Pathophysiological concentrations of lysophosphatides and the slow response.
Pathophysiological concentrations of lysophosphatides and the slow response.
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溶血磷脂的病理生理浓度和反应缓慢。
DOI：
10.1152/ajpheart.1982.243.2.h187
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发表时间：
1982
期刊：
影响因子：
--
通讯作者：
Sobel,BE
中科院分区：
文献类型：
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作者：
Corr,PB;Snyder,DW;Lee,BI;Gross,RW;Keim,CR;Sobel,BE
方法动物制备。用高速抽吸钻（15）对猫心脏正常区和缺血区的活体组织进行了LPG含量分析。活检组织在液氮中快速冷冻。如前所述，在左前降支（LAD）冠状动脉近端闭塞后10分钟对缺血区进行采样（15）。缺血区很容易通过LAD冠状动脉供血区域的心外膜发绀确定。将动物维持在受控的生理条件下，并且在用α-氯蔗糖（75 mg/kg）诱导麻醉后，将全身pH和CO2和O2分压（分别为Pcoz和POT）维持在猫的正常限度内。用食管温度探头控制的红外线灯将体温维持在37 ℃。如前所述（15），通过将加湿（100%）和加热（40 ℃）的室内空气连续循环通过放置在胸部上方的树脂玻璃室，将心脏的表面温度保持在正常范围内。对于电生理学研究，用硫喷妥钠（10 mg/kg）麻醉成年杂种犬，快速取出心脏并置于充氧Krebs溶液中。取下右束支和左束支的远端部分及附着的心室肌，固定在7.5 mm的底部。ml蜡衬浴中，并在37.5 ℃下用含有以下物质（以meq/l计）的改良Krebs溶液连续灌流：Na +150，K+ 4.0，Mg+ 2.0，Ca 2 + 2.4，Cl-141，POT 3 0.9，HCO：22.0，以及葡萄糖（5.0 mmol/l）。通过用95%0 ~ 5%C02连续充气将pH调节至7.4。组织受到刺激。在光纤的相对端处脉冲阳极和阴极。使用2.0 ms持续时间的脉冲，舒张阈值的两倍，并使用800 ms的基本周期长度。记录细胞内电位。用填充有3 M KCl的玻璃微电极（6-20 mA DC电阻）进行，并且所有实验都使用单次保持的穿刺进行。信号首先通过高阻抗单位增益静电计（型号M4A，WP Instruments）、恒定增益运算放大器（x50）和可变电阻器进行处理。在FM模拟磁带上存储之前的ble-gain放大器（15 ips，频率响应1.6 kHz，3dB）。使用专门设计的VmaX分析仪进行电子微分，以获得如前所述的每个动作电位的0相的最大上升速率（I i，i，i）（43）。用一个完全自动化的系统离线分析存储在模拟磁带上的动作电位记录，该系统便于统计分析和验证连续信号的再现性，所有程序均在前文中详细描述（43）。电生理学研究中使用的LPG浓度的选择。细胞膜仅含有约2.5%的总细胞蛋白质，但细胞磷脂的比例要大得多（6）。假设LPG在所有细胞磷脂中均匀分布，则肌膜中LPG与蛋白质的比例将超过总细胞LPG与总细胞蛋白质的比例。因此，肌膜LPG浓度可能超过本研究中发现的总体细胞浓度（见结果）在缺血组织中为1.65 mM（由7.5nmol/mg蛋白质，220 mg蛋白质/g湿重，我们以前已经发现，蛋白结合的LPG的电生理效应约为10%，与游离LPG（14）一样大。因此，目前的电生理学研究进行了200和100 μ M…
METHODSAnimal preparations. Tissue from normal and ischemic zones-of cat hearts in vivo was assayed for LPG content in biopsies obtained with a high-speed suction drill (15). Biopsies were fast frozen in liquid Ne. Ischemic zones were sampled 10 min after the onset of proximal left anterior descending (LAD) coronary occlusion as previously described (15). The ischemic zone was readily identified by epicardial cyanosis in the region supplied by the LAD coronary artery. Animals were maintained under controlled physiological conditions, and systemic pH and CO2 and 02 partial pressures (Pcoz and POT, respectively) were maintained within normal limits for the cat after induction of anesthesia with a-chlorolose (75 mg/kg). Body temperature was maintained at 37OC with an infrared lamp controlled by an esophageal temperature probe. The surface temperature of the heart was maintained within normal limits by continuous circulation of humidified (100%) and warmed (40 C) room air through a Plexiglas chamber placed over the thorax as previously described (15). For electrophysiological studies, adult mongrel dogs were anesthetized with sodium thiopental (10 mg/kg), and the hearts were removed quickly and placed in oxygenated Krebs solution. The distal portions of the right and left bundle branches with attached ventricular muscle were removed, pinned to the bottom of a 7.5. ml wax-lined bath, and continuously superfused with a modified Krebs solution at 37.5 OC containing the following (in meq/l): Na’150, K+ 4.0, Mg+ 2.0, Ca2’2.4, Cl-141, POT3 0.9, HCO: 22.0, as well as glucose (5.0 mmol/l). The pH was adjusted to 7.4 by continuous gassing with 95% 0~~ 5% C02. The tissues were stimulated with. pulses anode and cathode at opposite ends of the fiber. Pulses of 2.0 ms d uration at twice the diastolic threshold were utilized, and a basic cycle length of 800 ms was used. Intracellular potentials were recorded. with glass microelectrodes filled with 3M KC1 (6-20 Ma DC resistance), and all experiments were performed with the use of single maintained impalements. Signals were processed first through a high-impedence unity gain electrometer (model M4A, WP Instruments), a constant-gain operational amplifier (x50), and a varia. ble-gain amplifier prior to storage on FM analog tape (15 ips, frequency response 1.6 kHz at 3dB). A specially designed VmaX analyzer was used for electronic differentiation to obtain the maximal rate of rise (\i,,;) of phase 0 of each action potential as previously described (43). Action potential recordings stored on analog tape were analyzed off-line with a completely automated system facilitating statistical analyses and verification of the reproducibility of consecutive signals, all procedures previously’described in detail (43). Selection of concentrations of LPG for use in electrophysiological studies. Cell membranes contain only approximately 2.5% of total cellular protein but a substantially larger fraction of cellular phospholipid (6). With the assumption of a homogeneous distribution of LPG throughout all cellular phospholipids, the ratio of LPG to protein in sarcolemma would exceed that of total cellular LPG to total cellular protein. Thus sarcolemmal LPG concentrations are likely to exceed the overall cellular concentrations found in this study (see RESULTS) of 1.65 mM in ischemic tissue (calculated from 7.5 nmol/mg protein, 220 mg protein/g wet wt, and hence 1650 nmol/ml or 1.65 mM).We have previously found that the electrophysiological effects of protein-bound LPG are approximately 10% as great as those of free LPG (14). Accordingly, the present electrophysiological studies were performed with 200 and 100, uM …