Vasopressin stimulates action potential firing by protein kinase C-dependent inhibition of KCNQ5 in A7r5 rat aortic smooth muscle cells
Vasopressin stimulates action potential firing by protein kinase C-dependent inhibition of KCNQ5 in A7r5 rat aortic smooth muscle cells
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DOI：
10.1152/ajpheart.00065.2006
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发表时间：
2007-03-01
影响因子：
4.8
通讯作者：
Byron, Kenneth L.
中科院分区：
文献类型：
--
作者：
Brueggemann, Lioubov I.;Moran, Christopher J.;Byron, Kenneth L.
[Arg(8)]-加压素(AVP)在低浓度(10-500 pm)时，可刺激A7r5大鼠主动脉平滑肌细胞内钙离子浓度(钙峰)的振荡。我们以前的研究提供了生化证据，表明蛋白激酶C(PKC)的激活和电压敏感K+(K-v)通道的磷酸化是这一过程中的关键步骤。本研究采用膜片钳技术测定了Kv电流(I-Kv)和膜电位。100 PM AVP对A7r5细胞的I-Kv有明显的抑制作用。这种效应与同一细胞中逐渐的膜去极化、膜电阻增加和动作电位(AP)的产生有关。对AVP敏感的I-Kv对4-氨基吡啶、伊比利亚毒素和格列本脲耐药，但可被选择性KCNQ通道阻断剂linopirdine(10mU M)和XE-991(10mU M)完全抑制，并被KCNQ通道激动剂flupirtin(10mU M)增强。BaCl2(100 MU M)或利诺匹定(5 MU M)模拟AVP对K+电流、AP产生和Ca~(2+)峰的影响。RT-PCR检测KCNQ5在A7r5细胞和新鲜分离的大鼠主动脉平滑肌中的表达。针对KCNQ5的RNA干扰降低了KCNQ5蛋白的表达，并导致A7r5细胞的I-Kv显著降低。PKC激活剂4β-佛波醇12-肉豆蔻酸酯13-乙酸酯(10 NM)也抑制I-Kv，而PKC抑制剂Calphostin C(250 NM)可阻断AVP对I-Kv的抑制作用。这些结果提示，生理浓度的AVP对钙离子峰电流的刺激涉及对KCNQ5通道的PKC依赖性抑制和对A7r5细胞AP放电的增加。
[Arg(8)]-vasopressin (AVP), at low concentrations (10-500 pM), stimulates oscillations in intracellular Ca2+ concentration (Ca2+ spikes) in A7r5 rat aortic smooth muscle cells. Our previous studies provided biochemical evidence that protein kinase C (PKC) activation and phosphorylation of voltage-sensitive K+ (K-v) channels are crucial steps in this process. In the present study, K-v currents (I-Kv) and membrane potential were measured using patch clamp techniques. Treatment of A7r5 cells with 100 pM AVP resulted in significant inhibition of I-Kv. This effect was associated with gradual membrane depolarization, increased membrane resistance, and action potential (AP) generation in the same cells. The AVP-sensitive I-Kv was resistant to 4-aminopyridine, iberiotoxin, and glibenclamide but was fully inhibited by the selective KCNQ channel blockers linopirdine (10 mu M) and XE-991 (10 mu M) and enhanced by the KCNQ channel activator flupirtine (10 mu M). BaCl2 (100 mu M) or linopirdine (5 mu M) mimicked the effects of AVP on K+ currents, AP generation, and Ca2+ spiking. Expression of KCNQ5 was detected by RT-PCR in A7r5 cells and freshly isolated rat aortic smooth muscle. RNA interference directed toward KCNQ5 reduced KCNQ5 protein expression and resulted in a significant decrease in I-Kv in A7r5 cells. I-Kv was also inhibited in response to the PKC activator 4 beta-phorbol 12-myristate 13-acetate (10 nM), and the inhibition of I-Kv by AVP was prevented by the PKC inhibitor calphostin C ( 250 nM). These results suggest that the stimulation of Ca2+ spiking by physiological concentrations of AVP involves PKC-dependent inhibition of KCNQ5 channels and increased AP firing in A7r5 cells.