抗钠-钙交换体α-1(106-145)抗体对大鼠离体心功能的影响及其机制

Effects of antibody against α-1_(( 106-145)) on cardiac function in isolated rat hearts

目的观察1～103nmol·L-1抗钠-钙交换体(NCX)α-1(106-145)肽段抗体对离体大鼠心功能的影响并分析其作用机制。方法用化学合成的NCXα-1(106-145)肽段主动免疫新西兰大耳白兔制备并纯化抗体,利用Langendorff离体灌流系统观察其对大鼠离体心功能的影响;利用全细胞膜片钳技术观察其对心肌细胞钠-钙交换电流、L-型钙电流、瞬时外向钾电流和内向整流钾电流的影响。结果经主动免疫后兔体内抗α-1(106-145)抗血清滴度明显升高,Protein A纯化后抗体浓度为13.1 g.L-1。与对照组相比,1～10 nmol·L-1α-1抗体可使大鼠离体心脏左室发展压(LVDP)和左室压最大上升速率(+dp/dtmax)明显增加(P<0.01),并使左室压最大下降速率(-dp/dtmax)减小(P<0.01),且KB-R7943(2μmol·L-1)可使α-1抗体(1 nmol·L-1)的上述效应进一步增强。然而,102～103nmol·L-1α-1抗体则可使大鼠离体心脏LVDP、±dp/dtmax呈不同程度地下降(P<0.01)。全细胞膜片钳实验结果表明,在1～103nmol·L-1浓度范围内,抗α-1抗体对大鼠心肌细胞外向和内向Na+/Ca2+交换电流均表现出剂量依赖性的抑制效应,对瞬时外向钾电流和内向整流钾电流均无明显影响。此外,102～103nmol·L-1α-1抗体对L-型钙电流也具有抑制作用。结论低浓度α-1抗体(1～10 nmol·L-1)可明显增强心肌收缩,这一效应与其在低浓度时专一性抑制Na+/Ca2+交换电流有关;高浓度α-1抗体(102～103nmol·L-1)则可同时抑制心肌收缩和舒张,该作用与其同时抑制Na+/Ca2+交换电流和L-型钙电流有关。

Aim To investigate the effects of antibody against α-1（106-145） of Na＋/Ca2＋ exchanger on cardiac function in isolated rat hearts and its mechanism. Methods Rabbits were actively immunized with synthesized α-1（106-145） peptide and the positive antisera were affinity-purified. Effects of the antibody against α-1 （106-145） on cardiac function were observed in Lange- ndorff-perfused isolated hearts. Whole-cell patch clamp technique was used to investigate the effects of the antibody against α-1 （106-145） on INa/Ca ,ICa-L, Ito and IK1 in rat ventricular myocytes. Results The titer of the antibody increased obviously in α-1 （106-145） immunized rabbits and the concentration of the purified antibody was 13.1 g·L^-1. Compared with control group, the antibody at 1 -10 nmol·L-1 increased left ventricular developed pressure （LVDP） , the maximal rising rate of left ven- tricular pressure （ ＋ dp/dtmax ） , and decreased the maximal falling rate of left ventricular pressure （ - dp/ dtmax） in Langendorff-perfused isolated hearts （P 〈 0.01 ）. The above effects of α-1 antibody （ 1 nmol·L-l） were further strengthened by application of KB- R7943（2 μmol · L-1）. At the concentration of 102 - 103 nmol · L-1 of the antibody, however, values of LVDP and ＋ dp/dtmax all diminished（P 〈0.01 ）. Electrophysiological experimental results showed that at 1 - 103 nmol · L-1, the antibody against α-1（106-145） decreased INa/Ca concentration-dependently in rat ventricu- lar myocytes without obvious effects on Ito and Ikl. At the concentration of 102 - 103 nmol·L-1 , however, the antibody also inhibited /Ca-L besides decreasing IN＋/Ca＋. Conelusion The antibody against α-1 （106-145） of Na＋/Ca2＋ exchanger promotes cardiac contraction via inhibiting Na＋/Ca2＋ exchange activity specifically in Langendorff - perfused rat hearts at low concentration of 1 -10 nmol·L-1, whereas at high concentration of 102 - 103 nmol·L-1 , the antibody depresses both cardiac contraction and relaxation through inhibition of Na＋/Ca2＋ exchanger and ICa-L simultaneously.