Measurement of mitochondrial and non-mitochondrial Ca2+ in isolated intact hepatocytes: a critical re-evaluation of the use of mitochondrial inhibitors.

Isolated rat hepatocytes treated with mitochondrial inhibitors FCCP or antimycin A release discrete amounts of Ca2+ in a Ca(2+)-free extracellular medium as revealed by changes in the absorbance of the Ca2+ indicator arsenazo III. The process is completed in 2 min and the amount of Ca2+ released is not affected by the type of the mitochondrial poison employed. The subsequent treatment with the cation ionophore A23187 causes a further release of Ca2+ that does not appear related to the specificity of the previous treatment with FCCP or antimycin A. Both FCCP and antimycin A cause a progressive loss of cellular ATP associated with a decrease in the ATP/ADP ratio from 6 to 2-1.5. However, this decrease does not significantly prevent 45Ca2+ accumulation in isolated liver microsomes. Moreover, the decrease of the ATP/ADP ratio to 1, does not promote a significant release of 45Ca2+ from 45Ca(2+)-preloaded microsomes. Finally, experiments with Fura-2-loaded hepatocytes reveal that agents specifically releasing Ca2+ from non-mitochondrial stores (vasopressin and 2,5-di-tert-butyl-1-4-benzohydroquinone) are still able to increase the cytosolic Ca2+ concentration in FCCP-treated cells. Taken together, these findings demonstrate that, in freshly isolated hepatocytes, FCCP specifically releases Ca2+ from mitochondrial stores without significantly affecting active Ca2+ sequestration in other cellular pools. For these reasons, FCCP can be used to release and quantitate mitochondrial Ca2+ in liver cells.     

Multiple effects of the phenylhydrazone derivative FCCP on the secretory pathway in rat plasma cells

We studied the sensitivity of the last steps of the secretory process of antibody-producing cells to carbonylcyanide-p-trifluoromethoxyphenylhydrazone (FCCP) and sodium azide (NaN3), agents which lower the cellular ATP content by inhibiting oxidative phosphorylation and mitochondrial electron transport, respectively. Popliteal lymph node cells or purified plasma cells from rats immunized against horseradish peroxidase were incubated with the drugs. The rate of secretion of anti-HRP antibodies was measured by an enzyme-linked immunoadsorbent assay or after biosynthetic labeling with L-[3H]fucose. FCCP as well as NaN3 were shown to rapidly inhibit (in less than 5 min) the secretion of immunoglobulins (Ig) and to partially block the release of fucosylated Ig. This indicates that the drugs inhibit the transport of Ig from the Golgi apparatus (GA) (where fucose is added to Ig) to the plasma membrane. However, the degree of inhibition reached 40 to 50% with NaN3 and 70 to 80% with FCCP, whereas both drugs similarly depleted ATP stores by 45 to 55%. These results are consistent with multiple effects of FCCP on the secretion pathway of Ig. As a tentative explanation, we suggest that FCCP, because of its protonophore properties, not only reduces cellular ATP levels but may also neutralize the Golgi or post-Golgi acidic compartments recently shown to be involved in the transport of plasma membrane and secretory proteins.     

Calcium buffering in presynaptic nerve terminals. II. Kinetic properties of the nonmitochondrial Ca sequestration mechanism

The kinetic properties of the nonmitochondrial ATP-dependent Ca sequestering mechanism in disrupted nerve terminal (synaptosome) preparations have been investigated with radioactive tracer techniques; all solutions contained DNP, NaN3, and oligomycin, to block mitochondrial Ca uptake. The apparent half-saturation constant, KCa, for the nonmitochondrial Ca uptake is approximately 0.4 micrometer Ca; the Hill coefficient is approximately 1.6. Mg is also required for the Ca uptake, and the apparent KMg is approximately 80 micrometer. ATP and deoxy-ATP, but not CTP, GTP, ITP, UTP, ADP, or cyclic AMP, promote Ca uptake; the KATP, is approximately 10 micrometer. ATP analogs with blocked gamma-phosphate groups are unable to replace ATP. Particulate fractions from the disrupted synaptosomes possess Ca-dependent ATPase activity in the presence of Mg; the apparent KCa for this activity is 0.4--0.8 micrometer Ca, and the Hill coefficient is approximately 1.6. The Ca uptake and ATPase kinetic data suggest that the hydrolysis of 1 ATP may energize the transport of two Ca2+ ions into the storage vesicles. The second part of the article concerns the intraterminal distribution of Ca in "intact" terminals. When the terminals are disrupted after 45Ca loading, about one-half of the 45Ca is retained in the particulate material; some of this Ca, presumably stored in mitochondria, is released by the uncoupler, FCCP. Some of the 45Ca is released by A-23187, but not by FCCP; this fraction may be Ca stored in the nonmitochondrial sites described above. The proportion of 45Ca stored in the nonmitochondrial sites is increased when the Ca load is reduced or when the mitochondria are blocked with ruthenium red. These data indicate that the nonmitochondrial Ca storage sites are involved in intraterminal Ca buffering; they may play an important role in synaptic facilitation and post-tetanic potentiation, which result from Ca retention after neural activity.     

Ca2+ Homeostasis in the Agonist-sensitive Internal Store: Functional Interactions Between Mitochondria and the ER Measured In Situ in Intact Cells

Mitochondria have a well-established capacity to detect cytoplasmic Ca²⁺ signals resulting from the discharge of ER Ca²⁺ stores. Conversely, both the buffering of released Ca²⁺ and ATP production by mitochondria are predicted to influence ER Ca²⁺ handling, but this complex exchange has been difficult to assess in situ using conventional measurement techniques. Here we have examined this interaction in single intact BHK-21 cells by monitoring intraluminal ER [Ca²⁺] directly using trapped fluorescent low-affinity Ca²⁺ indicators. Treatment with mitochondrial inhibitors (FCCP, antimycin A, oligomycin, and rotenone) dramatically prolonged the refilling of stores after release with bradykinin. This effect was largely due to inhibition of Ca²⁺ entry pathways at the plasma membrane, but a significant component appears to arise from reduction of SERCA-mediated Ca²⁺ uptake, possibly as a consequence of ATP depletions in a localized subcellular domain. The rate of bradykinin-induced Ca²⁺ release was reduced to 51% of control by FCCP. This effect was largely overcome by loading cells with BAPTA-AM, highlighting the importance of mitochondrial Ca²⁺ buffering in shaping the release kinetics. However, mitochondria-specific ATP production was also a significant determinant of the release dynamic. Our data emphasize the localized nature of the interaction between these organelles, and show that competent mitochondria are essential for generating explosive Ca²⁺ signals.     

Studies on the role of calcium ions in the stimulation by adrenaline of amylase release from rat parotid

1. Mitochondrial and microsomal fractions were prepared from rat parotid glands. Both fractions were able to take up (45)Ca. The mitochondrial (45)Ca-uptake system could be driven by ATP (energy-coupled Ca(2+) uptake) or by ADP+succinate (respiration-coupled Ca(2+) uptake). Energy-coupled Ca(2+) uptake was blocked by oligomycin but not by carbonyl cyanide m-chlorophenylhydrazone; respiration-coupled Ca(2+) uptake was blocked by carbonyl cyanide m-chlorophenylhydrazone but not by oligomycin. Microsomal Ca(2+) uptake was dependent on the presence of ATP; the ATP-dependent Ca(2+) uptake was not affected by oligomycin or carbonyl cyanide m-chlorophenylhydrazone. Ca(2+) uptake by both fractions was inhibited by Ni(2+). 2. Incubation of parotid pieces with adrenaline increased the rate of release of amylase and the uptake of (45)Ca. The adrenaline-stimulated release of amylase was not dependent on the presence of extracellular Ca(2+). 3. The effect of adrenaline on the subcellular distribution of (45)Ca in parotid pieces incubated with (45)Ca was studied. In parotid tissue incubated with (45)Ca, both mitochondrial and microsomal fractions contained (45)Ca. Incubation with adrenaline increased the amount of (45)Ca incorporated into the mitochondrial fraction but not the microsomal fraction. In parotid tissue preloaded with (45)Ca subsequent incubation with adrenaline caused a decrease in the amount of (45)Ca found in both the mitochondrial and microsomal fractions. 4. From these data we conclude that the regulation of the cytosolic Ca(2+) concentration in the parotid may involve both mitochondrial and microsomal Ca(2+)-uptake systems. We suggest that the action of adrenaline on the parotid may be to increase the movement of Ca(2+) to the cytosol by increasing the flux of Ca(2+) across mitochondrial, microsomal and plasma membranes.     

Stoichiometry of contraction and Ca2+ mobilization by inositol 1,4,5-trisphosphate in isolated gastric smooth muscle cells

Smooth muscle cells were isolated from the circular muscle layer of guinea pig stomach and permeabilized by brief exposure to saponin. Both permeabilized and intact muscle cells contracted in response to cholecystokinin octapeptide (CCK-8) and acetylcholine, but only permeabilized muscle cells contracted in response to inositol 1,4,5-trisphosphate (InsP3). The contractile response to InsP3 was prompt (peak less than 5 s), concentration-dependent (EC50-0.3 microM), and insensitive to antimycin or oligomycin. Contraction induced by either InsP3 or CCK-8 was accompanied by a concentration-dependent increase in free Ca2+ that was directly correlated with the magnitude of contraction. Both InsP3 and CCK-8 caused rapid net efflux of Ca2+ from cells preloaded with 45Ca2+. Contraction, increase in free Ca2+ concentration, and net 45Ca2+ efflux elicited by a combination of maximal concentrations of InsP3 and CCK-8 were not significantly different from those elicited by maximal concentrations of either agent alone. Repeated stimulation of single muscle cells with either InsP3 or CCK-8 in Ca2+-free medium caused eventual loss of the contractile response to all agents. The response to all agents was restored upon re-exposure of the cell to a cytosol-like concentration of Ca2+, implying equal access of InsP3 and receptor-linked agonists to the same intracellular Ca2+ store. The results demonstrate that InsP3 mimics the effects of receptor-linked agonists on contraction and mobilization of intracellular Ca2+ in permeabilized smooth muscle cells that retain the functional properties of intact smooth muscle cells and support a role for InsP3 as membrane-derived messenger responsible for mobilization of intracellular Ca2+ in smooth muscle cells.     

Calcium pools in saponin-permeabilized guinea pig hepatocytes

The plasma membranes of isolated guinea pig hepatocytes were made permeable with saponin. The cells were then suspended in a medium resembling cytosol in which the level of ATP was kept constant with an ATP-regenerating system. Intracellular ATP-dependent 45Ca and 40Ca sequestration was then followed at various concentrations of Ca2+ in the medium. It was found that ATP-dependent Ca uptake could be divided into two mechanisms: a low affinity high capacity uptake sensitive to 2,4-dinitrophenol (DNP) and oligomycin, thought to be mitochondrial, and a low capacity high affinity uptake, which was insensitive to DNP and oligomycin, thought to be mainly endoplasmic reticulum (ER). The threshold for ATP-dependent Ca uptake by the latter pool was about 20 nM Ca2+. The process had an EC50 value of 0.3 microM (for 45Ca) and a capacity of 2.7 nmol/45Ca/mg of protein. The "ER" mechanism also had a high affinity for ATP (EC50, about 43 microM). There was no significant accumulation of Ca by the postulated mitochondrial pool until the [Ca2+] of the medium was greater than 1 microM. The concentration of Ca2+ in the cytosol of normal unstimulated hepatocytes was estimated from measurements of phosphorylase a activity to be about 0.18 microM. At this [Ca2+], the ER pool of the saponin-treated hepatocytes accumulated Ca but there was no evidence of any Ca uptake into the "mitochondrial" pool. This suggests that most of the exchangeable Ca in a normal cell may be in DNP and oligomycin-insensitive pools (presumably the ER or possibly the plasma membrane) and suggests that these pools are likely to be involved in the increase in cytosolic [Ca2+] which occurs after stimulation by Ca-mobilizing hormones.     

Bax translocation to mitochondria subsequent to a rapid loss of mitochondrial membrane potential

Bax, a pro-apoptotic member of the Bcl-2 family, is a cytosolic protein that inserts into mitochondrial membranes upon induction of cell death. Using the green fluorescent protein fused to Bax (GFP-Bax) to quantitate mitochondrial binding in living cells we have investigated the cause of Bax association with mitochondria and the time course relative to endogenous and induced changes in mitochondrial membrane potential (DeltaPsi(m)). We have found that staurosporine (STS) induces a loss in DeltaPsi(m) before GFP-Bax translocation can be measured. The onset of the DeltaPsi(m) loss is followed by a rapid and complete collapse of DeltaPsi(m) which is followed by Bax association with mitochondria. The mitochondria uncoupler FCCP, in the presence of the F(1)-F(0) ATPase inhibitor oligomycin, can trigger Bax translocation to mitochondria suggesting that when ATP levels are maintained a collapse of DeltaPsi(m) induces Bax translocation. Neither FCCP nor oligomycin alone alters Bax location. Bax association with mitochondria is also triggered by inhibitors of the electron transport chain, antimycin and rotenone, compounds that collapse DeltaPsi(m) without inducing rapid ATP hydrolysis that typically occurs with uncouplers such as FCCP. Taken together, our results suggest that alterations in mitochondrial energization associated with apoptosis can initiate Bax docking to mitochondria.     

Somatostatin and lanthanum discriminate two Ca2+ mobilizing mechanisms regulated by bombesin receptors in peptic cells

Bombesin (BB)-stimulated pepsinogen secretion from frog esophageal peptic acini was inhibited 40% by somatostatin (SS) (IC50 = 1 nM), and by 30-50% by low concentrations (10(-7)-10(-4)M) of lanthanum chloride. SS inhibited basal secretion as well as both early (0-2 min) and late (2-30 min) BB-stimulated secretory phases. By contrast, LaCl3 selectively inhibited the late secretory phase and was without effect on basal secretion. SS (100 nM) and LaCl3 (30 microM) attenuated BB-stimulated 45Ca2+ uptake, and the combination resulted in additive inhibition. High K+ media decreased basal secretion, abolished SS but not LaCl3 inhibition of BB-stimulated secretion, and blocked SS inhibition of BB-mediated 45Ca2+ uptake. These findings suggest the existence on peptic cells of distinct La3+-sensitive, and somatostatin-sensitive, K+ dependent, Ca2+ mobilizing mechanisms which contribute to BB receptor-mediated secretion.     

Calcium uptake and calcium release by subcellular fractions of smooth muscle. II. Kinetics of calcium uptake by microsomes and mitochondria from pig coronary artery and guinea pig ileum.

Calcium uptake by the microsomal and mitochondrial fractions of pig coronary artery and guinea pig ileum was studied in the presence of ATP, ATP plus oxalate and without ATP and oxalate. Microsomes and mitochondria of both smooth muscles were found to be unable to accumulate appreciable amounts of calcium in the absence of ATP. Oxalate noticeably stimulated the calcium uptake of the mitochondrial fraction from pig coronary artery but had little effect on calcium uptake by the microsomal fraction of this smooth muscle. The calcium uptake of microsomes and mitochondria from guinea pig ileum was not or only slightly enhanced by oxalate. There are typical kinetics regarding the time course and the extent of calcium uptake by microsomes and mitochondria from pig coronary artery and guinea pig ileum. In comparison, considerable qualitative and quantitative differences between both smooth muscles are observed. The high ATP-dependent calcium uptake capacity of the mitochondria from pig coronary artery and guinea pig ileum are a further argument for the hypothesis that these organelles may play an important role in the contraction-relaxation mechanism of smooth muscle.     

Relationships between the exchange of calcium and phosphate in isolated fat-cells.

The uptake and the washout of 45Ca2+ and 32Pi is described in free fat-cells and whole epididymal fat-pads from fed rats. 2. In isolated fat-cells, the uptake of 45Ca2+ proceeds with an initial rapid phase of about 1 min duration, followed by a slower subsequent accumulation. In contrast with the rapid phase, the slow phase is inhibited by 2,4-dinitrophenol, warfarin, oligomycin and verapamil, shows saturation, and presumably represents transport across the plasma membrane. 3. The washout of 45Ca2+ from preloaded cells consists of a rapid (1 min) initial phase and a slow phase which is non-monoexponential, suggesting that the radioactive isotope is released from several cellular pools. 4. When Pi is omitted from the incubation medium, the slow phase of 45Ca uptake is almost abolished, and the washout of 45Ca from preloaded fat-cells is markedly accelerated. At elevated extracellular concentrations of Pi (2,4-6.2mM), the uptake of 45Ca is stimulated by 2-10-fold, and the release of the radioactive isotope from preloaded cells is inhibited. In whole epididymal fat-pads, variations in the extracellular concentration of Pi have no detectable effect on the uptake or the washout of 45Ca. 5. In isolated fat-cells, the accumulation of 32Pi is inhibited by 2,4-dinitrophenol or the omission of glucose from the incubation medium. In a Ca2+-depleted buffer, the uptake of 32Pi is diminished, and hyperosmolarity, which stimulates 45Ca uptake, also accelerates the accumulation of 32Pi. 6. It is concluded that in free fat-cells, the uptake and release of Ca2+ and Pi take place by closely interrelated processes, which are dependent on mitochondrial energy production.     

Synchronous oscillation of the cytoplasmic Ca2+ concentration and membrane potential in cultured epithelial cells (Intestine 407)

Cultured epithelial Intestine 407 cells exhibit regular oscillations of the membrane potential with repeated hyperpolarizations. These hyperpolarizations were inhibited not only by K+ channel blockers (tetraethylammonium and nonyltriethylammonium) but also by inhibitors of the Ca2+-activated K+ channel (quinine and quinidine). Using Ca2+-selective microelectrodes, cyclic increases in the cytosolic free Ca2+ concentration of more than 1 X 10(-6) M were found to coincide with the cyclic membrane hyperpolarizations. Thus, it appears that the potential oscillation is brought about by the oscillation of the intracellular free Ca2+ level which induces periodic activation of the Ca2+-dependent K+ channels. Neither the deprivation of extracellular Ca2+ nor the application of Ca2+ channel blockers (Co2+ and Ni2+) abolished the potential oscillation. Mitochondrial inhibitors (KCN, NaN3, antimycin A, FCCP and dinitrophenol) inhibited the potential oscillation, whereas glycolytic inhibitors (iodoacetic acid and NaF) had no effects. Caffeine and oxalate, which affect the microsomal Ca2+ transport, failed to exert any effect upon the potential oscillation. It is concluded that the cytosolic Ca2+ oscillation results from cyclic releases of Ca2+ from the intracellular storage site, which depends upon mitochondrial activities.     

Cross-talk between the sarcoplasmic reticulum and the mitochondrial calcium handling systems may play an important role in the regulation of contraction in anococcygeus smooth muscle

Mitochondrial Ca(2+) and its relation with the contraction induced by phenylephrine was investigated. In normal Ca(2+), carbonyl cyanide p-(trifluoro-methoxy)phenyl-hydrazone (FCCP) and oligomycin produced contraction similar to that promoted by phenylephrine. Phenylephrine-induced contraction was reduced by FCCP+oligomycin. In Ca(2+)-free, FCCP+oligomycin did not induce contraction. Response to FCCP+oligomycin was reduced upon Ca(2+) repletion and this response was lower than that to phenylephrine. Ca(2+) concentration was increased by FCCP+oligomycin. Since a profuse net of sarcoplasmic reticulum encloses mitochondria, a cross-talk between the two organelles may play an important role in the phenylephrine-induced contraction in presence of Ca(2+) encountered in both sarcoplasmic reticulum and extracellular medium of anococcygeus cells.     

The KATP channel is critical for calcium sequestration into non-ER compartments in mouse pancreatic beta cells.

K(ATP) channel activity influences beta cell Ca(2+) homeostasis by regulating Ca(2+) influx through L-type Ca(2+) channels. The present paper demonstrates that loss of K(ATP) channel activity due to pharmacologic or genetic ablation affects Ca(2+) storage in intracellular organelles. ATP depletion, by the mitochondrial inhibitor FCCP, led to Ca(2+) release from the endoplasmic reticulum (ER) of wildtype beta cells. Blockade of ER Ca(2+) ATPases by cyclopiazonic acid abolished the FCCP-induced Ca(2+) transient. In beta cells treated with K(ATP) channel inhibitors FCCP elicited a significantly larger Ca(2+) transient. Cyclopiazonic acid did not abolish this Ca(2+) transient suggesting that non-ER compartments are recruited as additional Ca(2+) stores in beta cells lacking K(ATP) channel activity. Genetic ablation of K(ATP) channels in SUR1KO mice produced identical results. In INS-1 cells transfected with a mitochondrial-targeted Ca(2+)-sensitive fluorescence dye (ratiometric pericam) the increase in mitochondrial Ca(2+) evoked by tolbutamide was 5-fold larger compared to 15 mM glucose. These data show that genetic or pharmacologic ablation of K(ATP) channel activity conveys Ca(2+) release from a non-ER store. Based on the sensitivity to FCCP and the property of tolbutamide to increase mitochondrial Ca(2+) it is suggested that mitochondria are the recruited store. The change in Ca(2+) sequestration in beta cells treated with insulinotropic antidiabetics may have implications for beta cell survival and the therapeutic use of these drugs.     

Reaction mechanism of (Ca2+, Mg2+)-ATPase of sarcoplasmic reticulum vesicles. II. (ATP, ADP)-dependent Ca2+-Ca2+ exchange across the membranes

Sarcoplasmic reticulum vesicles were preloaded with unlabeled CaCl2, and 45Ca2+ incorporation into the vesicles was determined by adding 45CaCl2 to the external medium in the presence of ATP and ADP. In the absence of added MgCl2, the steady state rate of the (ATP, ADP)-dependent 45Ca2+ incorporation was extremely low, being in good agreement with that of the Ca2+-dependent ATP hydrolysis which was catalyzed by the membrane-bound (Ca2+, Mg2+)-ATPase. In contrast, it was greatly increased by addition of MgCl2 and became much higher than the steady state rate of the Ca2+-dependent ATP hydrolysis. The kinetic analysis of the results gave support to the probability that the MgCl2 addition markedly shifted the equilibrium of the reaction of Caout . EP and Cain . EP represent phosphoenzymes with bound Ca2+ which is exposed to the external medium and to the internal medium, respectively).     

Role of calcium in the secretion of leptin from white adipocytes

The mechanism by which calcium regulates leptin secretion was studied in adipocytes isolated from rat white adipose tissue. Incubation of adipocytes in a medium containing glucose, but no calcium, markedly inhibited insulin-stimulated leptin secretion (ISLS) and synthesis, without affecting basal leptin secretion or lipolysis. However, when pyruvate was used as a substrate, ISLS was insensitive to the absence of calcium. Likewise, the stimulatory effects of insulin were completely prevented by phloretin, cytochalasin B, and W-13 (3 agents that interfere with early steps of glucose metabolism) in the presence of glucose, but not in the presence of pyruvate. Thus calcium appears to be specifically required for glucose utilization. On the other hand, (45)Ca uptake and leptin secretion were not affected by insulin or by inhibitors of L-type calcium channels. However, agents increasing plasma membrane permeability to calcium (high calcium concentrations, A-23187, and ATP) increased (45)Ca uptake and concomitantly inhibited ISLS. Similarly, release of endogenous calcium stores by thapsigargin inhibited ISLS in a dose-dependent manner. ATP, A-23187, calcium, and thapsigargin inhibited ISLS, even in the presence of pyruvate. These results show that 1) extracellular calcium is necessary for ISLS, mainly by affecting glucose uptake, 2) insulin does not affect extracellular calcium uptake, and 3) increasing cytosolic calcium by stimulating its uptake or its release from endogenous stores inhibits ISLS at a level independent of glucose metabolism. Thus calcium regulates leptin secretion from adipocytes in a manner that is markedly different from its role in the exocytosis of many other polypeptidic hormones.     

Studies on the mechanism of action of guinea pig lymphotoxin. II. Increase of calcium uptake rate in LT-damaged target cells.

In the previous paper it was suggested that the primary action of guinea pig lymphotoxin (LT) involved creation of ionic imbalances within the target L cells. The nature of these ionic disturbances is explored in this paper. The exogenous addition of CaCl2, but not KCl or NaCl, inhibited the cytotoxic action of LT. Cellular uptake rates of 45Ca++, but not 86Rb+, increased in LT-damaged L cells. The factor responsible for increasing the 45Ca++ uptake rate cochromatographed on a hydroxyapatite column with the cytotoxic activity of LT. Ouabain prevented the LT-mediated lysis and, concomitantly, depressed the LT-induced increase of 45Ca++ uptake rate. The LT-damaged L cells excluded trypan blue to the same extent as the normal cells. The addition of LT to and LT-resistant L cell mutant affected neither the 45Ca++ uptake rate nor the viability. From these observations, damage to the calcium transport system in the L cell plasma membrane is proposed as a mechanism of LT action.     

Effect of inhibitors and uncouplers of oxidative phosphorylation during compaction and blastulation of bovine embryos cultured in vitro

The effect of inhibiting ATP production via oxidative phosphorylation during pericompaction of in vitro produced bovine embryos was investigated. This was achieved by: (i) varying the atmospheric O2 concentration (0, 1, 2, 4 and 7%); (ii) addition of oxidative phosphorylation inhibitors, NaN3 and antimycin A; and (iii) addition of 2,4-dinitrophenol, an uncoupler of oxidative phosphorylation from electron transport. The development of embryos under various O2 concentrations from day 5 to day 7 of development indicated that an optimal concentration occurred at about 2%. Addition of NaN3 revealed that doses above 100 mumol l-1 were toxic to embryo development, but that concentrations of 5-10 mumol l-1 stimulated embryo development by 10-25%. A similar result was observed after addition of 2,4-dinitrophenol, whereas antimycin A was inhibitory at doses as low as 1 mumol l-1. At concentrations of NaN3 or 2,4-dinitrophenol that stimulated embryo development, the number of cells of the resulting blastocysts was also significantly increased. Addition of NaN3 from day 1 of development inhibited subsequent development. Metabolic data of NaN3-treated embryos revealed that O2 uptake was significantly lower at inhibitory doses (100 mumol l-1). A significant (P < 0.05) log linear increase in glucose uptake was measured between the three concentrations of NaN3 (0, 10 and 100 mumol l-1). These results demonstrate that ATP production via oxidative phosphorylation is essential for bovine embryo development in vitro. However, transient (subacute) inhibition appears to be beneficial to embryo development and the number of cells, perhaps by creating a more favourable intracellular environment.     

The abnormal-shaped mitochondria in thymus lymphocytes treated with inhibitors of mitochondrial energetics

The effects of uncouplers (DNP, FCCP), oligomycin, and rotenone on the energetics and mitochondrial ultrastructure in lymphocytes have been studied. We confirmed the previous observations done on Ehrlich ascites and cardiomyocyte culture cells that uncouplers and respiratory inhibitors cause the appearance of ringlike and dumbbell-like mitochondria. It is shown that this effect does not correlate with decrease in ATP concentration, changes in oxygen consumption, or condensation of the mitochondrial matrix. FCCP (2 µM) is more effective in the induction of abnormal-form mitochondria than 240 µM DNP, oligomycin, or rotenone. Combined treatment with DNP, oligomycin, and rotenone or with DNP and rotenone produces an effect as strong as 2 µm FCCP. DNP (240 µM) and FCCP (2 µM) have a similar effect on respiration and intracellular ATP, but only the latter induces condensation of the mitochondrial matrix.     

Silver ion triggers Ca2+ release from intracellular store sites in saponin-treated HL-60 cells

We examined the effect of silver ion on Ca2+ mobilization from intracellular stores in permeabilized HL-60 cells using a filtration method and 45Ca2+. In HL-60 cells preloaded with Ca2+ in the presence of ATP, micromolar concentrations of AgNO3 elicited marked Ca2+ release within 1 min. The AgNO3-induced Ca2+ release was not affected by the free Ca2+ concentration in the medium. Equivalent concentrations of AgNO3 inhibited energy-dependent Ca2+ uptake as well as oxalate-supported Ca2+ uptake. In passive Ca2+ release experiments when ATP was completely depleted in the solution, AgNO3 also triggered Ca2+ release. Sulfhydryl protecting agents such as 2-mercaptoethanol, dithiothreitol, and glutathione (reduced form) blocked the AgNO3-induced Ca2+ release. From these results, we conclude that the apparent Ca2+ release induced by AgNO3 is mainly due to inhibition of the Ca2+ pump with increased permeability for Ca2+ and partly due to a direct effect on the Ca2+ release channel, probably by modification of sulfhydryl groups on these proteins.