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?150-260?m?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?40?m?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(0.3?mol/L,an L-type voltage-gated Ca²⁺channel blocker)?PD98059?3?mol/L,an ERK1/2 inhibitor??SB239063?3?mol/L,a p38MAPK inhibitor??cyclooxygenase inhibitor indomethacin?10,30,100?mol/L??chloride channel blockers NFA?10,30,100?mol/L?and NPPB?10,30,100?mol/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?10,30,100?mol/L?,NPPB?10,30,100?mol/L?shifted the concentration-contraction curve rightwards.NFA?30,100?mol/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 56.34?mol/L.NPPB?30,100?mol/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 37.81?mol/L?4.Indomethacin?10,30,100?mol/L?shifted the concentration curve rightwards concentration dependently and inhibited the maximal of the contraction.Indomethacin?100?mol/L?decreased the BaCl₂-induced maximal contraction by?73.23±5.47?%?P<0.01?.The value of IC₅₀0 was 93.42?mol/L.5.PD98059?3?mol/L??SB239063?3?mol/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.