Roles Of Alveolar Macrophage Potassium Ion Channels In Silica-induced Cell Inflammatory Response

Silicosis is a lung disease that develops subsequent to inhalation of silica dust.Amechanism of action to explain these consequences of silica exposure is not established.It isgenerally accepted that persistent lung inflammation is the basis of silicosis.Alveolarmacrophages (AM) are main target cells after dust flow into the bronchoalveolar lavage,andthe interaction between AM and dust is a key process to silicosis,which is an early event ininflammatory response induced by silica.The mechanism of the early response of AM tosilica,however,is still not clear.Recently some research has confirmed that potassium ionchannels in macrophages associated with cells activation and functional response under LPStreatment.Potassium ion channel is a transmembrane proteins stimulated by extracellularsignal,which also can be conversed to the intracellular signal through membrane potentialchanges.This suggested that potassium ion channels in alveolar macrophage may beinvolved in dust-induced activation and response,which maybe an early transmembranesignal in the interaction between AM and dust.According to this hypnosis the voltage-gatedpotassium ion channels in alveolar macrophages was choused in this study so as to exploreits' roles in silica induced lung inflammation.Part One Patch clamp study on voltage dependent potassium ionchannel in AMObjective:To explore voltage-gated potassium channel electrophysiological characteristicsin rat alveolar macrophage cell by method of the whole-cell patch clamp recording mode, perforated-patch clamp recording mode,and compare the differences among those variousrecord modesMethods:Alveolar macrophages were isolated from Sprague-Dawley rats bybronchoalveolar lavage,and seed at the 24-well plate with cover-slip.After culture 2h underthe conditions of 5% CO2,37℃,cover-slips were removed to the cell pool.Standardborosilicate glass electrode were used as microelectrode for whole-cell patch clampfabricated by two steps,and the resistance were 4-6MΩafter filling with inner solution;thin-walled borosilicate glass electrode were used as microelectrode for perforatedpatch-clamp also fabricated by two-steps,the impedance were 2-4 MΩafter filling withinner solution.A square pulse with duration of 500ms and range of-160-60 mV in step of20mV depolarization were given after recording mode was established.The current signalwas analysis using pClamp 9.0 software.Potassium channel activation curve were fitted byBoltzmann equation:G/G_max=1/{ 1+ exp[(V_0.5-V_m)/k]}.Results:AM voltage-dependent inward rectifier potassium current and delayed outwardpotassium current can be recorded both in whole-cell patch clamp recording mode and inperforated-patch clamp recording mode,both recorded successfully eight cells respectively.At the condition of the largest stimulating voltage,current density of delayed outwardpotassium in whole-cell patch clamp recording mode was 8.32.24 pA/pF,current densityof inward rectifier potassium was-6.77±3.89 pA/pF respectively;half activation voltage of-28.69±2.65 mV and-90.04 2.15 mV were found respectively;slope factor was 28.27±1.27 and 19.57±2.14 respectively.Current density of outward potassium inperforated-patch clamp recording mode was 9.42±4.41 pA/pF,and the current density ofinward potassium was-8.49±4.71 pA/ pF,which is slightly larger than the one ofwhole-cell recording;the corresponding half of the activation voltage was-15.38±2.30mVand-99.04±2.45mV respectively,the slope factor was 15.62±2.89 and 11.97±2.97respectively,which was significantly different from the whole cell recording mode,thedifference was statistically significant (P＜0.05).Membrane current in whole-cell patchclamp recording mode decay beginning at 12min,and the current was decay almostcompletely in 20min.There was no significant attenuation of membrane current inperforated-patch clamp recording mode during recording period,and the longest recordingtime was close to 40 min. Conclusions:1.Rat AM can express voltage-dependent inward rectifier potassium currentand voltage-dependent outward potassium currents.There were different among individualmacrophages.In a summary,there are three types of potassium expressing mode:the oneexpress mainly the delayed K⁺ currents,the one express mainly the rectifier potassiumcurrent,and the one express both inward and outward potassium current.2.Both models can be used to record voltage-gated potassium channel activity,butwhole-cellpatch clamp mode is more suitable for records of a large number of cells inshort-term,and perforated-patch clamp mode is suitable for the observation of the effect oftoxins or drugs on channel activity in real-time.3.The experimental Parameters of whole-cell patch clamp recording mode are:standardwall thickness of the electrodes,impedance is controlled in the range of 4-6 MΩ,range ofvoltage stimulate pulse is-160-60 mV,and more importantly the record should becompleted in 10 min;The experimental parameters perforated-patch clamp recording modeare:thin-walled electrode,final concentration of nystatin is 400μg/ml,electrode impedanceis controlled in the range of 2-4 MΩ,range of voltage stimulating pulse is-160～60 mV,and record time should not be more than 40min.Part two The effect of silica particles on voltage-gated potassiumion channel in AMObjective:To study the effect and its dynamic changes of silica particles onvoltage-dependent potassium channel in AM,and explore the response mechanism ofpotassium channel in AM to silica particles after immediate or long (24 h)treatment.Methods:The collection and purification of AM was same to the first part.cells wereplated 24-well plate with cover-slip,cell concentration was 1×10⁵/ml.Differentconcentrations of silica particles:0μg/ml,25μg/ml,50μg/ml,100μg/ml,200μg/ml,amorphous silica particles and 100μg/ml were added to plate.The cover-slips were used forwhole-cell patch clamp experiments after 24h culture.AMs without any treatment were used for perforated-patch clamp experiments. extracellular fluid flow slowly through cell pool at 2 ml/min by ALA perfusion system afterestablishment of perforated patch-clamp,silica particles of 25μg/ml,50μg/ml,100μg/ml,200μg/ml and 100μg/ml amorphous silica particles were injected to cell pool immediateusing self made tool,and thechange of potassium channel were recorded.Cation or anionoperated currents were tested by means of continuous recording mode after injection ofsilica particles.For another excrement,cells were plated at 96-well plates,cell concentration adjustedto 5×10⁵/ml,then adding 0μg/ml,25μg/ml,50μg/ml,100μg/ml,200μg/ml of standardquartz particles and 100μg/ml amorphous silica particles to each well,cell lactatedehydrogenase (LDH) leakage rate and cell survival (MTT method) were detected after 24hculture.Results:1.24 h treatment of 100μg/ml silica particles enabled the AM outward potassiumcurrent increased significantly (P＜0.05),but the inward rectifier potassium current did notchange significantly (P＞0.05).25μg/ml,50μg/ml silica particles and 100μg/mlamorphous silica particles had no effect on AM outward and inward potassium currents.itwas not recorded successfully in 200μg/ml silica group,but the same results could beobserved after shorten the treating time to 2h.2.In addition to 100μg/ml silica group,the activation curve of the other treatment groupswere shifted to right compared with the control group (P＞0.05) after co-cultured 24 h.Activation curve in 100μg/ml silica group shifted significant to left (P＜0.05);and slopefactor in 100μg/ml silica group was less significantly than that in 100μg/ml amorphoussilica group (P＜0.05).3.With the increase of silica particles concentration,AM delayed outward potassiumcurrent and inward rectifier potassium current increased compared with the control groupunder real-time treatment,and the current increased significantly in 50μg/ml,100μg/ml,200μg/ml group (P＜0.05).The treatment of 100μg/ml amorphous silica to AM have nosignificant effect on outward and inward potassium currents.4.The activation curve of delayed outward potassium current in all treatment group shiftto the left at the immediate treatment method,and half activation voltage was significantlyless than that of the control group (P＜0.05),and slope factor was also significantly lessthan the control group(P＜0.05);Activation curve of inward rectifier potassium current in all treatment group shifted to right,and half activation voltage was greater significantlythan that of the control group,the difference was statistically (P＜0.05),slope factor wassignificantly less than that of the control group (P＜0.05).100μg/ml amorphous silicaparticles treatment decreased statistically slope factor of outward potassiumcurrentcompared with the control group (P＜0.05),but activation curve of inward potassiumcurrent was not significantly affected.Half activation voltage of outward potassium currentin amorphous silica particles group was greater than that of the same dose of silicatreatment(P＜0.05),but the half activation voltage of inward potassium current inamorphous silica particles group were less significantly than the same dose of silicatreatment (P＜0.05).5.Cationic or anionic membrane current in AM could not be induced by silica particles.6.With the increase of silica particles concentration the AM LDH leakage was significantlyincreased,and the viability decreased significantly compared with the control (P＜0.05).100μg/ml amorphous silica particles treatment had no significant effect on the viability of AM,but the LDH leakage rate reached 46.0%,which was less than the result of same dosetreatment of silica (P＜0.05).Conclusions:Silica particles have activation effect on AM delayed outward potassiumchannel and inward rectifier potassium channel,with the main kinetics of high probabilityand flow rate.Silica particles can not affect AM electrophysiological activity in method ofreceptor operation.These resultes suggested that AM membrane voltage-dependentpotassium channels may be one of the early signaling proteins in cell activation and damageinduced by silica particles.Part three Roles of voltage-dependent K⁺ channel in AM activationand injury induced by silicaObjective:Blocker or agonist of voltage-gated potassium channel were used in this studyto observe voltage-gated potassium channel activity in activation,damage and secretion ofinflammatory mediators of AM of rat treated by silica particles. Methods:SD rat AM obtained by lavage,cells with concentration were adjusted to 5×10⁵/ml or 1×10⁶/ml (for detection of TNF-α) platedin 96-weli plates,each well fill with200μl.Purified cells were co-treated by potassium channel blocker tetraethyl ammonium(TEA,final concentration of 2.5mM,5mM,10mM,20mM),4 aminopyridine (4-AP,thefinal concentration 0.625mM,1.25mM,2.SmM,5mM),and agonist K⁺ (final concentration15 mM,30 mM,60 mM,120 mM) with 100μg/ml silica particles at the same time,cellswere cultured 24 h and the culture supernatants were used for determination of the rate ofLDH leakage,cell viability (MTT method),TNF-αcontent of culture supernatantsdetermined by ELISA.Results:1.The leakage rate of LDH in groups of blockers TEA and silica particles wereabout 51.6%-69.8% of that of silica particles dealing with alone (P＜0.01).The leakagerate of LDH in groups of blocker 4-AP and silica particles were about 18.1%-39.1% ofthat of silica particles dealing with alone (P＜0.01).The leakage rate of LDH in groups ofagonist K⁺ and silica particles were about 101.1%-108.3% of that of silica particles dealingwith alone (P＞0.05).2.Cell viability in group of blockers TEA and silica particles were about 152.5%-213.2%of that of group silica alone (P＜0.01).Cell viability in group of blockers 4-AP and silicaparticles were about 152.5%-213.2% of that of group silica alone (P＜0.01).Cell viabilityonly in group of 1.25 mM 4-AP with silica particles was higher than that of group of silicaparticles,the viability change in other groups were not obvious.Cell viability in group ofagonist of K⁺ and silica particles (60mM and 120mM)were significantly less than theresults of individual quartz treatment (P＜0.05).3.TNF-αrelease in group of blocker TEA and silica were 47.6%-79.3% of that of silicatreatment alone (P＜0.01).TNF-αrelease in group of blocker 4-AP and silica were 44.1%-56.9% of that of silica treatment alone (P＜0.01).TNF-αrelease in group of K⁺ and silicawere 132.8%-217.2% of that of silica treatment alone (P＜0.01).Conclusions:Voltage gated potassium ion channel activity in AM may be related tomembrane damage and cell necrosis of AM induced by silica particles,and can modulate therelease of TNF-αinduced by silica particles.Outward and inward potassium channel in AM may be an early signaling protein in response to silica treatment.Part four Relationship between potassium ion channels inmembrane of AM and intracellular calcium induced by silicaObjective:Blocker and agonist of voltage-gated potassium channel were used to studyrelationship between potassium ion channels in membrane of AM and intracellular calciumsignal induced by silica,to explore signaling mechanism of cell activation and injuryinduced by silica particles.Methods:Collecting and pre-treatment of alveolar macrophage were same to the first part.Cell concentration were adjusted to 1×10⁵/ml and plated to 6-well plate containing glass slip,each well contain 2 ml median.Purified cells continued to culture for 2h,and Fluo-3/AMwas used to load cells for 30min,remove to laser scanning confocal microscope,then usingexcitation wavelength 488 nm and radiation wavelength of 526 nm to record fluorescencechanges in cell,scanning frequency was 0.5 Hz.After fluorescence changes was in a stablebaseline (about 100 s),immediately injected different treatment liquid containing:100μg/ml silica particles,100μg/ml amorphous silica particles,100μg/ml silica particles and20 mM TEA,100μg/ml silica particles and 5mM 4-AP,100μg/ml silica particles and 120mM K⁺,then continuous scanning for 20 min.Fluorescence intensity changes in each groupwere analyzed and compared.Standardized changes in calcium signal were calculated byratio of calcium ion fluorescence intensity in excremental group to calcium ion fluorescenceintensity in control group were as the basis of value.Results:Intracellular calcium fluorescence intensity were increased most in silica group inthese five testing group,reaching more than 2 times,and signal intensity has beenmaintained at a higher level during recording period,range of fluorescence fluctuationswere 2371.98±378.55,which was significantly large than that of amorphous silica particlestreatment (P＜0.05);amorphous silica particles treatment had no effect on calciumconcentration,which even was in a trend of lower,range of fluorescence fluctuations was447.62±164.36;K⁺ and silica particles co-treatment increased calcium signal,the amplitude was less than the results of silica particles along,but the difference was notstatistic significant(P＞0.05),range of fluorescence fluctuations was 2455.27±536.39,which was slightly larger than silica particles treatment alone,and there are obvious calciumfluorescence intensity peaks appear in 150 s-300 s;potassium channel blockers 4-AP andTEA co-treatment with silica respectively have no obvious effect on intracellular calciumconcentration changes,which was similar to amorphous silica particles treatment,butfrequent of signal fluctuations was high with the scope significantly less than silica particlestreatment (P＜0.05).Conclusion:Silica particles have the effect to induce AM initiated calcium signal quikly,suggesting calcium signal is one of most important mechanism in inflammtory response tosilica particles.Blockers of K⁺ channel can significantly reduce the increase of calciumconcentration induced by silica particles,which suggested that outward and inwardpotassium channel potassium channels in membranes of cell may be involved in rugulationof intracellar calcium signal.