EFFECTS OF ACIDOSIS ON RESTING CYTOSOLIC AND MITOCHONDRIAL CA2+ IN MAMMALIAN MYOCARDIUM
EFFECTS OF ACIDOSIS ON RESTING CYTOSOLIC AND MITOCHONDRIAL CA2+ IN MAMMALIAN MYOCARDIUM
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DOI：
10.1085/jgp.102.3.575
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发表时间：
1993-09-01
影响因子：
3.8
通讯作者：
CAPOGROSSI, MC
中科院分区：
文献类型：
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作者：
GAMBASSI, G;HANSFORD, RG;CAPOGROSSI, MC
酸中毒可增加心肌组织静息细胞内[Ca~(2+)]，(Ca(I))，但Ca~(2+)升高的钙来源及酸中毒对线粒体游离[Ca~(2+)]，(Ca(M))的影响尚不清楚。在这项研究中，监测了成年大鼠左室肌细胞内负载snarf-1的乙酰氧甲酯(AM形式)的胞浆pH(pH(I))。从平衡了5%二氧化碳的碳酸氢盐缓冲液切换到平衡了20%二氧化碳的缓冲液，pH(I)稳定下降了0.52+/-0.05U(n=16)。0.5或1.5赫兹的电刺激对5%CO2中的pH(I)无影响，也不影响高碳酸酸中毒引起的pH(I)下降的幅度。用MnCl2猝灭细胞内Indo-I荧光后，检测Indo-1/游离酸负载的心肌细胞的Ca(I)和负载Indo-1/AM的细胞的Ca(M)。在静息状态下浸泡在1.5 mM[Ca~(2+)]中的完整心肌细胞，高碳酸血症使Ca(I)从130+/-5增加到221+/-13 nM。但在酸中毒时，静息心肌细胞舒张期Ca(I)增加幅度大于静息Ca(I)，1.5 Hz起搏时舒张期Ca(I)(285+/-17 nM)高于0.5 Hz(245+/-18 nM；P<；0.05)。肌浆网(SR)钙离子耗竭或肌浆网钙离子耗竭及无钙缓冲液灌流均不影响静息心肌细胞Ca(I)升高的幅度。在未受刺激的完整心肌细胞中，高碳酸血症使Ca(M)从95+/-12 nM增加到147+/-19 nM，这一反应不能被兰尼定和无钙缓冲液改变，也不能被50微米的Ru红改变以阻断线粒体单一转运蛋白。在负载BCECF/AM或Indo-1/AM的线粒体悬液中，乳酸引起的酸中毒降低了线粒体内外的pH，增加了Ca(M)。对浸泡在Indo-1/游离酸液中的线粒体悬浮液的研究表明，添加乳酸后线粒体外钙离子增加。因此，在静止的心肌细胞中，胞浆和线粒体内的缓冲液，而不是跨肌膜的钙内流或肌浆网钙的释放，可能分别是钙(I)和钙(M)增加的来源；此外，线粒体的钙外流可能有助于钙(I)的增加。相反，对酸中毒的反应，电刺激心肌细胞的舒张期Ca(I)比静息状态的Ca(I)增加得更多，这表明在起搏过程中，细胞净钙增加有助于舒张期Ca(I)的增加。
Acidosis increases resting cytosolic [Ca2+], (Ca(i)) of myocardial preparations; however, neither the Ca2+ sources for the increase in Ca(i) nor the effect of acidosis on mitochondrial free [Ca2+], (Ca(m)) have been characterized. In this study cytosolic pH (pH(i)) was monitored in adult rat left ventricular myocytes loaded with the acetoxymethyl ester (AM form) of SNARF-1. A stable decrease in the pH(i) of 0.52 +/- 0.05 U (n = 16) was obtained by switching from a bicarbonate buffer equilibrated with 5% CO2 to a buffer equilibrated with 20% CO2. Electrical stimulation at either 0.5 or 1.5 Hz had no effect on pH(i) in 5% CO2, nor did it affect the magnitude of pH(i) decrease in response to hypercarbic acidosis. Ca(i) was measured in myocytes loaded with indo-1/free acid and Ca(m) was monitored in cells loaded with indo-1/AM after quenching cytosolic indo-I fluorescence with MnCl2. In quiescent intact myocytes bathed in 1.5 mM [Ca2+], hypercarbia increased Ca(i) from 130 +/- 5 to 221 +/- 13 nM. However, when acidosis was effected in electrically stimulated myocytes, diastolic Ca(i) increased more than resting Ca(i) in quiescent myocytes, and during pacing at 1.5 Hz diastolic Ca(i) was higher (285 +/- 17 nM) than at 0.5 Hz (245 +/- 18 nM; P < 0.05). The magnitude of Ca(i) increase in quiescent myocytes was not affected either by sarcoplasmic reticulum (SR) Ca2+ depletion with ryanodine or by SR Ca2+ depletion and concomitant superfusion with a Ca2+-free buffer. In unstimulated intact myocytes hypercarbia increased Ca(m) from 95 +/- 12 to 147 +/- 19 nM and this response was not modified either by ryanodine and a Ca2+-free buffer or by 50 muM ruthenium red in order to block the mitochondrial uniporter. In mitochondrial suspensions loaded either with BCECF/AM or indo-1/AM, acidosis produced by lactic acid addition decreased both intra- and extramitochondrial pH and increased Ca(m). Studies of mitochondrial suspensions bathed in indo-1/free acid-containing solution showed an increase in extramitochondrial Ca2+ after the addition of lactic acid. Thus, in quiescent myocytes, cytoplasmic and intramitochondrial buffers, rather than transsarcolemmal Ca2+ influx or SR Ca2+ release, are the likely Ca2+ sources for the increase in Ca(i) and Ca(m), respectively; additionally, Ca2+ efflux from the mitochondria may contribute to the raise in Ca(i). In contrast, in response to acidosis, diastolic Ca(i) in electrically stimulated myocytes increases more than resting Ca(i) in quiescent cells; this suggests that during pacing, net cell Ca2+ gain contributes to enhance diastolic Ca(i).