Complete hunting cycle of Dionaea muscipula: Consecutive steps and their electrical properties

The total hunting cycle of the Venus flytrap consists of five stages: 1. Open state → 2. Closed state → 3. Locked state → 4. Constriction and digestion → 5. Semi-open state → 1. Open state. The opening of the trap after digestion consists of two steps: opening of the lobes, and changing of their curvature from concave to convex shape. Uncouplers carbonylcyanide-4-trifluoromethoxyphenyl hydrazone (FCCP) and carbonylcyanide-3-chlorophenylhydrazone (CCCP) inhibit the trap from opening for two weeks and antracene-9-carboxylic acid inhibits the trap from constricting. Different stages of the hunting cycle have different electrical characteristics. The biologically closed electrochemical circuits in the Venus flytrap are analyzed using the charged capacitor method. If the initial voltage applied to the Venus flytrap is 0.5 V or greater, changing the polarity of the electrodes between the midrib and one of the lobes results in a rectification effect and in different kinetics of discharge capacitance. These effects can be caused by the fast transport of ions through ion channels. The electrical properties of the Venus flytrap were investigated and equivalent electrical circuits within the upper leaf were proposed to explain the experimental data.     

Effects of Protonophores on the Synthesis of Catecholamines and the Intracellular pH in Cultured Bovine Adrenal Medullary Cells

The protonophores carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP) and carbonyl cyanide m-chlorophenylhydrazone (CCCP) stimulated the synthesis of 14C-catecholamines from [14C]tyrosine in cultured bovine adrenal medullary cells. The stimulatory effect of CCCP but not of FCCP was partially dependent on extracellular Ca2+. CCCP but not FCCP increased the influx of 45Ca2+ to the cells. When cells were incubated with either CCCP or FCCP (0.01-0.2 microgram/ml), the intracellular pH fell from 7.2 to 6.3-6.5 and catecholamine synthesis increased. Tyrosine hydroxylase activity in a soluble fraction prepared from cultured adrenal medullary cells was measured after incubation of the cells with FCCP or CCCP. Although FCCP did not affect the activity of the enzyme, CCCP caused a stable activation of it which was dependent on extracellular Ca2+. Since the optimal pH of soluble tyrosine hydroxylase is around 6.0 in adrenal medullary cells, FCCP may increase the synthesis of catecholamines by shifting the intracellular pH toward it. In addition to this mechanism, CCCP may enhance the synthesis of catecholamines by a Ca2+-dependent mechanism.     

The effect of carbonyl cyanide m-chlorophenylhydrazone on steroid transport in membrane vesicles of Pseudomonas testosteroni

The uncoupler carbonyl cyanide chlorophenylhydrazone (CCCP) was an effective inhibitor of steroid transport in membrane vesicles of Pseudomonas testosteroni between 10 microM and 1 microM CCCP. At these concentrations the inhibition of steroid transport was not due to an inhibition of the 3 beta and 17 beta-hydroxysteroid dehydrogenase enzyme. CCCP also affected testosterone-dependent oxygen consumption at concentrations up to 100 microM and inhibited respiration at 0.5 and 1 microM. The effect of CCCP on testosterone-dependent oxygen consumption indicated that CCCP was acting as an uncoupler. The concurrent inhibition of testosterone transport and stimulation of testosterone-dependent oxygen consumption at 10-100 microM CCCP supported the conclusion that transport and metabolism were tightly coupled processes. When membrane vesicles were pre-incubated with CCCP for 15 min, CCCP did inhibit transport and the 3 beta and 17 beta-hydroxysteroid dehydrogenase activity. However, both transport and enzyme inhibition could be prevented by the addition of NAD+ to the incubation mixture. This indicated that CCCP exhibits the properties of a sulfhydryl reagent under pre-incubated conditions.     

Staining of mitochondrial membranes with 10-nonyl acridine orange, MitoFluor Green, and MitoTracker Green is affected by mitochondrial membrane potential altering drugs

BACKGROUND: We set out to develop an assay for the simultaneous analysis of mitochondrial membrane potential and mass using the probes 10-nonyl acridine orange (NAO), MitoFluor Green (MFG), and MitoTracker Green (MTG) in HL60 cells. However, in experiments in which NAO and MFG were combined with orange emitting mitochondrial membrane potential (DeltaPsi(m)) probes, we found clear responses to DeltaPsi(m) altering drugs for both probes. METHODS: The three probes were titrated to determine whether saturation played a role in the response to drugs. The effects of a variety of DeltaPsi(m) altering drugs were tested for MFG and MTG at probe concentrations of 20 nM and 200 nM and for NAO at 0.1 microM and 5 microM, using rhodamine 123 at 0.1 microM as a reference probe. RESULTS: Incubation of GM130, HL60, and U937 cells with 2,3-butanedione monoxime (BDM), nigericin, carbonyl cyanide 3-chlorophenylhydrazone (CCCP), carbonyl cyanide p-(trifluoromethoxy)phenylhydrazone (FCCP), 2,4-dinitrophenol (DNP), gramicidin, ouabain, and valinomycin resulted in increases of the fluorescence intensity for MFG or MTG with only a few exceptions. The fluorescence intensity of cells stained with 0.1 microM NAO increased following incubation with BDM, nigericin, and decreased for FCCP, CCCP, DNP, gramicidin, and valinomycin. The results with 5 microM NAO were similar. CONCLUSIONS: MFG, MTG, and NAO appeared poor choices for the membrane potential independent analysis of mitochondrial membrane mass. Considering the molecular structure of these probes that favor accumulation in the mitochondrial membrane because of a positive charge, our results are not surprising. Cytometry 39:203-210, 2000. Published 2000 Wiley-Liss, Inc.     

The significance of hydrogenase activity for the energy metabolism of green algae: anaerobiosis favours ATP synthesis in cells of Chlorella with active hydrogenase

In cells of the green alga Chlorella fusca, which contain active hydrogenase(s), the concentration of ATP, NADH and NADPH were measured during a 5 h period of anaerobiosis in the dark and upon subsequent illumination with high light intensities (770 W/m²), conditions which favour optimal hydrogen photoproduction.ATP concentrations were also determined in cells of Chlorella fusca, whose hydrogenase was inactivated prior to illumination, and in cells of Chlorella vulgaris which do not contain hydrogenase. In the dark, the ATP concentration increased slightly during anaerobiosis in cells with active hydrogenase. This increase in ATP concentration was accompanied by an increase of NADH and a decrease of NADPH content.Upon illumination, the ATP content increased in cells with an active hydrogenase, whereas the NADH content decreased. The rate of phosphorylation was twice that observed in cells without active hydrogenase.This ATP synthesis in the light was not inhibited by 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU) (10 mol/l) nor by carbonylcyanide-3-chlorophenyl-hydrazone (CCCP) (1 mol/l) but was diminished by 500 mol/l dibromothymoquinone (DBMIB) and 6 mol/l carbonylcyanide-3-chlorophenyl-hydrazone (CCCP).It was concluded that an active hydrogenase can support ATP production under anaerobic conditions in the dark as well as in the light. NADH might serve in vivo as electron donor for a fermentative production of hydrogen in the light.Possible mechanisms underlying ATP production under anaerobiosis and hydrogen productive conditions are discussed.Abbreviations CCCP Carbonylcyanide-3-chlorophenyl-hydrazone - DBMIB dibromothymoquinone - DCMU 3-(3,4-dichlorophenyl)-1,1-dimethylurea - FCCP carbonylcyanide-p-trifluormethoxyphenyl-hydrazone - HEPES N-2-hydroxyethylpiperazin-N-2-ethan-sulfonic acid - PSI II, photosystem I, II respectively - PQ plastoquinone     

Effect of carbonylcyanide-4-(trifluoromethoxy)phenylhydrazone (FCCP) on the interaction of 1-anilino-8-naphthalene sulfonate (ANS) with phosphatidylcholine liposomes

The weak hydrophobic acid carbonylcyanide-4-(trifluoromethoxy)phenylhydrazone (FCCP) is a protonophoric uncoupler of oxidative phosphorylation in mitochondria. It dissipates the electrochemical proton gradient (Δμ_H ⁺) increasing the mitochondrial oxygen consumption. However, at concentrations higher than 1 μM it exhibits additional effects on mitochondrial energy metabolism, which were tentatively related to modifications of electrical properties of the membrane. Here we describe the effect of FCCP on the binding of 1-anilino-8-naphthalene sulfonate (ANS) to 1, 2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) and 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) unilamellar vesicles. FCCP inhibited the binding of ANS to liposomes either in the gel or in the liquid crystalline phase, by increasing the apparent dissociation constant of ANS. Smaller effect on the dissociation constant was observed at high ionic strength, suggesting that the effect of FCCP is through modification of the electrostatic properties of the membrane interface. In addition, FCCP also decreased (approximately 50 %) the quantum yield and increased the intrinsic dissociation constant of membrane-bound ANS, results that suggest that FCCP makes the environment of the ANS binding sites more polar. On those grounds we postulate that the binding of FCCP: i) increases the density of negative charges in the membrane surface; and ii) distorts the phospholipid bilayer, increasing the mobility of the polar headgroups making the ANS binding site more accessible to water.     

ATP-dependent calcium accumulation by non-mitochondrial organelles of axoplasm isolated from Myxicola giant axons

Axoplasm from freshly isolated Myxicola giant axons was mixed with small volumes of 'artificial axoplasm' containing 45Ca and either CaEGTA/EGTA or CaDTPA/DTPA buffers giving various nominal values of [Ca2+]. The axoplasm samples were centrifuged at 100 000 X g for 30 min to form a pellet and the percentage of 45Ca bound to the pellet was determined. The fraction of bound calcium rose with increasing values of [Ca2+] along an S-shaped curve. Carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP) was used to reveal the presence of mitochondrial Ca uptake. At physiological values of [Ca2+], around 100 nM, Ca uptake was insensitive to FCCP. As [Ca2+] was elevated, increasing sensitivity to FCCP was noted above [Ca2+] = 0.5 microM. At low values of [Ca2+], including the physiological range, Ca binding was significantly reduced by vanadate and quercetin, agents known to inhibit Ca uptake mediated by Ca2+-activated ATPase reactions. Inhibition of Ca binding by these agents was approximately 50% at physiological values of [Ca2+]. ATP depletion decreased the percentage of Ca binding by the pellet at physiological [Ca2+]. The results suggest that about 50% of the Ca buffering by particulate matter in axoplasm is via organelles requiring intact Ca2+-ATPase reaction at physiological values of [Ca2+].     

Role of dark respiration in photoinhibition of photosynthesis and its reactivation in the cyanobacterium Anacystis nidulans

Photoinhibition of photosynthesis and its reactivation was studied in the cyanobaterium A. nidulans in the presence of the respiratory inhibitor sodium azide, the uncouplers carbonyl cyanide p -(trifluoromethoxy)-phenylhydrazone (FCCP) and carbonyl cyanide m -chlorophenylhydrazone (CCCP) and the photosystem I elicitor phenazine methosulphate (PMS). Inhibition of dark respiration by azide increased the susceptibility of the cyanobacterium to photoinhibition. Both FCCP and CCCP also remarkably affected the process of photoinhibition in A. nidulans. The PMS at lower photoinhibitory light intensity partially protected A. nidulans from photoinhibition. The recovery from photoinhibition in the presence of azide or FCCP was slow and normal photosynthesis could not be resumed even after a longer period of incubation under suitable reactivating condition. Thus dark respiration has a key function in the process of photoinhibition of photosynthesis and its reactivation in the cyanobacterium A. nidulans.     

Cytoplasmic reorganization during the resumption of meiosis in cultured preovulatory rat oocytes

Changes in organelle topography and microtubule configuration have been studied during the resumption and progression of meiosis in cultured preovulatory rat oocytes. Germinal vesicle breakdown (GVBD) was reversibly inhibited by dibutyryl cAMP (DcAMP) or nocodazole, a microtubule-disrupting agent. The microtubule stabilizing agent taxol did not inhibit GVBD, but did impair further maturation. The migration of acidic organelles and chromatin in living oocytes was analyzed using the vital stains acridine orange and Hoechst 33258, respectively. Germinal vesicle stage oocytes undergo perinuclear aggregation of acidic organelles during GVBD and these organelles subsequently disperse into the cell cortex as the first meiotic spindle migrates to the oocyte periphery. DcAMP and nocodazole block the perinuclear aggregation of acidic organelles, whereas, in taxol-treated oocytes, organelle aggregation and GVBD occur but the dispersion of acidic organelles was arrested. Dose-response studies on the effects of nocodazole showed that GVBD was generally retarded and that a 50% inhibition of GVBD was achieved at concentrations in excess of 1.0 microM. Concentrations of taxol at 10 microM or above effectively inhibited both chromatin condensation and meiotic spindle formation. Indirect immunofluorescence microscopy with anti-tubulin antibodies revealed dissolution of microtubules with 1.0 microM nocodazole. Taxol had little effect on microtubule organization in germinal vesicle or chromatin condensation stage oocytes; however, when oocytes that had formed first meiotic spindles were treated with taxol, numerous microtubule asters appeared which were preferentially associated with the oocyte cortex. The changes in organelle topography, microtubule configuration, and drug sensitivity are discussed with respect to the regulation of cytoplasmic reorganization during the meiotic maturation of rat preovulatory oocytes.     

Photoreactions of cytochrome b-559 and cyclic electron flow in Photosystem II of intact chloroplasts

The high potential cytochrome b-559 of intact spinach chloroplasts was photooxidized by red light with a high quantum efficiency and by far-red light with a very low quantum efficiency, when electron flow from water to Photosystem II was inhibited by a carbonyl cyanide phenylhydrazone (FCCP or CCCP). Dithiothreitol, which reacts with FCCP or CCCP, reversed the photooxidation of cytochrome b-559 and restored the capability of the chloroplasts to photoreduce CO₂ showing that the FCCP/CCCP effects were reversible. The quantum efficiency of cytochrome b-559 photooxidation by red or far-red light in the presence of FCCP was increased by 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone which blocks oxidation of reduced plastoquinone by Photosystem I. When the inhibition of water oxidation by FCCP or CCCP was decreased by increased light intensities, previously photooxidized cytochrome b-559 was reduced. Red light was much more effective in photoreducing oxidized high potential cytochrome b-559 than far-red light. The red/far-red antagonism in the redox state of cytochrome b-559 is a consequence of the different sensitivity of the cytochrome to red and far-red light and does not indicate that the cytochrome is in the main path of electrons from water to NADP. Rather, cytochrome b-559 acts as a carrier of electrons in a cyclic path around Photosystem II. The redox state of the cytochrome was shifted to the oxidized side when electron transport from water became rate-limiting, while oxidation of water and reduction of plastoquinone resulted in its shifting to the reduced side.     

Rapidly transported organelles containing membrane and cytoskeletal components: their relation to axonal growth

We have examined the movements, composition, and cellular origin of phase-dense varicosities in cultures of chick sympathetic and sensory neurons. These organelles are variable in diameter (typically between 0.2 and 2 microns) and undergo saltatory movements both towards and away from the neuronal cell body. Their mean velocities vary inversely with the size of the organelle and are greater in the retrograde than the anterograde direction. Organelles stain with the lipophilic dye 1, 1'-dioctadecyl-3,3,3',3'-tetramethyl-indocarbocyanine and with antibodies to cytoskeletal components. In cultures double-stained with antibodies to alpha-tubulin and 70-kD neurofilament protein (NF-L), approximately 40% of the organelles stain for tubulin, 30% stain for NF- L, 10% stain for both tubulin and NF-L, and 40% show no staining with either antibody. The association of cytoskeletal proteins with the organelles shows that these proteins are able to move by a form of rapid axonal transport. Under most culture conditions the predominant direction of movement is towards the cell body, suggesting that the organelles are produced at or near the growth cone. Retrograde movements continue in culture medium lacking protein or high molecular mass components and increase under conditions in which the advance of the growth cone is arrested. There is a fourfold increase in the number of organelles moving retrogradely in neurites that encounter a substratum-associated barrier to elongation; retrograde movements increase similarly in cultures exposed to cytochalasin at levels known to block growth cone advance. No previously described organelle shows behavior coordinated with axonal growth in this way. We propose that the organelles contain membrane and cytoskeletal components that have been delivered to the growth cone, by slow or fast anterograde transport, in excess of the amounts required to synthesize more axon. In view of their rapid mobility and variable contents, we suggest that they be called "neuronal parcels."     

Light-induced redox changes of cytochrome b-559

Dark incubation of spinach or pea chloroplasts with 10 μm carbonylcyanide m-chlorophenylhydrazone (CCCP) had a negligible effect either on the redox state or the redox potential of the high potential form of cytochrome b-559 (cytochrome b-559_hp). A similar result was obtained with spinach chloroplasts on incubation with 3.3 μm carbonylcyanide p-trifluoromethoxyphenylhydrazone (FCCP), but pea chloroplasts showed a decrease of 10–20% in the amount of reduced cytochrome b-559.Light-induced redox changes of cytochrome b-559 were not observed in untreated spinach chloroplasts. In the presence of CCP or FCCP, cytochrome b-559 was photooxidized both in 655 nm actinic light and in far-red light. Addition of the plastoquinone antagonist, 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone (DBMIB) to CCCP- or FCCP-treated chloroplasts had only a small effect on the photooxidation of cytochrome b-559 in 655 light, but it completely inhibited the oxidation in far-red light.Electron flow from water to 2,3′,6-trichlorophenolindophenol was partly inhibited by CCCP or FCCP, but the degree of inhibition does not appear to be sufficient to account for the photooxidation of cytochrome b-559.The photooxidation of cytochrome b-559 by 655 nm light at liquid nitrogen temperature was not influenced by prior treatment of the chloroplasts at room temperature with CCCP, DBMIB, or CCCP + DBMIB.The results cannot be explained by the presence of two independent pools of cytochrome b-559 in CCCP-treated chloroplasts, one photooxidized by Photosystem II and the other photooxidized by Photosystem I and photoreduced by Photosystem II.     

Interaction of oxidized and reduced N-methylphenazonium methosulfate (PMS) with Photosystem II

In 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU) poisoned chloroplasts, the restoration of the fluorescence induction is presumed to be due to a back reaction of the reduced primary acceptor (Q⁻) and the oxidized primary donor (Z⁺) of Photosystem II. Carbonylcyanide m-chlorophenylhydrazone (CCCP) is known to inhibit this back reaction. The influence of reduced N-methylphenazonium methosulfate (PMS) in the absence of CCCP and of oxidized PMS in the presence of CCCP on the back reaction was investigated and the following results were obtained:

1. (1) Reduced PMS at the concentration of 1 μM inhibits the back reaction as effectively as hydroxylamine, suggesting an electron donating function of reduced PMS for System II.

2. (2) The inhibition of the back reaction by CCCP is regenerated to a high degree by oxidized PMS which led to assume a cyclic System II electron flow catalysed by PMS.

3. (3) At concentrations of reduced PMS higher than 1 μM it is shown that both the fast initial emission and more significantly the variable emission are quenched.

Inhibition of nitrate uptake by ammonia in a blue-green alga,Anabaena cylindrica

Ammonia at concentrations above 1×10^-5 M inhibits uptake of nitrate in the nitrogen-fixing blue-green alga, Anabaena cylindrica. This inhibition takes place both in the light and in the dark. The rate of nitrate uptake is stimulated by light. Addition of relatively high concentrations of nitrate (1–10 mM) reversibly inhibits ammonia uptake. FCCP, an uncoupler of phosphorylation, inhibits both nitrate and ammonia uptake. Ammonia may inhibit nitrate uptake by reducing the supply of energy (ATP) for active nitrate transport.Abbreviations FCCP carbonyl cyanide p-trifluoromethoxy-phenylhydrazone - CCCP carbonyl cyanide m-chlorophenyl-hydrazone     

Changes in FAD and NADH Fluorescence in Neurosecretory Terminals Are Triggered by Calcium Entry and by ADP Production

We measured changes in the intrinsic fluorescence (IF) of the neurosecretory terminals of the mouse neurohypophysis during brief (1-2 s) trains of stimuli. With fluorescence excitation at either 350 +/- 20 or 450 +/- 50 nm, and with emission measured, respectively, at 450 +/- 50 or > or = 520 nm, DeltaF/F(o) was approximately 5-8 % for a 2 s train of 30 action potentials. The IF changes lagged the onset of stimulation by approximately 100 ms and were eliminated by 1 microM tetrodotoxin (TTX). The signals were partially inhibited by 500 microM Cd(2+), by substitution of Mg(2+) for Ca(2+), by Ca(2+)-free Ringer's with 0.5 mM EGTA, and by 50 microM ouabain. The IF signals were also sensitive to the mitochondrial metabolic inhibitors CCCP (0.3 microM), FCCP (0.3 microM), and NaN(3) (0.3 mM), and their amplitude reflected the partial pressure of oxygen (pO(2)) in the bath. Resting fluorescence at both 350 nm and 450 nm exhibited significant bleaching. Flavin adenine dinucleotide (FAD) is fluorescent, while its reduced form FADH(2) is relatively non-fluorescent; conversely, NADH is fluorescent, while its oxidized form NAD is non-fluorescent. Thus, our experiments suggest that the stimulus-coupled rise in [Ca(2+)](i) triggers an increase in FAD and NAD as FADH(2) and NADH are oxidized, but that elevation of [Ca(2+)](i), alone cannot account for the totality of changes in intrinsic fluorescence.     

Differences in release of endogenous sugars and amino acids from attached and detached seed coats of developing pea seeds

The effect of p -chloromercuribenzenesulfonic acid (PCMBS), carbonylcyanide- m -chlorophenylhydrazone (CCCP) and a high apoplastic pH (pH 7.5 compared with pH 5.5) on the release of sugars (sucrose and glucose) and amino acids from attached and detached seed coats of Pisum sativum L. cv. Marzia into a bathing solution was measured by means of the 'empty seed coat technique'. PCMBS reduced the release of sugars and amino acids from attached as well as from detached seed coats, suggesting that carrier-mediated transport might be involved. CCCP reduced sugar release from attached seed coats while amino acid release was hardly affected. In experiments with detached seed coats CCCP had no effect on release of either sugar or amino acids, suggesting that it is not energy-dependent. Raising the pH of the bathing solution from pH 5.5 to pH 7.5 slightly increased sugar release from both attached and detached seed coats while amino acid release was not affected. This might indicate a role of the apoplastic pH in regulating sugar release from the seed coat via a retrieval mechanism. The presented data indicate that there are important differences between sugars and amino acids with respect to transport processes in the seed coat. This is supported by the observation that the rate of amino acid release from the seed coat was higher than the rate of sugar release. The release data of detached seed coats were subjected to compartmental analysis in order to calculate rate constants for release from cell compartments. In the case of sugars, the half-times for emptying the cytoplasmic and vacuolar compartment were 0.8 h and 12.5 h. respectively. For amino acids the half-times were 0.5 h for emptying the cytoplasmic and 3.8 h for emptying the vacuolar compartment.     

Clomiphene, an ovulation-inducing agent, mobilizes intracellular Ca2+ and causes extracellular Ca2+ influx in bladder female transitional carcinoma cells

BACKGROUND/METHODS: The effect of clomiphene, an ovulation-inducing agent, on cytosolic free Ca2+ levels ([Ca2+]i) in populations of BFTC human bladder cancer cells was explored by using fura-2 as a Ca2+ indicator. RESULTS: Clomiphene at concentrations between 10 and 75 microM increased [Ca2+]i in a concentration-dependent manner and the signal saturated at 50 microM. The [Ca2+]i signal was biphasic with an initial rise and a slow decay. Ca2+ removal inhibited the Ca2+ signal by about 40-50% in maximum [Ca2+]i. Adding 3 mM Ca2+ increased [Ca2+]i in cells pretreated with 50 microM clomiphene in Ca2+-free medium, suggesting that clomiphene induced capacitative Ca2+ entry. In Ca2+-free medium, pretreatment with 50 microM brefeldin A (to disrupt the Golgi complex Ca2+ store), 1 microM thapsigargin (to inhibit the endoplasmic reticulum Ca2+ pump), and CCCP (to uncouple mitochondria) inhibited 85% of clomiphene-induced intracellular Ca2+ release. Conversely, pretreatment with 50 microM clomiphene in Ca2+-free medium abolished the [Ca2+]i increase induced by brefeldin, thapsigargin or CCCP. The intracellular Ca2+ release was unaltered by inhibiting formation of inositol-1,4,5-trisphosphate (IP3) with 2 mM 1-(6-((17beta-3-methoxyestra-1,3,5(10)-trien-17-yl)amino)hexyl)-1H-pyrrole-2,5-dione (U73122; a phospholipase C inhibitor). CONCLUSION: The [Ca2+]i increase induced by 50 microM clomiphene was not affected by 10 microM of nifedipine, verapamil or diltiazem. Collectively, the results suggest that clomiphene releases intracellular Ca2+ in an IP3-independent manner and also activates extracellular Ca2+ influx.     

Maintenance of a neutral cytoplasmic pH is not obligatory for growth of Escherichia coli and Streptococcus faecalis at an alkaline pH

Both Escherichia coli and Streptococcus faecalis grew in an alkaline medium containing 100 microM carbonyl cyanide m-chlorophenylhydrazone (CCCP). Our data suggested that CCCP functioned as a protonophore at a high pH and that the proton-motive force was dissipated almost completely under such conditions. The pH gradient measured with dimethyloxazolidine-2,4-dione and acetylsalicylic acid was very small in both bacteria at a high pH above 8, and was not affected significantly by the addition of CCCP. Based on these findings, we propose that the maintenance of neutral cytoplasmic pH is not obligatory for the growth of E. coli and S. faecalis in an alkaline medium.     

Decrease of intracellular ATP content downregulated UCP2 expression in mouse hepatocytes

Mitochondrial uncoupling protein 2 (UCP2) plays an important role in regulating energy metabolism. We previously reported that UCP2 expression in steatotic livers is increased which leads to diminished hepatic ATP stores and renders steatotic hepatocytes vulnerable to ischemic damage. In this study, reagents that inhibit the production of ATP were used to mimic an ischemic state in the liver in order to investigate the effects of decreased intracellular ATP levels on UCP2 expression in a murine hepatocyte cell line (HEP6-16). Carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP), an oxidative phosphorylation uncoupler, was found to decrease intracellular ATP levels in a dose- and time-dependent manner. Relatively high concentrations of FCCP from 8 to 80 microM were required to reduce the intracellular concentration of ATP. The inhibitory effect of FCCP on intracellular ATP was significantly potentiated by 2-deoxy-D-glucose, an inhibitor of glycolysis that when administered alone had no negative effect on cellular ATP levels in mouse hepatocytes. Decreased intracellular ATP levels were accompanied by lower UCP2 mRNA expression. Upon removal of FCCP and/or 2-deoxy-D-glucose and reculture with normal medium, ATP and UCP2 mRNA levels returned to normal within a few hours. Mitochondrial membrane potential in HEP6-16 cells was dissipated by 80 microM FCCP but not 8 microM FCCP, suggesting that the downregulation of UCP2 expression by FCCP was not related to mitochondrial potential changes. Consequently, the in vitro manipulation of ATP stores is consistent with the in vivo observations associated with ischemia/reperfusion injury.     

Effects of Carbonyl Cyanide Phenylhydrazones on Two Mitochondrial Ion Channel Activities

The respiratory uncouplers carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP) and carbonyl cyanide m-chlorophenylhydrazone (CCCP) affect the activities of two mitochondrial ion channels from mouse liver. At micromolar concentrations, the phenylhydrazones block the voltage-dependent 100-pS channel, mCS, and induce the multiple-conductance-level channel, MCC. The binding site(s) involved in perturbation of channel activities are probably distinct from the sites involved in uncoupling of oxidative phosphorylation which occurs at nanomolar concentrations of the phenylhydrazones. The effects of FCCP and CCCP on the mitochondrial ion channels could be partially reversed by washing with fresh media and were always reversed by perfusion with dithiothreitol. These results indicate that the effects of the phenylhydrazones on mitochondrial ion channels may be related to the ability of these compounds to act as sulfhydryl reagents and not to their protonophoric and uncoupling activity.