EFFECT OF REMOVAL OF EXTERNAL CALCIUM ON
PHOSPHOINOSITIDE HYDROLYSIS IN CULTURED
MYOTUBES OF EMBRYONIC CHICKEN^*

WU　DI，ZHANG　XIAO-HUI,ZHU　PEI-HONG

ABSTRACT：The effect of removal of external Ca²⁺ on phosphoinositide hydrolysis was investigated in cultured myotubes from 9-day-old Leghorn embryonic chicken. In the myotubes exposed to Ca²⁺ -free Ringer′s solution, the turnover of phosphoinositide was exponentially decreased with a time constant of about 26 min. In the presence of external Ca²⁺, the hydrolysis of phosphoinositide was significantly increased by exposure to 80 mmol/L K⁺ solution. After removal of external Ca²⁺, 80 mmol/L K⁺ exposure caused a slight decrease of phosphoinositides hydrolysis in comparison with the control (normal Ringer). It is indicated that hydrolysis of phosphoinositide in cultured myotubes can be enhanced by high K⁺ exposure. External Ca²⁺ is essential for this effect, which is different from mature muscle fibres.
Key words: high K⁺; Ca²⁺ -free; phosphoinositide; myotube

去细胞外钙对培养鸡胚肌管磷脂酰肌醇水解的影响^*

吴　迪　章晓辉　朱培闳

摘　　要：本工作研究了去细胞外钙对取自9天来亨鸡胚的培养肌管磷脂酰肌醇水解的影响。80 mmol/L K⁺暴露可以显著升高磷脂酰肌醇的水解。在暴露于无钙任氏液的肌管中，磷脂酰肌醇的水解随时间延长而呈指数递减，时间常数约为26分钟。在胞外无钙时，80 mmol/L K⁺ 引起的磷脂酰肌醇水解与对照(正常任氏液)相比略有下降。结果表明，高钾暴露能够增强培养肌管的磷脂酰肌醇的水解；但与成熟的肌纤维不同，细胞外钙是必须的。
关键词: 高钾；无钙；磷脂酰肌醇；肌管
学科分类号: Q445

　　Inositol phospholipids play an important role in signal transduction of various cells^［1］. Under the action of phosphatidylinositol specific phospholipase C (PI-PLC), the phospholipids are hydrolyzed into two important second messengers^［2］: diacylglycerol (DAG) and 1,4,5-trisphosphate (IP₃), which can activate PKC^［3］ and mobilize Ca²⁺ from intracellular Ca²⁺ store^［1］, respectively. Several subtypes of PI-PLC have been identified, some of which are Ca²⁺-dependent^［4］. Thus, increasing cytoplasmic free calcium (［Ca²⁺］_i) may elicit hydrolysis of phosphoinositide. Recently, we have shown that, besides the increase of ［Ca²⁺］_i, depolarization may also enhance phosphoinositide hydrolysis in frog skeletal muscle^［5,6］. However, phosphoinositide hydrolysis in immature muscle cells may be regulated differently, since cultured myotubes exposed to Ca²⁺-free high K⁺ medium showed a decrease of phosphoinositide hydrolysis^［7］. Owing to the fact that about 75% of the embryonic myotubes cultured for 3 days could respond to Ca²⁺-free high K⁺ exposure with raised ［Ca²⁺］_i (unpublished data), an increase of phosphoinositide hydrolysis should be expected, considering Ca²⁺ dependence of PI-PLC. In the present study, the effect of removal of external Ca²⁺ on phosphoinositide hydrolysis was investigated in cultured myotubes of embryonic chicken.
　　The methods of cell culture and extraction of inositol phosphates were as described pre-viously^［7,8］. In brief, the cells were prepared from the thigh muscles of 9-day-old embryos of Leghorn chickens. After cultured in DMEM (GIBCO) with 10% fetal cattle serum and 5% embryo extract at 37℃ in 5% CO₂ and 95% air for 3 days, the cells were incubated in 2 ml DMEM containing 10 μCi ［³H］myo-inositol (Shanghai Institute of Nuclear Research) for another 14 hr. Then, the cells were washed with DMEM 3×15 min at 37℃ and loaded with Li⁺ by incubating the cells in 20 mmol/L Li⁺ Ringer (see below) for 20 min. After provided with different challenges, the cells were extracted with trichloroacetic acid (TCA). The TCA extract was loaded on a column of Dowex 1X8 (Bio-Rad), and the total labeled inositol phosphates (IPs) were eluted with 16 ml of 1.0 mol/L ammonium formate/0.1 mol/L formate acid. The radioactivity was determined with a liquid scintillator (Beckman LS6000IC). Student t-test was used for statistical comparison between groups.
　　The composition of Ringer′s solution was (in mmol/L): 155 NaCl, 5 KCl, 5 CaCl₂, 2 MgCl₂, and 5 HEPES. It was titrated to pH 7.2 with NaOH. 20 mmol/L Li⁺ Ringer was made by equivalent replacement of Na⁺ in Ringer with Li⁺. To examine the effect of high K⁺ exposure on phosphoinositide turnover, the K⁺ concentration was elevated to 80 mmol/L and the ionic strength of the solution was maintained by reducing Na⁺. Cl^－ was partially replaced by CH₃SO^－₃ to keep the product of ［K⁺］_o and　［Cl^－］_o constant. For preparing Ca²⁺-free solution, CaCl₂ was omitted, but 2 mmol/L EGTA was added. Moreover, MgCl₂ was increased to 5 mmol/L. The free Ca²⁺ con～cen～tration in the Ca²⁺-free solution was about 10^－8 mol/L.
　　In consistence with the previous result^［7］, the labeled IPs of the myotubes exposed to 80 mmol/L K⁺ for 15 min increased to 346.3±132% (mean±SD, n=12) of the control (Ringer′s solution for 15 min), indicating that in the presence of external Ca²⁺ high K⁺ exposure can increase phosphoinositide hydrolysis in cultured myotubes. Moreover, as seen previously^［7］, the hydrolysis of phosphoinositide was decreased in the myotubes exposed to Ca²⁺-free Ringer′s solution for 15 min. As a new finding of the present study, this decrease of phosphoinositide hydrolysis induced by removal of external Ca²⁺ was observed to be time dependent (Fig.1). The time course of this reduction could be fit with a single exponential, as shown in Fig.2. The time constant was about 26 min, which may represent a translocation of Ca²⁺ ions from cytoplasm into extracellular compartment. Since it is difficult to measure slow change of ［Ca²⁺］_i, whether the decrease of phosphoinositide turnover is due to reduction of ［Ca²⁺］_i remains to be clarified.

Fig.1　Phosphoinositide hydrolysis in the myo～tu～bes exposed to Ca²⁺-free Ringer′s so～lu～tion (■) and Ca²⁺-free 80 mmol/L K⁺ (●)
Control: the labeled IPs of the myotubes exposed to Ringer′s solution for the same time. The bar represents S.D. of the mean. The figure near each data point indicates the number of experiments. ～^*　P<0.05; ～^*　*　P<0.01; ～^*　*　*　P<0.001 vs control (Ringer′s solution).

Fig.2　Time course of the decrease of pho～spho～ino～sitide hydrolysis in the myo～tubes exposed to Ca²⁺-free Ringer′s solution
The ordinate is represented by logarithm. The error bar indicates S.D. of the mean. The figure near each data point represents the number of experiments.

　　Different from matured muscle fibres^［5,6］, the present study clearly indicated that the presence of external Ca²⁺ was essential for high K⁺ induced increase of phosphoinositide hydrolysis in the cultured myotubes. In comparison with the control (normal Ringer), the exposure to Ca²⁺-free 80 mmol/L K⁺ medium caused a slight decrease of phosphoinositides hydrolysis (Fig.1). But, if the myotubes treated with Ca²⁺-free Ringer′s solution for the same period of time were used as a control, different results can be seen. For instance, the labeled IPs of the myotubes exposed to Ca²⁺-free Ringer′s solution and Ca²⁺-free 80 mmol/L K⁺ for 15 min was 57.1±11.6% and 82.5±21.1% of the control (normal Ringer), respectively. Their difference became significant (P<0.01), representing the effect of high K⁺ exposure in the absence of external Ca²⁺. The difference was still significant (P<0.01) when the exposure time was decreased to 5 min. However, with an exposure of 2 min, their difference was no longer significant (P>0.05). Thus, it is evident that, being different from mature skeletal muscle fibers, depolarization alone can not increase the hydrolysis of phosphoinositide in the myotubes. The coupling between depolarization and activation of PI-PLC which probably exists in mature fibers may be absent in cultured myotubes. As shown in Fig.1, the time dependence of the effect of high K⁺ exposure mainly resulted from that phosphoinositide hydrolysis in the myotubes exposed to Ca²⁺-free Ringer′s solution was decreased with a rate faster than that in the myotubes exposed to Ca²⁺-free 80 mmol/L K⁺.
　　In conclusion, it is indicated that the hydrolysis of phosphoinositide in cultured myotubes can be enhanced by exposure to high K⁺ medium, but external Ca²⁺ is essential for this effect.

^*This work was granted by the National Natural Science Foundation of China (No.39670274).
^*国家自然科学基金 (No.39670274) 资助项目
作者单位：中国科学院上海生理研究所，上海　200031
Shanghai Institute of Physiology, Chinese Academy of Sciences, Shanghai 200031

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Received 1998-09-01　　Revised 1998-10-20