Mechanism of Free Fatty Acids-Induced Mitochondrial Na+ and Ca2+ Overloads Resulting in Cell Apoptosis

• Main Authors: Kwang-Ming Fang, 方光明
• Other Authors: 吳美玲
• Format: Others
• Language: en_US
• Published: 2008
• Online Access: http://ndltd.ncl.edu.tw/handle/35989824570461765369

博士 === 國立臺灣大學 === 生理學研究所 === 96 === There is ample evidence that marked accumulation of free fatty acids, including arachidonic acid (AA), linoleic acid and oleic acid, during ischemia - reperfusion in the brain. In the first part of my study, I found that AA induced cytosolic Na+ ([Na+]cyt) and Ca2+ ([Ca2+]cyt) overload via a non-selective cation conductance (NSCC), resulting in mitochondrial [Na+]m and [Ca2+]m overload in cerebellar neurons. Moreover, oleic acid and eicosapentaenoic acid also induced a small increase in the [Ca2+]i and [Na+]i. A selective inhibitor of the mitochondrial Ca2+ uniporter, RU360, inhibited the AA-induced [Ca2+]m and [Na+]m overload, but not the [Ca2+]cyt and [Na+]cyt overload. The [Na+]m overload was also markedly inhibited by either Ca2+-free medium or CGP3715, a selective inhibitor of the mitochondrial Na+cyt-Ca2+m exchanger. Furthermore, RU360, Ca2+-free medium, Na+-free medium, or cyclosporin A (CsA) largely prevented AA-induced opening of the mitochondrial permeability transition pore (mPTP), cytochrome c (cytC) release, and caspase-3-dependent neuronal apoptosis. Importantly, Na+-ionophore/Ca2+-free medium, which induced [Na+]m overload, but not [Ca2+]m overload, also caused CsA-sensitive mPTP opening, resulting in caspase-3 dependent apoptosis, indicating that [Na+]m overload per se induced apoptosis. Our results therefore suggest that AA-induced [Na+]m overload, acting via activation of the NSCC, is an important upstream signal in the mitochondrial-mediated apoptotic pathway. The NSCC may therefore act as a potential neuronal death pore which is activated by AA accumulation under pathological conditions. Disturbances in lipid metabolism have been suggested to play an important role in myocardial damage. Marked accumulation of free fatty acids (FFAs), including arachidonic acid (AA), palmitic acid, oleic acid, and linoleic acid, also occurs during post-ischemia and reperfusion (post-I/R) in the heart. In the second part of my study is to investigate possible mechanism of the myocyte apoptosis mediated by free fatty acids. Here, I shows that AA also activated a novel non-selective cation conductance (NSCC), resulting in both intracellular Ca2+ and Na+ overload in cultured ventricular myocytes. The EC50 of the AA-induced Ca2+ and Na+ overload was 8 microM and 5 microM, respectively. A higher concentration (30 microM) of other FFAs was required to induce significant Na+/Ca2+ overload. Overproduction of reactive oxygen species (ROS) and AA metabolites was not involved. Using confocal microscopy, I showed that AA caused sustained [Na+]cyt and [Ca2+]cyt overload, resulting in [Na+]m and [Ca2+]m overload, which induced opening of the mPTP, cytochrome c release, and caspase-3-mediated apoptosis. Similar apoptotic effects were seen using Na+-ionophore cocktail/Ca2+-free medium, which induced [Na+]cyt and [Na+]m, but not [Ca2+]cyt and [Ca2+]m overload. Electron microscopy showed that inhibition of [Na+]m overload prevented disruption of the mitochondrial membrane, indicating that [Na+]m overload is also an important upstream signal in AA- and FFAs-induced myocyte apoptosis. In summary, FFAs including AA may therefore act as endogenous ionophores, which activate an NSCC, resulting in both myocyte and neuronal cell death seen in post-I/R.