FLUORIDE ACTION NETWORK

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mitochondria fluoride - page 2

hypoxia, apotosis

MITOCHONDRIA:
A part of the cell (organelle) that is responsible for energy production. The organelle consists of two sets of membranes, a smooth continuous outer coat and an inner membrane arranged in tubules or in folds that form plate-like double membranes (cristae); the principal energy source of the cell, containing the cytochrome enzymes of terminal electron transport and the enzymes of the citric acid cycle, fatty acid oxidation, and oxidative phosphorylation. Responsible for converting nutrients into energy as well as many other specialized tasks.

Mitochondrial Disease

Scientists believe that the aging process itself may be due to a lifetime of damage to the mitochondria. Research also suggests that genetic defects in the mitochondria may be linked to many chronic diseases associated with age-related degenerative diseases such as cancer, Alzheimer's, Parkinson's, and heart disease. http://www.umdf.org/mitodisease/facts.html

Basis of the Disease
Mitochondrial diseases result from failures of the mitochondria, specialized compartments present in every cell of the body except red blood cells. Mitochondria are responsible for creating more than 90% of the energy needed by the body to sustain life and support growth. When they fail, less and less energy is generated within the cell. Cell injury and even cell death follow. If this process is repeated throughout the body, whole systems begin to fail, and the life of the person in whom this is happening is severely compromised. The disease primarily affects children, but adult onset is becoming more and more common. Diseases of the mitochondria appear to cause the most damage to cells of the brain, heart, liver, skeletal muscles, kidney and the endocrine and respiratory systems... http://www.umdf.org/mitodisease/diagnosis.html

Symptoms & Diagnosis
When to Suspect Mitochondrial Dysfunction There is no one identifying feature of mitochondrial disease. Patients can have combinations of problems whose onset may occur from before birth to late adult life. Mitochondrial diseases should be considered in the differential diagnosis when there are these unexplained features, especially when these occur in combination. Encephalopathy Seizures, Developmental Delay or Regression (including early and late-onset dementia), Myoclonus, Movement Disorders (dystonia, dyskinesias, chorea, etc.), Complicated Migraine ,Stroke http://www.umdf.org/mitodisease/diagnosis.html

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Hexafluoroacetone - (Not a pesticide)
Non-Human Toxicity Excerpts : Testicular atrophy was induced in rats by dermal application of hexafluoroacetone (HFA) at 39 or 130 mg/kg/day for 14 days, but not at a dosage of 13 mg/kg/day. Affected germ cells were mostly spermatids and to a lesser extent spermatocytes; spermatogonia were unaffected. Late spermatids were retained in Sertoli cells and showed degenerative changes. Sertoli cells exhibited cytoplasmic vacuolation, distended endoplasmic reticulum and a marked increase ln lipid droplets. Leydig cells exhibited a slight increase in lipid droplets, fewer mitochondria and diminution and segregation of the agranular endoplasmic reticulum from mitochondria. A correlation between ultrastructural and biochemical changes in HFA induced testicular atrophy was presented. Ref: Lee KP, Gullies PJ; Exp Mol Pathol 40 (1): 29-37 (1994)

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Sodium fluoride
PubMed Abstract: OBJECTIVE--To address the role of fluoride in causing defects to spermatids and epididymal spermatozoa. METHODS--Male rabbits were treated with 10 mg NaF/kg body weight daily for 18 months and maintained under identical laboratory conditions along with the control rabbits not given NaF. Testis and epididymis (caput) were investigated for ultrastructural details of spermatids and spermatozoa. RESULTS--A wide variety of structural defects were observed in the flagellum, the acrosome, and the nucleus of the spermatids and epididymal spermatozoa of fluoride-treated rabbits. Abnormalities included absence of outer microtubules, complete absence of axonemes, structural and numeric aberrations of outer dense fibers, breakdown of the fibrous sheath, and structural defects in the mitochondria of the middle piece of the flagellum. Detachment and peeling off of the acrosome from the flat surfaces of the nucleus were also observed. CONCLUSION--The abnormalities observed render the sperm nonfunctional and ineffective, and thus there is a possible role of fluoride in causing infertility. Ref: Int J Fertil Menopausal Stud 1994 May-Jun;39(3):164-71 Ultrastructural studies of spermiogenesis in rabbit exposed to chronic fluoride toxicity. Kumar A, Susheela AK PMID: 7920753, UI: 95004768

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CFC-12 and CFC-114 do not markedly affect oxygen consumption or oxidative phosphorylation in mitochondria isolated from the liver, lung, brain, heart, or kidney of rats exposed to about 7.5% chlorofluorocarbons prior to mitochondrial isolation. Further in vitro studies were conducted with liver and heart mitochondria in which measurements were taken during exposure of the mitochondria to CFC-12 at 990 g/cu m (20%) (time of exposure not specified). No effects on either oxidation or phosphorylation were noted. [WHO; Environmental Health Criteria 113: Fully Halogenated Chlorofluorocarbons p.87 (1990)]**PEER REVIEWED** Dichlorodifluoromethane. TOXNET http://www.fluoridealert.org/pesticides/Dichlorodifluorometh.TOXNET.htm

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D. Cumulative Effects The pyrrole insecticides represent a new class of chemistry with a unique mechanism of action. The parent molecule, AC303,630 is a pro- insecticide which is converted to the active form, CL303,268, via rapid metabolism by mixed function oxidases (MFOs). The active form uncouples oxidative phosphorylation in the insect mitochondria by disrupting the proton gradient across the mitochondrial membrane. The production of adenosine triphosphate (ATP) is inhibited resulting in the cessation of all cellular functions. Because of this unique mechanism of action, American Cyanamid believes that it is highly unlikely that toxic effects produced by chlorfenapyr would be cumulative with those of any other pesticide chemical. In mammals, there is a lower titer of MFOs, and chlorfenapyr is metabolized by different pathways (including dehalogenation, oxidation, and ring hydroxylation) to other polar metabolites without any significant accumulation of the potent uncoupler, CL303,268. In the rat, approximately 85% of the administered dose is excreted in the feces within 48 hours, thereby reducing the levels of AC303,630 and CL303,268 that are capable of reaching the mitochondria. This differential metabolism of AC303,630 to CL303,268 in insects, versus to other polar metabolites in mammals, is responsible for the selective insect toxicity of the pyrroles.
Chlorfenapyr (American Cyanamid). February 5, 1997, Pesticide Petition. Federal Register. http://www.fluoridealert.org/pesticides/Chlorfenapyr.FR.Feb.1997.htm
also http://www.fluoridealert.org/pesticides/Chlorfenapyr.FR.Aug.26.1999.htm
also http://www.fluoridealert.org/pesticides/Chlorfenapyr.FR.Mar.27.1998.htm
also http://www.fluoridealert.org/pesticides/Chlorfenapyr.FR.Sept30.1998.htm

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Running title: Pharmacological implications of aluminofluoride complexes Corresponding author: Anna Struneck‡ Department of Physiology and Developmental Physiology, Faculty of Sciences, Charles University, Vini�n‡ 7, 128 00, Prague 2, Czech Republic Telephone: (42) - 02/21953239 Fax no.: (42) - 02/299713 E-mail: astrun@cesnet.cz
Energy metabolism. ATP generation in mitochondria requires the association of F1 subunit with F0 transmembrane subunit transporting protons. The binding of ADP and Pi in a catalytic site of F1 triggers conformational changes which lock both of them into the site and induce the formation of pyrophosphate bonds by eliminating a water molecule (Chabre 1990). Lunardi et al. (1988) reported the inhibition of mitochondrial ATPase activity in the presence of [AlF4]-. This inhibition is not reversed by elution of fluoride from solution or by addition of strong chelators of aluminium. No significant release of the complex occured over a period of days. [AlF4]- inhibits many ATPases, phosphatases and phosphorylases. The intervention of aluminofluoride complexes in the energy transformation processes may thus affect the energy metabolism of the entire organism.
http://www.fluoridealert.org/ifin-28.htm

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Other organohalides used to induce anesthesia,'such as fluoroxene (CF3-CH2-0-CH=CH2), isoflurane (CF3-0-CClH-CF3) and halothane (CF3-CBrClH), release little inorganic fluoride during oxidative metabolism (Loew et al. 1974; Gion et al. 1974.) (NOTE: Metabolites other than inorganic fluoride compounds may, however, be toxic, e.g. trifluoroethanol (Gion et al. 1974; Tucker et al. 1973; Fiserova-Bergerova 1977).) It appears that this relatively greater stability is attributable to the bonding position of fluorine in these compounds (i.e. entirely on CF3 groups). However, Hitt et al. (1974) noted that isoflurane is approximately one-tenth as soluble as methoxyflurane, and suggested that the substrate concentration in vivo may limit its metabolic degradation to inorganic fluoride. Hitt et al. (1974) observed a release of inorganic fluoride from isoflurane by preparations of rat-liver mitochondria in vitro, especially if the live rats had been preconditioned (enzyme induction) by exposure to phenobarbital.
National Research Council of Canada NRC Associate Committee on Scientific Criteria for Environmental Quality Environmental Fluoride 1977 by Dyson Rose & John R. Marier National Research Council of Canada NRCC NO. 16081 ISSN 0316-0114
http://www.fluoridealert.org/NRC-fluoride.htm

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Inactive until consumed, chlorfenapyr kills by interrupting the manufacture of the energy storage molecule ATP in cells' mitochondria. The action of chlorfenapyr depends on several of the ATP cycle's enzymes, which are common to all living organisms. The company says, however, that only insects have adequate amounts of the necessary enzymes to be affected; most organisms will be safe.
Scientific American October 1999
Science and the Citizen PIRATE FEAR: Controversy heats up about chlorfenapyr, a.k.a. Pirate--a pesticide some claim is the next DDT

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Non-Human Toxicity Excerpts: NON-PHYTOTOXIC TO CEREALS. BEES EXPOSED TO SPRAY WERE UNAFFECTED BY 1000 PPM PURE CHLORFLURAZOLE AS AQUEOUS SUSPENSION OR SODIUM SALT SOLN, BUT WERE KILLED BY 4000 PPM. ... AN UNCOUPLER OF OXIDATIVE PHOSPHORYLATION CAUSING 50% UNCOUPLING OF RAT LIVER MITOCHONDRIAL OXIDATIVE PHOSPHORYLATION AT 6X10-7 MOLAR ALSO STIMULATING ATPASE ACTIVITY & CELL RESPIRATION. [Spencer, E. Y. Guide to the Chemicals Used in Crop Protection. 7th ed. Publication 1093. Research Institute, Agriculture Canada, Ottawa, Canada: Information Canada, 1982. 107]**PEER REVIEWED**
Chlorfluorazole (Chlorofluorazole). TOXNET profile from Hazardous Substances Data Bank.
http://www.fluoridealert.org/pesticides/Chlorfluorazole.TOXNET.HSDB.htm

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The report later observes that work by others in the 1940s and 1950s "ƒshowed that fluoride has an inhibitive effect on the activity of succinate dehydrogenase. These studies indicate that under the effect of fluoride intake, a serious metabolic distress may develop in the kidneys." In concluding, the report notes that "Earlier, some workers had also indicated that inorganic fluorides have a strongly adverse effect on the activity of some enzymes and of these, mitochondrial enzymes, acid and alkaline phosphatases and ATP-utilizing enzymes and aldolase may be the most affected (Batenburg & Van den Bergh, 1972; Katz & Tenenhouse, 1973)."
Ref: The Kinetics of Acetylcholinesterase Inhibition and the Influence of Fluoride and Fluoride Complexes on the Permeability of Erythrocyte Membranes Dissertation to receive Ph.D. in Chemistry from the University of Hamburg By Johannes Westendorf Hamburg, Germany - 1975
http://www.fluoridealert.org/westendorf-foreword.htm

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http://www.ncbi.nlm.nih.gov:80/entrez/query.fcgi?cmd=Retrieve&db=PubMed&list_uids=12095014&dopt=Abstract

Eur J Anaesthesiol 2002 May;19(5):341-9

Fluoride ion toxicity in rabbit kidney thick ascending limb cells.

Cittanova ML, Estepa L, Bourbouze R, Blanc O, Verpont MC, Wahbe E, Coriat P, Daudon M, Ronco PM.

Departement d'Anesthesie-Reanimation, Hjpital Pitie-Salpetriere, Paris, France. marie-laure.cittanova@psl.ap-hop-paris.fr

BACKGROUND AND OBJECTIVE: Some halogenated agents, especially methoxyflurane, because of a higher level of fluoride production, induce a renal concentrating defect that could be related to an ascending limb impairment. We investigated the mechanisms of fluoride toxicity on an immortalized cell line.
METHODS: Cells were cultured for 2, 6 or 24 h in the presence of fluoride. Toxicity evaluation was based on: cell numbers, protein content, leucine-incorporation, lactate dehydrogenase (LDH) and N-acetyl-beta-glucosaminidase (NAG) releases, Na-K-ATPase and Na-K-2Cl activities, electron microscope studies. Infrared analysis and fluoride microdetermination allowed crystal components.
RESULTS: At 5 mmol after 24 h, fluoride decreased cell numbers (-14%, *P < 0.05), protein content (-16%*), leucine incorporation (-54%*), Na-K-2Cl activity (-84%*), increased LDH (+145%*) and NAG release (+190%*). Na-K-ATPase was more sensitive and impaired from 1 mmol for 24h and after 2 h at 5 mmol. Crystal formation in mitochondria occurred after 6 h at 5 mmol. Infra-red analysis and fluoride microdetermination established that crystals contained sodium, phosphate and fluoride.
CONCLUSIONS: The results suggest that the Na-K-ATPase pump is a major target for fluoride toxicity in Henle's loop.

PMID: 12095014 [PubMed - in process]

http://www.ncbi.nlm.nih.gov:80/entrez/query.fcgi?cmd=Retrieve&db=PubMed&list_uids=12022883&dopt=Abstract
Biochemistry 2002 May 28;41(21):6789-97 Mitochondrial aconitase modification, functional inhibition, and evidence for a supramolecular complex of the TCA cycle by the renal toxicant S-(1,1,2,2-tetrafluoroethyl)-L-cysteine.

James EA, Gygi SP, Adams ML, Pierce RH, Fausto N, Aebersold RH, Nelson SD, Bruschi SA.

Department of Medicinal Chemistry, University of Washington, Seattle, WA 98195, USA.

Metabolism of the common industrial gas tetrafluoroethylene in mammals results in the formation of S-(1,1,2,2)-tetrafluoroethyl-L-cysteine (TFEC), which can be bioactivated by a mitochondrial C-S lyase commonly referred to as beta-lyase. The resultant "reactive intermediate", difluorothioacetyl fluoride (DFTAF), is a potent thioalkylating and protein-modifying species. Previously, we have identified mitochondrial HSP70, HSP60, aspartate aminotransferase, and the E2 and E3 subunits of the alpha-ketoglutarate dehydrogenase (alphaKGDH) complex as specific proteins structurally modified during this process. Moreover, functional alterations to the alphaKGDH complex were also detected and implicated in the progression of injury. We report here the identification, by tandem mass spectrometry, and functional characterization of the final remaining major protein species modified by DFTAF, previously designated as P99(unk), as mitochondrial aconitase. Aconitase activity was maximally inhibited by 56.5% in renal homogenates after a 6 h exposure to TFEC. In comparison to alphaKGDH, aconitase inhibition (up to 79%) in a cell culture model for TFEC-mediated cytotoxicity was greater and preceded alphaKGDH inhibition, indicating that aconitase modification may constitute an early event in TFEC-mediated mitochondrial damage and cell death. These findings largely define the initial lesion of TFEC-mediated cell death and also have implications for the modeling of mitochondrial enzymatic architecture and the localization and identity of renal mitochondrial cysteine S-conjugate beta-lyase.

PMID: 12022883 [PubMed - indexed for MEDLINE]

Acta Pharmacol Sin 2001 Mar;22(3):249-52

A phospholipase C inhibitor, phenylmethylsulfonyl fluoride, ameliorates ischemic injury to brain mitochondria in rats.

Wang QS, Zhou JN, Xu TL, Li LS.

Department of Neurobiology, Life Science School, University of Science and Technology of China, Hefei 230027, China. wqss@china.com

AIM: To study the effect of phenylmethylsulfonyl fluoride (PMSF), a phospholipase C inhibitor, on ischemic injury to brain mitochondria in rats.
METHODS: The phospholipid level, membrane fluidity, and respiratory control ratio of brain mitochondria were measured. The effect of phenylmethylsulfonyl fluoride was tested.
RESULTS: The phospholipid level, membrane fluidity, and respiratory control ratio of brain mitochondria in ischemia group decreased but increased in the PMSF treatment group (P<0.05).
CONCLUSION: PMSF ameliorated ischemic injury to brain mitochondria in rats.

PMID: 11742572 [PubMed - in process]

Cytokine 2001 Aug 7;15(3):166-70

Tumor necrosis factor-related apoptosis inducing ligand (TRAIL)-induced apoptosis is dependent on activation of cysteine and serine proteases.

Park IC, Park MJ, Woo SH, Lee KH, Lee SH, Rhee CH, Hong SI.

Laboratory of Cell Biology, Korea Cancer Center Hospital, 215-4 Gongneung-dong, Nowon-ku, 139-240 Seoul, South Korea.

We examined the role of caspases and serine protease(s) in cell death induced by tumour necrosis factor-related apoptosis-inducing ligand (TRAIL). After incubation of adenocarcinoma cells with TRAIL, caspase-3, -8 were activated and the cleavage of Bid induced the release of cytochrome c, from the mitochondria to the cytosol. Tetrapeptide inhibitors of caspase-1, -2, -3, and -8 suppressed DNA fragmentation and attenuated the release of cytochrome c, whereas inhibitors of caspase-5 did not. Interestingly, the general serine protease(s) inhibitor 4-(2-aminoethyl)benzylsulfonyl fluoride (AEBSF) resulted in the arrest of apoptosis. However, the AEBSF did not prevent the release of mitochondrial cytochrome c during TRAIL-induced apoptosis. From these results, we postulate that serine protease(s) may be involved in post-mitochondrial apoptotic events, that lead to the activation of the initiator, caspase-9. Copyright 2001 Academic Press.

PMID: 11554786 [PubMed - indexed for MEDLINE]

Cell 2001 Aug 10;106(3):331-41

Structure of bovine mitochondrial F(1)-ATPase with nucleotide bound to all three catalytic sites: implications for the mechanism of rotary catalysis.

Menz RI, Walker JE, Leslie AG.

Medical Research Council Laboratory of Molecular Biology, Hills Road, Cambridge CB2 2QH, United Kingdom.

The crystal structure of a novel aluminium fluoride inhibited form of bovine mitochondrial F(1)-ATPase has been determined at 2 A resolution. In contrast to all previously determined structures of the bovine enzyme, all three catalytic sites are occupied by nucleotide. The subunit that did not bind nucleotide in previous structures binds ADP and sulfate (mimicking phosphate), and adopts a "half-closed" conformation. This structure probably represents the posthydrolysis, pre-product release step on the catalytic pathway. A catalytic scheme for hydrolysis (and synthesis) at physiological rates and a mechanism for the ATP-driven rotation of the gamma subunit are proposed based on the crystal structures of the bovine enzyme.

PMID: 11509182 [PubMed - indexed for MEDLINE]

Free Radic Biol Med 2001 Aug 1;31(3):367-73

Oxidative damage to mitochondria is a preliminary step to caspase-3 activation in fluoride-induced apoptosis in HL-60 cells.

Anuradha CD, Kanno S, Hirano S.

Regional Environment Division, National Institute for Environmental Studies, 16-2 Onogawa, Tsukuba, Ibaraki 305 8506, Japan.

It has been suggested that oxidative stress plays a major role in various forms of cell death, including necrosis and apoptosis. We have previously reported that fluoride (NaF) induces apoptosis in HL-60 cells by caspase-3 activation. The main focus of this investigation was to arrive at a possible pathway of the apoptosis induced by NaF upstream of caspase-3, because the mechanism is still unknown. The present study showed that after exposure to NaF, there was an increase in MDA and 4-HNE and a loss of mitochondrial membrane potential (deltaPsi(m)) was also observed in NaF-treated cells.There was a significant increase in cytosolic cytochrome c, which is released from the mitochondria. We have reported a downregulation of Bcl-2 protein in NaF-treated cells. The antioxidants N-acetyl cysteine (NAC), glutathione (GSH) protected the cells from loss of deltaPsi(m), and there was no cytochrome c exit or Bcl-2 downregulation, and we suggest that these antioxidants prevent apoptosis induced by NaF. These results suggested that perhaps NaF induced apoptosis by oxidative stress-induced lipid peroxidation, causing loss of deltaPsi(m), and thereby releasing cytochrome c into the cytosol and further triggering the caspase cascade leading to apoptotic cell death in HL-60 cells.

PMID: 11461774 [PubMed - indexed for MEDLINE]

Oral Surg Oral Med Oral Pathol Oral Radiol Endod 2001 Feb;91(2):230-4

Cytotoxicity of fluoride on human pulp cell cultures in vitro.

Chang YC, Chou MY.

School of Dentistry, Chung Shan Medical and Dental College, Department of Dentistry, Chung Shan Medical and Dental College Hospital, Taichung, Taiwan, ROC.

OBJECTIVES: Numerous studies have revealed that conventional glass-ionomer cements might release fluoride into an aqueous environment. The objective of this study was to examine the effects of fluoride on human pulp cells in vitro.
STUDY DESIGN: H33258 fluorescence, cell proliferation, protein synthesis, and mitochondrial activity assay were used to investigate the pathobiological effects of fluoride on cultured human pulp cells.
RESULTS: Fluoride was found to be a cytotoxic agent to cultured human pulp cells by inhibiting cell growth, proliferation, mitochondrial activity, and protein synthesis.
CONCLUSIONS: Fluoride release has significant potential for pulpal toxicity.

PMID: 11174602 [PubMed - indexed for MEDLINE]

Am J Physiol Cell Physiol 2000 Oct;279(4):C1220-9

Bioenergetics and mitochondrial transmembrane potential during differentiation of cultured osteoblasts.

Komarova SV, Ataullakhanov FI, Globus RK.

Life Sciences Division, NASA Ames Research Center, Moffett Field, California 94035-1000, USA.

To evaluate the relationship between osteoblast differentiation and bioenergetics, cultured primary osteoblasts from fetal rat calvaria were grown in medium supplemented with ascorbate to induce differentiation. Before ascorbate treatment, the rate of glucose consumption was 320 nmol. h(-1). 10(6) cells(-1), respiration was 40 nmol. h(-1). 10(6) cells(-1), and the ratio of lactate production to glucose consumption was approximately 2, indicating that glycolysis was the main energy source for immature osteoblasts. Ascorbate treatment for 14 days led to a fourfold increase in respiration, a threefold increase in ATP production, and a fivefold increase in ATP content compared with that shown in immature cells. Confocal imaging of mitochondria stained with a transmembrane potential-sensitive vital dye showed that mature cells possessed abundant amounts of high-transmembrane-potential mitochondria, which were concentrated near the culture medium-facing surface. Acute treatment of mature osteoblasts with metabolic inhibitors showed that the rate of glycolysis rose to maintain the cellular energy supply constant. Thus progressive differentiation coincided with changes in cellular metabolism and mitochondrial activity, which are likely to play key roles in osteoblast function.

PMID: 11003602 [PubMed - indexed for MEDLINE]

Structure Fold Des 2000 Jun 15;8(6):567-73
 
Structure of bovine mitochondrial F(1)-ATPase inhibited by Mg(2+) ADP and aluminium fluoride.

Braig K, Menz RI, Montgomery MG, Leslie AG, Walker JE.

Institut fur Organische Chemie und Biochemie, Albert-Ludwigs Universitat Freiburg, Freiburg in Breisgau, D-79104, Germany.

BACKGROUND: The globular domain of the membrane-associated F(1)F(o)-ATP synthase complex can be detached intact as a water-soluble fragment known as F(1)-ATPase. It consists of five different subunits, alpha, beta, gamma, delta and epsilon, assembled with the stoichiometry 3:3:1:1:1. In the crystal structure of bovine F(1)-ATPase determined previously at 2.8 A resolution, the three catalytic beta subunits and the three noncatalytic alpha subunits are arranged alternately around a central alpha-helical coiled coil in the gamma subunit. In the crystals, the catalytic sites have different nucleotide occupancies. One contains the triphosphate form of the nucleotide, the second contains the diphosphate, and the third is unoccupied. Fluoroaluminate complexes have been shown to mimic the transition state in several ATP and GTP hydrolases. In order to understand more about its catalytic mechanism, F(1)-ATPase was inhibited with Mg(2+)ADP and aluminium fluoride and the structure of the inhibited complex was determined by X-ray crystallography.
RESULTS: The structure of bovine F(1)-ATPase inhibited with Mg(2+)ADP and aluminium fluoride determined at 2.5 A resolution differs little from the original structure with bound AMP-PNP and ADP. The nucleotide occupancies of the alpha and beta subunits are unchanged except that both aluminium trifluoride and Mg(2+)ADP are bound in the nucleotide-binding site of the beta(DP) subunit. The presence of aluminium fluoride is accompanied by only minor adjustments in the surrounding protein.
CONCLUSIONS: The structure appears to mimic a possible transition state. The coordination of the aluminofluoride group has many features in common with other aluminofluoride-NTP hydrolase complexes. Apparently, once nucleotide is bound to the catalytic beta subunit, no additional major structural changes are required for catalysis to occur.

PMID: 10873854 [PubMed - indexed for MEDLINE]

Biochem J 2000 May 1;347 Pt 3:669-77
 
Serine protease inhibitors suppress cytochrome c-mediatedcaspase-9 activation and apoptosis during hypoxia-reoxygenation.

Dong Z, Saikumar P, Patel Y, Weinberg JM, Venkatachalam MA.

Department of Pathology, University of Texas Health Science Center, 7703 Floyd Curl Drive, San Antonio, TX 78229, USA. dong@uthscsa.edu

We have shown that reoxygenation of hypoxic rat kidney proximaltubule cells leads to apoptosis. This is mediated by translocation ofBax from the cytosol to mitochondria, accompanied by release of mitochondrial cytochrome c (cyt.c). The present study has examined the proteolytic mechanisms responsible for apoptosis during hypoxia-reoxygenation. Caspases were activated during hypoxia, as shown by cleavage of fluorogenic peptide substrates. By 5 h caspase-3-like activity to cleave carbobenzoxy-Asp-Glu-Val-Asp-7-amino-4-trifluoromethyl coumarin was increased approx. 30-fold. This was accompanied by specific processing of pro-caspase-3, -8 and -9 intoactive forms. Caspase activation during hypoxia was blocked by carbobenzoxy-Val-Ala-Asp-fluoromethyl ketone and overexpression of Bcl-2. Of particular interest, caspase activation was also suppressed by the chymotryptic inhibitors N-tosyl-L-phenylalaninechloromethyl ketone (TPCK) and Ala-Pro-Phe chloromethyl ketone (APF),and the general serine protease inhibitor 4-(2-aminoethyl)benzenesulphonyl fluoride. Inhibition of caspase activation by these compounds resulted in arrest of apoptosis. On the other hand,the serine protease inhibitors did not prevent release of mitochondrial cyt.c during hypoxia, suggesting that these compounds blocked a critical step in post-mitochondrial caspase activation. Further studies using an in vitro reconstitution model showed that cyt. c/dATP stimulated caspase-9 processing and downstream caspase activation were significantly suppressed in the presence of TPCK and APF. Based on these results, we speculate that serine proteases may be involved in post-mitochondrial apoptotic events that lead to activation of the initiator, caspase-9.

PMID: 10769169 [PubMed - indexed for MEDLINE]

Am J Physiol 1999 Mar;276(3 Pt 1):C611-20

Rat liver GTP-binding proteins mediate changes in mitochondrial membrane potential and organelle fusion.

Cortese JD.

Department of Cell Biology and Anatomy, University of North Carolina, Chapel Hill, North Carolina 27599-7090, USA.

The variety of mitochondrial morphology in healthy and diseased cells can be explained by regulated mitochondrial fusion. Previously, a mitochondrial outer membrane fraction containing fusogenic, aluminum fluoride (AlF4)-sensitive GTP-binding proteins (mtg) was separated from rat liver (J. D. Cortese, Exp. Cell Res. 240: 122-133, 1998). Quantitative confocal microscopy now reveals that mtg transiently increases mitochondrial membrane potential (DeltaPsi) when added to permeabilized rat hepatocytes (15%), rat fibroblasts (19%), and rabbit myocytes (10%). This large mtg-induced DeltaPsi increment is blocked by fusogenic GTPase-specific modulators such as guanosine 5'-O-(3-thiotriphosphate), excess GTP (>100 microM), and AlF4, suggesting a linkage between DeltaPsi and mitochondrial fusion. Accordingly, stereometric analysis shows that decreasing DeltaPsi or ATP synthesis with respiratory inhibitors limits mtg- and AlF4-induced mitochondrial fusion. Also, a specific G protein inhibitor (Bordetella pertussis toxin) hyperpolarizes mitochondria and leads to a loss of AlF4-dependent mitochondrial fusion. These results place mtg-induced DeltaPsi changes upstream of AlF4-induced mitochondrial fusion, suggesting that GTPases exert DeltaPsi-dependent control of the fusion process. Mammalian mitochondrial morphology thus can be modulated by cellular energetics.

PMID: 10069988 [PubMed - indexed for MEDLINE]

Exp Cell Res 1998 Apr 10;240(1):122-33
 
Stimulation of rat liver mitochondrial fusion by an outer membrane-derived aluminum fluoride-sensitive protein fraction.

Cortese JD.

Department of Cell Biology and Anatomy, University of North Carolina at Chapel Hill 25799-7099, USA. jcortes@med.unc.edu

In normal livers, hepatocytes contain a large number of spheroidal mitochondria. Mitochondrial morphology changes drastically in liver disease, but the underlying fusion-fission mechanisms are not known. We detected GTP- and aluminum fluoride-dependent membrane fusion events between rat liver mitochondria. Separation of outer mitochondrial membrane-derived proteins led to a subfraction containing a 60-kDa protein band that is detected by specific antibodies directed to common amino acid sequences of the GTP-binding site or carboxyl-terminus of eukaryotic heterotrimeric G-protein alpha subunits. Addition of this subfraction and aluminum fluoride to permeabilized rat hepatocytes triggered a substantial morphological change in hepatic mitochondria, giving them the three-dimensional appearance of a tubulovesicular network. Comparative stereology using electron and confocal microscopy showed that these morphological changes represent true mitochondrial fusion. Addition of aluminum fluoride alone induces a more limited change in mitochondrial morphology, from spheroidal organelles to short rods. Mitochondria maintained their normal membrane potential and overall membrane ultrastructure after all these morphological changes. Our results reveal that mammalian liver mitochondria contain proteins that stimulate mitochondrial fusion and suggest that members of the GTPase superfamily control the normalcy of mitochondrial morphology, which is closely linked to physiological cellular energetics.

PMID: 9570927 [PubMed - indexed for MEDLINE]

J Physiol 1998 Jan 1;506 ( Pt 1):109-25

Submaximal stimulation of porcine endothelial cells causes focal Ca2+ elevation beneath the cell membrane.

Graier WF, Paltauf-Doburzynska J, Hill BJ, Fleischhacker E, Hoebel BG, Kostner GM, Sturek M.

Department of Medical Biochemistry, University of Graz, Austria. wolfgang.graier@kfunigraz.ac.at

1. Endothelial cell activation is correlated with increased cytosolic Ca2+ concentration, often monitored with cytoplasmic Ca2+ dyes, such as fura-2 and Calcium Green-1. We tested the hypothesis that during weak stimulation of porcine coronary artery endothelial cells, focal, subplasmalemmal Ca2+ elevations occur which are controlled by cell membrane Na(+)-Ca2+ exchange near mitochondrial membrane and superficial endoplasmic reticulum (SER).
2. Bulk Ca2+ concentration ([Ca2+]b) was monitored using fura-2 or Calcium Green-1 and subplasmalemmal Ca2+ concentration ([Ca2+]sp) was determined with FFP-18. The distribution of the SER network was estimated using laser scanning and deconvolution microscopy.
3. Sodium fluoride (10 mmol l-1) and submaximal concentrations of bradykinin (Bk; 1 nmol l-1) stimulated Ca2+ entry with no increase in [Ca2+]b. Although inositol 1,4,5-trisphosphate formation and intracellular Ca2+ release in response to both stimuli were similar, Ca2+ entry in response to NaF exceeded that in response to 1 nmol l-1 BK by fourfold, suggesting additional effects of NaF on Ca+ entry pathways but stimulation via intracellular Ca2+ release.
4. Prevention of Na(+)-Ca2+ exchange activity by decreasing extracellular Na+ unmasked intracellular Ca2+ release in response to NaF and 1 nmol l-1 Bk, indicated by an increase in [Ca2+]b. Thereby, NaF depleted Bk-releasable Ca2+ pools, while mitochondrial Ca2+ content (released with FCCP or oligomycin) and the amount of Ca2+ stored within the cells (released with ionomycin) was increased compared with cells treated with NaF under normal Na+ conditions. The NaF-initiated increase in [Ca2+]b and depletion of Bk-releasable Ca2+ pool(s) in the low-Na+ condition was diminished by 25 mumol l-1 ryanodine, indicating the involvement of Ca(2+)-induced Ca2+ release (CICR).
5. In simultaneous recordings of [Ca2+]sp (with FFP-18) and [Ca2+]b (with Calcium Green-1), 1 nmol l-1 Bk or 10 mmol l-1 NaF yielded focal [Ca2+] elevation in the subplasmalemmal region with no increase in the perinuclear area.
6. Treatment with 10 mumol-1 nocodazole caused the SER to collapse and unmasked Ca2+ release in response to 1 nmol l-1 Bk and 10 mmol l-1 NaF, similar to low-Na+ conditions, while the effect of thapsigargin was not changed.
7. These data show that in endothelial cells, focal, subplasmalemmal Ca2+ elevations in response to small or slow IP3 formation occur due to vectorial Ca2+ release from the SER towards the plasmalemma followed by Ca2+ extrusion by Na(+)-Ca2+ exchange. While these local Ca2+ elevations are not detectable with Ca2+ dyes for the determination of [Ca2+]b, prevention of Ca2+ extrusion or SER disruption yields increases in [Ca2+]b partially due to CICR.
8. All of the data support our hypothesis that in weakly stimulated endothelial cells, intracellular Ca2+ release and [Ca2+] elevation are limited to the subplasmalemmal region. We propose that the SER co-operates with associated parts of the plasma membrane to control Ca2+ homeostasis, Ca2+ distribution and Ca2+ entry. The existence of such a subplasmalemmal Ca2+ control unit (SCCU) needs to be considered in discussions of Ca2+ signalling, especially when cytoplasmic Ca2+ dyes, such as fura-2 or Calcium Green-1, are used.

PMID: 9481676 [PubMed - indexed for MEDLINE]

Anesthesiology 1996 Feb;84(2):428-35
 
Fluoride ion toxicity in human kidney collecting duct cells.

Cittanova ML, Lelongt B, Verpont MC, Geniteau-Legendre M, Wahbe F, Prie D, Coriat P, Ronco PM.

Departement d'Anesthesie-Reanimation, Hopital Pitie-Salpetriere, Paris, France.

BACKGROUND: Several halogenated anesthetics induce a urinary concentrating defect, partly related to fluoride ion toxicity in collecting duct cells. The aim of this study was to investigate the effects of fluoride ion in human kidney cells.
METHODS: Immortalized human collecting duct cells were used. In a first set of experiments, the toxicity threshold concentration was determined by exposing cell cultures for 24 h to increasing concentrations of fluoride ion in the medium: 0, 1, 5, and 10 mM. The second set of experiments was a time- effect study in which cells were exposed to 5 mM fluoride for 2, 6, and 24 h. Assessment of toxicity was based on several endpoints: cell number, protein content, (3)H-leucine incorporation in newly synthesized proteins, extracellularly released lactate dehydrogenase, Na-K-ATPase pump activity, and electron microscope studies.
RESULTS: After 24 h of exposure, fluoride ion decreased cell number (-23%, P<0.05), total protein content (-30%, P<0.05) and increased lactate dehydrogenase release (+236%, P<0.05) at a threshold concentration of 5mM. Fluoride ion also inhibited Na-K- ATPase activity at 5 mM (-58%, P<0.05). Major morphologic alterations of mitochondria, including crystal formation, were detected from 1 mM fluoride concentration. Time-effect studies showed that, after only 6 h of exposure at 5 mM, fluoride decreased cell number (-13%, P<0.05), (3)H-leucine incorporation (-48%, P<0.05), and Na-K-ATPase activity (- 20%, P<0.05) and increased lactate dehydrogenase release (+145%, P<0.05). Crystal deposits in mitochondria again were a more sensitive marker of cell injury, detectable after only 2 h of exposure.
CONCLUSIONS: these results suggest that the mitochondrion is a target of fluoride toxicity in human collecting duct cells, and its alteration is partly responsible for the sodium and water disturbances observed in patients.

PMID: 8602675 [PubMed - indexed for MEDLINE]

Microsc Res Tech 1995 Jul 1;31(4):317-25

Comparative methodological investigations on the cytochemical localization of calcium in brain and inner ear of cichlid fish.

Vohringer P, Nindl G, Aich B, Kortje KH, Rahmann H.

Institute of Zoology, University of Hohenheim, Stuttgart, Germany.

Four different methods for calcium precipitation are compared in the optic tectum and the inner ear of the cichid fish, Oreochromis mossambicus. Several parameters are investigated concerning their influences on the reaction product. Three procedures (bichromate, fluoride, and oxalate-pyroantimonate) produce fine-grained deposits, often flocculent in the latter method. The fourth method (potassium-pyroantimonate) generates predominantly coarse-grained reaction product. The calcium content of the deposits is always proven with energy-filtering transmission electron microscopy (EFTEM). In both tissues fine-grained reaction product is found in endoplasmic reticulum and synaptic vesicles, and in addition in some mitochondria and at the cytoskeleton. The coarse-grained deposits of the potassium-pyroantimonate method have a more unspecific distribution. This is the only method which produces extracellular deposits in the inner ear, whereas in the optic tectum extracellular precipitates are always present except with the oxalate-pyroantimonate procedure. Two factors have an influence on the reaction product: the duration of fixation and the type of resin. The prolongation of the fixation time up to 24 hours leads to an increase of the reaction product, which also becomes coarse-grained. These observations are corroborated by quantification with image analysis. Furthermore the use of an epoxy resin compared to acrylic resins decreases the amount of reaction product produced. We show that the application of several methods is meaningful in order to understand the calcium properties of the investigated tissue, but it is necessary to optimize a certain method for a given tissue.

PMID: 7549006 [PubMed - indexed for MEDLINE]

J Membr Biol 1996 Nov;154(2):183-95

Role and source of ATP for activation of nonselective cation channels by AlF complex in guinea pig chromaffin cells.

Inoue M, Ogawa K, Fujishiro N, Yano A, Imanaga I.

Department of Physiology, School of Medicine, Fukuoka University, Japan.

Intracellular dialysis with the solution containing the G protein activator, AlF complex, induced an inward nonselective cation current (INS) at -55 mV in chromaffin cells. Amplitudes of INS induced by dialysis with ATP-free AlF solutions progressively diminished as cells were pretreated with cyanide, a mitochondrial inhibitor. After a 10-min pretreatment, generation of INS by the AlF complex depended on exogenous ATP delivered from pipette solution. The relationship between amplitudes of INS and concentrations of MgATP was well expressed by a rectangular hyperbola with an EC50 of 0.265 mM. This result suggests that the cyanide treatment almost depleted ATP near the plasma membrane. On the other hand, a similar cyanide treatment of adrenal medullary preparations did not induce a marked decrease in cellular ATP content. GTP, ITP, or UTP could not substitute for ATP in generation of INS by the AIF complex. Similarly, the substitution of ATP with non- or poorly hydrolyzable ATP analogues did not aid in generating INS. Bath application of the kinase inhibitor, H-7 (100 microM), suppressed AlF-induced INS in a manner depending on intracellular Mg2+. We conclude that ATP is a prerequisite for generation of INS as a phosphoryl donor and that mitochondria is the main source of ATP.

PMID: 8929292 [PubMed - indexed for MEDLINE]