Mechanisms Underlying Rat Coronary Vasoconstriction Induced By Blockade Of Inward Rectifier Potassium Channels With Bacl₂

Objectives:1.The coronary vasoconstriction induced by blockade of inward rectifier K⁺channels(K_ir)was studied by observing the myogenic response of isolated quiescent rat coronary artery（RCA）to K_irr blocker BaCl₂（0.1-1.0 mmol/L）.2.The dependence of BaCl₂-induced RCA contraction on intracellular Ca²⁺([Ca²⁺]_i)release and extracellular Ca²⁺([Ca²⁺]_o)influx was studied by Ca²⁺-deprivation and restoration.3.The mechanisms underlying the BaCl₂-induced RCA contraction were further explored by studying with inhibitors including chloride channel blockers（NFA and NPPB）,calcium channel blocker（Nifedipine）,cyclooxygenase inhibitor（indomethacin）and MAPK inhibitors（PD98059 and SB239063）.Methods:1.Male Sprague-Dawley rats（200²50 g）were used.Animals were anaesthetized by intraperitoneal administration of sodium pentobarbital（40 mg/kg）.After exsanguination,the rat heart was removed and transferred immediately into chilled（4℃）physiological salt solution（PSS）.The RCAs（150-260μm）were isolated carefully and cut into 2-mm-long rings.The rings were then mounted on a wire myograph（Multi Myograph System-610M,DMT）using two tungsten wires（40μm）in a tissue containing 5.0 mL of PSS maintained at 37℃.The tension changes of RCAs were recorded by PowerLab system.After normalization,the RCAs were equilibrated for at least 1 h and then contracted with KCl（60 mmol/L）for three times.When the successive contractions induced by KCl（60mmol/L）were repeatable and the tension restored to the resting tone,BaCl₂（0.1,0.3,1.0mmol/L）was cumulatively added to the bath and concentration-response curve was constructed.The maximal contraction of KCl（60 mmol/L）was taken as 100%.2.The dependence of BaCl₂-induced RCA contraction on the infux of extracelluar Ca²⁺([Ca²⁺]_o)and the release of intracellular Ca²⁺([Ca²⁺]_i)was studied by the Ca²⁺-deprivation and restoration.The RCAs were rinsed with Ca²⁺-free PSS solution（containing 1.0 mmol/L EGTA）for three times and then the solution in the chamber was replaced by Ca²⁺-free PSS solution（without EGTA）.After 20 min,the RCAs were stimulated by BaCl₂（0.3 mmol/L）or KCl（60 mmol/L）in Ca²⁺-free PSS.10 min later,concentration of Ca²⁺in the chamber was restored to 2.5 mmol/L.The contractions induced by KCl（60 mmol/L）and BaCl₂（0.3 mmol/L）were recorded in the absence or presence of Ca²⁺（2.5 mmol/L）,respectively.KCl（60 mmol/L）-induced contraction served as a reference.3.When the concentration-contraction curve for BaCl₂ was repeatable,the mechanisms underlying the contraction were investigated with specific inhibitors.Following inhibitors were used:Nifedipine(0.3μmol/L,an L-type voltage-gated Ca²⁺channel blocker)、PD98059（3μmol/L,an ERK1/2 inhibitor）、SB239063（3μmol/L,a p38MAPK inhibitor）、cyclooxygenase inhibitor indomethacin（10,30,100μmol/L）、chloride channel blockers NFA（10,30,100μmol/L）and NPPB（10,30,100μmol/L）.The different inhibitors were added to the bath 30 min before BaCl₂（0.1-1.0 mmol/L）addition,respectively.The contractions induced by BaCl₂ were recorded in the absence or presence of the inhibitors.The maximal contraction of KCl（60 mmol/L）was taken as 100%.Results:1.In isolated quiescent RCA,BaCl₂（0.1-1.0 mmol/L）induced contraction in a concentration-dependent manner.Contractions induced by BaCl₂（0.1,0.3,1.0 mmol/L）were（0.42±0.12）mN,（2.63±0.51）mN,（5.68±1.62）mN,accounting for（7.64±2.13）%,（49.32±6.46）%,（104.56±10.15）%,respectively,with 60 mmol/L KCl-induced contraction as 100%.The value of EC₅₀0 was 0.34 mmol/L.2.In the normal PSS,the contraction of BaCl₂（0.3 mmol/L）was（2.83±0.98）mN,equal to（49.92±5.83）%of KCl（60 mmol/L）.The contractions induced by BaCl₂ in Ca²⁺-free solution and by followed restoration of Ca²⁺（2.5 mmol/L）were（31.63±5.23）%and（68.37±5.94）%,respectively,with overall contraction induced by BaCl₂（0.3 mmol/L）as100%.The contraction of KCl（60 mmol/L）was（5.78±1.21）mN.The above two components were（5.58±1.63）%and（94.42±11.27）%,respectively,with overall contraction induced by KCl（60 mmol/L）as 100%。3.Nifedipine（0.3 mmol/L）inhibited the BaCl₂-induced contraction by（87.82±5.43）%（P<0.01）.NFA（10,30,100μmol/L）,NPPB（10,30,100μmol/L）shifted the concentration-contraction curve rightwards.NFA（30,100μmol/L）depressed the BaCl₂-induced contraction by（28.29±5.37）%（P<0.05）,（82.24±7.69）%（P<0.01）,respectively.The value of IC₅₀0 was 56.34μmol/L.NPPB（30,100μmol/L）depressed the BaCl₂-induced maximal contraction by（40.45±6.91）%（P<0.05）,（90.83±10.42）%（P<0.01）,respectively.The value of IC₅₀0 was 37.81μmol/L。4.Indomethacin（10,30,100μmol/L）shifted the concentration curve rightwards concentration dependently and inhibited the maximal of the contraction.Indomethacin（100μmol/L）decreased the BaCl₂-induced maximal contraction by（73.23±5.47）%（P<0.01）.The value of IC₅₀0 was 93.42μmol/L.5.PD98059（3μmol/L）、SB239063（3μmol/L）inhibited the BaCl₂-induced maximal contraction by（40.62±5.29）%（P<0.05）and（54.95±8.32）%（P<0.05）,respectively.Conclusions:1.BaCl₂（0.1-1.0 mmol/L）induces vasoconstriction in quiescent RCA concentration-dependently.2.Ca²⁺needed for BaCl₂-induced coronary vasoconstriction is mainly dependent on[Ca²⁺]_o influx and small part of it is released from[Ca²⁺]_i stores.3.Prostanoid synthesis increase,activation of chloride and L-type voltage-gated Ca²⁺channels,as well as activation of MAPK pathway may be involved in BaCl₂-induced contraction.