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1993 Fluoride Abstracts. Part 1.
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Fluoride 1993; 26(2):79-82
Guest Editorial: Fluoridation and bone cancer
JH Lee M.D.
Excerpt: The NTP (National Toxicology Program) fluroide/cancer study of rats and mice (1) found a statistically significant dose-related increase of osteosarcoma incidence in male rats and, in addition, found fluoride correlations with thyroid follicular cell adenomas, oral and nasal squamous dysplasia, a rare type of liver cancer (hepatocholangiocarcinoma), and, as might have been expected, extensive osteosclerosis...
[In his editorial Dr Lee included the following 2 tables:]
Table 1.
Age-/sex-specific osteosarcoma incidence in fluoridated vs non-fluoridated muicipalities in seven counties in the central New Jersey study area. Number of cases (1979-1987) population and average annual incidence rate (cases per million), all races. NJDOH, 1992.
[Cohn PD (1992). A brief report on the association of drinking water fluoridation and the incidence of osteosarcoma among young males. New Jersey Department of Health, Trenton, NJ. November 8.]Sex Age Population Rates Males 0-9 Fluoridated 48,129 4.6 Non-fluoridated 102,123 1.0 10-19 Fluoridated 62,990 17.6 Non-fluoridated 151,384 5.1 20-49 Fluoridated 141,439 3.9 Non-fluoridated 348,570 1.5 50-69 Fluoridated 65,126 0 Non-fluoridated 161,459 4.8 70+ Fluoridated 21,614 5.1 Non-fluoridated 48,649 9.1 Females 0-9 Fluoridated 45,936 0 Non-fluoridated 103,462 2.1 10-19 Fluoridated 61,533 5.4 Non-fluoridated 145,790 3.8 20-49 Fluoridated 152,173 1.4 Non-fluoridated 362,616 1.5 50-69 Fluoridated 76,461 1.4 Non-fluoridated 182,912 1.2 70+ Fluoridated 37,634 14.7 Non-fluoridated 77,708 5.7
Table 2.
Age-/sex-specific osteosarcoma incidence in fluoridated vs non-fluoridated municipalities in Mercer, Middlesex, and Monmouth Counties. Number of cases (1979-1987), population and average annual incidence rate (cases per million), all races; NJDOH, 1992.
[Cohn PD (1992). A brief report on the association of drinking water fluoridation and the incidence of osteosarcoma among young males. New Jersey Department of Health, Trenton, NJ. November 8.]Sex Age Population Rates Males 0-9 Fluoridated 38,654 5.7 Non-fluoridated 46,708 2.3 10-19 Fluoridated 50,297 22.0 Non-fluoridated 67,678 3.2 20-49 Fluoridated 115,367 3.8 Non-fluoridated 153,713 1.4 50-69 Fluoridated 51,853 0 Non-fluoridated 66,607 3.3 70+ Fluoridated 16,930 0 Non-fluoridated 18,478 18.0 Females 0-9 Fluoridated 36,956 0 Non-fluoridated 44,247 ..0 10-19 Fluoridated 48,976 6.8 Non-fluoridated 65,120 5.1 20-49 Fluoridated 122,936 0 Non-fluoridated 157,545 0.7 50-69 Fluoridated 60,427 1.8 Non-fluoridated 74,846 1.4 70+ Fluoridated 29,068 15.2 Non-fluoridated 28,524 11.6
http://www.ncbi.nlm.nih.gov:80/entrez/query.fcgi?cmd=Retrieve&db=PubMed&list_uids=7679201&dopt=AbstractMutat Res 1993 Mar;301(3):183-8
Clastogenic activity of sodium fluoride in great ape cells.
Kishi K, Ishida T.
School of Health Sciences, Kyorin University, Tokyo, Japan.
Conflicting evidence has been reported concerning the mutagenicity of sodium fluoride (NaF), especially clastogenicity at concentrations of more than 1 mM. NaF is known to induce chromosome aberrations at these concentrations in human cells, but not in most rodent cells. We considered that such species-specific difference in chromosomal sensitivity would be derived from the phylogenetic distance between rodents and man. To clarify the role of interspecies differences, we investigated the chromosomal sensitivity to NaF in cell lines from various primates, which diverged into many species, including rodent-like prosimians and human-like great apes. The results showed that the clastogenicity of NaF was limited to human and great ape cells.
PMID: 7679201 [PubMed - indexed for MEDLINE]
http://www.ncbi.nlm.nih.gov:80/entrez/query.fcgi?cmd=Retrieve&db=PubMed&list_uids=8190041&dopt=AbstractMetab Brain Dis 1993 Dec;8(4):217-34
Astrocytes and the entry of circulating ammonia into the brain: effect of fluoroacetate.
Szerb JC, Redondo IM.
Department of Physiology and Biophysics, Dalhousie University, Halifax, N.S., Canada.
Chronic hyperammonemia is known to lead to pathological forms of astrocytes. To test the influence of these changes on the neurotoxicity of ammonia, the glial metabolic poison fluoroacetate (FA) was applied locally, through microdialysis to the hippocampal dentate gyrus. The penetration of ammonia into the brain following the i.p. injection of 7.8 mmol/kg NH4 acetate was evaluated by measuring the ammonia and glutamine content of the microdialysate. Field EPSPs (fEPSPs) evoked by perforant path stimulation were recorded 1.5 mm from the microdialysis probe. When 20 mM FA was perfused, NH4 acetate injection increased the ammonia efflux by 300% and decreased fEPSPs by 40%, but glutamine concentration remained low. With no FA in the microdialysate, NH4 acetate treatment increased the efflux of ammonia by only 60%, did not affect fEPSPs but doubled glutamine efflux. Arterial ammonia content, as measured by microdialysis in the common carotid, increased 4-5 fold following i.p. administration of NH4 acetate, while arterial glutamine was not elevated. Systemically administered FA did not affect either of these changes significantly, but slightly reduced arterial pH. These observations indicate that FA applied by microdialysis acted locally on astrocytes and therefore impaired astrocytic function contributes to the development of hepatic encephalopathy by facilitating the entry of ammonia into the brain. Inhibition of excitatory synaptic transmission by elevated brain ammonia may underlay CNS depression in hepatic encephalopathy.
PMID: 8190041 [PubMed - indexed for MEDLINE]
Fluoride 1993; 26(1):57-60Changes of serotonin content and turnover rate in hypothalamus of female rat during fluorosis
Yuan SD, Xie QW, Lu FY
Dr ShuDe Yuan, Neuroendocrine Research Laboratory, China Medical University, Shenyang 110001, China
Summary: Animal models of subacute and chronic fluorosis in female rats were developed with injection of large doses of NaF(IP) and with drinking water containing 100 ppm F-, respectively. The serotonin or 5-hydroxytryptamine (5-HT) content and turnover rate in the hypothalamus were determined with spectrofluorometry combined with degradation blockade. The 5-HT turnover rate decreased during both subacute and chronic fluorosis. The 5-HT content increased during subacute fluorosis, but decreased during chronic fluorosis. These results suggest that the influences of 5-HT metabolism of the two kinds of fluorosis are not completely identical. The decrease of 5-HT turnover rate in hypothalamus may be one of possible mechanisms of deficiency of pituitary prolactin (PRL) and milk secretion during fluorosis.
http://www.ncbi.nlm.nih.gov:80/entrez/query.fcgi?cmd=Retrieve&db=PubMed&list_uids=7903892&dopt=AbstractEur J Neurosci 1993 Oct 1;5(10):1334-8
Alterations of G-protein coupling function in phosphoinositide signalling pathways of rat hippocampus by ischaemic brain injury.
Lu YM, Lu BF, Yan YL, Yan TH, Ho XP, Wang WJ.
Division of Molecular Pharmacology, Naval Neurobiology Research Centre, Nanjing, China.
The activation of membrane-associated phospholipase C is rapidly and transiently induced in the central nervous system by a variety of stimuli. Ischaemic brain injury is one of the situations that leads to a dramatic increase in polyphosphoinositide (PPI) turnover. In this study, stimulation of PPI hydrolysis by glutamate (500 microM) was measured in hippocampal slices from rats up to 21 days after an ischaemic insult of 30 min. Ischaemia was induced using the four-vessel occlusion method. PPI hydrolysis elicited by glutamate was significantly increased in the slices prepared from ischaemic rats 24 h after reperfusion, the accumulation of inositol phosphates (InsPs) and inositol 1,4,5-trisphosphate (Insp3) was 614 +/- 74% (n = 8) and 182 +/- 11% (n = 9) of the basal level respectively. This potentiation was also observed 21 days after ischaemia. Hyper-responsiveness to glutamate was also accompanied by an increase in AIF4(-)-stimulated formation of [3H]inositol phosphates. In addition, global ischaemia did not change either high-affinity [3H]glutamate binding in hippocampal membranes or the stimulation of PPI hydrolysis by carbachol or noradrenaline in hippocampal slices. The present results suggest that the increased responsiveness to glutamate is the result, at least in part, of functional changes at the G-protein level, and may contribute to the pathophysiology of ischaemic brain injury or to the regenerative phenomena that accompany ischaemic damage.
PMID: 7903892 [PubMed - indexed for MEDLINE]
http://www.ncbi.nlm.nih.gov:80/entrez/query.fcgi?cmd=Retrieve&db=PubMed&list_uids=8287268&dopt=AbstractBrain Res 1993 Nov 26;629(1):133-40
Aluminum decreases muscarinic, adrenergic, and metabotropic receptor-stimulated phosphoinositide hydrolysis in hippocampal and cortical slices from rat brain.
Shafer TJ, Mundy WR, Tilson HA.
Cellular and Molecular Toxicology Branch, United States Environmental Protection Agency, Research Triangle Park, NC 27711.
Effects of aluminum chloride (AlCl3) (0.1 to 1000 microM) on inositol phosphate (IP) accumulation stimulated by carbachol (CARB), norepinephrine (NE) or quisqualate (QUIS) were examined in rat hippocampal and cortical slices. In the absence of agonist, only 1000 microM AlCl3 significantly reduced basal accumulation of IPs. For CARB-stimulated IP accumulation, 100 microM and greater AlCl3 significantly inhibited IP accumulation. In cortical slices, 1000 microM AlCl3 reduced CARB-stimulated IP accumulation by 55% and in hippocampal slices 1000 microM AlCl3 inhibited IP accumulation by 40%. Similar effects of AlCl3 were observed for NE-stimulated IP accumulation. In cortical slices, the concentration-response for AlCl3 effects on agonist-stimulated IP accumulation was significantly different from that in hippocampal slices. For QUIS-stimulated accumulation of IPs, 1000 microM AlCl3 significantly inhibited IP accumulation in hippocampal slices. However, in cortical slices a biphasic effect of AlCl3 was observed. 500 and 1000 microM AlCl3 significantly inhibited IP accumulation, whereas 10 and 50 microM AlCl3 significantly enhanced QUIS-stimulated IP accumulation. In both hippocampal and cortical slices, 500 microM AlCl3 significantly inhibited CARB-, NE- or QUIS-stimulated IP accumulation at all agonist concentrations (0.1 to 10000 microM) tested, indicating a post-receptor effect on agonist-mediated IP accumulation. Stimulation of G-proteins with NaF (5-30 mM) resulted in accumulation of IPs in hippocampal and cortical slices in the absence of added agonists. NaF (5-30 mM) plus 1 mM CARB produced increased accumulation of IPs over CARB or NaF alone.(ABSTRACT TRUNCATED AT 250 WORDS)
PMID: 8287268 [PubMed - indexed for MEDLINE]
http://www.ncbi.nlm.nih.gov:80/entrez/query.fcgi?cmd=Retrieve&db=PubMed&list_uids=8345784&dopt=AbstractMagn Reson Imaging 1993;11(5):697-703
Uptake and life time of fluoride ion in rats by 19F-NMR.
Scarpa M, Vianello F, Rigo A, Viglino P, Bracco F, Battistin L.
Department of Biological Chemistry, University of Padova, Italy.
19F nuclear magnetic resonance (NMR) was utilized to obtain information on the uptake and half-life time of fluoride ion in rats. Changes in tissue fluoride level after acute loading were monitored over time in blood and tissue homogenates obtained from liver and brain. The rate of fluoride elimination from various tissues was roughly similar, following in all cases a first-order kinetic rate law. The F- concentration in brain was about 20% of that found in liver, indicating a reduced fluoride diffusion across the blood-brain barrier. In vivo F- spectra were obtained in rat brain in few minutes with a good signal-to-noise ratio; this confirms the possibility of extending the use of F- as a probe of biomolecules to in vivo applications.
PMID: 8345784 [PubMed - indexed for MEDLINE]
http://www.ncbi.nlm.nih.gov:80/entrez/query.fcgi?cmd=Retrieve&db=PubMed&list_uids=8242380&dopt=AbstractBrain Res 1993 Sep 17;622(1-2):35-42
Alterations in the activity of adenylate cyclase and high affinity GTPase in Alzheimer's disease.
Ross BM, McLaughlin M, Roberts M, Milligan G, McCulloch J, Knowler JT.
Wellcome Neuroscience Group, Wellcome Surgical Institute & Hugh Fraser Neuroscience Labs., Glasgow UK.
The aim of this study was to assess the effect of Alzheimer's disease has on the functional integrity of several signal transduction proteins. The relative levels of the G-protein alpha subunits Gs alpha-L, Gs alpha-S, Gi alpha-2 and G(o) alpha were measured by western blotting and found to be unchanged in membranes prepared from Alzheimer-diseased frontal cortex or hippocampus compared to control brains. However the activity of the G-protein associated enzyme, high affinity GTPase, was found to be reduced in the frontal cortex (reduced by 25%) and by a similar magnitude in the hippocampus (reduced by 27%) of Alzheimer subjects. The same membrane preparations were also assayed for the activity of adenylate cyclase. Basal enzyme activity was not significantly altered in Alzheimer diseased hippocampus, but was markedly reduced (by 45%) in the frontal cortex. The ability of fluoride and aluminium ions to stimulate adenylate cyclase was not significantly changed in either brain region. This suggests that G-proteins, especially Gs, are still able to interact with this enzyme. These results indicate that although the presence of Alzheimer's disease does not significantly alter G-protein levels, changes have taken place in the overall activity of these proteins. However this alteration does not affect their ability to stimulate adenylate cyclase activity.
PMID: 8242380 [PubMed - indexed for MEDLINE]
http://www.ncbi.nlm.nih.gov:80/entrez/query.fcgi?cmd=Retrieve&db=PubMed&list_uids=8491210&dopt=AbstractEnviron Mol Mutagen 1993;21(4):309-18
Genetic toxicity of fluoride.
Zeiger E, Shelby MD, Witt KL.
Experimental Carcinogenesis and Mutagenesis Branch, National Institute of Environmental Health Sciences, Research Triangle Park, North Carolina 27709.
F- is not mutagenic in standard bacterial systems, but produces chromosome aberrations and gene mutations in cultured mammalian cells. Although there is disagreement in the literature concerning the ability of F- to induce chromosome aberrations in cultured human and rodent cells, the weight of the evidence leads to the conclusion that F- exposure results in increased chromosome aberrations in these test systems. NaF induced primarily chromatid gaps and chromatid breaks, indicating that the rodent cells are responsive in the G2 stage of the cell cycle. In contrast, studies with synchronized human cells indicated that the S phase was the most sensitive. If F- does have a cell cycle-specific effect, it could be expected that differences in the cell treatment and harvest protocols could lead to conflicting results for the induction of chromosome aberrations. Gene mutations were produced in cultured rodent and human cells in the majority of the studies. Unfortunately, a number of the in vitro and in vivo cytogenetic studies are of questionable utility because of the protocols used, the quality of the responses reported, or the interpretations of the data. The conflicting results in the in vivo cytogenetic studies are difficult to reconcile. There are reports of increased chromosome aberrations in rat bone marrow and testes, but other studies, using similar protocols and dose ranges, have reported no induced chromosome damage. Although some of the studies were performed at toxic levels of F-, other studies, including those that showed positive results, were at F- concentrations (1-5 ppm) equivalent to human exposure levels. In the majority of studies that were reported to be positive, there were high background frequencies, or the investigators reported categories of nuclear or chromosome damage that are difficult to interpret. Interestingly, many of the positive results were obtained when anaphase cells were scored, whereas similar treatment protocols in other laboratories yielded negative results when metaphase cells were the only cell type examined. It is difficult, without additional data, to determine the reasons for finding chromosome breaks in anaphase, but not metaphase, cells. Other reports have presented insufficient information to allow adequate evaluations. Therefore, at this time, the question of whether F- produces chromosome damage in vivo should be considered unresolved.
PMID: 8491210 [PubMed - indexed for MEDLINE]
http://www.ncbi.nlm.nih.gov:80/entrez/query.fcgi?cmd=Retrieve&db=PubMed&list_uids=7694121&dopt=AbstractMutat Res 1993 Dec;290(2):293-302
Cell cycle dependence of cytotoxicity and clastogenicity induced by treatment of synchronized human diploid fibroblasts with sodium fluoride.
Hayashi N, Tsutsui T.
Department of Pharmacology, School of Dentistry, Nippon Dental University, Tokyo, Japan.
To study the cell cycle dependence of cytotoxicity and clastogenicity of sodium fluoride (NaF), synchronized human diploid fibroblasts were treated with NaF during different phases of the cell cycle and analyzed. Exponentially growing cells were synchronized by the following two procedures.
(1) The cells were synchronized at G0/G1 phase by a period of growth in medium containing 1% serum (low serum medium).
(2) The cells were synchronized at the G1/S boundary by growth in low serum medium, followed by hydroxyurea treatment (Tsutsui et al., 1984a). Synchronized cells were treated with NaF for 3 h during the G1 phase or G2 phase, and for each of three 3-h periods during the S phase which lasted 9 h.
Cytotoxicity, as determined by a decrease in colony-forming ability, was dependent upon the phase of the cell cycle during which NaF treatment was administered. The highest lethality was induced in cultures treated with NaF during the first 3 h of S phase (early S phase). Moderate lethality was observed when the cultures were treated with NaF during the second or third 3 h of S phase (middle or late S phase, respectively), or G2 phase. Little lethality was observed in cultures in G1 phase. Inducibility of chromosome aberrations of the cells following treatment with NaF was also dependent upon the phase of the cell cycle. A significant increase in the incidence of chromosome aberrations was observed only in cultures treated with NaF during early and/or middle S phases of cell cycle. These results suggest that cytotoxicity and clastogenicity of NaF to cultured human diploid fibroblasts are cell cycle dependent, and that the cells in early and middle S phases are more sensitive to the effects.
PMID: 7694121 [PubMed - indexed for MEDLINE]
http://www.ncbi.nlm.nih.gov:80/entrez/query.fcgi?cmd=Retrieve&db=PubMed&list_uids=8168199&dopt=AbstractZhonghua Bing Li Xue Za Zhi 1993 Oct;22(5):299-301
[Promotive action of sodium fluoride on precancerous lesions of hepatocellular carcinoma induced by diethylnitrosamine (DEN) in rats--stereologic study of enzyme histochemistry]
[Article in Chinese]
Liu YQ.
Department of Pathology, Tianjin Medical College.
In 10 Wistar male rats, partial hepatectomy was performed and followed by intraperitoneal injection of DEN (30 mg/kg) once and giving drinking water containing 80 ppm sodium fluoride (36 ppm F) for 14 weeks. By stereologic method of enzyme histochemistry, it was observed for the first time that the foci of hepatocytic precancerous enzyme alterations (gamma-GT positive, ATPase negative and G-6-Pase negative) were significantly increased in number and size compared with the negative control group. The results suggest that sodium fluoride promoted the growth of precancerous lesions of the liver induced by DEN in rats, and this has provided some data to the understanding of the relationship between fluorosis and neoplasms.
PMID: 8168199 [PubMed - indexed for MEDLINE]
http://www.ncbi.nlm.nih.gov:80/entrez/query.fcgi?cmd=Retrieve&db=PubMed&list_uids=8278638&dopt=AbstractRegul Toxicol Pharmacol 1993 Oct;18(2):154-68
Confounded carcinogenicity study of sodium fluoride in CD-1 mice.
Maurer JK, Cheng MC, Boysen BG, Squire RA, Strandberg JD, Weisbrode SE, Seymour JL, Anderson RL.
Procter & Gamble Co., Cincinnati, Ohio 45239-8707.
To determine its carcinogenic potential, sodium fluoride (NaF) was fed to CD-1 mice for up to 97 weeks. Mice given NaF at a dose of 4, 10, or 25 mg/kg of body weight per day added to a low-fluoride diet were compared to controls given either an unsupplemented low-fluoride diet or laboratory chow. Nonneoplastic changes consistent with those previously recognized from fluoride toxicity were observed in teeth, bones, and joints. Unexpectedly, osteomas occurred in all groups. The incidence of osteomas was similar in groups given the low-fluoride control diet, laboratory chow, or NaF doses of 4 or 10 mg/kg per day. The incidence of osteomas in these groups was increased over that historically experienced at the laboratory and reported in the literature for CD-1 mice. The incidence of osteomas in the mice given 25 mg NaF/kg per day added to a low-fluoride diet was increased over that in the other groups. Osteomas were first observed at Week 55. No malignant bone tumors were observed during the course of the study. The locations, multiplicity, and morphologic features of the osteomas in all groups were similar to those associated with virus-induced bone tumors. Electron microscopic examination revealed abundant retrovirus particles in all osteomas examined from control and test mice. It was concluded that the study was confounded by a retrovirus which contributed to the induction of the osteomas. Because the study was confounded, it cannot be considered a valid bioassay to be used for risk assessment.
PMID: 8278638 [PubMed - indexed for MEDLINE]
http://www.ncbi.nlm.nih.gov:80/entrez/query.fcgi?cmd=Retrieve&db=PubMed&list_uids=8351288&dopt=AbstractProc Soc Exp Biol Med 1993 Sep;203(4):480-4
Low zinc status in rats impairs calcium uptake and aggregation of platelets stimulated by fluoride.
Emery MP, O'Dell BL.
Department of Biochemistry, University of Missouri, Columbia 65211.
Platelets from rats of low zinc status exhibit impaired aggregation in response to ADP stimulation. The abnormality has been traced to defective uptake of calcium from the external medium. This study was designed to determine the location of the molecular defect and whether or not the ADP receptor is involved. Washed platelets were collected from rats fed a low zinc diet (< 1 mg/kg) and control groups that consumed a zinc-adequate diet (100 mg/kg), ad libitum- and pair-fed. Fluoride, a G-protein stimulant, was used to bypass the ADP receptor. F- stimulated platelet aggregation and calcium uptake; both of these functions were impaired by zinc deficiency. At 10 mM F-, the time to half maximal aggregation was increased from 1.8 min in platelets from control to 2.8 min in zinc deficient rats. At 8 mM F-, the uptake of calcium was decreased from 170 to 85 nM cytosolic free calcium. At this concentration of F- there was no release of internal calcium. The results show that the molecular defect in the zinc-deficient platelet is located in the aggregation pathway beyond the ADP receptor and suggest a point between, or including, a G-protein and the plasma membrane calcium channel.
PMID: 8351288 [PubMed - indexed for MEDLINE]
Fluoride 1993; 26(1):45-56Beneficial effects of ascorbic acid and calcium on reversal of fluoride toxicity in male rats
Chinoy NJ, Sharma M, Michael M
Dr NJ Chinoy, Professor and Head, Department of Zoology, Gujarat University, Ahmedabad 380 009, India
Summary: The present study was undertaken to investigate the therapeutic effects of simultaneous ascorbic acid and calcium ingestion along with sodium fluoride (NaF) treatment on the structure and metabolism of vital organs like liver, adrenal, gastrocnemius muscle and serum parameters of male rats. The decrease in muscle and serum proteins suggested inhibition of protein synthesis by fluoride and a possible change in osmotic balance. Alterations in the activities of succinate dehydrogenase (SDH), adenosine triphosphatase (ATPase) and cholinesterase (ChE) in gastrocnemius muscle elucidate disturbanes in its oxidative metabolism, neuromuscular transmission and contractility. The accumulation of glycogen in liver and muscle indicate reduction in the activities of enzymes of glycolytic pathway. The total ascorbic acid in liver and adrenal were enhanced to overcome the stress imposed by the treatment. However, the fluorotic rats did not suffer from hypercholesterolemia. The levels of Na+ and K+ in serum showed a significant increase, thus suggesting alteration in the electrolyte balance of the body due to fluoride intake. The liver of fluoride treated rats revealed zonal necrosis and pycnosis of nucleus of the hepatocytes. The enhanced levels of serum transaminases, which are considered as markers for liver structure and function, also support this observation. The altered histology of the adrenal gland is related to adrenal hypofunction in NaF treated rats. However, simultaneous administration of ascorbic acid (AA) and calcium (Ca+2) to the NaF treated rats revealed marked recovery from fluoride toxicity in all the parameters. The recovery was more pronounced in the NaF + AA treated rats than in the NaF + Ca+2 treated ones. We consider the data of the present study are significant since the beneficial therapeutic effects of AA and Ca+2 in overcoming and reversing fluoride toxicity have far reaching implications the world over.
http://www.ncbi.nlm.nih.gov:80/entrez/query.fcgi?cmd=Retrieve&db=PubMed&list_uids=8253721&dopt=AbstractJ Biol Chem 1993 Dec 15;268(35):26075-8
The regulation of store-dependent Ca2+ influx in HL-60 granulocytes involves GTP-sensitive elements.
Jaconi ME, Lew DP, Monod A, Krause KH.
Division of Infectious Diseases, University Hospital, Geneva, Switzerland.
In granulocytes, emptying of intracellular Ca2+ stores activates Ca2+ influx across the plasma membrane. To study the putative role of GTP-binding proteins in this process, we have introduced non-hydrolyzable guanosine phosphate analogues into the cytosol of non-permeabilized HL-60 granulocytes using an endocytosis-hypoosmotic shock procedure. At the cytosolic concentrations obtained (100-500 microM), neither guanosine 5'-3-O-(thio)triphosphate (GTP gamma S) nor guanosine 5'-3-O-(thio)diphosphate (GDP beta S) affected basal [Ca2+]i. Ca2+ release in response to the receptor agonist fMet-Leu-Phe, the Ca(2+)-ATPase inhibitor thapsigargin, or the Ca2+ ionophore ionomycin was also unaffected by GTP gamma S or GDP beta S. In contrast, the activation of the Ca2+ influx pathway by fMet-Leu-Phe or by thapsigargin was blocked by GTP gamma S but not by GDP beta S. The GTP gamma S effect was mimicked by NaF. The GTP gamma S and NaF effects were independent of protein kinase C activation and actin polymerization. Our results demonstrate that a GTP-sensitive element is involved in the signaling between intracellular Ca2+ stores and plasma membrane Ca2+ channels. The identical effects of GTP gamma S and NaF suggest that the GTP-sensitive element is a heterotrimeric G-protein.
PMID: 8253721 [PubMed - indexed for MEDLINE]
http://www.ncbi.nlm.nih.gov:80/entrez/query.fcgi?cmd=Retrieve&db=PubMed&list_uids=8312137&dopt=AbstractCell Signal 1993 Sep;5(5):633-41
Mechanisms of aluminum fluoride- and insulin-stimulated p33 mRNA accumulation in rat hepatoma cells: involvement of a G protein and kinase action and demonstration of effects on mRNA turnover.
Nagasaka Y, Okubo M, Larner J.
Department of Pharmacology, University of Virginia Health Sciences Center, Charlottesville 22908.
The insulin signalling pathway to control nuclear p33 gene expression was examined. An AlF4-stimulated pertussis toxin-insensitive G protein was shown to be involved. The action of AlF4- was completely blocked by deferoxamine. Insulin action was markedly stimulated in the presence of AlF4-. cAMP and diacylglycerol concentrations were examined as possible regulators but no increases were detected. The effects of AlF4- and of insulin were completely inhibited by the general kinase inhibitor H-7. A second calcium calmodulin protein kinase inhibitor, W-7, had no detectable effect. Insulin and AlF4- were shown to stabilize p33 mRNA.
PMID: 8312137 [PubMed - indexed for MEDLINE]
http://www.ncbi.nlm.nih.gov:80/entrez/query.fcgi?cmd=Retrieve&db=PubMed&list_uids=8312133&dopt=AbstractCell Signal 1993 Sep;5(5):555-64
Evidence for involvement of a GTP-binding protein in activation of Ca2+ influx by epidermal growth factor in A431 cells: effects of fluoride and bacterial toxins.
Kuryshev YA, Naumov AP, Avdonin PV, Mozhayeva GN.
Institute of Cytology, Russian Academy of Sciences, St Petersburg.
Aluminium fluoride (AlF4-), a G protein activator, was used to study a possible role of G protein in the control of the pathways for Ca2+ influx through plasma membrane of human carcinoma A431 cells. Fluorimetric measurements with the Ca2+ indicator Indo-1 have shown that addition of fluoride induces an increase in concentration of cytosolic free calcium ([Ca2+]in) due to both release of Ca2+ from intracellular stores and Ca2+ influx from the extracellular medium. The cells stimulated by fluoride became unresponsive to subsequent addition of epidermal growth factor (EGF), histamine and bradykinin. The Ca2+ signal induced by fluoride as well as one induced by EGF was inhibited by the pretreatment of cells with protein kinase C activator, phorbol myristate acetate (PMA). The pretreatment of the cells with pertussis toxin produced no effect on EGF-induced calcium response. In contrast, the pretreatment with cholera toxin (CTX) increased the basal level of [Ca2+]in and abolished the effect of EGF. The effects of CTX could not be reproduced by treating the cells with forskolin or IBMX, agents known to elevate cAMP content in the cell. Patch clamp experiments have shown that fluoride increases the activity of Ca(2+)-permeable channels identical to those activated by EGF from the extracellular side of the membrane [Mozhayeva et al. (1991) J. Membr. Biol. 124, 113-126]. The results obtained suggest the involvement of GTP-binding protein in signal transduction from the EGF receptor to Ca(2+)-permeable channel of plasma membrane in A431 cells.
PMID: 8312133 [PubMed - indexed for MEDLINE]
http://www.ncbi.nlm.nih.gov:80/entrez/query.fcgi?cmd=Retrieve&db=PubMed&list_uids=8298492&dopt=AbstractBiochem Mol Biol Int 1993 Nov;31(4):613-25
Guanine nucleotide-binding protein stimulates arachidonic acid metabolism in TEA3A1 thymic epithelial cells by stimulating release and inhibiting incorporation of arachidonic acid.
Liu P, Wen M, Hayashi J.
W. Alton Jones Cell Science Center, Lake Placid, NY 12946.
Guanosine 5'-O-(thiotriphosphate) (GTP gamma S), an activator of guanine nucleotide binding protein (G protein), increased prostaglandin E2 (PGE2) production in saponin permeabilized rat thymic epithelial cells, TEA3A1. Aluminum fluoride (A1F4-), a cell permeable G protein activator, also stimulated PGE2 production and arachidonic acid (AA) release from TEA3A1 cells. Using A1F4- instead of GTP gamma S as a G-protein activator, we have investigated the mechanism of G-protein mediated stimulation of PGE2 production in TEA3A1 cells. Results from our experiments indicate that G protein mediated activation of AA metabolism in TEA3A1 cells is regulated by two independent mechanisms. One is by the stimulation of AA release via the activation of PLA2 enzymatic activity through PLC and PKC mediated pathway and the other is by a concomitant inhibition of AA incorporation into membrane phospholipids.
PMID: 8298492 [PubMed - indexed for MEDLINE]
Full report available free at: http://jcs.biologists.org/cgi/reprint/106/4/1239.pdfJ Cell Sci 1993 Dec;106 ( Pt 4):1239-48
Binding of the cytosolic p200 protein to Golgi membranes is regulated by heterotrimeric G proteins.
de Almeida JB, Doherty J, Ausiello DA, Stow JL.
Renal Unit, Massachusetts General Hospital, Charlestown.
The formation of vesicles for protein trafficking requires the dynamic binding of cytosolic coat proteins onto Golgi membranes and this binding is regulated by a variety of GTPases, including heterotrimeric G proteins. We have previously shown the presence of the pertussis toxin-sensitive G alpha i-3 protein on Golgi membranes and demonstrated a functional role for G alpha i-3 in the trafficking of secretory proteins through the Golgi complex. We have also described a brefeldin A-sensitive phosphoprotein, p200, which is found in the cytoplasm and on Golgi membranes. The present study investigates the role of heterotrimeric G proteins in the regulation of p200 binding to Golgi membranes. An in vitro binding assay was used to measure the binding of cytosolic p200 to LLC-PK1 cell microsomal membranes and to purified rat liver Golgi membranes in the presence of specific activators of G proteins. The binding of p200 to Golgi membranes was compared to that of the coatomer protein beta-COP, for which G protein-dependent membrane binding has previously been established. Membrane binding of both p200 and beta-COP was induced maximally by activation of all G proteins in the presence of GTP gamma S. More selective activation of the heterotrimeric G proteins, with AlFn or mastoparan, also induced membrane binding of p200 and beta-COP. Pertussis toxin pretreatment of Golgi membranes, to selectively inactivate G alpha i-3, reduced the AlFn and mastoparan-induced binding of p200 to Golgi membranes, whereas no significant effect of pertussis toxin on beta-COP binding was found in this assay. The effect of pertussis toxin thus implicates G alpha i-3, as one component of a regulatory pathway, in the binding of cytosolic p200 to Golgi membranes. The effects of AlFn and pertussis toxin on p200 membrane binding were also shown in intact cells by immunofluorescence staining. AlFn treatment of cells induced translocation of p200 from the cytoplasm onto the Golgi complex, resulting in a conformational change in some Golgi membranes. The translocation of p200 was blocked by pretreatment of intact NRK cells with pertussis toxin. The data presented here support the conclusion that the binding of the p200 protein to Golgi membranes involves regulation by the pertussis toxin-sensitive heterotrimeric G proteins, specifically the G alpha i-3 protein.
PMID: 8126104 [PubMed - indexed for MEDLINE]
Full report available free at: http://jcs.biologists.org/cgi/reprint/106/3/789.pdfJ Cell Sci 1993 Nov;106 ( Pt 3):789-802
The small GTP-binding protein rab6p is redistributed in the cytosol by brefeldin A.
Roa M, Cornet V, Yang CZ, Goud B.
Unite de Genetique Somatique (URA CNRS 361), Institut Pasteur, Paris, France.
Rab6 protein belongs to the Sec4/Ypt/rab subfamily of small GTP-binding proteins involved in intracellular membrane trafficking in yeast and mammalian cells. Its localization both in medial and trans-Golgi network prompted us to study the effects of brefeldin A (BFA) on rab6p redistribution. By two techniques, indirect immunofluorescence and cell fractionation, we investigated the fate of rab6p and compared it to other Golgi or trans-Golgi network markers in BHK-21 and NIH-3T3 cells. BFA, at 5 micrograms/ml, induced redistribution of rab6p according to a biphasic process: during the first 10-15 minutes, tubulo-vesicular structures--colabelled with a bona fide medial Golgi marker called CTR 433--were observed; these structures were then replaced by punctate diffuse staining, which was stable for up to 3 hours. The 110 kDa peripheral membrane protein beta-COP was released much more rapidly from the Golgi membranes, whereas the trans-Golgi network marker TGN 38 relocated to the microtubule organizing center. The kinetics of reversion of BFA action on these antigens was also followed by immunofluorescence. Consistent with these results, rab6 antigen, originally found as 40% in the cytosolic versus 60% in the particulate (P 150,000 g) fraction, became almost entirely cytosolic; moreover, it partitioned in the aqueous phase of Triton X-114 whereas the membrane fraction was detergent-soluble. Rab6p did not become part of the coatomers after its BFA-induced release from Golgi structures. Three requirements seemed to be necessary for such a release: integrity of the microtubules, presence of energy, and a hypothetical trimeric G protein, as revealed by the respective roles of nocodazole, ATP depletion, and sensitivity to aluminium fluoride. Finally, we have shown that BFA does not prevent attachment of newly synthesized rab6p to membranes.
PMID: 8308062 [PubMed - indexed for MEDLINE]
http://www.ncbi.nlm.nih.gov:80/entrez/query.fcgi?cmd=Retrieve&db=PubMed&list_uids=8269958&dopt=AbstractEur J Biochem 1993 Dec 1;218(2):669-77
Nanomolar arachidonic acid influences the respiratory burst in eosinophils and neutrophils induced by GTP-binding protein. A comparative study of the respiratory burst in bovine eosinophils and neutrophils.
Aebischer CP, Pasche I, Jorg A.
Institute of Biochemistry, University of Fribourg, Switzerland.
To investigate a possible role of phospholipase A2 (PLA2) in the respiratory burst in bovine eosinophilic and neutrophilic leukocytes dependent on GTP-binding protein (G-protein), we permeabilized these cells with Staphylococcus aureus alpha-toxin and induced NADPH oxidase activity with the non-hydrolysable GTP analogue GTP[S] or the aluminium tetrafluoro complex AlF4-. Under same experimental conditions, cells responded with different onset times. The onset time for eosinophils was 50-200 s, for neutrophils it was only a few seconds. GTP[S] stimulated in neutrophils only 5% of the respiratory burst compared to eosinophils, whereas AlF4(-)-induced comparable responses (neutrophils 120% of eosinophils). GDP inhibited these responses with an IC50 value of 2.4 mM. Arachidonic acid showed, with the exception of AlF4- stimulated neutrophils, on both stimuli and cell types an enhancing effect (150%) that reached its maximum at 0.1-1 microM. The PLA2 inhibitor 4-bromophenacylbromide reduced the GTP[S]- and AlF4(-)-induced response almost completely (10 microM) and the inhibition was not significantly different for eosinophils and neutrophils (IC50 1-3 microM). If the respiratory burst was reduced with 4-bromophenacylbromide to 1-4% of the original value, 10% of the basal NADPH oxidase activity could be restored by addition of only 20-100 nM arachidonic acid. In addition, the PLA2 activator adriamycin enhanced the response in a dose-dependent manner and in the same order as arachidonic acid did. The results presented above suggest that the respiratory burst may be regulated by different low-molecular-mass and/or heterotrimeric G-proteins and an active role for arachidonic acid or its metabolites in the activation and the maintenance of the direct G-protein-stimulated respiratory burst in bovine eosinophils and neutrophils.
PMID: 8269958 [PubMed - indexed for MEDLINE]
http://www.ncbi.nlm.nih.gov:80/entrez/query.fcgi?cmd=Retrieve&db=PubMed&list_uids=8307614&dopt=AbstractImmunology 1993 Dec;80(4):633-9
The role of the phosphatidylinositol turnover in 12-hydroxyeicosatetraenoic acid generation from human platelets by Escherichia coli alpha-haemolysin, thrombin and fluoride.
Konig B, Konig W.
Lehrstuhl fur Med. Mikrobiologie und Immunologie, AG Infektabwehrmechanismen, Ruhr-Universitat Bochum, Germany.
Activation of human platelets with either the Escherichia coli alpha-haemolysin or thrombin, or with pharmacological agonists such as sodium fluoride (NaF), Ca-ionophore A23187 or phorbol myristate acetate (PMA), induced a similar pattern of serotonin release, unlike 12-hydroxyeicosatetraenoic acid (12-HETE) generation. In the presence of neomycin (0.1, 1, 10 mM), an inhibitor of the phosphatidylinositol-specific phospholipase C (PIP2-PLC), the E. coli alpha-haemolysin-induced 12-HETE generation was enhanced up to threefold in a dose-dependent manner. 12-HETE generation by NaF and thrombin was slightly inhibited at high neomycin concentrations (10 mM). Treatment of human platelets with E. coli alpha-haemolysin induced a different activation pattern of PIP2-PLC and phosphatidylinositol4-kinase (PI4-kinase), compared to NaF and thrombin. Haemolysin treatment resulted in a down-regulation of PIP2-PLC and PI4-kinase enzymatic activities by 20 +/- 9/40 +/- 8% compared to unstimulated cells; the decrease in enzymatic activities was observed within 2 min of stimulation and was still apparent after 60 min of stimulation. In NaF- and thrombin-stimulated platelets, dose- and time-dependent increases in PIP2-PLC (by up to 21 +/- 10%, 34 +/- 11%, respectively) and PI4-kinase (by up to 71 +/- 18, 54 +/- 14) activities were measured. Maximal enzymatic activities were reached after 5-20 min of stimulation (NaF, thrombin) followed by a decline to baseline levels (thrombin) or below baseline levels (NaF). Our results indicate that the phosphatidylinositolphosphate metabolism plays an important role in the regulation of 12-HETE release from human platelets by E. coli alpha-haemolysin.
PMID: 8307614 [PubMed - indexed for MEDLINE]
http://www.ncbi.nlm.nih.gov:80/entrez/query.fcgi?cmd=Retrieve&db=PubMed&list_uids=8223434&dopt=AbstractEMBO J 1993 Nov;12(11):4191-8
Tryptophan W207 in transducin T alpha is the fluorescence sensor of the G protein activation switch and is involved in the effector binding.
Faurobert E, Otto-Bruc A, Chardin P, Chabre M.
CNRS, Institut de Pharmacologie Moleculaire et Cellulaire, Valbonne, France.
We have produced a recombinant transducin alpha subunit (rT alpha) in sf9 cells, using a baculovirus system. Deletion of the myristoylation site near the N-terminal increased the solubility and allowed the purification of rT alpha. When reconstituted with excess T beta gamma on retinal membrane, rT alpha displayed functional characteristics of wild-type T alpha vis a vis its coupled receptor, rhodopsin and its effector, cGMP phosphodiesterase (PDE). We further mutated a tryptophan, W207, which is conserved in all G proteins and is suspected to elicit the fluorescence change correlated to their activation upon GDP/GTP exchange or aluminofluoride (AlFx) binding. [W207F]T alpha mutant displayed high affinity receptor binding and underwent a conformational switch upon receptor-catalysed GTP gamma S binding or upon AlFx binding, but this did not elicit any fluorescence change. Thus W207 is the only fluorescence sensor of the switch. Upon the switch the mutant remained unable to activate the PDE. To characterize better its effector-activating interaction we measured the affinity of [W207F]T alpha GDP-AlFx for PDE gamma, the effector subunit that binds most tightly to T alpha. [W207F]T alpha still bound in an activation-dependent way to PDE gamma, but with a 100-fold lower affinity than rT alpha. This suggests that W207 contributes to the G protein effector binding.
PMID: 8223434 [PubMed - indexed for MEDLINE]
http://www.ncbi.nlm.nih.gov:80/entrez/query.fcgi?cmd=Retrieve&db=PubMed&list_uids=8107023&dopt=AbstractJ Reprod Fertil 1993 Nov;99(2):415-9
Activation of temperature-dependent flagellar movement of demembranated fowl spermatozoa: involvement of an endogenous serine protease.
Ashizawa K, Katayama S, Wishart GJ, Shiraishi A, Tsuzuki Y.
Laboratory of Animal Reproduction, Faculty of Agriculture, Miyazaki University, Japan.
In the presence of ATP, the motility of demembranated fowl spermatozoa was vigorous at 30 degrees C, but negligible at 40 degrees C. Motility could be restored at 40 degrees C by the addition of 10-100 ng trypsin ml-1. Chymotrypsin also stimulated the motility, but neither papain nor carboxypeptidase B appreciably affected motility. Conversely, at 30 degrees C sperm motility was inhibited by aprotinin or phenylmethylsulfonyl fluoride. These results suggest that endogenous protease, presumably serine protease, activity is instrumental in the regulation of fowl sperm motility. It seems likely that the site of action of this protease is axonemal, but a direct effect of added protease on dynein ATPase activity could not be demonstrated.
PMID: 8107023 [PubMed - indexed for MEDLINE]
http://www.ncbi.nlm.nih.gov:80/entrez/query.fcgi?cmd=Retrieve&db=PubMed&list_uids=8240404&dopt=AbstractBiochem Pharmacol 1993 Oct 19;46(8):1511-4
Effects of sodium fluoride and cobalt chloride on the enantioselectivity of microsomal and cytosolic esterases in rat intestinal mucosa.
Yang SK, Chang WC, Huang JD.
Department of Pharmacology, F. Edward Hebert School of Medicine, Uniformed Services University of the Health Sciences, Bethesda, MD 20814-4799.
The effects of sodium fluoride (NaF) and cobalt chloride (CoCl2) on the enantioselective hydrolysis of racemic oxazepam 3-acetate (rac-OXA) by microsomal and cytosolic esterases in rat intestinal mucosa were studied. Microsomal and cytosolic esterases hydrolyzed S-OXA and R-OXA in approximately 1:19 and 4:1 ratios, respectively. The hydrolysis of R-OXA by microsomal esterases was inhibited by NaF with an IC50 of 13.4 +/- 1.5 mM. Hydrolyses of both S-OXA and R-OXA by cytosolic esterases were inhibited by NaF with a similar IC50 value (approximately 3 mM). The hydrolysis of S-OXA by cytosolic esterases was inhibited by CoCl2 (IC50 = approximately 5 mM), whereas the hydrolysis of R-OXA by cytosolic esterases was stimulated by approximately 10% in the presence of 1 mM CoCl2. In comparison, the hydrolysis of R-OXA by microsomal esterases was stimulated by approximately 55% in the presence of 1 mM CoCl2. These results not only revealed the effects of NaF and CoCl2 on the catalytic activities of enantioselective cytosolic and microsomal esterases, but also indicated that microsomal and cytosolic esterases that selectively hydrolyzed R-OXA were distinctly different protein entities.
PMID: 8240404 [PubMed - indexed for MEDLINE]
NOTE: AG Gilman, co-author of this report, shared the 1994 Nobel Prize for the discovery of "G-proteins and the role of these proteins in signal transduction in cells."Full report available free at http://www.pnas.org/cgi/reprint/90/19/9176.pdf
Proc Natl Acad Sci U S A 1993 Oct 1;90(19):9176-80
Purification and characterization of recombinant G16 alpha from Sf9 cells: activation of purified phospholipase C isozymes by G-protein alpha subunits.
Kozasa T, Hepler JR, Smrcka AV, Simon MI, Rhee SG, Sternweis PC, Gilman AG.
Department of Pharmacology, University of Texas Southwestern Medical Center, Dallas 75235.
A cDNA encoding G16 alpha, the alpha subunit of a heterotrimeric guanine nucleotide-binding protein, was expressed in Sf9 cells using recombinant baculovirus. G16 alpha in membrane extracts of Sf9 cells activated phospholipase C-beta 1 (PLC-beta 1) in the presence of guanosine 5'-[gamma-thio]triphosphate; the system could not be activated by Al3+, Mg2+, and F-. The G16 alpha in the cytosolic fraction of Sf9 cells did not stimulate PLC-beta 1. Concurrent expression of the G-protein beta gamma subunit complex increased the amount of G16 alpha in Sf9 cell membranes. The guanosine 5'-[gamma-thio]triphosphate-activated form of G16 alpha was purified from cholate extracts of membranes from cells expressing G16 alpha, and the G-protein beta 2 and gamma 2 subunits. G16 alpha activated PLC-beta 1, PLC-beta 2, and PLC-beta 3 in a manner essentially indistinguishable from that of Gq alpha. G16 alpha-mediated activation of PLC-beta 1 and PLC-beta 3 greatly exceeded that of PLC-beta 2. G16 alpha did not activate PLC-gamma 1 or PLC-delta 1. Thus, two distantly related members of the Gq alpha family, Gq alpha and G16 alpha, have the same ability to activate the known isoforms of PLC-beta.
PMID: 8415674 [PubMed - indexed for MEDLINE]
http://www.ncbi.nlm.nih.gov:80/entrez/query.fcgi?cmd=Retrieve&db=PubMed&list_uids=8399129&dopt=AbstractBiochemistry 1993 Sep 28;32(38):10021-6
Stoichiometry of tight binding of magnesium and fluoride to phosphorylation and high-affinity binding of ATP, vanadate, and calcium in the sarcoplasmic reticulum Ca(2+)-ATPase.
Daiho T, Kubota T, Kanazawa T.
Department of Biochemistry, Asahikawa Medical College.
We previously showed that, when the purified Ca(2+)-ATPase from sarcoplasmic reticulum (SR) is treated with fluoride (F-) in the presence of Mg2+, a complete inactivation of the enzyme is induced by tight binding of approximately 2 mol of Mg2+ and 4 mol of F- to the catalytic site per mole of phosphorylation site [Kubota, T., Daiho, T., & Kanazawa, T. (1993) Biochim. Biophys. Acta 1163, 131-143]. Contradictorily, on the basis of the postulated content of the Ca(2+)-ATPase in F(-)-treated SR vesicles, Coll and Murphy [(1992) J. Biol. Chem. 267, 21584-21587] suggested that each inactivated enzyme contains one tightly-bound Mg2+ and two tightly-bound F-. The present study has been made to resolve this conflict. The contents of phosphorylation site, high-affinity ATP-binding site, high-affinity vanadate-binding site, and high-affinity Ca(2+)-binding site in the SR vesicles used were 3.33 +/- 0.06, 3.54 +/- 0.12, 3.34 +/- 0.04, and 6.98 +/- 0.16 nmol/mg, respectively. When the vesicles were incubated with F- in the presence of Mg2+, the Ca(2+)-ATPase was inactivated progressively. After removal of unbound Mg2+ and F- by gel filtration, tightly-bound Mg2+ and F- were determined by use of an atomic absorption spectrophotometer and a F(-)-selective electrode. A linear relationship existed between the extent of the enzyme inactivation and the contents of the tightly-bound ligands. The contents of tightly-bound Mg2+ and F- in the fully inactivated vesicles were 6.65 and 12.6 nmol/mg, respectively. The same stoichiometry was obtained with another preparation of SR vesicles.(ABSTRACT TRUNCATED AT 250 WORDS)
PMID: 8399129 [PubMed - indexed for MEDLINE]
http://www.ncbi.nlm.nih.gov:80/entrez/query.fcgi?cmd=Retrieve&db=PubMed&list_uids=8490045&dopt=AbstractBiochim Biophys Acta 1993 May 13;1163(2):131-43
Quasi-irreversible inactivation of the sarcoplasmic reticulum Ca(2+)-ATPase by simultaneous tight binding of magnesium and fluoride to the catalytic site.
Kubota T, Daiho T, Kanazawa T.
Department of Biochemistry, Asahikawa Medical College, Japan.
The sarcoplasmic reticulum Ca(2+)-ATPase was inactivated quasi-irreversibly by the treatment with KF in the presence of Mg2+ and absence of Ca2+. This inactivation was Mg(2+)-dependent, and prevented by high-affinity Ca2+ binding. The enzyme was completely protected by ATP against the inactivation with an affinity consistent with that of the catalytic site for ATP. The affinity for Mg2+ in this inactivation was in agreement with that for Mg2+ in phosphorylation of the enzyme with Pi. Mg.ATP did not bind to the inactivated enzyme, whereas metal-free ATP did bind to it with a high affinity. These findings suggest that the Mg2+ binding sub-site in the catalytic site of the inactivated enzyme is occupied by tightly-bound Mg2+. The enzyme was completely protected by Pi against the inactivation with an affinity consistent with that of the catalytic site for Pi. The inactivated enzyme showed neither phosphorylation with Pi nor high-affinity vanadate binding. These findings suggest that the phosphorylation site of the inactivated enzyme is occupied by tightly-bound F-. The contents of tightly-bound Mg2+ and F- in the inactivated enzyme were determined after unbound Mg2+ and F- were removed by gel filtration. 2.3 mol of Mg2+ and 3.7 mol of F- per mol of phosphorylation sites were tightly bound to the enzyme. The tight binding of these ligands depended on the presence of each other, and was completely prevented by high-affinity Ca2+ binding. Linear relationships were found between the contents of the tightly-bound ligands and the extent of the enzyme inactivation. The tightly-bound Mg2+ and F- were entirely released by low-affinity Ca2+ binding, and correspondingly the ATPase activity was restored. It is concluded that the observed enzyme inactivation is caused by simultaneous tight binding of Mg2+ and F- to the catalytic site.
PMID: 8490045 [PubMed - indexed for MEDLINE]
http://www.ncbi.nlm.nih.gov:80/entrez/query.fcgi?cmd=Retrieve&db=PubMed&list_uids=8466517&dopt=AbstractBiochem Biophys Res Commun 1993 Mar 31;191(3):802-10
Evidence for the involvement of G proteins in the modulation of 22Na and 45Ca fluxes in myogenic L6 and aortic smooth muscle cells.
Orlov SN, Sen CK, Kolosova IA, Bernhardt J, Hanninen O, Buhler FR.
Laboratory of Physical Chemistry of Biomembranes, Moscow State University, Russia.
Preincubation of rat skeletal muscle derived L6 myoblasts with 10 mM NaF increased, the activity of Na+/H+ exchanger, and the uptake of 45Ca by 2 and 5 folds, respectively. In cultured vascular smooth muscle cells (CVSMC) of rat aorta, NaF increased the activity of Na+,K(+)-pump, Na+/H+ exchanger, passive permeability for Na+, and 45Ca uptake by 1.6, 9, 2, and 9 folds, respectively. Both, in CVSMC and L6 cells, the effect of NaF on the Na+/H+ exchanger and 45Ca uptake were significantly augmented by 20 microM AlCl3. No effect of AlCl3 on the NaF dependent changes in ion flux was seen in rat erythrocytes. The results suggest that in L6 and CVSMC cells, the Na+/H+ exchanger and Ca++ uptake pathway(s) are activated by GTP-binding proteins.
PMID: 8466517 [PubMed - indexed for MEDLINE]
http://www.ncbi.nlm.nih.gov:80/entrez/query.fcgi?cmd=Retrieve&db=PubMed&list_uids=8485231&dopt=AbstractBiokhimiia 1993 Mar;58(3):456-60
[The role of GTP-binding proteins in regulating the activation of Na+/H+-exchange and Na+, K+, 2Cl- cotransport: effect of fluoride ion]
[Article in Russian]
Kolosova IA, Bernkhard I, Orlov SN, Biuller FR.
The effects of NaF and AlCl3 on basal and stimulated by hyperosmotic shock activities of Na+/H(+)-exchange and Na+, K+, 2Cl(-)-cotransport in rat smooth muscle and red blood cells have been studied. Preincubation of cells with 10 mM NaF leads to inhibition of basal and hyperosmotic shock-activated Na+, K+, 2Cl(-)-cotransport in smooth muscle cells--by 45% and 190%, respectively. Sodium fluoride causes 80% activation of basal Na+, K+, 2Cl(-)-cotransport in red blood cells with no effect on the transport induced by hyperosmotic shock. The effect of NaF on Na+, K+, 2Cl(-)-cotransport in red blood cells does not depend on Al3+. Basal Na+/H(+)-exchange is stimulated by NaF by 780% in smooth muscle cells and is insensitive to the fluoride in red blood cells. Under hyperosmotic conditions NaF activates Na+/H(+)-exchange by 100% in smooth muscle cells and by 130% in red blood cells; in the latter case its effect is potentiated by Al3+. The data obtained suggest that GTP-binding proteins are involved in the activation of Na+/H(+)-exchange under hyperosmotic conditions.
PMID: 8485231 [PubMed - indexed for MEDLINE]
http://www.ncbi.nlm.nih.gov:80/entrez/query.fcgi?cmd=Retrieve&db=PubMed&list_uids=8226854&dopt=AbstractJ Biol Chem 1993 Nov 5;268(31):23307-10
Formation of a stable inactive complex of the sarcoplasmic reticulum calcium ATPase with magnesium, beryllium, and fluoride.
Murphy AJ, Coll RJ.
Department of Biochemistry, School of Dentistry, University of the Pacific, San Francisco, California 94115.
Incubation of leaky or nonionic detergent-solubilized sarcoplasmic reticulum vesicles in solutions containing magnesium, beryllium, and fluoride caused a time-dependent complete inhibition of calcium ATPase activity. The inhibited state persisted through dialysis for 2 days versus EGTA and was reversed within minutes by the presence of 0.5 mM calcium. Calcium-independent ATPase activity was unaffected. Omission of magnesium or fluoride resulted in retention of activity, while omission of beryllium produced slower inactivation, as described previously (Murphy, A. J., and Coll, R. J. (1992) J. Biol. Chem. 267, 5229-5235). Incubation of nonleaky vesicles had a similar effect, although it occurred more than 10-fold more slowly, suggesting that a component, probably beryllium, must enter the vesicles for inhibition to occur. By contrast, inhibition of nonleaky vesicles by magnesium and fluoride developed less than 2-fold more slowly. Including calcium in the incubation mixtures resulted in partial protection, so that the time course of CaATPase activity leveled off at nonzero values (for example, at 0.1 mM calcium, the activity leveled off at 43% of control). This result is most simply accounted for by a model involving simultaneous binding of calcium and a form of fluoroberyllium to the CaATPase (e.g. Ca2EMgBeFn).
PMID: 8226854 [PubMed - indexed for MEDLINE]
http://www.ncbi.nlm.nih.gov:80/entrez/query.fcgi?cmd=Retrieve&db=PubMed&list_uids=7679793&dopt=AbstractPflugers Arch 1993 Jan;422(4):397-400
Aluminofluoride activates hyperpolarization- and stretch-activated cationic channels in single smooth muscle cells.
Hisada T, Singer JJ, Walsh JV Jr.
Department of Physiology, University of Massachusetts Medical Center, Worcester 01655.
Aluminofluoride (AF) has a variety of biological actions such as activation of GTP binding proteins and inhibition of phosphatases. In the present study, the effects of AF on hyper-polarization- and stretch-activated cationic channels (HA-SACs) were investigated in isolated gastric smooth muscle cells from the toad, Bufo marinus, using the patch-clamp technique. In cell-attached patches extracellular application of AF (20 mM KF plus 20 microM AlCl3) reversibly increased HA-SAC activity without changing its voltage sensitivity. The single channel current amplitude of HA-SACs was not affected during this procedure. The mechanism of AF-induced activation of HA-SACs remains unclear. However, this activation may play a role in contraction of smooth muscle induced by AF.
PMID: 7679793 [PubMed - indexed for MEDLINE]
http://www.ncbi.nlm.nih.gov:80/entrez/query.fcgi?cmd=Retrieve&db=PubMed&list_uids=8381408&dopt=AbstractJ Biol Chem 1993 Feb 5;268(4):2393-402
The mechanism of aluminum-independent G-protein activation by fluoride and magnesium. 31P NMR spectroscopy and fluorescence kinetic studies.
Antonny B, Sukumar M, Bigay J, Chabre M, Higashijima T.
Institut de Pharmacologie Moleculaire et Cellulaire, Centre National de la Recherche Scientifique, Valbonne, France.
With magnesium present, fluoride and aluminum ions activate heterotrimeric G-proteins by forming AlFx complexes that mimic the gamma phosphate of a GTP. We report compelling evidence for a newly proposed process of G-protein activation by fluoride and magnesium, without Al3+. With millimolar Mg2+ and F-, Gs and Gt activate adenylylcyclase and cGMP-phosphodiesterase, respectively. In 31P NMR, addition of magnesium to Gi1 alpha GDP or Gt alpha GDP solutions containing fluoride, but no Al3+, modifies the chemical shift of the GDP beta phosphorus, suggesting that magnesium interacts with the beta phosphate. Titration of this effect indicates that two Mg2+ are bound per G alpha. Biphasic activation kinetics, monitored by G alpha tryptophan fluorescence, suggests the rapid binding of one Mg2+ to G alpha GDP and the slow association of another Mg2+, in correlation with fluoride binding and G alpha activation. The deactivation rate upon fluoride dilution shows a second order dependence with respect to the residual F- concentration, suggesting the sequential release of at least three F-/G alpha. Thus, in millimolar Mg2+ and F-, and without Al3+, two Mg2+ and three F- bind sequentially to G alpha GDP and induce the switch to an active G alpha (GDP-MgF3)Mg state, which is structurally analogous to G alpha (GDP-AlFx)Mg and to G alpha (GTP)Mg.
PMID: 8381408 [PubMed - indexed for MEDLINE]
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