van Heugten H A, de Jonge H W, Bezstarosti K, Lamers J M
Department of Biochemistry, Cardiovascular Research Institute (COEUR), Faculty of Medicine, Erasmus University Rotterdam, The Netherlands.
J Mol Cell Cardiol. 1994 Aug;26(8):1081-93. doi: 10.1006/jmcc.1994.1128.
Cultured neonatal rat cardiac myocytes have been utilized as a model for the study of the effect of variations in cytoplasmic free Ca2+ on the activity of phospholipase C, a key enzyme in agonist-stimulated signal transduction through the phosphoinositide pathway. Cells prelabelled with [3H]inositol were exposed to various agents in an attempt to modulate the cytoplasmic free Ca2+ concentration and the formation of [3H]inositolphosphates (15-30 min) in the presence of Li+ was taken as a measure of phospholipase C activity. Not the basal but the endothelin-1 (10(-8) M) induced [3H]inositolphosphate production (15 min) was stimulated 1.54- and 1.43-fold by A23187 (10 microM external Ca2+) and 50 mM K+ (1.3 mM external Ca2+) treatment of cells, respectively. The phenylephrine (10(-4) M) induced response was also stimulated (1.35-fold) by A23187, however it was 43% inhibited by high K+. Ouabain (10 microM) treatment of cells did not affect either basal or agonist stimulated phosphoinositide turnover. On the other hand, total removal of external free Ca2+ by addition of 50 microM ethylene glycol bis(beta-aminoethyl ether) (N,N,N',N'-tetraacetic acid strongly inhibited (75%) the endothelin-1 induced but not the basal phospholipase C activity. Endothelin-1 binding to its receptor was shown not to be inhibited by the absence of external Ca2+ while resynthesis of [3H]phosphatidylinositol 4,5-bisphosphate was not rate-limiting under this condition. The lack of external Ca2+ eventually resulted in total standstill of the ET-1 induced PtdIns turnover after 30 min. Although not always as predicted, effects on basal and agonist-activated phospholipase C were observed too when cells were treated with low Ca2+ medium, Ca2+ entry blocker nifedipine (1 microM) or Ca(2+)-channel agonist Bay K8644 (1 microM) but most of these effects were only seen after 90 min incubation. Fluorometric (fura-2) measurements showed that total removal of external free Ca2+ for a short period decreased, while short exposure to high K+ increased cytoplasmic free Ca2+ but neither Ca2+ free buffer or nifedipine nor Bay K8644 had any effect. Furthermore, in saponin-permeabilized cardiomyocytes we could demonstrate that basal as well as GTP gamma S (30 microM) stimulated phospholipase C activity was strongly activated by free Ca2+ in the concentration range of 0.1-10 microM. We conclude that in the intact cardiomyocyte the signalling pathway through phospholipase C/phosphatidylinositol 4,5-bisphosphate, stimulated by agonist-receptor interaction that activates GTP-binding proteins as does GTP gamma S, is likely be a Ca2+ dependent process.
培养的新生大鼠心肌细胞已被用作模型，用于研究细胞质游离Ca2+变化对磷脂酶C活性的影响，磷脂酶C是通过磷酸肌醇途径进行激动剂刺激信号转导的关键酶。用[3H]肌醇预标记的细胞暴露于各种试剂中，试图调节细胞质游离Ca2+浓度，并将在Li+存在下[3H]肌醇磷酸酯的形成（15 - 30分钟）作为磷脂酶C活性的指标。不是基础而是内皮素-1（10(-8) M）诱导的[3H]肌醇磷酸酯产生（15分钟），分别通过用A23187（10 microM细胞外Ca2+）和50 mM K+（1.3 mM细胞外Ca2+）处理细胞而被刺激1.54倍和1.43倍。苯肾上腺素（10(-4) M）诱导的反应也被A23187刺激（1.35倍），然而它被高K+抑制43%。哇巴因（10 microM）处理细胞对基础或激动剂刺激的磷酸肌醇周转均无影响。另一方面，通过添加50 microM乙二醇双（β-氨基乙基醚）（N,N,N',N'-四乙酸）完全去除细胞外游离Ca2+强烈抑制（75%）内皮素-1诱导的但不抑制基础磷脂酶C活性。已表明缺乏细胞外Ca2+不会抑制内皮素-1与其受体的结合，而在此条件下[3H]磷脂酰肌醇4,5-二磷酸的重新合成不是限速步骤。缺乏细胞外Ca2+最终导致30分钟后ET-1诱导的磷脂酰肌醇周转完全停止。尽管并不总是如预期的那样，但当用低Ca2+培养基、Ca2+进入阻滞剂硝苯地平（1 microM）或Ca(2+)-通道激动剂Bay K8644（1 microM）处理细胞时，也观察到了对基础和激动剂激活的磷脂酶C的影响，但这些影响大多仅在孵育90分钟后才出现。荧光（fura-2）测量表明，短时间完全去除细胞外游离Ca2+会降低，而短时间暴露于高K+会增加细胞质游离Ca2+，但无Ca2+缓冲液、硝苯地平或Bay K8644均无任何影响。此外，在皂素通透的心肌细胞中，我们可以证明基础以及GTPγS（30 microM）刺激的磷脂酶C活性在0.1 - 10 microM的游离Ca2+浓度范围内被强烈激活。我们得出结论，在完整的心肌细胞中，通过磷脂酶C/磷脂酰肌醇4,5-二磷酸的信号通路，由激活GTP结合蛋白（如GTPγS）的激动剂-受体相互作用所刺激，可能是一个Ca2+依赖的过程。
