Centro de Investigaciones Cardiovasculares, Facultad de Ciencias Médicas, Universidad Nacional de La Plata, La Plata, Argentina.
J Mol Cell Cardiol. 2010 Nov;49(5):812-8. doi: 10.1016/j.yjmcc.2010.07.018. Epub 2010 Aug 6.
The Na(+)/HCO(3)(-) cotransporter (NBC) plays an important role in intracellular pH (pH(i)) regulation in the heart. In the myocardium co-exist the electrogenic (eNBC) and electroneutral (nNBC) isoforms of NBC. We have recently reported that angiotensin II (Ang II) stimulated total NBC activity during the recovery from intracellular acidosis through a reactive oxygen species (ROS) and ERK-dependent pathway. In the present work we focus our attention on eNBC. In order to study the activity of the eNBC in isolation, we induced a membrane potential depolarization by increasing extracellular K(+) K(+) from 4.5 to 45 mM (K(+) pulse). This experimental protocol enhanced eNBC driving force leading to intracellular alkalization (0.19 ± 0.008, n=6; data expressed as an increase of pH(i) units after 14 min of applying the K(+) pulse). This alkalization was completely abrogated by the NBC blocker S0859 (-0.004 ± 0.016*, n=5; * indicates p<0.05 vs control) but not by the Na(+)/H(+) exchanger blocker HOE642 (0.185 ± 0.04, n=4), indicating that we are exclusively measuring eNBC. The K(+) pulse induced alkalization was canceled by 100 nM Ang II (-0.008 ± 0.018*; n=5). This inhibitory effect was prevented when the myocytes were incubated with losartan (AT(1) receptor blocker, 0.18 ± 0.02; n=4) or SB202190 (p38 MAP kinase inhibitor, 0.25 ± 0.06; n=5). Neither chelerythrine (PKC inhibitor, -0.06 ± 0.04*; n=4), nor U0126 (ERK inhibitor, -0.07 ± 0.04*; n=4) nor MPG (ROS scavenger, -0.02 ± 0.05*; n=8) affected the Ang II-induced inhibition of eNBC. The inhibitory action of Ang II on eNBC was corroborated with perforated patch-clamp experiments, since no impact of the current produced by eNBC on action potential repolarization was observed in the presence of Ang II. In conclusion, we propose that Ang II, binding to AT(1) receptors, exerts an inhibitory effect on eNBC activity in a p38 kinase-dependent manner.
钠/碳酸氢盐共转运体（NBC）在心脏细胞内 pH 值（pH（i））调节中发挥重要作用。在心肌中同时存在电活性（eNBC）和电中性（nNBC）NBC 同工型。我们最近报道，血管紧张素 II（Ang II）通过活性氧（ROS）和 ERK 依赖性途径刺激细胞内酸中毒恢复期间的总 NBC 活性。在本工作中，我们将注意力集中在 eNBC 上。为了分离研究 eNBC 的活性，我们通过将细胞外 K+（[K+]（o））从 4.5 增加到 45 mM 来诱导膜电位去极化（K+脉冲）。该实验方案增强了 eNBC 的驱动力，导致细胞内碱化（0.19 ± 0.008，n=6；数据表示为施加 K+脉冲 14 分钟后 pH（i）单位的增加）。NBC 阻断剂 S0859 完全阻断了这种碱化（-0.004 ± 0.016*，n=5；表示与对照相比 p<0.05），而 Na+/H+交换体阻断剂 HOE642 则没有（0.185 ± 0.04，n=4），表明我们正在专门测量 eNBC。100 nM Ang II 可取消 K+脉冲诱导的碱化（-0.008 ± 0.018；n=5）。当心肌细胞用氯沙坦（AT1 受体阻断剂，0.18 ± 0.02；n=4）或 SB202190（p38 MAP 激酶抑制剂，0.25 ± 0.06；n=5）孵育时，这种抑制作用得到预防。Chelerythrine（PKC 抑制剂，-0.06 ± 0.04*；n=4）、U0126（ERK 抑制剂，-0.07 ± 0.04*；n=4）或 MPG（ROS 清除剂，-0.02 ± 0.05*；n=8）均不影响 Ang II 对 eNBC 的抑制作用。Ang II 对 eNBC 的抑制作用通过穿孔膜片钳实验得到证实，因为在 Ang II 存在下，eNBC 产生的电流对动作电位复极化没有影响。总之，我们提出 Ang II 与 AT1 受体结合，以依赖 p38 激酶的方式对 eNBC 活性发挥抑制作用。
