高糖环境中培养的小鼠心肌微血管内皮细胞蛋白激酶C活性、A激酶锚定蛋白150表达变化
|
摘要
目的观察高糖预处理的小鼠心肌微血管内皮细胞蛋白激酶C(PKC)活性变化和A激酶锚定蛋白150(AKAP150)表达变化,并探讨其意义。方法 8周龄雄性C57BL小鼠,分离并培养左心室心肌微血管内皮细胞。将心肌微血管内皮细胞分为观察组和对照组,分别为含有25 mmol/L葡萄糖、5 mmol/L葡萄糖培养基。培养24 h时采用非放射性PKC活性试剂盒检测两组细胞PKC活性,采用Western blotting法检测细胞AKAP150。运用免疫荧光技术观察细胞AKAP150与PKC结合情况。结果观察组、对照组细胞PKC活性分别为0.231±0.037、0.143±0.047,二者比较,P<0.05;观察组、对照组细胞AKAP150蛋白相对表达量分别为0.527±0.080、0.124±0.038,二者比较,P<0.05。免疫荧光显示观察组细胞AKAP150、PKC结合量多于对照组。结论 PKC活性水平、AKAP150表达在高糖预处理的小鼠心肌微血管内皮细胞中均升高,且结合明显增多。PKC活性水平及AKAP150表达升高可能在高糖导致的小鼠心肌微血管内皮细胞损伤中起重要作用。
Objective To observe the activation of protein kinase C(PKC) and expression of A-kinase anchor protein150(AKAP150) in cardiac microvascular endothelial cells of mice pretreated with high glucose,and to investigate its significance. Methods The left ventricular cardiac microvascular endothelial cells were isolated from 8-week old male C57 BL mice. The cultured cardiac microvascular endothelial cells were randomized into two groups: the control group(n = 5)which was cultured in the normal medium with 5 mmol/L glucose,and the observation group(n = 5) that was cultured in the medium supplemented with 25 mmol/L glucose for 24 h. We used non-radioactive PKC assay to detect PKC activity and Western blotting to measure AKAP150 expression. The subcellular co-localization of AKAP150 and PKC was examined by immunofluorescence. Results The PKC activity of cardiac microvascular endothelial cells in the observation group was0. 231 ± 0. 037,which was higher than that of the control group(0. 143 ± 0. 047)(P < 0. 05). The expression of AKAP150 protein in the observation group was 0. 527 ± 0. 080,which was higher than that in the control group(0. 124 ±0. 038)(P < 0. 01). The co-localizaiton of AKAP150 and PKC in the observation group was more than that in the control group. Conclusions The PKC activity and AKAP150 expression are both increased in the cardiac microvascular endothelial cells of mice pretreated with high glucose,and the co-localization of PKC and AKAP150 is also significantly increased.The increase of PKC activity and AKAP150 expression may play a major role in the injury of mouse cardiac microvascular endothelial cells induced by high glucose.
Objective To observe the activation of protein kinase C(PKC) and expression of A-kinase anchor protein150(AKAP150) in cardiac microvascular endothelial cells of mice pretreated with high glucose,and to investigate its significance. Methods The left ventricular cardiac microvascular endothelial cells were isolated from 8-week old male C57 BL mice. The cultured cardiac microvascular endothelial cells were randomized into two groups: the control group(n = 5)which was cultured in the normal medium with 5 mmol/L glucose,and the observation group(n = 5) that was cultured in the medium supplemented with 25 mmol/L glucose for 24 h. We used non-radioactive PKC assay to detect PKC activity and Western blotting to measure AKAP150 expression. The subcellular co-localization of AKAP150 and PKC was examined by immunofluorescence. Results The PKC activity of cardiac microvascular endothelial cells in the observation group was0. 231 ± 0. 037,which was higher than that of the control group(0. 143 ± 0. 047)(P < 0. 05). The expression of AKAP150 protein in the observation group was 0. 527 ± 0. 080,which was higher than that in the control group(0. 124 ±0. 038)(P < 0. 01). The co-localizaiton of AKAP150 and PKC in the observation group was more than that in the control group. Conclusions The PKC activity and AKAP150 expression are both increased in the cardiac microvascular endothelial cells of mice pretreated with high glucose,and the co-localization of PKC and AKAP150 is also significantly increased.The increase of PKC activity and AKAP150 expression may play a major role in the injury of mouse cardiac microvascular endothelial cells induced by high glucose.
引文
[1]Yang W,Lu J,Weng J,et al.Prevalence of diabetes among men and women in China[J].N Engl J Med,2010,362(12):1090-1101.
[2]Szuszkiewicz-Garcia MM,Davidson JA.Cardiovascular Disease in Diabetes Mellitus:Risk Factors and Medical Therapy[J].Endocrinol Metab Clin North Am,2014,43(1):25-40.
[3]Lam DW,LeRoith D.The worldwide diabetes epidemic[J].Curr Opin Endocrinol Diabetes Obes,2012,19(2):93-96.
[4]Adameova A,Dhalla NS.Role of microangiopathy in diabetic cardiomyopathy[J].Heart Fail Rev,2014,19(1):25-33.
[5]Verma SK,Deshmukh V,Liu P,et al.Reactivation of fetal splicing programs in diabetic hearts is mediated by protein kinase C signaling[J].Jour Bio Chem,2013,288(49):35372-35386.
[6]Zeng C,Wang J,Li N,et al.AKAP150 mobilizes c PKC-dependent cardiac glucotoxicity[J].Am J Physiol Endocrinol Metab,2014,307(4):384-397.
[7]Kuusisto J,Laakso M.Update on type 2 diabetes as a cardiovascular disease risk equivalent[J].Curr Cardiol Rep,2013,15(2):331.
[8]Geraldes P,King GL.Activation of protein kinase C isoforms and its impact on diabetic complications[J].Cir Res,2010,106(8):1319-1331.
[9]Hoque M,Rentero C,Cairns R,et al.Annexins-Scaffolds modulating PKC localization and signaling[J].Cell Signal,2014,26(6):1213-25.
[10]Mishra PK,Ying W,Nandi SS,et al.Diabetic cardiomyopathy:an immunometabolic perspective[J].Front Endo,2017,8:72-84.
[11]Gaudreault N,Perrin RM,Guo M,et al.Counter regulatory effects of PK Cbeta II and PKCdelta on coronary endothelial permeability[J].Arterioscler Thromb Vasc Biol,2008,28(8):1527-1533.
[12]Carnegie GK,Means CK,Scott JD.A-kinase anchoring proteins:from protein complexes to physiology and disease[J].IUBMB Life,2009,61(4):394-406.
[13]Greenwald EC,Saucerman JJ.Bigger,better,faster:principles and models of AKAP anchoring protein signaling[J].Jour Card P harm,2011,58(5):462-469.
[14]Sanderson JL,Gorski JA,Gibson ES,et al.AKAP150-anchored calcineurin regulates synaptic plasticity by limiting synaptic incorporation of Ca2+-permeable AMPA receptors[J].J Neurosci,2012,32(43):15036-15052.
[15]Efendiev R,Bavencoffe A,Hu H,et al.Scaffolding by A-kinase anchoring protein enhances functional coupling between adenylyl cyclase and TRPV1 channel[J].Jour Bio Chem,2013,288(6):3929-3937.
[16]Navedo MF,Nieves-Cintron M,Amberg GC,et al.AKAP150 is required for stuttering persistent Ca2+sparklets and angiotensin IIinduced hypertension[J].Cir Res,2008,102(2):1-11.
[2]Szuszkiewicz-Garcia MM,Davidson JA.Cardiovascular Disease in Diabetes Mellitus:Risk Factors and Medical Therapy[J].Endocrinol Metab Clin North Am,2014,43(1):25-40.
[3]Lam DW,LeRoith D.The worldwide diabetes epidemic[J].Curr Opin Endocrinol Diabetes Obes,2012,19(2):93-96.
[4]Adameova A,Dhalla NS.Role of microangiopathy in diabetic cardiomyopathy[J].Heart Fail Rev,2014,19(1):25-33.
[5]Verma SK,Deshmukh V,Liu P,et al.Reactivation of fetal splicing programs in diabetic hearts is mediated by protein kinase C signaling[J].Jour Bio Chem,2013,288(49):35372-35386.
[6]Zeng C,Wang J,Li N,et al.AKAP150 mobilizes c PKC-dependent cardiac glucotoxicity[J].Am J Physiol Endocrinol Metab,2014,307(4):384-397.
[7]Kuusisto J,Laakso M.Update on type 2 diabetes as a cardiovascular disease risk equivalent[J].Curr Cardiol Rep,2013,15(2):331.
[8]Geraldes P,King GL.Activation of protein kinase C isoforms and its impact on diabetic complications[J].Cir Res,2010,106(8):1319-1331.
[9]Hoque M,Rentero C,Cairns R,et al.Annexins-Scaffolds modulating PKC localization and signaling[J].Cell Signal,2014,26(6):1213-25.
[10]Mishra PK,Ying W,Nandi SS,et al.Diabetic cardiomyopathy:an immunometabolic perspective[J].Front Endo,2017,8:72-84.
[11]Gaudreault N,Perrin RM,Guo M,et al.Counter regulatory effects of PK Cbeta II and PKCdelta on coronary endothelial permeability[J].Arterioscler Thromb Vasc Biol,2008,28(8):1527-1533.
[12]Carnegie GK,Means CK,Scott JD.A-kinase anchoring proteins:from protein complexes to physiology and disease[J].IUBMB Life,2009,61(4):394-406.
[13]Greenwald EC,Saucerman JJ.Bigger,better,faster:principles and models of AKAP anchoring protein signaling[J].Jour Card P harm,2011,58(5):462-469.
[14]Sanderson JL,Gorski JA,Gibson ES,et al.AKAP150-anchored calcineurin regulates synaptic plasticity by limiting synaptic incorporation of Ca2+-permeable AMPA receptors[J].J Neurosci,2012,32(43):15036-15052.
[15]Efendiev R,Bavencoffe A,Hu H,et al.Scaffolding by A-kinase anchoring protein enhances functional coupling between adenylyl cyclase and TRPV1 channel[J].Jour Bio Chem,2013,288(6):3929-3937.
[16]Navedo MF,Nieves-Cintron M,Amberg GC,et al.AKAP150 is required for stuttering persistent Ca2+sparklets and angiotensin IIinduced hypertension[J].Cir Res,2008,102(2):1-11.