Hypercontractility Of Rat Coronary Artery To Extracellular Acidosis Owes To The Greater Activity Of TMEM16A/ANO1 In The Arterial Smooth Muscle Cell

Backgrounds:Acidosis is one of the most common and most difficult clinical problems.Severe acidosis can not only endanger human health,but also threaten people’s lives.Acidosis has an obvious deteriorating effect on the heart and cardiac muscle,and also has a great impact on vascular basic tension and vasomotor movement.Previous studies have found that extracellular acids and extracellular（o）acidosis（p H_o=6.8）only constricted rat coronary artery（RCA）and had no effect on rat intrarenal arteries（IRA）and mesenteric arteries（MA）,but the mechanism of acidosis is still unclear.To fully understand the mechanism of damage induced by acidosis is a prerequisite for proper prevention and accurate treatment of acidosis.ANO1,which is encoded by TMEM16A,is a Ca²⁺-activated Cl^-channel（Ca CC）.Studies have found that Ca CC played a particularly important role in vascular tone regulation,vascular smooth muscle cell proliferation,epithelial cell secretion,tumor cell vitality,etc.The effect of extracellular acidosis on CACC activity in coronary artery smooth muscle cell（ASMC）and its relationship with vascular muscle tone regulation have rarely been reported.The purpose of this study was to study the relationship between extracellular acidosis constriced RCA and extracellular Ca²⁺influx,intracellular Ca²⁺release and Ca CC.In this way,we can further understand the mechanism of acidosis and provide theoretical basis for clinical treatment of acidosis.Part 1 Role of Ca²⁺-activated Cl^-channel in the constriction of rat coronary artery induced by extracellular acidosis.Objectives:1.To observe the effects of extracellular acidosis on the muscle tone of different arteries in rat under different conditions.2.To observe the effect of extracellular acidosis on RCA and whether it is associated with Ca CC.3.To observe whether the effct of extracellular acidosis on RCA is associated with extracellular Ca²⁺influx and intracelluar Ca²⁺release.Methods:1.Adult healthy male Sprague-Dawley rats（12 weeks old,290～310 g）were used.Animals were killed by exsanguination from the left common carotid artery after anesthesia with sodium pentobarbital（40 mg/kg,ip）.RCA,cerebral basilar artery（CBA）,IRA,MA,intrapulmonary artery（IPA）and intramuscular artery（IMA）were quickly isolated and cut into about 2 mm of arterial rings,and fixed on the DMT.The tension of the vascular ring was then standardized.The vasomotor reactivity of the vascular ring to the constrictor and the diastolic agent was detected,and the success of endothelium removal was determined by observing the diastolic response of the vascular ring to acetylcholine.The vascular ring with good reactivity and successful endothelium removal was selected for the experiment.2.The effects of extracellular acidification（p H_o=7.2,7.0,6.8,6.6,6.4）on the resting state and U46619 0.3μmol/L and KCl 30 mmol/L pre-constricting state of RCA,CBA,IRA,MA,IPA and IMA were observed.3.The role of Ca CC in the constriction of RCA induced by extracellular acidosis was studied by using chloride channel inhibitors（NFA and NPPB）,Ca CC inhibitors(T16A_inh-A01,Ca CC_inh-A01 and Benzbromarone),Cl^--deprivation and ANO1 antibody（1:100）experiments.4.The effects of extracellular Ca²⁺influx and intracellular Ca²⁺release on the constriction of RCA induced by extracellular acidosis were studied by using L-type voltage gated Ca²⁺channel inhibitor（nifedipine）and sarcoplasmic reticulum Ca²⁺release channel inhibitors（ryanodine and tetracaine）.Results:1.Extracellular acidification（p H_o=7.2,7.0,6.8,6.6,6.4）concentration-dependently constricted RCA in resting state and preconstriction states of U46619 and KCl,but there was no significant effect on CBA,IRA,MA,IPA and IMA.2.Extracellular acidosis induced a reversible and repeatable vasoconstriction of 3.74±1.12 m N in the isolated RCA.Both chlorine channel inhibitors（NFA and NPPB）and Ca CC inhibitors(T16A_inh-A01,Ca CC_inh-A01 and Benzbromarone)concentration-dependently inhibited the constriction of RCA induced by extracellular acidosis,the IC₅₀values were 9.74±0.24,10.03±0.32,3.04±0.48,2.45±0.54 and 2.84±0.12μmol/L,respectively.Cl^--deprivation also time-dependently inhibited the constriction of RCA induced by extracellular acidosis.ANO1 antibody（1:100）also inhibited the extracellular acidosis-induced constriction of RCA by 41.17±18.64%.3.Extracellular acidosis initiated a transient constriction（2.01±0.61 m N,phasic）,which is supposed to be caused by intracellular Ca²⁺release.The following restoration of2.5 mmol/L Ca²⁺in the bath produced a sustained constriction（4.52±1.66 m N,tonic）.Sarcoplasmic reticulum Ca²⁺release channel inhibitors（ryanodine 50μmol/L and tetracaine 1 mmol/L）weakened the phasic constriction by 90.93±3.50%and 87.27±7.04%,respectively.L-type voltage gated Ca²⁺channel inhibitor（nifedipine）inhibited the constriction of rat coronary artery induced by extracellular acidosis,the IC₅₀ value was 9.85±0.44 nmol/L.Part 2 Effect of extracellular acidosis on Ca²⁺-activated Cl^-current in rat arterial smooth muscle cellsObjectives:1.To observe the effects of extracellular acidosis on Ca²⁺-activated Cl^-current and membrane potential in ASMCs from RCA,CBA,IRA and MA.2.To observe the effects of Ca CC inhibitors(T16A_inh-A01,Ca CC_inh-A01),Cl^--deprivation and ANO1 antibody on the change of Ca²⁺-activated Cl^-current in RCA ASMC induced by extracellular acidosis.3.To observe the effects of L-type voltage gated Ca²⁺channel inhibitor（nifedipine）and sarcoplasmic reticulum Ca²⁺release channel inhibitors（ryanodine and tetracaine）on the change of Ca²⁺-activated Cl^-current in RCA ASMC induced by extracellular acidosis.Methods:1.The isolated de-endothelium arteries were respectively transferred to the total volume of 1 m L of enzymatic solution I,with a constant temperature of 37℃.O₂ was continuously injected for about 30 min until the vascular tissues became soft.Then the arteries in the enzymatic hydrolysate I were transferred to the total volume of 0.5 m L of the enzymatic solution II,with a constant temperature of 37℃.O₂ was continuously injected for about 5 min.After that,it was added to the enzymatic solution II with 0.5 m L of the same volume of salt solution at 37℃,which was continued for about 3 min.After the arteries became thinner,the arteries were gently blown with a glass pipette to mechanically separate the cells.Finally,to terminate digestion,the cell solution was added 4℃salt solution and centrifuged twice at 6 min,800 rpm.The supernatant was removed and 1 m L of cell solution was stored at 4℃for whole-cell patch clamp experiment.2.The effects of extracellular acidosis on Ca²⁺-activated Cl^-current in ASMCs from RCA,CBA,IRA and MA by whole-cell patch clamp technique were observed.3.By whole-cell patch-clamp technique,Ca CC inhibitors(T16A_inh-A01 10μmol/L and Ca CC_inh-A01 10μmol/L),Cl^-deprivation and ANO1 antibody（1:100）were used to observe the effects on the change of Ca²⁺-activated Cl^-current in RCA ASMC induced by extracellular acidosis.4.The effects of L-type voltage gated Ca²⁺channel inhibitor（nifedipine 30 nmol/L）and sarcoplasmic reticulum Ca²⁺release channel inhibitors（ryanodine 50μmol/L and tetracaine 1 mmol/L）on the change of Ca²⁺-activated Cl^-current in RCA ASMC induced by extracellular acidosis were observed by whole-cell patch-clamp technique.5.By whole-cell patch clamp technique,the effects of extracellular acidosis on membrane potential of ASMCs from RCA,CBA,IRA,MA and the effect of ANO1antibody（1:100）on them were observed.Results:1.Extracellular acidosis increased the Ca²⁺-activated Cl^-current of RCA ASMC,but had no significant effect on the Ca²⁺-activated Cl^-current of ASMCs from RCA,IRA and MA.2.Ca CC inhibitors(T16A_inh-A01 10μmol/L and Ca CC_inh-A01 10μmol/L)inhibited the increase of Ca²⁺-activated Cl^-current in RCA ASMC induced by extracellular acidosis,At test potentials of+100 m V,the inhibition rates were 78.95±20.66%and73.39±18.02%,respectively;At test potentials of-100 m V,the inhibition rates were87.49±32.33%and 55.50±15.25%,respectively.Cl^--deprivation and ANO1 antibody（1:100）also inhibited the increase of Ca²⁺-activated Cl^-current in RCA ASMC induced by extracellular acidosis,At test potentials of+100 m V,the inhibition rates were 99.44%±2.54%and 91.54±7.66%,respectively;At test potentials of-100 m V,the inhibition rates were 99.75%±1.74%and 91.61±18.20%,respectively.3.L-type voltage gated Ca²⁺channel inhibitor（nifedipine 30 nmol/L）inhibited the increase of Ca²⁺-activated Cl^-current in RCA ASMC induced by extracellular acidosis,At test potentials of-100 m V and+100 m V,the inhibition rates were 86.63±15.70%and82.29±12.37%,respectively.Sarcoplasmic reticulum Ca²⁺release channel inhibitors（ryanodine 50μmol/L and tetracaine 1 mmol/L）inhibited the increase of Ca²⁺-activated Cl^-current in RCA ASMC induced by extracellular acidosis in varying degrees,At test potentials of+100 m V,the inhibition rates were 90.88±8.81%and 95.21±11.11%,respectively;At test potentials of-100 m V,the inhibition rates were 96.15±20.41%and89.68±11.00%,respectively.4.Extracellular acidosis depolarized all ASMCs freshly isolated from RCA,CBA,IRA and MA,but the depolarization was greater in RCA ASMC as compared with ASMCs from MA,CBA and IRA.ANO1 antibody（1:100）inhibited extracellular acidosis-induced RCA ASMC membrane potential elevation by 79.16±5.25%.Part 3 Effects of extracellular acidosis on intracellular Ca²⁺and Cl^-levels in rat arterial smooth muscle cellsObjectives:1.To observe the effects of extracellular acidosis on intracellular Ca²⁺and Cl^-levels in ASMCs from RCA,CBA,IRA and MA.2.To observe the effects of Ca CC inhibitors(T16A_inh-A01,Ca CC_inh-A01),Cl^-deprivation and ANO1 antibody on the change of intracellular Cl^-level in RCA ASMC induced by extracellular acidosis.3.To observe the effects of sarcoplasmic reticulum Ca²⁺release channel inhibitors（ryanodine and tetracaine）and ANO1 antibody on the change of intracellular Ca²⁺levels in RCA ASMC induced by extracellular acidosis.Methods:1.ASMCs were seeded into wells,incubated for 40 min in the cell bath solution to allow the cells to settle and adhere to the bottom of the wells and divided into different treatment groups.Afterward,the cells were loaded with either the Cl^-indicator dye（6-methoxy-N-（3-sulfopropyl）quinolinium,SPQ,10μmol/L）or the Ca²⁺indicator dye（fluo-4-AM,5μmol/L）in a normal bath solution for 1 h（37°C）.For intracellular Cl^-level measurement,the excitation and emission wavelengths were 350 and 470 nm,respectively.For intracellular Ca²⁺level measurement,the excitation and emission wavelengths were 488 nm and>510 nm,respectively.Increments in fluorescence fractions（F/F₀,intracellular fluorescence/background fluorescence）were recorded continually.The concentrations of intracellular Cl^-were calculated according to intracellular Cl^-（mol）=[F₀/（F-F₀）-1]/Ksv,Ksv=7.61.2.The effects of extracellular acidosis on intracellular Cl^-level in ASMCs from RCA,CBA,IRA and MA were observed with Cl^-probe.3.The effect of Cl^-deprivation for 1 h on intracellular Cl^-level was observed.4.Ca CC inhibitors(T16A_inh-A01 10μmol/L and Ca CC_inh-A01 10μmol/L),Cl^-deprivation and ANO1 antibody（1:100）were used to observe their effects on intracellular Cl^-level change in RCA ASMC induced by extracellular acidosis.5.The effects of extracellular acidosis on intracellular Ca²⁺level induced by intracellular Ca²⁺release and extracellular Ca²⁺influx in RCA ASMC were observed with Ca²⁺probe.6.The effects of sarcoplasmic reticulum Ca²⁺release channel inhibitors（ryanodine50μmol/L and tetracaine 1 mmol/L）on the changes of intracellular Ca²⁺release and extracellular Ca²⁺influx in RCA ASMC induced by extracellular acidosis and ANO1antibody（1:100）on intracellular Ca²⁺level change in RCA ASMC induced by extracellular acidosis were observed.Results:1.Extracellular acidosis reduced the intracellular Cl^-level in RCA ASMC,but had no significant effect on the intracellular Cl^-level in ASMCs from CBA,IRA and MA.2.Ca CC inhibitors(T16A_inh-A01 10μmol/L and Ca CC_inh-A01 10μmol/L)inhibited the reduction of intracellular Cl^-level in RCA ASMC induced by extracellular acidosis,the inhibition rates were 64.29±12.16%and 57.14±8.45%,respectively.Cl^-deprivation reduced intracellular Cl^-level,and Cl^-deprivation and ANO1 antibody also inhibited the reduction of intracellular Cl^-level in RCA ASMC induced by extracellular acidosis,the inhibition rates were 75.00±7.75%and 92.36±8.25%,respectively.3.Extracellular acidosis increased intracellular Ca²⁺level in RCA ASMC by intracellular Ca²⁺release and extracellular Ca²⁺influx.4.Sarcoplasmic reticulum Ca²⁺release channel inhibitors ryanodine 50μmol/L and tetracaine 1 mmol/L depressed extracellular acidosis-induced intracellular Ca²⁺elevation by 90.93±19.56%和87.27±7.04%in Ca²⁺-free bath solution and they also depressed intracellular Ca²⁺elevation induced by the restoration of 2.5 mmol/L Ca²⁺in the bath by20.56±14.67%and 40.63±6.62%,respectively.ANO1 antibody（1:100）inhibited extracellular acidosis-induced intracellular Ca²⁺elevation in RCA ASMC by 75.45±2.39%.Part 4 Expression of TMEM16A/ANO1 in rat different arteries and their smooth muscle cellsObjectives:1.To observe the expression of TMEM16A/ANO1 in RCA,CBA,IRA,MA smooth muscles and their ASMCs.Methods:1.ANO1 protein in RCA,CBA,IRA,MA smooth muscles and their ASMCs was observed by immunofluorescence staining.2.Total m RNA and Protein was extracted from the endothelium-denuded arterial lysates.TMEM16A/ANO1 expression differences between RCA,CBA,IRA,MA smooth muscles was analysed by real-time fluorescence quantitative polymerase chain reaction and western blottingResults:1.Immunofluorescence,western blotting and real-time fluorescent quantitative polymerase chain reaction revealed that TMEM16A m RNA and ANO1 protein were expressed in RCA,CBA,IRA,MA smooth muscles and their ASMCs,but RCA smooth muscle and its ASMC expressed a greater amount of TMEM16A/ANO1 as compared with CBA,IRA,MA smooth muscles and their ASMCsConclusions:1.Extracellular acidosis constricts RCA,increases Ca²⁺-activated Cl^-current,reduces the intracelluar Cl^-level,increases intracelluar Ca²⁺level in RCA ASMC,and these effects are inhibited by Cl^-channel inhibitors,Ca CC inhibitors,Cl^-deprivation and calcium antagonists.Extracellular acidosis has no significant effect on the IRA,MA,CBA and their ASMC.2.Extracellular acidosis depolarizes the membrane potential of ASMCs in the RCA,IRA,MA and CBA,but it has a greater membrane potential depolarization in RCA ASMC.3.TMEM16A/ANO1 is expressed in RCA,CBA,IRA,MA smooth muscles and their ASMCs,but RCA smooth muscle and its ASMC express a greater amount of TMEM16A/ANO1.ANO1 antibody attenuates all observed changes induced by extracellular acidosis in RCA and its ASMC.