Effects of the mitochondrial probe rhodamine 123 and related analogs on the function and viability of pulsating myocardial cells in culture

Rhodamine 123, a cationic fluorescent dye, has previously been shown to specifically localize in or on mitochondria in living cells. Since it has also been shown to be relatively non-toxic in a variety of cell types it has been a useful tool for probing mitochondriain vitro. In this report, using cardiac cells in culture, we demonstrate that rhodamine 123 and 6G both positively charged compounds, quickly inhibit beating and kill cardiac-muscle cells while uncharged or neutral rhodamines, 116 and B, produce neither effect. We also present data which illustrate that the cationic rhodamines inhibit oxidative phosphorylation in isolated mitochondria while the neutral dyes do not. It is suggested that both phenomena may be related.     

A new method for testing cell ageing using two mitochondria specific fluorescent probes

Cell culture techniques have considerably improved our understanding of the numerous changes related to aging. For instance, murine lymphocytes obtained from animals older than 6 months progressively lose their, in vitro, proliferative capacity. Numerous studies have shown that this loss is due to changes in the mitochondrial compartment such as reduction in the transmembrane potential and/or membrane mass. Using two mitochondria specific probes with a potential-dependent (Rhodamine 123) or independent (Nonyl Acridine Orange) uptake, we found that the decline in the respiratory activity in the mouse occurred approximately 6 months prior to the decrease in mitochondrial membrane mass. The analysis of the Rh 123/NAO fluorescence ratio measured in splenocytes obtained from mice aged more than 6 months, showed that there was a linear loss of respiratory efficiency per unit of mitochondrial membrane mass. Moreover, cells with a ratio of less than 0.85 were incapable of proliferating and remained quiescent. The time separating the infection points of the two dye uptake curves might provide informations about the regulation and coordination of nuclear and/or mitochondrial genomes. Moreover, the ratio between the two fluorescent probes, in particular during the linear phase, may also have a predictive value.     

Mitochondrial adenosine triphosphatase in the oxyphil cells of a renal oncocytoma

Oxyphil cells are characterized by cytoplasm packed with large numbers of mitochondria. Study of these unusual cells may provide information about the regulation of mitochondrial biogenesis. Although it has been suggested that this is a compensatory proliferation due to a mitochondrial dysfunction, no such dysfunction has been well documented. In this study we considered the possibility of dysfunction in the mitochondrial enzyme F1/Fo-adenosine triphosphatase(ATPase) as a stimulating factor involved in the mitochondrial proliferation of oxyphil cells. Mitochondria isolated from frozen tissue of a renal oncocytoma showing structural integrity and purity by electron microscopy were studied. Submitochondrial particles formed by sonic disruption showed the presence of the F1 component of mitochondrial ATPase with electron microscopy which was functionally active. The oligomycinsensitive ATPase activity from the renal oncocytoma was 0.133 mumol/min.mg submitochondrial particle protein which was higher than the readings obtained from normal kidney tissue (0.091 mumol/min.mg SMP protein) obtained from hamsters. Normal human renal tissue obtained at autopsy contained only nonfunctional mitochondria and therefore could not be used as control tissue. Mitochondrial ATPase dysfunction does not appear to be the inciting factor in the proliferation of mitochondria seen in oxyphil cell metaplasia and future studies should consider other possibilities. Preliminary functional studies of this nature can be performed with properly prepared frozen surgical tissue.     

Flow cytometry as a tool to discriminate respiratory-competent and respiratory-deficient yeast cells

Summary The cationic lipophilic dye Rhodamine 123 (Rh123) is selectively enriched in mitochondria in a membrane potential-dependent manner. Application of drugs which interfere with the electron flow of the respiratory chain lead to a severe reduction of mitochondrial dye uptake. In this communication we show that the same effect is observed after Rh123-staining of respiratory-deficient yeast mutants. Based on this observation we used flow cytometry to discriminate respiratory-compentent and respiratory-deficient yeast cells. Combined with a cell sorter we were able to selectively enrich respiring and non-respiring yeast cells, repectively, from a mixture of cells.     

Mitochondria as the site of action of tetracycline on Plasmodium falciparum

Rhodamine 123 (Rh 123) was used as a fluorescent probe for the mitochondria of the malarial parasite Plasmodium falciparum. On treatment with tetracycline in vitro, a marked decrease in the percentage of parasites with Rh123 fluorescence in the mitochondria was observed in parallel with an increase in the percentage of parasites with abnormal morphology during onset of decrease in parasitemia. Similar results were obtained, over a shorter time period, with 2,4-dinitrophenol. However, the percentage of parasites with fluorescence did not decrease with increase in parasite abnormal morphology or decrease in parasitemia on treatment with pyrimethamine or cycloheximide. Isoelectric focusing-SDS gel electrophoresis of radiolabelled parasite proteins showed two components of 95 and 85 kDa, the synthesis of which was sensitive to tetracycline, but not cycloheximide. It is concluded that tetracycline exerts its action through the effect on parasite mitochondria and mitochondrial protein synthesis.     

Maternal antioxidant treatments prevent diabetes-induced alterations of mitochondrial morphology in rat embryos

Previous studies have suggested that production of reactive oxygen species by embryonic mitochondria may have a role in the induction of both high-amplitude mitochondrial swelling and embryonic dysmorphogenesis in diabetic pregnancy. The present study analyzed the relationships between a putative metabolite-induced production of free oxygen radicals, mitochondrial lipid peroxidation, and high-amplitude mitochondrial swelling in embryos during organogenesis. For studies in vitro, day 9 embryos of normal rats were cultured for 48 h with a high concentration of glucose in the absence or presence of α-cyano-4-hydroxycinnamic acid (CHC), a mitochondrial pyruvate transport inhibitor. The morphology of mitochondria in the neuroepithelium of the embryos was studied with the aid of transmission electron microscopy. For studies in vivo, normal and diabetic pregnant rats were fed a diet supplemented with the antioxidants α-tocopherol (vitamin E) or 2,6-di-tert-butyl-4-methylphenol (BHT), and the ultrastructure of mitochondria in the embryonic neuroepithelium and in the visceral yolk sac was investigated on gestational day 11. Exposure to a high concentration of glucose in vitro or to maternal diabetes in vivo induced high-amplitude swelling of mitochondria in the neuroepithelium of the embryos. The swelling of mitochondria was prevented by addition of CHC to the culture media or by maternal ingestion of antioxidant-supplemented food. In diabetic pregnancy, embryonic mitochondria during organogenesis produce free oxygen radicals that cause mitochondrial lipid peroxidation and swelling and furthermore embryonic dysmorphogenesis. Dietary supplementation with antioxidants to the mother may prevent embryonic malformations in diabetic pregnancy by inhibition of mitochondrial dysfunction. Anat. Rec. 251:303–315, 1998. © 1998 Wiley-Liss, Inc.     

银杏达莫注射液抑制大鼠离体心脏缺血/再灌注损伤的机制研究

 目的：观察银杏达莫注射液预处理对大鼠离体心脏缺血/再灌注损伤的影响,并探讨其可能的作用机制。方法：SD雄性大鼠40只随机分成5组（n=8）：正常对照（NC）组、缺血/再灌注（I/R）组、缺血预处理（IPC+I/R）组、银杏达莫注射液预处理（GD+I/R）组和银杏达莫+氯化镧预处理（GD+LaCl3+I/R）组。观察各组相同时点（预灌30 min稳定点,缺血30 min,再灌5 min、30 min、60 min）的心功能指标,包括心率(HR)、左室收缩压(LVSP)和室内压变化速率（±dp/dtmax）,同时收集各时点冠脉流出液,检测其中乳酸脱氢酶（LDH）和肌酸激酶（CK）活性。实验结束后检测心肌线粒体Ca2+浓度和α-酮戊二酸脱氢酶（α-OGDH）含量。结果：与I/R组比较,IPC+I/R组和GD+I/R组在心脏再灌注期各项心功能指标均得到改善（P<0.05）;心肌LDH和CK的释放量降低（P<0.01）;线粒体内Ca2+超载降低（P<0.01）,且线粒体内α-OGDH含量升高（P<0.05）;而GD+I/R组中银杏达莫对心肌的保护作用被LaCl3抑制（P<0.05）。结论：银杏达莫可能通过抑制钙超载、增强线粒体酶活性以稳定线粒体能量代谢,从而缓解缺血/再灌注诱导的心肌细胞损伤。     

Mitochondrial polarisation status and [Ca2+]i signalling in rat cerebellar granule neurones aged in vitro

Mitochondrial membrane potential is a major factor that controls, ultimately, the cellular energy supply. By use of a mitochondrial membrane potential dye (rhodamine 123, R123) and image analysis we show that during long-term (>3 weeks) culture of primary neurones (cerebellar granule neurones) there is a gradual and time-dependent depolarisation of neuronal mitochondria. This process was demonstrated by analysing the changes in the heterogeneity of the cytosolic rhodamine 123 fluorescent signal as a function of the age in culture and by measuring the amplitude of the rhodamine 123 fluorescence evoked by the addition of a mitochondrial protonophore (FCCP). The relationship between cytosolic [Ca(2+)](i) and mitochondrial membrane potential was assessed by recording both parameters simultaneously, in neurones loaded with fura-2 and rhodamine 123. Neuronal stimulation (KCl-evoked depolarisation) induced a mitochondrial depolarisation response resulting from the entry of cytosolic Ca(2+) into mitochondria. In young cultures (10 DIV), the mitochondrial membrane potential recovered fully within 30s from the start of the stimulation, despite the continuous presence of the depolarisation stimulus and the maintained cytosolic [Ca(2+)](i) signal. In contrast, in older neurones (DIV 22), the mitochondrial response was of smaller amplitude and displayed a much longer repolarization period. Also, in these older neurones, the threshold [Ca(2+)](i) level required for the initiation of the mitochondrial depolarisation response was increased by 50%. Thus, the present results indicate that neuronal maturation and ageing in conditions of long-term in vitro culture determine significant changes in the mitochondrial polarisation status that are manifest both in resting conditions and during stimulation and could explain some of the reported changes in neuronal homeostasis in long-term neuronal cultures.     

Increased uptake and retention of rhodamine 123 by mitochondria of old human fibroblasts

Binding of the fluorescent dye R123 by a variety of mammalian cells has been shown to be dependent on the high transmembrane potential maintained in functional mitochondria. Recent studies in our laboratory have shown that old human fibroblasts (HF) bind and retain more R123 than young HF. In an effort to determine whether this difference in R123 uptake indeed reflected a difference in mitochondrial transmembrane potential, drugs known to disrupt the transmembrane potential of mitochondria were used to monitor the R123-mitochondria interaction of young and old HF. Distinct differences indicating that old HF maintain a higher mitochondrial transmembrane potential were observed. More significantly, perhaps this difference reflects an age-related change(s) in the structure and/or function of mitochondria.     

A novel system for assigning the mode of inheritance in mitochondrial disorders using cybrids and rhodamine 6G.

When normal human cultured skin fibroblasts were treated with the fluorescent dye rhodamine 6G (R6G), there was a drastic reduction in numbers of intact mitochondria and electron transport chain enzyme activities, despite the fact that mitochondrial DNA (mtDNA) was still present in treated cells. We used this observation to develop a novel system for generating cybrids. When cultured skin fibroblast cells from a patient with the mitochondrial encephalopathy and ragged-red fibers (MERRF) syndrome harboring the A8344G mtDNA mutation and which showed a severe reduction in cytochrome c oxidase activity were treated with R6G and fused to enucleated HeLaCOT cells, the resulting cybrid clones showed recovery of cytochrome c oxidase activity, and were shown to have mtDNA derived solely from the HeLaCOT cell line. R6G has significant advantages over ethidium bromide in removing the mitochondrial elements from cultured cells, and the results reported here demonstrate that this strategy can be used to determine the origin of the genetic defect in patients with electron transport chain abnormalities.     

Action of bacterial endotoxin and lipid A on mitochondrial enzyme activities of cells in culture and subcellular fractions.

Escherichia coli O127:B8 lipopolysaccharide (LPS), prepared by the Westphal procedure, caused a marked decrease in the activities of mitochondrial malate dehydrogenase, succinate dehydrogenase, and adenylate kinase in African green monkey kidney (Vero) cells and primary cultures of mouse liver cells within 2 h after exposure to 10 micrograms of LPS/ml of culture medium. These three enzyme activities leaked into the supernatant fraction, and cytochrome oxidase activity was lost from the mouse liver mitochondrial particulate fraction within 45 min after exposure to 10 micrograms of LPS/mg of protein. Loss malate dehydrogenase activity from isolated mitochondria was also accelerated by LPS from E. coli O26:B6 (Boivin preparation) or Salmonella typhosa O901 (Westphal preparation), and by lipid A from Salmonella minnesota or Shigella sonnei. In addition, LPS and lipid A inhibited state 3 respiration by isolated mitochondria with attendant loss of respiratory control, but adenosine 5'-diphosphate/O ratios were relatively unchanged. Impaired mitochondrial function is an early event after exposure to biologically relevant amounts of LPS or lipid A.     

Studies on mitochondrial ATPase of Leishmania donovani using digitonin-permeabilized promastigotes

Mitochondrial ATPase of Leishmania donovani was characterized using digitonin-permeabilized promastigotes and the results were compared with those from isolated mitochondria. Maximum mitochondrial ATPase activity was obtained in promastigotes permeabilized with digitonin at a final concentration of 20 μM and the specific activity of the enzyme was 46% and 57% higher than that of homogenized and sonicated promastigotes, respectively. At concentrations above 20 μM digitonin inhibited ATPase activity and the degree of inhibition increased with increasing concentrations of the detergent. The ATPase activity of promastigotes remained DCCD-sensitive when permeabilized with digitonin at concentrations up to 120 μM but the enzyme became increasingly resistant to this inhibitor as digitonin concentrations were increased to 140 μM and more, indicating the loss of functional activity of the enzyme. The pH and temperature optima for mitochondrial ATPase were determined to be 7.5 and 30°C, respectively. Mg²⁺ ions were essential for ATPase activity but free Mg²⁺ ions were found to be inhibitory. A Mg²⁺/ATP ratio of 1:3 supported the optimum ATPase activity. Sulfite and hexanol activated the enzyme but failed to prevent the inhibition by free Mg²⁺ ions. The results indicate that digitonin-permeabilized promastigotes provide an ideal system for studying the mitochondrial ATPase of L. donovani.     

Human cell variants resistant to rhodamine 6G

Two variants have been isolated from the cultured human cell line VA₂-B which are resistant in vivo to the mitochondrial-specific fluorescent dyes rhodamine 6G and rhodamine 123. Both mutants are cross-resistant to ethidium bromide but are sensitive to both colchicine and chloramphenicol. When either mutant is treated with low levels of rhodamine 6G, mitochondrial-associated fluorescence is significantly lower than in wild-type cells. Furthermore, when cell cultures are treated with high concentrations of either rhodamine 123 or 6G, and then washed free of the dye, mitochondrial-associated rhodamine fluorescence rapidly diminishes in the parental cell line. In hybrid cell fusions between resistant and sensitive cell lines, rhodamine resistance is gradually expressed, reaching maximal expression approximately 11 days after fusion. Cytoplasmic transmission of rhodamine resistance has not been clearly demonstrated in cytoplast-cell fusions, and thus resistance is probably due to a mutation of a nuclear, rather than mitochondrial DNA gene(s). These observations indicate that mitochondria of both rhodamine-resistant variants, unlike wild type, have a significantly decreased ability to bind and retain rhodamine, and thus their mitochondrial tramsmembrane electrical potential may be significantly reduced.     

Mitochondrial antibodies in primary biliary cirrhosis. VI. Association of the complement fixing antigen with a component of the mitochondrial F1-ATPase complex.

The complement fixing antigen of the inner mitochondrial membrane previously shown to be associated with the mitochondrial ATPase could be further purified by subjecting the ATPase extracted from beef heart and brown fat mitochondria to ion exchange and gel filtration chromatography. Although the ATPase activity could be clearly dissociated from the complement fixing activity, subunits of the F1-ATPase complex were always found in the purified fractions. The alpha, gamma, delta and epsilon subunits of the complex could be excluded with high probability as target antigens in contrast to the beta band which was always found in association with the antigen activity. These findings imply that the active centre of the ATPase enzyme is not involved in the antibody reaction but molecules of the ATPase complex may have antigen binding capacity. Treatment of ATPase associated antigen with trypsin did not markedly affect the complement binding, while SMP's treated in the same way lost their antigen activity indicating that sera from patients with primary biliary cirrhosis (PBC) may have mitochondrial antibodies of different specificities reacting with trypsin sensitive as well as trypsin insensitive components of the inner membrane. The purified antigen reacted exclusively with sera from patients with PBC and may be therefore used as a marker antigen.     

Measurement of ATP synthesis and flocculent matrix densities in mitochondria as a function of 'in vitro' ischemia in the heart and liver of rats

We intended to determine the levels of adenosine triphosphate (ATP) synthesis at the time when mitochondria ultrastructurally show flocculent densities in the matrix space. For this purpose, mitochondria were isolated from rat heart and rat liver after the tissues have been maintained under controlled ischemic conditions in vitro at 37 degrees C for intervals of 15, 30, 45, 60, 120, 180, and 240 (heart) min. The isolated mitochondria were tested for new ATP synthesis by luciferin/luciferase luminescence in the presence of substrate and adenosine 5'-diphosphate (ADP). The luminescence peaks were standardized and related to an external measure by measuring absorbance of ATP at 259 nm where the extinction coefficient is 15,400. Mitochondrial yield was monitored by measuring succinate dehydrogenase activity in the first homogenate and in the final mitochondrial pellet. Alternatively, cytochrome oxidase activity was used and the protein in the mitochondrial pellet was also determined. We found that the yield of mitochondria was above 53-54% in both liver and heart at 2 h of ischemia. Longer intervals were accompanied by lower yields. The ability to synthesize new ATP declined at different time intervals in ischemia of the heart compared to the liver. After 30 min ischemia, the synthesis in heart mitochondria is 18% of control, while the synthesis of liver mitochondria reaches 16% of control after 45 min of in vitro ischemia. Flocculent densities in heart mitochondria appeared at 45 min ischemia in vitro and in vivo, and at 60 min in liver mitochondria. We conclude that the decline of ATP synthesis is a significant early change in mitochondria and antedates the appearance of flocculent densities.     

Effect of hyperthermia on electron transport in Ehrlich ascites tumor mitochondria

The effect of hyperthermia (1 hr, 41 degrees C) on the functional properties of Ehrlich ascites tumor mitochondria was investigated. Mitochondria isolated from Ehrlich ascites tumor after exposure of whole cells to 41 degrees C for 1 hr still phosphorylate and maintain a normal acceptor control ratio (ACR). The temperature decreases state 4 and ADP-and FCCP-stimulated respiration on various substrates entering at three energy-conserving sites of the respiratory chain. The inhibition of oxygen consumption by NAD- and FAD-linked substrates was 40% for state 4 and 70% for ADP- or FCCP-stimulated respiration. State 4 and FCCP-stimulated respiration of mitochondria on TMPD + ascorbate was affected 38% and 45%, respectively. ATPase activity was unaffected by hyperthermia, indicating that under these experimental conditions, the inhibition of ADP-stimulated respiration does not depend on an effect on either Fo F1-ATPase or adenine translocase, the activity of which is required for ATP entry prior to ATPase activity. Because of the inability to detect a specific site of action of temperature, it is conceivable that hyperthermia might inhibit substrate oxidation by altering some components of the inner mitochondrial membrane, which regulates the kinetic properties of the membrane-associated enzymes.     

鱼藤酮致多巴胺能神经细胞早期凋亡模型的研究

目的 :探讨鱼藤酮引起多巴胺能神经细胞早期凋亡的时间和浓度规律 ,为进一步研究其机制建立平台。方法 :不同浓度鱼藤酮作用多巴胺能神经细胞系SH SY5Y细胞不同时间 ,用四唑盐比色法检测细胞活性 ,用流式细胞术检测细胞的线粒体膜电位。结果 :低剂量 ( 5nmol/L和 2 0nmol/L)鱼藤酮处理 4 8h内对细胞活性无明显影响 (P >0 .0 5) ;超过 10 0nmol/L的鱼藤酮明显抑制细胞活性 (P <0 .0 5) ,并且抑制的程度呈剂量依赖性。 2 0nmol/L及更高浓度的鱼藤酮作用 2 4h可以引起细胞线粒体膜电位明显下降 (P <0 .0 5)。结论 :采用2 0nmol/L鱼藤酮作用 2 4h可以建立鱼藤酮多巴胺能神经细胞早期凋亡模型。     

The effects of ethanol and acetaldehyde on the products of protein synthesis by liver mitochondria

Ethanol and acetaldehyde, alone or in combination, at physiologic concentrations, significantly inhibit mitochondrial protein synthesis in vitro. Mitochondria from rats chronically fed ethanol also display a reduced rate of mitochondrial protein synthesis in vitro. This effect is further aggravated by addition of ethanol to the incubation medium. Sodium dodecyl sulfate-gel electrophoresis of mitochondria fractionated with acetic acid-lubrol, which were incubated in the presence of ethanol or acetaldehyde, revealed a modest over-all decrease in labeling. However, a polypeptide fraction in the molecular weight range of 36,000 to 40,000 was conspicuously decreased. This range includes subunits of cytochrome oxidase, cytochrome b, and ATPase. Liver mitochondria from rats fed ethanol chronically showed a comparable decrease in the 36,000- to 40,000-molecular weight peak after incubation with radioactive leucine in vitro and fractionation with acetic acid-lubrol. Similar results were obtained when mitochondrial protein synthesis was determined in vivo in chronically treated rats. The data suggest that chronic ethanol consumption interferes with mitochondrial membrane biogenesis and that several products are more sensitive to this effect than others.     

Staining of cellular mitochondria with LDS-751.

We have found the dye LDS-751 to bind almost exclusively to mitochondria when incubated with viable, nucleated cells. Treatment of cells with the nuclear stain acridine orange and LDS-751 revealed little colocalization when the cells were examined by confocal microscopy. Staining with the dye rhodamine 123, which is known to bind polarized mitochondria, was virtually identical to the pattern observed with LDS-751. This staining pattern was observed to be consistent over a range of 0.02-20 microg/ml LDS-751 and was consistent between both fibroblasts and monocytes. Depolarization of mitochondria with the mitochondrial depolarizing agents phenyl arsine oxide and carbonyl cyanide m-chlorophenylhydrazone (CCCP) dramatically reduced both LDS-751 staining, and rhodamine 123 fluorescence. Taken together, these results suggest that LDS-751 is excluded from the nucleus and binds the polarized membranes of mitochondria. Given this, interpretation of LDS-751 fluorescence as being indicative of nuclear status, as is commonly done to discriminate between leukocytes and erythrocytes, is unwarranted and may lead to erroneous conclusions if mitochondria become depolarized upon processing.