Prostanoids Contribute To Regulation Of Inwardly Rectifying K~+ Channels In Intrarenal Arterial Smooth Muscle Cells

Part 1.Factors affecting BaCl₂-induced constriction of intrarenal arteries in ratObjective:1.To study the effects of different types of potassium channel blockers on the vascular ring tension in isolated rat intrarenal arteries（RIRAs）:inward rectifier potassium channels（Kir）inhibitor（BaCl₂）,voltage-dependent K⁺channels（K_V）inhibitor4-aminopyridine（4-AP）,calcium-activated K⁺channels(K_Ca)inhibitor tetraethylamine（TEA）,and ATP-sensitive K⁺channels(K_ATP)inhibitor glibenclamide（GLI）.2.To observe the effect of endothelium denudation,nitric oxide synthase（NOS）inhibition（NG-nitro-L-arginine methylester ester,L-NAME）and cyclooxygenase（COX）inhibition indomethacin（INDO）,COX1 inhibitor S2064 and COX2 inhibitor N194 and thromboxane-prostanoid receptor（TPR）antagonism S18886 and L-655240 on BaCl₂-induced RIRA contraction.3.Observe the influence of various vasoconstrictors for BaCl₂-induced contraction in RIRAs:use potassium chloride（KCl,30 m M）,thromboxane A2 analogue（U46619,0.03（?）M）or phenylephrine（PE,0.1（?）M）make the blood vessel produces mild stimulus beforehand（equivalent to the biggest contraction of 5～20%）.Then,on the basis of pre-contraction,to study the effect of BaCl₂-induced constriction in rat intrarenal arterial.Methods:Adult male SD rats（280～320 g）by intraperitoneal injection of sodium pentobarbital（40 mg/kg）severed head bleeding to death after anesthesia,immediately open the abdominal cavity to take out the kidney tissue,and put it in 4℃,p H=7.40 cold HEPES fluid.Under a microscope to isolate the second and third in the renal artery,blood vessel diameter is about 150～280（?）m,and will be isolated from the blood vessels are divided into 2 mm vascular ring,after using two 40（?）m diameter stainless steel wire（length is 2cm）through the ring,try to avoid touching the blood vessel walls,reduce endothelial damage,and should be fixed to the microvascular ring tension recorder（DMT）in the bath（gently rubbing repeatedly with steel wire is clingy wall vessels can achieve the goal of removing endothelial）,after adjusting the distance between the two steel wire to standardization of vascular ring,make the ring to achieve equivalent to an in vivo blood pressure of 80 mm Hg preload.The blood vessels in the bath were incubated at 37℃,p H7.40,in normal physiological saline solution（PSS）saturated with 95%O₂+5%CO₂ for30 min,and the blood vessels were continuously stimulated with 60 m M KCl for 2 times.If the difference in the systolic intensity of RIRAs before and after two times was less than 10%,and the systolic intensity was greater than 2 m N,it indicated good vascular activity,and drug experiments could be conducted.After the blood vessels were stabilized,BaCl₂（0.03,0.1,0.3,1.0 m M）,4-AP（0.03,0.1,0.3,1.0 m M）,TEA（0.03,0.1,0.3,1.0 m M）,and GLI（3,10,30,100 m M）were added in the bath,and the tension changes were recorded.The accumulation of the next concentration can be carried out after the contraction of the previous concentration reaches the level table.Results of concentration-contraction curve were established with the maximum contraction induced by 60 m M KCl as 100%,and the tension changes of blood vessels induced by different potassium channel blockers at the maximum concentration were observed.BaCl₂（0.03,0.1,0.3,1.0 m M）was added in the bath of RIRAs with and without endothelium,respectively.The maximum contraction of BaCl₂ was set as 100%to make a concentration-contraction curve,and the percentage of vessel contraction caused by each concentration was calculated to observe the effect of endothelium on BaCl₂contraction.After the vessels were stabilized in the bath for 30 min,the concentration contraction curves of BaCl₂（0.03,0.1,0.3,1.0 m M）were constructed repeatedly.When the contraction curves were repeatable,Inhibitors INDO（0.01 m M）,L-NAME（0.01m M）,S2064（10（?）M）,N194（0.1（?）M）or vasoconstrictors PE（0.1（?）M）,KCl（30 m M）,U46619（0.03（?）M）or INDO+PE,L-NAME+PE were added into the bath 10 min before the next concentration-contraction curve of BaCl₂.The maximum contraction induced by BaCl₂ before incubation inhibitor or agitation was 100%to evaluate the effect of inhibitors and vasoconstrictors on the contraction induced by 0.1 m M BaCl₂.Respectively in the bath to add U46619（0.03,0.1,0.3,1.0（?）M）,PE（0.03,0.1,0.3,1.0（?）M）,KCl（20,40,60,80 m M）,BaCl₂（0.03,0.1,0.3,1.0 m M）,PE（0.1（?）M）+BaCl₂（0.03,0.1,0.3,1.0 m M）,when vascular contraction curve can be repeated,with normal PSS liquid washing blood vessels to restore its basic tension,Then the blood vessels were incubated in TPR antagonists L-655240（1.0（?）M）and S18886（10（?）M）for 10 min to reconstruct the concentration-contraction curves.The maximum contraction caused by each contractile agent before L-655240 and S18886 was 100%,and the changes of vascular tension corresponding to different concentrations of contractile agents after pre-incubation L-655240 and S18886 were calculated.Results:1.0.03～1.0 m M BaCl₂,K_V channel blocker 4-AP and K_Ca channel blocker TEA contracted the RIRA rings in a concentration-dependent manner,while K_ATP blocker GLI did not significantly affect the basal tone at up to 100μM.Compared with 4-AP,TEA and GLI,BaCl₂ contracted the artery most potently.The contractions induced by BaCl₂（1m M）,4-AP（1 m M）,TEA（1 m M）and GLI（0.1 m M）were 6.75±0.86 m N（70.5%of 60m M KCl induced contraction）,1.03±0.54 m N,1.87±0.75 m N and 0.35±0.21 m N respectively（P<0.05）.2.The result of endothelium denudation,L-NAME,COX inhibitors on BaCl₂ curves in the quiescent RIRAs shows that denudation increased the contraction induced by 0.1m M BaCl₂ from 1.07±0.42 m N to 2.01±0.03 m N（P<0.05）.In the quiescent endothelium-intact RIRAs,L-NAME（0.01 m M）shifted BaCl₂ curve upwards and increased the contraction induced by 0.1 m M BaCl₂ to 3.15±0.24 m N（P<0.05）.Contrary to L-NAME,indomethacin（10μM）shifted the curve downwards to the right and decreased the contraction induced by 0.1 m M BaCl₂ to 0.21±0.08 m N（P<0.05）.Similar to indomethacin,both COX1 inhibitor S2064 and COX2 inhibitor N194significantly depressed Ba²⁺-induced contraction（P<0.05）.3.Mild stimulations with vasoconstrictors tremendously boosted BaCl₂-induced RIRA contraction,especially at low concentrations of BaCl₂.The mild stimulation with PE（0.1μM）,KCl（30 m M）or U46619（0.03μM）increased 0.1 m M BaCl₂-induced contraction by 2.31-fold,2.15-fold and 2.89-fold（P<0.05）,respectively.Again,L-NAME potentiated,while indomethacin depressed BaCl₂-induced contraction in the RIRAs stimulated mildly with PE.4.TPR antagonists L-556240 and S18886 markedly depressed the contraction induced by TPR agonist U46619,while,in contrast,they showed much less inhibition on the contractions induced by PE or KCl.In the quiescent RIRAs,L-556240 and S18886reduced 1 m M BaCl₂-induced contraction from 6.03±1.78 m N to 3.14±0.67 m N and3.89±1.15 m N,respectively（P<0.05）.In the RIRAs stimulated with PE,L556240 and S18886 also significantly decreased BaCl₂-induced contraction.Conclusions:1.0.03～1.0 m M BaCl₂ concentration-dependently contracted RIRAs.2.Mild stimulations with various vasoconstrictors at low concentrations significantly potentiated RIRA contraction induced by Kir closure with BaCl₂.3.In both the quiescent and the stimulated RIRAs,COX inhibition and TPR antagonism depressed BaCl₂-induced RIRAs contraction,while NOS inhibition and endothelium-denudation enhanced the contraction.Part 2.Function and expression of Kir channel in smooth muscle cells of rat internal renal arteryObjective:1.To observe the effect of BaCl₂ on the intracellular calcium concentration([Ca²⁺]_i)in acutely isolated rat intrarenal artery smooth muscle cells（RIASMCs）.2.To study the effect of BaCl₂,TPR agonist U46619,NO donor sodium nitroprusside（Nitro）,and Kir2.1 Antibody（Kir2.1-Ab）on Kir current in RIASMCs.3.Western blotting was used to observe the expression of Kir channels in RIRAs.Methods:1.Cell preparation:The RIRAs were dissected into enzymolysis liquidⅠcontaining1 mg/ml bovine serum albumin,0.5 mg/ml papain,1 mg/ml albumin and incubated for28～30 min at 37°C bubbled with O₂.And then,the arteries were transferred to the enzymolysis liquidⅡcontaining 0.5 mg/ml collagen H,0.5 mg/ml collagen F and 1mg/ml bovine serum albumin incubated for 5 min at 37°C and bubbled with O₂.Single cells were dispersed mechanically by gently triturating through a Pasteur pipette.Morphologically identified ASMCs were used immediately for[Ca²⁺]_i measurement or patch clamp study.2.[Ca²⁺]_imeasurement:Freshly isolated RIASMCs were loaded with fluo4-AM（5μM）in normal HBSS solution for 30 min at 37°C.ASMCs were placed in a laminar flow cover glass cell chamber,mounted on the stage of the Nikon inverted microscope and perfused with normal oxygen-saturated HBSS solution for 15 min at 37°C to remove the extracellular dye.The images of ASMCs were acquired with Live-Scan Field Swept Field Confocal Microscope（Nikon,Japan）at a frequency of 10 frames/min for a period of 40 min.The excitation beam of 488 nm was produced by a solid-state diode laser.Emitted fluorescence intensities were measured by averaging 10 frames in areas（2.1μm×2.1μm）at wavelengths 516～530 nm and analyzed with NIS-Elements Ar.Fluorescence ratios（F/F_o,fluorescence intensity after treatment vs the base fluorescence intensity before the treatment）were calculated.3.Patch clamp study:Kir currents were recorded at 23～26°C.Briefly,the cells were held at a holding potential of-60 m V and subjected to step depolarization of 500ms from-140 m V to+20 m V in 20 m V increment.Whole-cell currents were recorded in the absence or the presence of BaCl₂,U46619 or sodium nitroprusside in the bath solution.The effect of Kir2.1 antibody on Kir currents was also observed with pipette-filling solution containing Kir2.1 antibody（1:500）.The current density was calculated by a ratio of the average plateau phase Kir currents to the cell capacitance（p A/p F）.4.Western blotting:Endothelium-denuded RIRAs with an inner diameter of150～200（?）m and 200～280μm were used to measure Kir2.1 protein with Western blot assay.Collective data were presented as means±SD of 6 pooled samples.Each pooled sample was collected from 8 arteries of 8 separate animals.RIRA sample lysis was performed for 30 min at 4℃with RIPA,PMSF,protease inhibitor cocktail and phosphatase inhibitor mixture.The homogenate was cleared of debris by centrifugation at 13,000×g at 4°C for 20 min.Protein concentration was determined using Bio-Rad protein assay solution with BSA as standard.Total protein（24μg）was separated on a 15%Tris-HCl polyacrylamide gel and transferred onto nitrocellulose blotting membranes.The membranes were blocked in a blocking buffer,TBST containing 5%non-fat milk,at room temperature for 2 h.The membranes were then incubated with anti-Kir2.1 antibody（1:5000,rabbit monoclonal）at 4°C overnight.Then,the membranes were washed three times with TBST for 10 min and incubated with horseradish peroxidase-conjugated secondary antibody（1:5000）for 1 h.Then the membranes were washed three times with10 min intervals in TBST,the bound antibodies were detected with an enhanced chemiluminescent（ECL）reagent using a chemiluminescence detection system（Bio-Rad,USA）.The amount of Kir2.1 protein was expressed as relative abundance normalized toβ-actin（1:4000,anti-rabbit Ig G antibody）.Results:1.0.03～1.0 m M,BaCl₂ concentration-dependently increased[Ca²⁺]_i-sensitive fluorescence intensity.Fluorescence intensity increased rapidly after each addition of BaCl₂ and declined slightly in most cells.Taking the intensity prior to the treatment with BaCl₂ as 1.0（control）,the maximal fluorescence intensity within the cell exposed to 1.0m M BaCl₂ was 2.27±0.48 folds（P<0.05）.Effect of BaCl₂ on[Ca²⁺]_iwas reversible,since the intracellular fluorescence intensity restored to the baseline when BaCl₂ was washed out with fresh drug-free HEPES solution.2.Western blotting show that Kir2.1 was expressed in RIRAs and the expression was more abundant in the smaller RIRAs（<200μm in diameter）.Patch clamp measurement detected the inwardly rectifying and Ba²⁺-sensitive currents in RIASMCs.The study show that at-140 m V,the Kir current density was 8.81±0.19 p A/p F.BaCl₂100（?）M and TPR agonist U46619 3μM depressed the current density to 2.62±0.15p A/p F,4.48±0.21 p A/p F（P<0.05）respectively,while NO donor sodium nitroprusside10μM augmented the current density to 10.93±0.62 p A/p F（P<0.05）.Kir2.1 antibody（1:500 in the pipette solution）reduced the current density to 5.61±0.42 p A/p F（P<0.05）.Conclusions:1.0.03～1.0 m M BaCl₂ concentration-dependently elevated[Ca²⁺]_i levels.2.Kir2.1 expression was significantly more abundant in smaller RIRAs.Ba²⁺-sensitive Kir currents were depressed by TPR agonist U46619 while increased by NO donor sodium nitroprusside.