microM Carbonyl Cyanide M-chlorophenylhydrazone Research Articles

Cadmium‐induced Cytosolic Ca2+ Elevation and Subsequent Apoptosis in Renal Tubular Cells

Cadmium (Cd2+) is an industrial and environmental metal. The effect of Cd2+ on intracellular free-Ca2+ levels ([Ca2+](i)) and viability in Madin Darby canine kidney cells was explored. Cd2+increased [Ca2+](i) in a concentration-dependent manner with an EC50 of 85 microM. Cd2+-induced Mn2+ entry demonstrated Ca2+ influx. Removal of extracellular Ca2+ decreased the [Ca2+](i) signal by 60%. The [Ca2+](i) signal was inhibited by La3+ but not by L-type Ca2+ channel blockers. In Ca2+-free medium, Cd2+-induced [Ca2+]i signal was abolished by pre-treatment with 1 microM thapsigargin (an endoplasmic reticulum Ca2+pump inhibitor) and 2 microM carbonylcyanide m-chlorophenylhydrazone (CCCP; a mitochondrial uncoupler). Cd2+-induced Ca2+ release was not altered by inhibition of phospholipase C. At concentrations between 10 and 100 microM, Cd2+killed cells in a concentration-dependent manner. The cytotoxic effect of 100 microM Cd2+was reversed by pre-chelating cytosolic Ca2+with BAPTA. Cd2+-induced apoptosis was demonstrated by propidium iodide. Collectively, this study shows that Cd2+ induced a [Ca2+](i) increase in Madin Darby canine kidney cells via evoking Ca2+ entry through non-selective Ca2+ channels, and releasing stored Ca2+ from endoplasmic reticulum and mitochondria in a phospholipase C-independent manner.

Role of membrane potential on artificial transformation of E. coli with plasmid DNA

The standard method of transformation of Escherichea coli with plasmid DNA involves two important steps: cells are first suspended in 100mM CaCl(2) at 0 degrees C (in which DNA is added), followed by the administration of a heat-pulse from 0 to 42 degrees C for 90s [Cohen, S., Chang, A., Hsu, L., 1972. Nonchromosomal antibiotic resistance in bacteria. Proc. Natl. Acad. Sci. U.S.A., 69, 2110-2114]. The first step makes the cells competent for uptake of DNA and the second step is believed to facilitate the DNA entry into the cells by an unknown mechanism. In this study, the measure of membrane potential of the intact competent cells, at different steps of transformation process, either by the method of spectrofluorimetry or that of flow cytometry, indicates that the heat-pulse step (0-->42 degrees C) heavily decreases the membrane potential. A subsequent cold shock (42-->0 degrees C) raises the potential further to its original value. Moreover, the efficiency of transformation of E. coli XL1 Blue cells with plasmid pUC19 DNA remains unaltered when the heat-pulse step is replaced by the incubation of the DNA-adsorbed competent cells with 10 microM carbonyl cyanide m-chlorophenyl hydrazone (CCCP) for 90s at 0 degrees C. Since the CCCP, a well-known protonophore, reduces membrane potential by dissipating the proton-motive-force (PMF) across E. coli plasma membrane, our experimental results suggest that the heat-pulse step of the standard transformation procedure facilitates DNA entry into the cells by lowering the membrane potential.

Sulfhydryl modification by 4,4'-dithiodipyridine induces calcium mobilization in human osteoblast-like cells.

The effect of oxidants on Ca(2+) movement in osteoblasts is unclear. In this study, we show that 4,4'-dithiodipyridine (4,4'-DTDP), a reactive disulphide that mobilizes Ca(2+) in muscle, induces an increase in cytoplasmic free-Ca(2+) concentrations ([Ca(2+)](i)) in MG63 human osteosarcoma cells loaded with the Ca(2+)-sensitive dye fura-2. 4,4'-DTDP acted in a concentration-dependent manner with an EC(50) of 10 microM. Removing extracellular Ca(2+) reduced the Ca(2+) signal by 35%. In Ca(2+)-free medium, the 4,4'-DTDP-induced [Ca(2+)](i) increase was not changed by depleting store Ca(2+) with 50 microM brefeldin A (a Golgi apparatus permeabilizer), by 2 microM carbonylcyanide m-chlorophenylhydrazone (CCCP, a mitochondrial uncoupler), by 1 microM thapsigargin (an inhibitor of the endoplasmic reticulum Ca(2+) pump) or by 5 microM ryanodine. Ca(2+) signals induced by 4,4'-DTDP in Ca(2+)-containing medium were not affected by modulation of protein kinase C activity or suppression of phospholipase C activity. However, 4,4'-DTDP-induced Ca(2+) release was inhibited by a thiol-selective reducing reagent, dithiothreitol (0.05-2.5 mM), in a concentration-dependent manner. Collectively, this study shows that 4,4'-DTDP induced [Ca(2+)](i) increases in human osteosarcoma cells via releasing store Ca(2+) from multiple stores in a manner independent of protein kinase C or phospholipase C activity. The store Ca(2+) release induced by 4,4'-DTDP appears to be associated with thiol oxidation. Furthermore, overnight incubation with 4,4'-DTDP inhibited cell activity in a concentration-dependent manner.

The relationship between intracellular [Ca(2+)] and Ca(2+) wave characteristics in permeabilised cardiomyocytes from the rabbit.

Spontaneous sarcoplasmic reticulum (SR) Ca(2+) release and propagated intracellular Ca(2+) waves are a consequence of cellular Ca(2+) overload in cardiomyocytes. We examined the relationship between average intracellular [Ca(2+)] and Ca(2+) wave characteristics. The amplitude, time course and propagation velocity of Ca(2+) waves were measured using line-scan confocal imaging of beta-escin-permeabilised cardiomyocytes perfused with 10 microM Fluo-3 or Fluo-5F. Spontaneous Ca(2+) waves were evident at cellular [Ca(2+)] > 200 nM. Peak [Ca(2+)] during a wave was 2.0-2.2 microM; the minimum [Ca(2+)] between waves was 120-160 nM; wave frequency was approximately 0.1 Hz. Raising mean cellular [Ca(2+)] caused increases in all three parameters, particularly Ca(2+) wave frequency. Increases in the rate of SR Ca(2+) release and Ca(2+) uptake were observed at higher cellular [Ca(2+)], indicating calcium-sensitive regulation of these processes. At extracellular [Ca(2+)] > 2 microM, the mean [Ca(2+)] inside the permeabilised cell did not increase above 2 microM. This extracellular-intracellular Ca(2+) gradient could be maintained for periods of up to 5 min before the cardiomyocyte developed a sustained and irreversible hypercontraction. Inclusion of mitochondrial inhibitors (2 microM carbonyl cyanide m-chlorophenylhydrazone and 2 microM oligomycin) while perfusing with > 2 microM Ca(2+) abolished the extracellular-intracellular Ca(2+) gradient through the generation of Ca(2+) waves with a higher peak [Ca(2+)] compared to control conditions. Under these conditions, cardiomyocytes rapidly (< 2 min) developed a sustained and irreversible contraction. These results suggest that mitochondrial Ca(2+) uptake acts to delay an increase in [Ca(2+)] by blunting the peak of the Ca(2+) wave.

Effect of the anti-breast cancer drug tamoxifen on Ca(2+) movement in human osteosarcoma cells.

The anti-breast cancer drug tamoxifen has recently been shown to cause an increase in [Ca(2+)]i in renal tubular cells, breast cells and bladder cells. Because tamoxifen is known to interact with oestrogens leading to modulation of bone metabolism, the present study was aimed at exploring whether tamoxifen could alter Ca(2+) signaling in human osteoblast-like MG63 cells. Cytosolic free Ca(2+) levels were recorded by using the Ca(2+)-sensitive dye fura-2. Tamoxifen induced a sustained [Ca(2+)]i increase at concentrations above 1 microM with an EC(50) of 8 microM. Removal of extracellular Ca(2+) reduced the response by 40%, suggesting that tamoxifen induced both Ca(2+) influx and store Ca(2+) release. Tamoxifen-induced Ca(2+) influx was confirmed as tamoxifen caused Mn(2+) influx-induced quench of fura-2 fluorescence. In Ca(2+)-free medium, pretreatment with 10 microM tamoxifen abolished the [Ca(2+)]i increase induced by 1 microM thapsigargin (an endoplasmic reticulum Ca(2+) pump inhibitor), and by 2 microM carbonylcyanide m-chlorophenylhydrazone (a mitochondrial uncoupler). Conversely, pretreatment with thapsigargin and carbonylcyanide m-chlorophenylhydrazone only reduced 64% of tamoxifen-induced [Ca(2+)]i increases. Addition of 2 microM U73122 to inhibit phospholipase C activity abolished the [Ca(2+)]i increase induced by 1 microM histamine, a phospholipase C-dependent Ca(2+) mobilizer, without affecting 10 microM tamoxifen-induced Ca(2+) release. The [Ca(2+)]i increase induced by 10 microM tamoxifen was not altered by 10 microM of nifedipine, verapamil and diltiazem. Together, the data show that tamoxifen induced a lasting increase in [Ca(2+)]i in human osteoblast-like cells by causing Ca(2+) influx and releasing Ca(2+) from multiple stores in a phospholipase C-independent manner.

Mechanism of estrogens-induced increases in intracellular Ca(2+) in PC3 human prostate cancer cells.

The effect of estrogens (diethylstilbestrol [DES], 17 beta-estradiol) on intracellular Ca(2+) concentrations ([Ca(2+)](i)) in hormone-insensitive PC3 human prostate cancer cells was examined. [Ca(2+)](i) changes in suspended cells were measured by using the Ca(2+)-sensitive fluorescent dye fura-2. Estrogens (1--20 microM) increased [Ca(2+)](i) concentration-dependently with DES being more potent. Ca(2+) removal inhibited 50 +/- 10% of the signal. In Ca(2+)-free medium, pretreatment with 20 microM estrogens abolished the [Ca(2+)](i) increases induced by 2 microM carbonylcyanide m-chlorophenylhydrazone (CCCP, a mitochondrial uncoupler) and 1 microM thapsigargin (an endoplasmic reticulum Ca(2+) pump inhibitor), but pretreatment with CCCP and thapsigargin did not alter DES-induced Ca(2+) release and partly inhibited 17 beta-estradiol-induced Ca(2+) release. Addition of 3 mM Ca(2+) increased [Ca(2+)](i) in cells pretreated with 1- 20 microM estrogens in Ca(2+)-free medium. Pretreatment with 1 microM U73122 to block phospholipase C-coupled inositol 1,4,5-trisphosphate formation did not alter estrogens-induced Ca(2+) release. The effect of 20 microM estrogen on [Ca(2+)](i) was not affected by pretreatment with 0.1 microM estrogens. Estrogen induced significant Ca(2+) release and Ca(2+) influx in an inositol 1,4,5-trisphosphate-independent manner in PC3 cells. These effects of estrogens on Ca(2+) signaling appear to be nongenomic. Prostate 47:141-148, 2001.

Accumulation of norfloxacin by Bacteroides fragilis.

The accumulation of norfloxacin by Bacteroides fragilis NCTC 9343 was determined by the modified fluorescence method. The time required to achieve a steady-state concentration (SSC) after allowing B. fragilis to accumulate norfloxacin in an aerobic or an anaerobic environment was approximately 2 min; the SSC achieved in air was 90.28 +/- 9.32 ng of norfloxacin/mg (dry weight) of cells, and that achieved anaerobically was 98.45 +/- 3.7 ng of norfloxacin/mg (dry weight) of cells. Initial rates of accumulation were determined with a range of external concentrations, as up to 8 microg/ml the concentration of norfloxacin accumulated increased proportionally to the external concentration, 12.13 ng/mg (dry weight) of cells per microg of exogenous norfloxacin per ml. At concentrations above 10 microg/ml no increase in the rate of norfloxacin accumulation was observed. From the kinetic data, a Lineweaver-Burk plot calculated a K(m) of 5.03 microg/ml and a V(max) of 25.1 ng of norfloxacin/s. With an increase in temperature of between 0 and 30 degrees C, the concentration of norfloxacin accumulated also increased proportionally at 4.722 ng of norfloxacin/mg (dry weight) of cells/ degrees C. At low concentrations of glucose (<0.2%; 11 mM), the concentration of norfloxacin accumulated was decreased. With the addition of 100 microM carbonyl cyanide m-chlorophenylhydrazone (CCCP) the mean SSC of norfloxacin was increased to 116 +/- 7.01 ng of norfloxacin/mg (dry weight) of cells; glucose had no significant effect in the presence of CCCP. Magnesium chloride (20 mM) decreased the SSC of norfloxacin to 40.5 +/- 3.76 ng of norfloxacin per mg (dry weight) of cells. These data suggest that the mechanism of accumulation of norfloxacin by B. fragilis is similar to that of aerobic bacteria and that the fluoresence procedure is suitable for use with an anaerobic bacterium.

Nutrient modulation of polarized and sustained submembrane Ca2+ microgradients in mouse pancreatic islet cells.

The intracellular calcium concentration ([Ca2+]i) near the plasma membrane was measured in mouse pancreatic islet cells using confocal spot detection methods. Whereas small cytosolic Ca2+ gradients were observed with 3 mM glucose, a steeper sustained gradient restricted to domains beneath the plasma membrane (space constant, 0.67 micrometer) appeared with 16.7 mM glucose. When the membrane potential was clamped with increasing K+ concentrations (5, 20 and 40 mM), no [Ca2+]i gradients were observed in any case. Increasing glucose concentration (0, 5 and 16.7 mM) in the presence of 100 microM diazoxide, a K+ channel opener, plus 40 mM K+ induced steeper [Ca2+]i gradients, confirming the role of membrane potential-independent effects of glucose. Prevention of Ca2+ store refilling with 30 microM cyclopiazonic acid (CPA) or blockade of uniporter-mediated Ca2+ influx into the mitochondria with 1 microM carbonyl cyanide m-chlorophenyl hydrazone (CCCP) or 1 microM Ru-360 significantly reduced the steepness of the 16.7 mM glucose-induced [Ca2+]i gradients. Measured values of [Ca2+]i reached 6.74 +/- 0.67 microM at a distance of 0.5 micrometer from the plasma membrane and decayed to 0.27 +/- 0.03 microM at a distance of 2 micrometer. Mathematically processed values at 0.25 and 0 micrometer gave a higher [Ca2+]i, reaching 8.18 +/- 0.86 and 10.05 +/- 0.98 microM, respectively. The results presented indicate that glucose metabolism generates [Ca2+]i microgradients, which reach values of around 10 microM, and whose regulation requires the involvement of both mitochondrial Ca2+ uptake and endoplasmic reticulum Ca2+ stores.

CAMP-mediated catabolite repression and electrochemical potential-dependent production of an extracellular amylase in Vibrio alginolyticus.

Vibrio alginolyticus, a halophilic marine bacterium, produced an extracellular amylase with a molecular mass of approximately 56,000, and the amylase appeared to be subject to catabolite repression mediated by cAMP. The production of amylase at pH 6.5, at which the respiratory chain-linked H+ pump functions, was inhibited about 75% at 24 hours following the addition of 2 microM carbonyl cyanide m-chlorophenylhydrazone (CCCP), while the production at pH 8.5, at which the respiratory chain-linked Na+ pump functions, was only slightly inhibited by the addition of 2 microM CCCP. In contrast, the production of amylase in a mutant bacterium defective in the Na+ pump was almost completely inhibited even at pH 8.5 as well as pH 6.5 by the addition of 2 microM CCCP.

Quinolone accumulation by Pseudomonas aeruginosa, Staphylococcus aureus and Escherichia coli.

The accumulation of nalidixic acid and 14 fluoroquinolones over a range of external drug concentrations (10-100 mg/L; c. 25-231 microM) into intact cells of Escherichia coli KL-16, Staphylococcus aureus NCTC 8532, Pseudomonas aeruginosa NCTC 10662 and spheroplasts of E. coli was investigated. The effect of 100 microM carbonyl cyanide m-chlorophenyl hydrazone (CCCP) upon the concentration of quinolone accumulated by intact cells and spheroplasts of E. coli was also determined. Except for pefloxacin, there was an increase in the concentration of the six quinolones examined accumulated by E. coli, despite a reduction in fluorescence at alkaline pH. For ciprofloxacin the partition coefficient (P(app)) was constant despite an increase in the pH; however, the P(app) for nalidixic acid decreased significantly with an increase in pH. The concentration of nalidixic acid, ciprofloxacin and enrofloxacin accumulated by E. coli and S. aureus increased with an increase in temperature up to 40 degrees C and 50 degrees C, respectively. Above these temperatures the cell viability decreased. With an increase in drug concentration there was, for intact E. coli and 12/15 agents, and for S. aureus and 10/15 agents, a linear increase in the concentration of drug accumulated. However, for P. aeruginosa and 13/15 agents there was apparent saturation of an accumulation pathway. Assuming 100% accumulation into intact cells of E. coli, for 10/14 fluoroquinolones < or = 40% was accumulated by spheroplasts. CCCP increased the concentration of quinolone accumulated but the increase varied with the agent and the bacterial species. The variation in the effect of CCCP upon accumulation of the different quinolones into E. coli could result from chemical interactions or from different affinities of the proposed efflux transporter for each quinolone. Overall, these data suggest that accumulation of most quinolones into E. coli and S. aureus proceeds by simple diffusion, but that P. aeruginosa behaves differently.

A single cell model of myocardial reperfusion injury: changes in intracellular Na+ and Ca2+ concentrations in guinea pig ventricular myocytes.

To investigate the contribution of the changes in intracellular Na+ and Ca2+ concentrations ([Na+]i and [Ca2+]i) to myocardial reperfusion injury, we made an ischemia/reperfusion model in intact guinea pig myocytes. Myocardial ischemia was simulated by the perfusion of metabolic inhibitors (3.3 mM amobarbital and 5 microM carbonyl cyanide m-chlorophenylhydrazone) with pH 6.6 and reperfusion was achieved by the washout of them with pH 7.4. [Na+]i increased from 7.9 +/- 2.0 to 14.0 +/- 3.4 mM (means +/- S.E., p < 0.01 ) during 7.5 min of simulated ischemia (SI) and increased further to 18.8 +/- 3.0 mM at 7.5 min after reperfusion. [Ca2+]i, expressed as the ratio of fluo 3 fluorescence intensity, increased to 133 +/- 8% (p < 0.01) during SI and gradually returned to the control level after reperfusion. Intracellular pH decreased from 7.53 +/- 0.04 to 6.31 +/- 0.04 (p < 0.01) and recovered quickly after reperfusion. Reperfusion with the acidic solution or the continuous perfusion of hexamethylene amiloride (2 microM) prevented the reperfusion-induced increase in [Na+]i. When the duration of SI was prolonged to 15 min, the cell response after reperfusion varied, 16 of 37 cells kept quiescent, 21 cells showed spontaneous Ca2+ waves, and 4 cells out of these 21 cells became hypercontracted. In quiescent cells, both [Na+]i and [Ca2+]i decreased immediately after reperfusion. In cells with Ca2+ waves, [Na+]i transiently increased further at the early phase of reperfusion, while [Ca2+]i declined. In hypercontracted cells, [Na+]i increased as much as in 'Ca2+ wave' cells, but [Ca2+]i increased extensively and both ion concentrations continued to increase. Reperfusion with the Ca2+-free solution prevented both the [Ca2+]i increase and morphological change. In the presence of ryanodine (10 microM), the increase in [Ca2+]i after reperfusion was augmented and some cells became hypercontracted. We concluded that (1) Na+/H+ exchange is active both during SI and reperfusion, resulting in the additional [Na+]i elevation on reperfusion, (2) the [Na+]i level after reperfusion and the following Ca2+ influx via Na+/Ca2+ exchange are crucial for reperfusion cell injury, and (3) the Ca2+ buffering capacity of sarcoplasmic reticulum would also contribute to the Ca2+ regulation and cell injury after reperfusion.

Background K+ currents and response to metabolic inhibition during early development in rat cardiocytes.

The effects of metabolic inhibition on K+ background currents and action potential duration were investigated in neonatal rat ventricle cells during early development. Action potentials and ionic currents were measured with the patch clamp technique in current and voltage clamp mode in cells isolated with collagenase from 1 day and 7 day old rats. During the first postnatal week, the cell surface increased from 1700 to 2210 microm2 and the membrane hyperpolarized from -66.1 to -72.0 mV. Concomitantly the action potential shortened and the plateau became more negative. Inhibition of oxidative phosphorylation (50 microM 2,4 DNP) or of glycolysis in 1 day old rats (5 mM 2-deoxyglucose, 2-DG) also shortened the action potential by about 50% after 5 min exposure. The background current measured in the absence of INa, ICa,L, and Ito included: (1) an inward rectifying component whose I/V curves crossed over when measured in 6, 15, or 30 mM [K]o and showed an increase in slope conductance when [K]o was raised. Inward rectification was abolished by 2.4 mM Ba2+ in 1 day old cells and by 0.2 mM one week after birth; (2) a glibenclamide (100 microM) sensitive component that developed with time after membrane rupture (5-10 min) showing a higher current density in 7 than in 1 day old animals (1.4 vs 0.2 microA x cm-2 at -50 mV); and (3) a small and almost linear leak component of comparable amplitude in both age groups. Inhibition of oxidative phosphorylation with 2.5 microM carbonylcyanide m-chlorophenylhydrazone induced the development of background currents with different properties in both age groups: An inwardly rectifying Ba2+ sensitive current in 1 day old cells and a glibenclamide sensitive outwardly rectifying current in the 7 day old group. In contrast, exposure to 5 mM 2-DG provoked in all cells the development of an outwardly rectifying current that was blocked by glibenclamide. We conclude that the electrophysiologic response to metabolic inhibition is determined by the relative importance of the metabolic pathways present which in turn depends on the developmental state of the cells.

An investigation on the role of vacuolar-type proton pumps and luminal acidity in calcium sequestration by nonmitochondrial and inositol-1,4,5-trisphosphate-sensitive intracellular calcium stores in clonal insulin-secreting cells.

To test whether in RINm5F rat insulinoma cells luminal acidity and the activity of a vacuolar-type proton pump are involved in calcium sequestration by intracellular calcium stores sensitive to inositol 1,4,5-trisphosphate (InsP3) we examined the effects of various proton-conducting ionophores and ammonium chloride, and of bafilomycin, a specific inhibitor of vacuolar proton pumps, on this parameter. Bafilomycin in concentrations up to 1 microM did not affect calcium sequestration by nonmitochondrial, InsP3-sensitive stores at all; 50 microM carbonylcyanide m-chlorophenylhydrazone, 50 microM monensin and 30 mM NH4Cl, which are diverse ways to dissipate transmembrane pH gradients, did not inhibit calcium sequestration. This argues against signficant involvement of internal acidity and vacuolar proton pumps in calcium sequestration by InsP3-sensitive stores in RINm5F cells. The proton-potassium-exchanging ionophore nigericin (20-100 microM), however, inhibited calcium sequestration by nonmitochondrial and InsP3-sensitive stores. This effect was dependent on the presence of potassium and could be reversed by inclusion of carbonylcyanide m-chlorophenylhydrazone or acetate in the incubation medium. Thus, the inhibitory effect of nigericin appears to be based on proton extrusion coupled to potassium influx across the membrane of calcium stores in RINm5F cells, creating an internal alkalinization of these stores. The effect of nigericin implies the continuous maintenance of an outside-to-inside potassium concentration gradient by nonmitochondrial calcium stores in RINm5F cells. This feature will be of potential interest in the identification of InsP3-sensitive calcium-storing organelles.

Biosynthesis and processing of mitochondrial glutaminase in HTC hepatoma cells

Rat HTC hepatoma cells were used to characterize the biosynthesis and processing of the renal isoenzyme of the mitochondrial glutaminase. Immunoblot analysis indicated that mitochondria isolated from HTC cells contained two prominent glutaminase peptides of 68 and 65 kDa and two minor peptides of 61 and 58 kDa. When the cells were labelled with [35S]methionine, the glutaminase-specific antibodies precipitated the same four polypeptides. However, when labelled in the presence of 5 microM-carbonyl cyanide m-chlorophenylhydrazone, an uncoupler of oxidative phosphorylation, only a 72 kDa cytoplasmic precursor of the mitochondrial glutaminase was immunoprecipitated. A comparison of the peptides generated by partial proteolysis of the precursor and the fully processed peptides indicates significant structural similarity. A 71 kDa form of the glutaminase was also observed when HTC cells were pulse-labelled for 2-6 min with [35S]methionine. Pulse-chase experiments indicate that the cytoplasmic precursor is quantitatively converted into the mature forms of the glutaminase. In addition, the observed kinetics established that the 71 kDa peptide is a true intermediate in the import of the mitochondrial glutaminase.

Biosynthesis and processing of renal mitochondrial glutaminase in cultured proximal tubular epithelial cells and in isolated mitochondria.

Primary cultures of rat renal proximal tubular epithelial cells were used to characterize the biosynthesis and processing of the mitochondrial glutaminase. When the cells were labeled with [35S]methionine in the presence of 20 microM carbonylcyanide m-chlorophenylhydrazone, only a 72-kDa peptide, which co-migrates with the primary translation product of the glutaminase mRNA, was immunoprecipitated. At lower concentrations of carbonylcyanide m-chlorophenylhydrazone, the 68- and 65-kDa peptides that are characteristic of the mature glutaminase and a 71-kDa peptide were synthesized. Pulse-chase experiments suggest that the 72-kDa cytosolic precursor could be quantitatively chased to generate the mature mitochondrial species. The observed kinetics indicate that the 71-kDa species is an intermediate in the import pathway. In addition, the 65-kDa glutaminase peptide was synthesized more rapidly than the 68-kDa peptide, and the two peptides were produced in a final ratio of 3:1, respectively. These results suggest that one subunit of the tetrameric glutaminase may be subject to covalent modification. In vitro processing was also characterized by incubating isolated rat liver mitochondria with the glutaminase precursor that was produced by in vitro translation of acidotic rat renal poly(A+) RNA. This system produced an identical sequence of processing reactions. The in vitro formation of the 71-kDa intermediate required a transmembrane potential. Both the intermediate and the mature forms of the glutaminase were recovered in the mitochondria and were resistant to trypsin digestion. Thus, the glutaminase precursor is rapidly translocated across the inner mitochondrial membrane and initially processed to yield an intermediate. The intermediate is subsequently processed to yield the two peptides that constitute the mature enzyme.

Kinetic mechanism of ATP action in Na(+)-K(+)-Cl- cotransport of HeLa cells determined by Rb+ influx studies

The kinetics of Na(+)-K(+)-Cl- cotransport were studied by measuring ouabain-insensitive furosemide-sensitive Rb+ influx (JRb) into HeLa cells while varying the cellular ATP and the extracellular Rb+ and Na+ concentrations. Results reveal that ATP stimulates JRb by increasing the affinity of the cotransporter for Rb+ (K+), and the apparent Michaelis constant (Km) for ATP was 0.95 +/- 0.03 mmol/l cell water. Two ATP molecules may relate to the uptake of one Rb+ by the cotransport pathway, as examined by the nonlinear least-squares method for goodness-of-fit and a Hill plot, JRb was strengthened by an increase in the inward chemical gradient associated with cell swelling on preincubation in a low-Na+ high-K+ medium, accompanying an increase in the affinity of the transporter for ATP. JRb was apparently activated by extracellular Na+, and the activation was enhanced by an increase in the cellular ATP concentration. Lactate production stimulated by 2 microM carbonylcyanide m-chlorophenyl hydrazone (CCCP) was reduced by 10 microM ouabain but not altered by further addition of 0.1 mM furosemide. Increases in cellular adenosine 3',5'-cyclic monophosphate (cAMP) caused by treatment with 0.1 mM isoproterenol plus 0.5 mM 3-isobutyl-1-methylxanthine or with 0.1 mM dibutyryl cAMP did not influence JRb. From this and previous studies, we propose a general and a specific model of Na(+)-K(+)-Cl- cotransport, which elucidate the order of binding of extracellular ions and reaction of cellular ATP.

Energy depletion-repletion and calcium transients in single cardiomyocytes.

Rapid fluctuations of intracellular free calcium in single adult rat heart myocytes were monitored by time-resolved fura-2 fluorescence microscopy. Under controlled aerobic conditions (35 degrees C, pH 7.3), electrical stimulation at 0.5 Hz produced a concave negative staircase of calcium transients. When the myocytes were challenged with 3 mM amobarbital (Amytal) and 2 microM carbonyl cyanide m-chlorophenylhydrazone (CCCP) to deplete ATP, the cells became unresponsive to electrical stimulation within 1 min but responded to 10 mM caffeine with a large increase in free calcium. After the development of rigor contracture, the cellular response to caffeine was blunted. Free calcium increased at a variable rate in individual cells, reaching values of 300-1,000 nM after 15 min. When the inhibitors were removed, calcium declined toward control values, and spontaneous contractile activity and calcium transients were invariably observed. During subsequent electrical stimulation, there was a decrease in the half-widths of the calcium transients and an attenuation of the negative staircase. Parallel experiments with cells in suspension indicated that Amytal and CCCP caused ATP to fall from 27.6 +/- 1.6 to 0.7 +/- 0.2 nmol/mg protein, and the percent rod-shaped cells to fall from 70 to 0% in 5 min. Removal of the inhibitors after 15 min caused a rebound in ATP to 5.3 +/- 1.5 nmol/mg within 2 min and 6.6 +/- 1.3 nmol/mg after 10 min.

Intracellular vacuoles in experimental acute pancreatitis in rats and mice are an acidified compartment.

The appearance of vacuoles inside acinar cells characterizes an early stage of development in different models of acute pancreatitis and, possibly, also in human disease. The vacuoles have been shown to contain both digestive and lysosomal enzymes. This abnormal admixture may have important implications for the pathogenesis of pancreatitis because the lysosomal enzyme cathepsin B can activate trypsinogen and may, by this way, trigger pancreatic autodigestion. For the activation process of trypsinogen by cathepsin B, however, an acidic pH is required. This study, therefore, looked for evidence of vacuole acidification in two different models of acute pancreatitis. Edematous pancreatitis was induced in rats by hyperstimulation with cerulein and hemorrhagic pancreatitis was induced in mice by feeding a choline-deficient, ethionine-supplemented diet. Pancreatic acinar cells were isolated at different times after induction of pancreatitis and incubated with 50 microM of acridine orange to identify acidic intracellular compartments. As shown in previous work, zymogen granules are the main acidic compartment of normal acinar cells; they remained acidic throughout the course of pancreatitis in both models. Vacuoles became increasingly more frequent in both models as pancreatitis progressed. Throughout development of pancreatitis, vacuoles accumulated acridine orange indicating an acidic interior. Addition of a protonophore (10 microM monensin or 5 microM carbonyl cyanide m-chlorophenylhydrazone [CCCP] or a weak base (5 mM NH4Cl) completely and rapidly abolished acridine orange fluorescence inside both zymogen granules and vacuoles providing further evidence for an acidic interior. The acidification of vacuoles seen in two different models of pancreatitis may be an important requirement for activation of trypsinogen by cathepsin B and thus for the development of acute pancreatitis.

Iron respiration-driven proton translocation in aerobic bacteria.

Washed cell suspensions of Aquaspirillum magnetotacticum MS-1, A. itersonii E12639, Bacillus subtilis 6633, and Escherichia coli CSH27 translocated protons in response to the added oxidant O2 or NO3-, with triphenylmethylphosphonium bromide as the permeant ion. Iron respiration-driven proton translocation was observed in A. magnetotacticum MS-1, B. subtilis, and E. coli but not in a nonmagnetic strain of A. magnetotacticum (strain NM-1A) or with A. itersonii. Proton translocation to Fe3+ was totally inhibited by 500 microM NaN3 or 0.5 microM carbonyl cyanide m-chlorophenylhydrazone.

Inhibitory effect of rhodamine 123 on the growth of the rodent malaria parasite, Plasmodium yoelii.

The effect of the cationic permeant fluorescent dye, rhodamine 123 (R123), on the in vivo growth of Plasmodium yoelii was examined. Plasmodium yoelii-infected mouse erythrocytes were incubated in vitro with R123 and injected intravenously into mice. Examination of daily parasitemias showed that R123 delayed parasite growth whereas rhodamine 110, a neutral compound, and fluorescein, a negatively charged fluorescent dye, did not. Infected erythrocytes treated with R123 were not cleared from the circulation even 7 h after injection. Quantitation of cell-associated R123 by spectrophotometry revealed that infected cells with increased levels of R123 considerably prolonged the 2% prepatent period, the time required for the parasite to develop a 2% parasitemia. Degenerating parasites within and outside the host erythrocytes were observed on day 1 of infection in the mice. Thus it follows that R123, which accumulated in infected erythrocytes, inhibits the growth of P. yoelii; moreover, when R123-labeled infected erythrocytes were treated with 1-10 microM carbonylcyanide m-chlorophenylhydrazone (CCCP), a proton ionophore, to release R123 from the cells, the inhibitory effect on the growth rate of P. yoelii was partially reversed.