Kang Misuk, Walker Jeffery W
Department of Physiology, Molecular and Cellular Pharmacology Training Program, University of Wisconsin, 1300 University Avenue, Madison, Wisconsin 53706, USA.
Exp Biol Med (Maywood). 2006 Jun;231(6):865-70.
It has been proposed that intracellular alkalinization underlies the enhanced contractility of ventricular myocytes exposed to endothelin (ET)-1. The effects of ET-1 on the contractility and intracellular pH (pH(i)) were examined here in cultured adult rat ventricular myocytes by employing the pH-sensitive fluorescent dye SNARF-1. Variable pH(i) changes were observed on ET-1 stimulation. Most myocytes (n = 20 of 32) did not alkalinize, but showed an approximate 60% increase in twitch amplitude in response to ET-1. In the remaining myocytes (12 of 32), ET-1 induced an increase in pH(i) by 0.05 +/- 0.02 pH units with a similar approximate 60% increase in twitch amplitude. Therefore, there was no strong correlation between ET-1-mediated positive inotropy (enhanced contractility) and intracellular alkalinization. To determine whether ET-1 contractile and pH(i) responses were mediated by protein kinase C (PKC), yellow fluorescent protein (YFP)-fused dominant negative (dn) PKC constructs were used as isoform specific inhibitors. In dn-PKC-epsilon-YFP-expressing myocytes, the ET-1-mediated positive inotropic response was greatly diminished to 13 +/- 15%, but alkalinization was still observed. Expression of dn-PKC-delta-YFP also did not block alkalinization, but in this case the positive inotropic response was still observed. In a previous study, we showed that expression of PKC-delta and PKC-epsilon caused a strong positive inotropy on stimulation with phorbol 12,13-dibutyrate (PDBu). Using this system, PDBu failed to affect pH(i) in the majority of PKC expressing myocytes despite increases in twitch amplitudes of >60%. Overall, the poor correlation of positive inotropic responses and alkalinization was observed for ET-1 with and without dn-PKC constructs and for PDBu with and without wild-type PKC constructs. These results suggest that ET-1 produces positive inotropy via PKC-epsilon by mechanisms other than intracellular alkalinization.
有人提出，细胞内碱化是内皮素（ET）-1作用下心室肌细胞收缩性增强的基础。在此，我们通过使用pH敏感荧光染料SNARF-1，研究了ET-1对培养的成年大鼠心室肌细胞收缩性和细胞内pH（pH(i)）的影响。在ET-1刺激下观察到了不同的pH(i)变化。大多数心肌细胞（32个中有20个）没有碱化，但对ET-1的反应中抽搐幅度增加了约60%。在其余的心肌细胞（32个中有12个）中，ET-1使pH(i)升高了0.05±0.02个pH单位，抽搐幅度也有类似的约60%的增加。因此，ET-1介导的正性肌力作用（增强的收缩性）与细胞内碱化之间没有很强的相关性。为了确定ET-1的收缩和pH(i)反应是否由蛋白激酶C（PKC）介导，将黄色荧光蛋白（YFP）融合的显性负性（dn）PKC构建体用作亚型特异性抑制剂。在表达dn-PKC-ε-YFP的心肌细胞中，ET-1介导的正性肌力反应大大降低至13±15%，但仍观察到碱化现象。dn-PKC-δ-YFP的表达也没有阻止碱化，但在这种情况下仍观察到正性肌力反应。在先前的一项研究中，我们表明PKC-δ和PKC-ε的表达在用佛波醇12,13-二丁酸酯（PDBu）刺激时会引起强烈的正性肌力作用。使用该系统，尽管抽搐幅度增加>60%，但PDBu在大多数表达PKC的心肌细胞中未能影响pH(i)。总体而言，无论有无dn-PKC构建体的ET-1以及无论有无野生型PKC构建体的PDBu，都观察到正性肌力反应与碱化之间的相关性较差。这些结果表明，ET-1通过PKC-ε产生正性肌力作用，其机制并非细胞内碱化。
