G-Proteins - Adverse Effects
Sodium Fluoride
CAS No. 7681-49-4

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Sodium fluoride and aluminium fluoride are G-protein activators.

The following is the 1994 Boston Globe's report on the awarding of the Nobel Prize in Medicine to the two scientists who cracked the code of G-proteins.

Two from US share Nobel Prize in Medicine
'G Proteins' seen keey to cell links

By Usha Lee McFarling, Boston Globe Staff
October 11, 1994, Page: 5

Two US scientists won the Nobel Prize in medicine yesterday for their discovery of an intricate internal "switchboard" that allows the body's billions of cells to communicate with one another -- and that unleashes cancer and cholera's devastating effects when it goes awry.

Alfred G. Gilman, of the University of Texas Southwestern Medical Center in Dallas, and Martin Rodbell, of the National Institute of Environmental Health Sciences in North Carolina, won the prize for work they conducted independently in the past three decades to discover the "G proteins" that act as the switchboard of the body's communication pathway... Gilman and Rodbell, 68, will share the prize's $930,000 award...

The realm the Nobel winners studied is the complex relay of cell communication, by which information from the outsides of cells is conveyed to the cells' interior machinery by a series of agents.

G proteins, the most crucial of those agents, have been described as ''biological traffic lights." Lying inside the cell, they can respond to signals from outside the cell -- light, smell, neurotransmitters and hormones -- and translate them into a frenzy of cellular action inside.

While G proteins are little known to the general public, research on them has been one of the hottest biological pursuits of the past decade.

"G proteins are one of the keys to all functions in every cell of the body," Reuters quoted Gosta Gahrton, a professor of medicine at the Karolinska Institute in Stockholm and a member of the prize panel that awarded the prize, as saying.

The proteins are implicated in a growing list of cellular activities -- from mating in yeast to thinking in humans. They permit sight and smell by converting light and scent into messages that can be taken to the brain.

When G proteins do not work, they can lead to symptoms of such diseases as diabetes, alcoholism, cholera and whooping cough. The proteins, which have been called "ubiquitous, influential, and enigmatic," will be the target of medical treatments, the Nobel institute predicted.

At least 17 of the proteins have been discovered. They are called G proteins because they bind to guanine nucleotides, a major component of the genetic molecules DNA and RNA.

Rodbell and his co-workers at the National Institutes of Health determined that guanine nucleotides were involved in cell communication -- a finding that led to the discovery of G proteins. His work in the 1960s and '70s also described how messages like light are converted inside cells, and showed that cells have different components that receive, transport and amplify outside messages.

Rodbell retired in May. He holds the title of scientist emeritus at the National Institutes of Health. Gilman chairs the pharmacology department at the University of Texas Southwestern Medical Center in Dallas and won biology's coveted Lasker award in 1989. Gilman is the fourth UT Southwestern faculty member to win the Nobel Prize.

Gilman and colleages, while working at the University of Virginia in Charlottesville in the 1970s, looked for the chemicals that made up the substances Rodbell described. In 1980, they discovered G proteins.

The field did not blossom until 1984, when the first genes for G proteins were cloned. Since then, more scientists began working with the proteins, and some pharmaceutical companies are now trying to develop drugs targeting G proteins.

In a 1992 Scientific American article on G proteins that Gilman co-wrote with Maurine E. Linder, he predicted that scientists would evenutally diagram the cellular players involved in communication and be able to predict how those cells will operate in response to different combinations of signals.

"For those who would hope to develop drug therapies," the authors said, ''such discoveries would be like giving a thief a wiring diagram to the alarm system at a bank."

Online at:

See: graphics from the Nobel Committeee for a more scientific understanding:
The Discovery of G Proteins
Signal Transduction in Cells
Activation and Inactivation of the G Protein
G Proteins and the Body
Cholera and G-proteins

Note from EC: I've added this abstract to this section as the authors concluded that flucythrinate "inhibits intercellular communication."

Flucythrinate - Acaricide, Insecticide - CAS No. 70124-77-5

Abstract: The effects of the pyrethroids flucythrinate (70124775), cyfluthrin (68359375), bioallethrin (584792) and resmethrin (10453868) were compared with the effects of tetradecanoyl-phorbol-acetate (TPA) and DDT on metabolic cooperation between Chinese-hamster-V79 cells to determine if pyrethroid structure was associated with ability to inhibit intercellular communication. Addition of flucythrinate (structurally related to fenvalerate and DDT) to cocultures of 6-thioguanine resistant cells and 6-thioguanine sensitive cells significantly increased mutant cell recovery, indicating inhibition of intercellular communication; other tested pyrethroids produced no such effect. Addition of TPA, together with increasing doses of flucythrinate or fenvalerate, produced a synergistic response. Various combinations of fenvalerate, flucythrinate, and DDT exposure indicated additive responses. The authors conclude that flucythrinate inhibits intercellular communication and that the p-substituted phenyl group may play an important role; there appear to be different pathways of action for TPA and the halogenated insecticides investigated in this study.
Effects of Tetradecanoyl Phorbol Acetate, Pyrethroids and DDT in the V79; by Warngard L, Flodstrom S. Cell Biology and Toxicology, Vol. 5, No. 1, pages 67-75, 27 references, 1989.

• See also section on PFOA and G-Proteins

Fluoride 2002; 35(4):244
XXVth ISFR Conference Abstract
Fluoride and aluminum: messengers of false information
Strunecka A.
Charles Univ. Prague, Faculty of Sciences, Dept. of Physiology and Developmental Biology, Vinicna 7, 128 00 Prague 2, Czech Republic. E-mail: strun@natur.cuni.cz

Intensive laboratory research on the mechanisms of signal transduction has produced experimental data that could change our understanding of the action of fluoride at the cellular level. After reflecting on these laboratory studies, we suggest that some of pathological changes are not produced by fluoride alone but by the synergistic action of fluoride and aluminum. Heterotrimeric G-proteins mediate the transfer of information from heptahelical receptors to effector molecules. The discovery of aluminofluoride complexes (AlFx) as a new class of phosphate analogues has been followed by demonstrations of their usefulness in laboratory investigations and their pharmacological efficacy. AlFx complexes interact with all known G-protein-activated effector enzymes. G-proteins take part in an enormous variety of biological signaling systems, helping control almost all important life processes. The family of cell-surface receptors that require coupling to G-protein transducers for functional signaling is vast and diverse. AlFx may clone or potentiate the action of numerous extracellular signals. It appears probable that we will not find any physiological process which is not potentially influenced by AlFx. The aluminofluoride complex acts as the first messenger triggering processes of neurotransmission and potentiating the action of various hormones. It is evident that AlFx are species that convey false information, which is then amplified by processes of signal transmission. Many human diseases have their origin in the malfunctioning of signaling components. Pharmacologists estimate that up to 60% of all medicines used today exert their effects through a G-protein signaling pathway. The synergistic action of fluoride and aluminum in the environment, water, and food can thus evoke multiple pathological symptoms. AlFx might induce alterations in homeostasis, metabolism, growth, and differentiation in living organisms. An awareness of the health risks of this new ecotoxicological phenomenon, an increasing load of aluminum ions and fluoride, would undoubtedly contribute significantly to reducing the risk of a decrease in intelligence of children and adults, and many other disorders in the 21 st century.


2003. Crit Rev Oral Biol Med;14(2):100-14
The biochemistry and physiology of metallic fluoride: action, mechanism, and implications.
Li L.
Faculty of Dentistry, University of Manitoba, 780 Bannatyne Avenue, Winnipeg R3E 0W2, MB, Canada; umlil@cc.umanitoba.ca

Fluoride is a well-known G protein activator. Activation of heterotrimeric GTP-binding proteins by fluoride requires trace amounts of Al3+ or Be2+ ions. AlFx mimics a gamma-phosphate at its transition state in a Galpha protein and is therefore able to inhibit its GTPase activity. AlFx also forms complexes with small GTP-binding proteins
in the presence of their GTPase-activating proteins (GAP). As phosphate analogs, AlFx or BeFx affect the activity of a variety of phosphoryl transfer enzymes. Most of these enzymes are fundamentally important in cell signal transduction or energy metabolism. Al3+ and F- tend to form stable complexes in aqueous solution. The exact structure and concentration of AlFx depend on the pH and the amount of F- and Al3+ in the solution. Humans are exposed to both F and Al. It is possible that Al-F complexes may be formed in vivo, or formed in vitro prior to their intake by humans. Al-F complexes may play physiological or pathological roles in bone biology, fluorosis, neurotoxicity, and oral diseases such as dental caries and periodontal disease. The aim of this review is to discuss the basic chemical, biochemical, and toxicological properties of metallic fluoride, to explore its potential physiological and clinical implications.


2003. Hum Exp Toxicol Mar;22(3):111-23
Fluoride-induced apoptosis in human epithelial lung cells (A549 cells): role of different G protein-linked signal systems.
Refsnes M, Schwarze PE, Holme JA, Lag M.
Division of Environmental Medicine, Norwegian Institute of Public Health, Geitmyrsvn. 75, PO Box 4404 Nydalen, N-0403 Oslo, Norway. magne.refsnes@fhi.no

In the present study, possible mechanisms involved in fluoride-induced apoptosis in a human epithelial lung cell line (A549) were examined. Sodium fluoride (NaF) induced apoptosis in the A549 cells, with a maximum at 5-7.5 mM after 20 hours of exposure. The number of cells with plasma membrane damage (PI-positive cells) increased moderately up to 5 mM, but markedly at 7.5 mM. Deferoxamine (an Al3+ chelator) almost completely prevented these NaF-induced responses, which may suggest a role for G protein activation. The apoptotic effect was partially reduced by the PKA inhibitor H89. NaF induced a weak but sustained increase in PKC activity, whereas the PKC activator TPA induced a transient effect. TPA, which enhanced the NaF-induced PKC activity, was not apoptotic when added alone, but facilitated the NaF-induced apoptosis and the increase in PI-positive cells. PKC downregulation induced by TPA pretreatment almost completely prevented the NaF-induced apoptosis and the increase in PI-positive cells. Pretreatment with the PKC inhibitor GF109203X, which abolished the PKC activity after 3 hours, enhanced the NaF-induced apoptosis. KN93 (a CaM kinase II inhibitor) and W7 (a calmodulin inhibitor) seem to reduce the apoptotic effect of NaF, whereas BAPTA-AM (a Ca2+ chelator) was without effect. The tyrosine kinase inhibitor genistein also markedly reduced the NaF-induced apoptosis, whereas the PI-3 kinase inhibitor wortmannin augmented the response. In conclusion, the present results suggest that NaF induces an apoptotic effect and an increase in PI-positive A549 cells via similar mechanisms, involving PKC, PKA, tyrosine kinase and Ca2+-linked enzymes, whereas PI-3 kinase seems to exert a counteracting effect.


2003. Brain Res. Jul 24 [Epub ahead of print].
GTP(gammaS) increases Na(v)1.8 current in small-diameter dorsal root ganglia neurons.
Saab CY, Cummins TR, Waxman SG.
Department of Neurology and PVA/Eastern Paralyzed Veterans Association Neuroscience Research Center, Yale Medical School, CT 06510, New Haven, USA.
Tetrodotoxin-resistant (TTX-R) sodium current in small-size dorsal root ganglia (DRG) neurons is upregulated by prostaglandin E(2) and serotonin through a protein kinase A (PKA)/protein kinase (PKC) pathway, suggesting G protein modulation of one or more TTX-R channels in these neurons. Recently, GTP(gammaS), a hydrolysis-resistant analogue of GTP, was shown to increase the persistent current produced by the TTX-R Na(v)1.9. In this study, we investigated the modulation of another TTX-R channel, Na(v)1.8, by GTP(gammaS) in small-diameter DRG neurons from rats using whole-cell voltage clamp recordings. Because it has been suggested that fluoride, often used in intracellular recording solutions, may bind to trace amounts of aluminum and activate G proteins, we recorded Na(v)1.8 currents with and without intracellular fluoride, and with the addition of deferoxamine, an aluminum chelator, to prevent fluoride-aluminum binding. Our results show that GTP(gammaS) (100 micro M) caused a significant increase in Na(v)1.8 current (67%) with a chloride-based intracellular solution. Although the inclusion of fluoride instead of chloride in the pipette solution increased the Na(v)1.8 current by 177%, GTP(gammaS) further increased Na(v)1.8 current by 67% under these conditions. While the effect of GTP(gammaS) was prevented by pretreatment with H7 (100 micro M), a non-selective PKA/PKC inhibitor, the fluoride-induced increase in Na(v)1.8 current was not sensitive to H7 (100 micro M), or to inclusion of deferoxamine (1 mM) in the intracellular solution. We conclude that G protein activation by GTP(gammaS) increases Na(v)1.8 current through a PKA/PKC mechanism and that addition of fluoride to the pipette solution further enhances the current, but is not a confounding variable in the study of Na(v)1.8 channel modulation by G proteins independent of a PKA/PKC pathway or binding to aluminum.


Brain Res Dev Brain Res. 2002 Jan 31;133(1):69-75.
Ontogenetic development of the G protein-mediated adenylyl cyclase signalling in rat brain.
Ihnatovych I, Novotny J, Haugvicova R, Bourova L, Mares P, Svoboda P.
Department of Developmental Epileptology, Institute of Physiology, Academy of Sciences, Vijdenska 1083, 142 20 Prague 4, Czech Republic.

Maturation of the brain adenylyl cyclase (AC) signalling system was investigated in the developing rat cortex, thalamus and hippocampus. Expression of AC type II, IV and VI measured by Western blot dramatically increased in all tested brain regions during the first 3 weeks after birth and these levels were maintained in adulthood. AC type I did not change during ontogenesis. In parallel, AC enzyme activities were determined in order to obtain the functional correlates to the preceding structural (immunoblot) analyses of trimeric G proteins [Ihnatovych et al., Dev. Brain Res. (2002) in press]. Surprisingly, basal, manganese-, fluoride-, forskolin- and GTP-stimulated adenylyl cyclase developed similarly. The relatively low enzyme activities, which were determined at birth, progressively increased (about four times) to a clear maximum around postnatal day PD 12. This was followed by a progressive regression to adulthood so that activity of AC at PD 90 was comparable with the low neonatal level. The peak of AC activities at PD 12 was detected in all tested brain regions. Stimulatory (isoproterenol) effect on basal AC activity as well as inhibitory (baclofen) effect on forskolin-stimulated AC activity were unchanged between PD 12 and PD 90. Thus, comparison of results of the structural and functional analyses of adenylyl cyclase signalling system revealed a clear dissociation between the increase in the amount protein of various AC isoforms and the decrease of total G-protein mediated enzyme activities between PD 12 and adulthood. As none of the complex changes in trimeric G protein levels can explain this difference, the future research has to be oriented to identification of potential negative regulators of AC in the course of brain development. Among these, the newly discovered group of GTPase activating proteins, RGS, appears to be of primary importance because these proteins represent potent negative regulators of any G protein-mediated signalling in brain.

Fluoride 2002; 35(4):244-245

XXVth ISFR Conference Abstract
Interactions between guanosine diphosphate (GDP) and aluminum fluoride (AlF3)
Machoy Z (1), Gutowska I (2), Straszko J (3), Machalinski B (2)
(1) Dept. of Biochemistry and Chemistry and
(2) Dept. of General Pathology, Pomeranian Academy of Medicine (PAM),
(3) Dept. of Physical Chemistry, Technical Univ., Szczecin, Poland.
For Correspondence: Machoy Z, Dept. of Biochemistry and Chemistry, PAM, Al. Powstancow Wlkp. 72, 70-111 Szczecin, Poland. Email: machalin@sci.pam.szczecin.pl

Fluorine-aluminum compounds are now being studied with increasing interest. The neurotoxic properties of AlF3 are most important medically because of reports connecting AlF3 with the pathogenesis of Alzheimer's disease. One hypothesis is that the complex of aluminum and fluorine can influence the G-protein receptors and the resulting phosphorylation. Moreover, AlF3 activates several guanine nucleotides mimicking the actions of some neurotransmitters and hormones. Some physico-chemical properties of AlF3 and GDP, as well as their antagonist interactions in many biochemical pathways, have been analyzed. The aim of this study was to investigate of interaction between AlF3 and GDP by virtual molecular modeling using the HyperChem computer program. The semi-empiric method (PM3), with Polak-Ribeier's optimization algorithm, was used. The PM3 method is suitable for modeling of the molecules containing elements of the main groups of the periodic table. The results indicated that the main sites of the reaction in GDP are phosphate groups. The computer analyses obtained from PM3 molecular modeling confirmed that the GDP molecule is attacked initially by one of the F - ions from AlF3. It gets near the phosphate moiety of the phosphate group and pulls it back from GDP (the O-P bond makes itself longer). Next, the remaining part of the AlF 2+ attacks the oxygen atom connecting the phosphate groups in GDP and causes breaking of the P-O bond. The two-stage nature of the reaction was confirmed by calculations con-cerning the length of the bonds, total energy E, and the molecular heat of formation. AlF3 can attack GDP in the space of the first and second phosphate rest, although according to our calculations, the outer one has priority. The mechanism presented clarifies the interactions between the inorganic AlF3 complex and the biologically important GDP nucleotide.


J Gen Virol 2002 Dec;83(Pt 12):3055-3066
Role of G protein and protein kinase signalling in influenza virus budding in MDCK cells.
Hui EK, Nayak DP.
Department of Microbiology, Immunology and Molecular Genetics, Jonsson Comprehensive Cancer Center (JCCC), UCLA School of Medicine, Los Angeles, CA 90095-1747, USA.

Recently, we have shown that influenza virus budding in MDCK cells is regulated by metabolic inhibitors of ATP and ATP analogues (Hui & Nayak, Virology 290, 329-341, 2001 ). In this report, we demonstrate that G protein signalling stimulators such as sodium fluoride, aluminium fluoride, compound 48/80 and mastoparan stimulated the budding and release of influenza virus. In contrast, G protein signalling blockers such as suramin and NF023 inhibited virus budding. Furthermore, in filter-grown lysophosphatidylcholine-permeabilized virus-infected MDCK cells, membrane-impermeable GTP analogues, such as guanosine 5'-O-(3-thiotriphosphate) or 5'-guanylylimidodiphosphate caused an increase in virus budding, which could be competitively inhibited by adding an excess of GTP. These results suggest that the G protein is involved in the regulation of influenza virus budding. We also determined the role of different protein kinases in influenza virus budding. We observed that specific inhibitors or activators of protein kinase A (H-89 and 8-bromoadenosine 3',5'-cyclic monophosphate) or of protein kinase C (bisindolylmaleimide I and Ro-32-0432) or of phosphatidylinositol 3-kinase (LY294002 and wortmannin) did not affect influenza virus budding. However, the casein kinase 2 (CK2) inhibitor 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole decreased virus budding. We further observed an increase in the CK2 activity during the replication cycle of influenza virus, although Western blot analysis did not reveal any increase in the amount of CK2 protein in virus-infected cells. Also, in digitonin-permeabilized MDCK cells, the introduction of CK2 substrate peptides caused a down-regulation of virus budding. These results suggest that CK2 activity also regulates influenza virus budding.

Biol Chem 2000 Feb;381(2):145-53
  • As cited and abstracted in Fluoride 2000; 33(2):96-97
    Two different mechanisms for activation of cyclic PIP synthase: by a G protein or by protein tyrosine phosphorylation
    Wasner HK, Gebel M, Hucken S, Schaefer M, Kincses M
    Diabetes-Forschungsinstitut, Dusseldorf, Germany

The biosynthesis of the functional, endogenous cyclic AMP antagonist, prostaglandylinositol cyclic phosphate (cyclic PIP) is performed by the plasma membrane-bound enzyme cyclic PIP synthase, which combines prostaglandin E (PGE) and activated inositol phosphate (n-IP) to cyclic PIP. The Km values of the enzyme for the substrates PGE and n-IP are in the micromolar range. The plasma membrane-bound synthase is activated by fluoride, by the stable GTP analog GMP-PNP, by protamine or biguanide, by noradrenaline, and by insulin. The activation by protamine or biguanide and fluoride (10 mM) is additive, which may indicate the presence of two different types of enzyme, comparable to phospholipase Cbeta and phospholipase Cgamma.

Plasma membrane-bound cyclic PIP synthase is inhibited by the protein tyrosine kinase inhibitor tyrphostin B46 with an IC50 of 1.7 microM. However, the solubilized and gel-filtrated enzyme is no longer inhibited by tyrphostin, indicating that the activity of cyclic PIP synthase is connected with the activity of a membrane-bound protein tyrosine kinase. Cyclic PIP synthase activity of freshly prepared plasma membranes is unstable. Upon freezing and rethawing of liver plasma membranes, this instability is increased about 2-fold. Protein tyrosine phosphatase inhibitors [vanadate, fluoride (50-100 mM)] stabilize the enzyme activity, but protease inhibitors do not, indicating that inactivation of the enzyme is connected with protein tyrosine dephosphorylation. Cyclic PIP synthase is present in all tissues tested, like brain, heart, intestine, kidney, liver, lung, skeletal muscle, spleen, and testis. Apart from liver, cyclic PIP synthase activity in most tissues is rather low, but it can be increased up to 5-fold when protein tyrosine phosphatase inhibitors like vanadate are present in the homogenization buffer. Preincubation of cyclic PIP synthase of liver plasma membranes with the tyrosine kinase src kinase causes a 2-fold increase of cyclic PIP synthase activity, though this is certainly not the physiological role played by src kinase in intact cells. The data indicate that cyclic PIP synthase can be activated by two separate mechanisms: by a G protein or by protein tyrosine phos-phorylation.

J Pharmacol Exp Ther 2000 Feb;292(2):761-8
  • As cited and abstracted in Fluoride 2000; 33(2):97-98
    Predominant contribution of the G protein-mediated mechanism to NaF-induced vascular contractions in diabetic rats: association with an increased level of G(qalpha) expression
    Hattori Y, Matsuda N, Sato A, Watanuki S, Tomioka H, Kawasaki H, Kanno M
    Department of Pharmacology, Hokkaido University School of Medicine, Sapporo, Japan. yhattori@med.hokudai.ac.jp

The purpose of this study was to determine the mechanism responsible for alterations in NaF-induced contractions of blood vessels from streptozotocin-induced diabetic rats. In the presence of AlCl3, NaF (7.5 mM) produced significantly greater contractions in diabetic aorta and mesenteric artery compared with age-matched controls. Pretreatment with 1 microM nifedipine eliminated the enhanced contractile responses of diabetic vessels to NaF, resulting in no difference in the magnitude of NaF-induced contractions between control and diabetic vessels. In the presence of 100 microM deferoxamine, an Al 3+ chela-tor, NaF-induced contractions of diabetic vessels were markedly attenuated, whereas only the responses to lower concentrations of NaF were reduced in control vessels. No significant difference was found in the peak amplitude of transient contractions induced by 10 microM cyclopiazonic acid between control and diabetic vessels. The addition of 10 microM okadaic acid produced attenuated contractions in diabetic vessels. These findings indicate no involvement of the inhibitory effects of NaF on endoplasmic reticular Ca 2+ -pump ATPase and protein phosphatases in the genesis of the enhanced responsive-ness of diabetic vessels to NaF. Western blot analysis showed a 2.5-fold in-crease in the expression of G(qalpha) in diabetic aortic membranes. In contrast, the G(ialpha) level was modestly decreased and the G(salpha) and G(betagamma) levels were unchanged in diabetes. The present results suggest that enhanced vascular contractions to NaF in diabetes is attributed predominantly to a G protein-mediated Ca 2+ channel acti-vation that results from markedly increased G(qalpha) expression in vascular tissues under this pathological state.


Int J Dev Neurosci 1999 Jul;17(4):357-67
Fluoride-induced depletion of polyphosphoinositides in rat brain cortical slices: a rationale for the inhibitory effects on phospholipase C.
Sarri E, Claro E.
Departament de Bioquimica i de Biologia Molecular, Facultat de Medicina, Universitat Autonoma de Barcelona, Spain.

Fluoride, which is used commonly as a pharmacological tool to activate phosphoinositide-phospholipase C coupled to the heterotrymeric Gq/11 proteins, inhibited the phosphorylation of phosphatidylinositol (PtdIns) to polyphosphoinositides (PtdIns4P and PtdIns4,5P2) in membranes from rat brain cortex. Fluoride enhanced basal production of 3H-inositol phosphates in membranes prepared from brain cortical slices that had been prelabeled with [3H]inositol, but inhibited the stimulation elicited by carbachol in the presence of GTPgammaS. However in both cases fluoride depleted [3H]PtdIns4P content by 95%. The inhibitory effects of fluoride on the release of 3H-inositol phosphates in slices were not apparent in a pulse [3H]inositol-labeling strategy, but became dramatic in a continuous labeling protocol, particularly at long incubation times. Prelabeling slices with [3H]inositol in the presence of fluoride precluded polyphosphoinositide labeling, and eliminated phospholipase C responsiveness to carbachol under normal or depolarizing conditions, and to the calcium ionophore ionomycin. The lack of response of 3H-polyphosphoinositide-depleted slices to phospholipase C stimuli was not due to fluoride poisoning, unaccessibility of the [3H]inositol label to phospholipase C or desensitization of Gq/11, as the effect of carbachol and GTPgammaS was restored, in the presence of ATP, in membranes prepared from slices that had been labeled in the presence of fluoride. In conclusion, our data show that fluoride, at a concentration similar to that used to stimulate directly Gq/11-coupled phospholipase C, effectively blocks the synthesis of phospholipase C substrates from PtdIns.


Brain Res Mol Brain Res 1998 Jan;53(1-2):196-205
Glutathione depletion exacerbates impairment by oxidative stress of phosphoinositide hydrolysis, AP-1, and NF-kappaB activation by cholinergic stimulation.
Li X, Song L, Jope RS.
Department of Psychiatry and Behavioral Neurobiology University of Alabama at Birmingham, Birmingham, AL 35294-0017, USA.

Oxidative stress appears to contribute to neuronal dysfunction associated with Alzheimer's disease and other CNS neurodegenerative disorders. This investigation examined if oxidative stress might contribute to impairments in cholinergic receptor-linked signaling systems and if intracellular glutathione levels modulated responses to oxidative stress. To do this the activation of the AP-1 and NF-kappaB transcription factors and of the phosphoinositide second-messenger system was measured in human neuroblastoma SH-SY5Y cells after exposure to the oxidants H2O2 or diamide, with or without prior depletion of cellular glutathione. H2O2 concentration-dependently inhibited carbachol-stimulated AP-1 activation and this inhibition was potentiated in glutathione-depleted cells. Carbachol-stimulated NF-kappaB activation was unaffected by H2O2 unless glutathione was depleted, in which case there was a H2O2 concentration-dependent inhibition. Glutathione depletion also potentiated the inhibition by H2O2 of carbachol- or G-protein (NaF)-stimulated phosphoinositide hydrolysis, whereas phospholipase C activated by the calcium ionophore ionomycin was not inhibited. The thiol-oxidizing agent diamide also inhibited phosphoinositide hydrolysis stimulated by carbachol or NaF, and glutathione depletion potentiated the diamide concentration-dependent inhibition. Unlike H2O2, diamide also inhibited ionomycin-stimulated phosphoinositide hydrolysis. Activation of both AP-1 and NF-kappaB stimulated by carbachol was inhibited by diamide, and glutathione depletion potentiated the inhibitory effects of diamide. Thus, diamide inhibited a wider range of signaling processes than did H2O2, but glutathione depletion increased the susceptibility of phosphoinositide hydrolysis and of transcription factor activation to inhibition by both H2O2 and diamide. These results demonstrate that the vulnerability of signaling systems to oxidative stress is influenced by intracellular glutathione levels, indicating that cell-selective susceptibility to inhibition of signal transduction systems by oxidative stress can arise from cellular variations in antioxidant capacity.


Prog Neurobiol 1996 Oct;50(2-3):255-73
Phosphoinositide signaling in human brain.
Pacheco MA, Jope RS.
Department of Psychiatry and Behavioral Neurobiology, University of Alabama at Birmingham 35294-0017, USA.

The phosphoinositide signal transduction system constitutes one of the primary means for intercellular communication in the central nervous system, but only recently has this system been studied in human brain. Although some investigations have studied phosphoinositide signaling in slices from biopsied human brain, due to the limited access to such material a greater number of studies have utilized membranes prepared from postmortem human brain. With membranes exposed to exogenous labeled phosphoinositides, activation of phospholipase C with calcium, with G-proteins stimulated by GTP gamma S or NaF, or with several receptor agonists, have demonstrated that all of the components of the phosphoinositide system are retained in human brain membranes and are responsive to appropriate stimuli. Investigators have begun to examine the effects of neurological (Alzheimer's disease, epilepsy, Parkinson's disease) and psychiatric (schizophrenia, major depression, bipolar affective disorder) diseases on the activity of the phosphoinositide system. Alzheimer's disease has been studied to the greatest extent and a severe deficit in phosphoinositide signaling has been identified in most studies. In addition, brain regionally selective deficits in G-protein function associated with phosphoinositide signaling have been reported in subjects with major depression or with bipolar affective disorder, and in the latter an ameliorative effect of the therapeutic drug lithium was identified. Although significant progress has been achieved in studying the phosphoinositide system in human brain, many issues remaining to be addressed are discussed in this review. With carefully controlled studies, it appears that much will be learned in the near future about the phosphoinositide signal transduction system in human brain and the effects of a variety of disorders on its function.


Brain Res 1996 Jun 3;723(1-2):37-45
Alterations in phosphoinositide signaling and G-protein levels in depressed suicide brain.
Pacheco MA, Stockmeier C, Meltzer HY, Overholser JC, Dilley GE, Jope RS.
Department of Psychiatry and Behavioral Neurobiology, University of Alabama at Birmingham 35294-0017, USA.

The function of the phosphoinositide signal transduction system and the levels of heterotrimeric G-protein alpha-subunits were examined in postmortem prefrontal cortex regions (8/9) and region (10) from suicide victims with major depression and matched control subjects without psychiatric illness. The hydrolysis of [3H]phosphatidylinositol (PI) stimulated by phospholipase C, GTP-gamma-S, NaF, and neurotransmitter receptor agonists was measured in membrane preparations from both groups. Phospholipase C-beta activity was similar in depressed suicide and control subjects in the two regions of prefrontal cortex. In prefrontal cortex (10), but not in (8/9), the GTP-gamma-S concentration-dependent stimulation of [3H]PI hydrolysis was significantly lower (30%) in the depressed suicide group compared to the control group. Receptor-coupled, G-protein-mediated [3H]PI hydrolysis induced with carbachol, histamine, trans-1-aminocyclopentyl-1, 3-dicarboxylic acid (ACPD, a glutamatergic metabotropic receptor agonist), serotonin, or 2-methylthio-adenosine triphosphate (2mATP, a purinergic receptor agonist) in the presence of GTP-gamma-S stimulated equivalent responses in the two groups of subjects in each brain region. In prefrontal cortex (10) there was a 68% increase in the level of the 45 kDa subtype of G alpha s and in prefrontal cortex (8/9) there was a significant decrease (21%) in the level of G alpha i2 in the depressed suicide group compared to the control group. Levels of other heterotrimeric G-protein alpha-subunits (G alpha q/11, G alpha i1, and G alpha o) were not different in depressed suicide and control subjects in either brain region. Moreover, there were no differences in the levels of phospholipase C-beta or protein kinase C-alpha in the two groups of subjects in either brain region examined. These results demonstrate that in the prefrontal cortex of suicide victims with major depression compared to normal control subjects there is a region-specific alteration of G-protein-induced activation of the phosphoinositide signal transduction system and in the levels of G-protein alpha-subunits involved in cyclic AMP synthesis. These findings provide direct evidence in human brain that these two important signal transduction systems are altered in suicide subjects with major depression.


J Neurosci 1996 Oct 1;16(19):5914-22
Cholinergic stimulation of AP-1 and NF kappa B transcription factors is differentially sensitive to oxidative stress in SH-SY5Y neuroblastoma: relationship to phosphoinositide hydrolysis.
Li X, Song L, Jope RS.
Department of Psychiatry and Behavioral Neurobiology, University of Alabama at Birmingham 35294-0017, USA.

Oxidative stress appears to contribute to neuronal dysfunction in a number of neurodegenerative conditions, notably including Alzheimer's disease, in which cholinergic receptor-linked signal transduction activity is severely impaired. To test whether oxidative stress could contribute to deficits in cholinergic signaling, responses to carbachol were measured in human neuroblastoma SH-SY5Y cells exposed to H2O2. DNA binding activities of two transcription factors that are respondent to oxidative conditions, AP-1 and NF kappa B, were measured in nuclear extracts. H2O2 and carbachol individually induced dose- and time-dependent increases in AP-1 and NF kappa B. In contrast, when given together, H2O2 concentration dependently (30-300 microM) inhibited the increase after carbachol in AP-1. Carbachol's stimulation of NF kappa B was not inhibited except with a high concentration (300 microM) of H2O2, which was associated with impaired activation of protein kinase C. Lower concentrations of H2O2 (30-300 microM) inhibited carbachol-induced [3H]phosphoinositide hydrolysis, and this inhibition correlated (r = 0.95) with the inhibition of carbachol-induced AP-1. Activation [3H]phosphoinositide hydrolysis by the calcium ionophore ionomycin was unaffected by H2O2, indicating that phospholipase C and phosphoinositides were impervious to this treatment. In contrast, activation with NaF of G-proteins coupled to phospholipase C was concentration dependently inhibited by H2O2, indicating impaired G-protein function. These effects of H2O2 are similar to signaling impairments reported in Alzheimer's disease brain, which involve deficits in receptor- and G-protein-stimulated phosphoinositide hydrolysis, but not phospholipase C activity. Thus, these findings indicate that oxidative stress may contribute to impaired phosphoinositide signaling in neurological disorders in which oxidative stress occurs, and that oxidative stress can differentially influence transcription factors activated by cholinergic stimulation.


Biol Psychiatry 1996 Apr 15;39(8):697-702
Platelet phosphoinositide signaling system: an overstimulated pathway in depression.
Karege F, Bovier P, Rudolph W, Gaillard JM.
University of Geneva Institutes of Psychiatry, Division of Neuropsychiatry, Chene-Bourg (Geneva), Switzerland.

In order to test a possible depression-associated defect in signal transduction, platelet alpha 2-adrenergic-mediated phosphoinositide (PI) hydrolysis was measured, both in drug-free major depressed patients and in control healthy subjects. Results that express phospholipase C activity have shown significant increase in the metabolites of epinephrine-stimulated tritiated phosphatidyl-4,5-biphosphate (3H-PIP2) with respect to basal activity (saline-stimulated). Thrombin (2 units) and 10 mM sodium fluoride (NaF) also induced an increase in 3H-PIP2 metabolites. These increases were potentiated in drug-free depressed patients both in epinephrine-and thrombin-stimulated platelets. In contrast, sodium fluoride, which directly stimulates G protein without receptor interaction, did not differentiate between patients and controls with respect to PI hydrolysis. This result suggests a possible depression-associated defect in heterologous receptor-G protein interaction.

Publication Types: Clinical Trial


Biochem Biophys Res Commun 1996 Dec 13;229(2):630-4
Evidence for G-protein-dependent and G-protein-independent activation of phospholipase D in lymphocytes.
Cao YZ, Reddy PV, Sordillo LM, Hildenbrandt GR, Reddy CC.
Environmental Resources Research Institute, Pennsylvania State University, University Park 16802, USA.

Previously we reported that tumor-promoting phorbol esters stimulate phospholipase D (PLD) independent of protein kinase C (PKC) activation in bovine lymph node lymphocytes. (Cao et al., Biochem. Biophys. Res. Commun. 171, 955-962, 1990; 217, 908-915, 1995). In the present study, we examined the effects of prostagladins (PGs), E2, F2 alpha, D2, and H2 on PLD activity as measured by conversion of [1-14C] arachidonic acid-labeled phospholipids into phosphatidylethanol (PEt) in bovine lymph node lymphocytes. Prostaglandins stimulated the formation of PEt at an optimal concentration of 10 microM with relative stimulatory effect on the order of PGE2 > PGF2 alpha > PGH2 > PGD2. The PGE2-stimulated formation of PEt was dose-dependent in the range of 0.1 to 10 microM and was not inhibited by PKC inhibitors staurosporine and K252a. When both PGE2 and 12-0-tetradecanoylphorbol-13-acetate (TPA) were included, their effect on the PLD activation was additive. Furthermore, NaF, a G-protein activator, stimulated the PEt formation. Interestingly, the stimulatory effects of PGE2 and NaF were not additive; however, the formation of PEt by NaF and TPA was additive. These results suggest that similar to TPA, PGs increase PLD activity independent of PKC and the stimulation by PGs and TPA in lymphocytes may involve both G-protein-dependent and G-protein-independent signaling pathways.


Microbios 1995;81(329):231-9
Effects of G-protein activator fluorides, protein kinase C activator phorbol ester and protein kinase inhibitor on insulin binding and hormonal imprinting of Tetrahymena.
Kovacs P, Csaba G.
Department of Biology, Semmelweis University of Medicine, Budapest, Hungary.

AlCl3, BeCl2 and NaF do not influence the insulin binding of Tetrahymena immediately after treatment, but 24 h later insulin binding is decreased or increased by NaF in a dose-dependent manner, AlCl3 barely influences the binding, and BeCl2 increases it. The effect of all the three fluorides is dose-dependent. While NaF and AlF4 decrease binding at low doses and increase the binding at higher doses, BeF3 increases the insulin binding enormously. NaF does not permit insulin imprinting to be developed, AlF4 inhibits or amplifies the imprinting in a dose-dependent manner, while BeF3 allows imprinting to develop. After 24 h the protein kinase C (PKC) activator phorbol ester (PMA) increases the insulin binding to a similar degree as does the insulin imprinting itself. There was only one dose of the three tested in which PMA inhibited the development of insulin imprinting, whereas the PKC inhibitor reduced insulin binding after 24 h, but could not inhibit insulin imprinting.


Mol Cell Biochem 1994 Nov 23;140(2):163-70
Alterations in G-proteins in congestive heart failure in cardiomyopathic (UM-X7.1) hamsters.
Sethi R, Bector N, Takeda N, Nagano M, Jasmin G, Dhalla NS.
Division of Cardiovascular Sciences, St. Boniface General Hospital Research Centre, Faculty of Medicine, University of Manitoba, Winnipeg, Canada.

In order to explain the attenuated sympathetic support during the development of heart failure, the status of beta-adrenergic mechanisms in the failing myocardium was assessed by employing cardiomyopathic hamsters (155-170 days old) at moderate degree of congestive heart failure. The norepinephrine turnover rate was increased but the norepinephrine content was decreased in cardiomyopathic hearts. The number and the affinity of beta receptors in the sarcolemmal preparations were not changed in these hearts at moderate stage of congestive heart failure. While the basal adenylyl cyclase activity was not altered in sarcolemma, the stimulation of enzyme activity by NaF, forskolin, Gpp(NH)p or epinephrine was depressed in hearts from these cardiomyopathic hamsters. Since G-proteins are involved in modifying the adenylyl cyclase activity, the functional and bioactivities as well as contents of both Gs and Gi proteins were determined in the cardiomyopathic heart sarcolemma. The functional stimulation of adenylyl cyclase by cholera toxin, which activates Gs proteins, was markedly depressed whereas that by Pertussis toxin, which inhibits Gi proteins, was markedly augmented in cardiomyopathic hearts. The cholera toxin and pertussis toxin catalyzed ADP-ribosylation was increased by 37 and 126%, respectively; this indicated increased bioactivities of both Gs and Gi proteins in experimental preparations. The immunoblot analysis suggested 74 and 124% increase in Gs and Gi contents in failing hearts, respectively. These results suggest that depressed adenylyl cyclase activation in cardiomyopathic hamsters may not only be due to increased content and bioactivity of Gi proteins but the functional uncoupling of Gs proteins from the adenylyl cyclase enzyme may also be involved at this stage of heart failure.


Toxicol Appl Pharmacol 1994 Dec;129(2):223-34
Beta-adrenergic receptor overexpression in the fetal rat: distribution, receptor subtypes, and coupling to adenylate cyclase activity via G-proteins.
Slotkin TA, Lau C, Seidler FJ.
Department of Pharmacology, Duke University Medical Center, Durham, North Carolina 27710.

Biogenic amines are hypothesized to play a role in the control of cell differentiation. We assessed the development of beta-adrenergic receptors and their linkage to adenylate cyclase activity in order to determine whether catecholaminergic cell signaling can take place early in development. On Gestational Day 12, beta-receptors were present in rat embryo in concentrations comparable to those in mature adrenergic target tissues; the concentrations climbed fivefold by Gestational Day 18. beta-Receptor expression was higher in liver than in heart and brain, as identified both by binding to isolated membrane preparations and by receptor autoradiography; nevertheless, receptor distribution was quite widespread, with labeling visible throughout the fetus. Receptor subtype selectivities (beta 2 in liver, beta 1 in heart, predominantly beta 2 in whole fetus) were already in place in early development, but receptor coupling to adenylate cyclase via G-proteins showed substantial developmental changes. Agonist-induced displacement of radioligand binding showed little or no GTP sensitivity on Gestational Day 12, suggesting relatively poor receptor linkage to Gs. In contrast, by Day 18, GTP produced a large shift in the agonist displacement curve. Receptor stimulation of adenylate cyclase by isoproterenol also showed a developmental spike by Gestational Day 18; the pattern for isoproterenol stimulation was distinct from the ontogeny of adenylate cyclase itself and from stimulation by forskolin-Mn2+ (which bypasses the need for receptors or G-proteins) or by fluoride (which activates G-proteins nonselectively). Thus, beta-receptors are highly expressed during fetal development and the receptors are readily capable of modulating intracellular cAMP production. Fetal catecholamines, which are produced and released by the adrenal medulla, extraadrenal chromaffin tissue, and cells that transiently express adrenergic phenotype, can thus have a direct impact on the differentiation of a wide variety of cells.


Biochem Mol Biol Int 1994 Nov;34(5):993-1001
Adenovirus infection of myocardial cells induces an enhanced sensitivity to beta-adrenergic agonists by increasing the concentration of the stimulatory G-protein.
Novotny J, Gustafson B, Kvapil P, Ransnas LA.
Wallenberg Laboratory for Cardiovascular Research, Gothenburg University, Sweden.

Neonatal rat cardiocytes were infected with a recombinant adenovirus type 5 containing the SV40 early promoter-Gsa fusion gene in order to evaluate the presumed role of the stimulatory G-protein (Gs) in hypertrophy of myocardial cells. In vitro infection of myocardial cells with the recombinant adenovirus induced a 79-fold increase in Gs alpha mRNA and a 5-fold increase in Gs alpha protein, which was accompanied by a pronounced cell hypertrophy but not cell proliferation. Interestingly, adenovirus-infected cells displayed features of cell hypertrophy, an increase in sodium fluoride-stimulatible membrane-bound activity of adenylyl cyclase, and an enhanced beta-adrenergic sensitivity irrespective of the presence or absence of the SV40 early promoter-Gs alpha fusion gene in the virus. While the recombinant adenovirus induced a 5-fold versus 3-fold increase for plain adenovirus in cellular Gs alpha, membrane-bound Gs alpha was increased about 2-fold in both instances, which can explain similar increase in the G-protein-modulated adenylyl cyclase activity determined in membranes derived from myocardial cells infected with both types of the virus. It is concluded that adenovirus infection per se can lead to overexpression of Gs alpha and myocardial hypertrophy and thus may be of importance in the pathogenesis of virus-induced cardiomyopathy.

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