using the fluorescent Ca2+ indicator fluo -3/ AM. lmg fluo -3/AM was dissolved in 886μL dimethyl sulfoxide ( DMSO) ( about 1mmol·L-1 ) , and mixed thoroughly. Then it was subdivided into ten vials and stored at -20℃.. 5 μL of vial of stock solution was diluted by D - Hanks with a proportion of 1: 200 ( V/V). The final concentration of fluo - 3 was about 5 μmol· L-1 . This loading solution should be used in 3 h, to maximize loading efficiency. Ca 2+ measurement; The TSM cells culture media was removed and washed for 10 min with D - Hanks solution. Remove the final wash solution, add the loading solution, incubate about 50 min at 37℃. Then remove the loading solution, wash the cells with D - Hanks, measure the fluorescence soon after loading. Single - cell fluorescence imaging at 37℃ was performed using a MetaFluor system ( Universal Imaging Corp. , West Chester, PA, U. S. A. ). Images were acquired on a 12 - bit, cooled charge - coupled device ( CCD) interlined camera controlled by the Metafluor 4. 0 software and stored on hard disks for later analysis and archiving. The data was treated at Compaq Pentium 1.7G. The peak excitation was about 488nm and the peak emission was about 528nm.Data AnalysisChanges in [ Ca 2+ ]; were expressed as a ratio ( F/Fo) of the fluorescence counts ( F) relative to baseline counts before stimulation ( Fo). △F/Fo indicates the magnitude of the change in F/Fo at the peak of the evoked transient relative to the baseline ratio. Original fluorescence records were not filtered, smoothed, or averaged. Background fluorescence was not subtracted. Statistical analyses were performed using either ANOVA tests or Students t tests. Summarized data are shown as means ± S. D. and taken to be statistically significant when p<0. 05. n indicates numbers of cells used.ResultsComparison of responsiveness of isolated tracheal smooth muscle [TSM) to Sco and calcium agonists; With or without extracellular Ca2+ , 5μM of ryano-dine could induce TSM contraction in the presence of neomycin (1 μM) , an inhibitor of phosphoinositide - phospholipase C. Compared with control group, TSM from asthmatic guinea - pigs show more remarkable contraction effect. Pretreatment of asthmatic guinea - pigs with Sco could remarkably decrease whose TSM contraction.In the presence of tetracaine (200 μM) , an inhibitor of the CICR mechanism , dose - response curve for histamine of asthmatic guinea - pigs was left and up shifted compared with control group. After pre - treated by Sco, dose - response curve for histamine both of normal and asthmatic guinea - pigs was down - shifted, Sco showed remarkable relaxation effect.Primary culture of enzyme - treated guinea - pig ASM cells: We checked whether α - smooth muscle actin was stained or not by using anti - asm - 1. The actin filaments of most cells at day 5 to day 7 of culture were observed, showing that most cells are smooth muscle cells. Therefore, we used smooth muscle cells at day 5 to day 7 of culture for measuring Ca2+ signaling.Comparison of the changing in [Ca2+]i of cultured TSM cells to Sco and calcium agonists; Scoparone significantly dose - dependent decreased [ Ca2+]i of cultured TSM cells. Compared with control group, change of [ Ca2+]i in cultured TSM cells of asthmatic guinea - pigs was more sensitive to 10-5 M and 10-4M Sco. Compared with control group, 10-4 M Sco significantly inhibited the histamine - induced increase of [ Ca2+ ]; in cultured TSM cells of asthmatic guinea - pigs of pretreatment with tetracaine. Compared with control group, 10-4M Sco significantly inhibited 5μM ryanodine - induced increase of [ Ca2+ ]; in cultured TSM cells of asthmatic guinea - pigs of pretreatment with neomycin.Conclusion1. Ca2+ releasing function of RyR and IP3R is enhanced in cultured tracheal smooth muscle cells of asthmatic guinea - pigs, remarkably resulted in increasing of [Ca2+]i.2. Scoparone significantly decreased [ Ca2+] i of cultured tracheal smooth...