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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
"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
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
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: http://www.boston.com/globe/search/stories/nobel/1994/1994l.html
See: graphics from the Nobel Committeee
for a more scientific understanding:
Discovery of G Proteins
Transduction in Cells
and Inactivation of the G Protein
Proteins and the Body
from EC: I've added this abstract to this section as
the authors concluded that flucythrinate "inhibits
Acaricide, Insecticide - CAS
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.
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,
• See also section
on PFOA and G-Proteins
Fluoride 2002; 35(4):244
XXVth ISFR Conference
and aluminum: messengers of false information
Charles Univ. Prague,
Faculty of Sciences, Dept. of Physiology and Developmental Biology,
Vinicna 7, 128 00 Prague 2, Czech Republic. E-mail: email@example.com
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
Crit Rev Oral Biol Med;14(2):100-14
The biochemistry and physiology of metallic fluoride: action,
mechanism, and implications.
Faculty of Dentistry, University of Manitoba, 780 Bannatyne
Avenue, Winnipeg R3E 0W2, MB, Canada; firstname.lastname@example.org
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
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.
Hum Exp Toxicol Mar;22(3):111-23
apoptosis in human epithelial lung cells (A549 cells): role
of different G protein-linked signal systems.
Refsnes M, Schwarze PE, Holme JA, Lag
Division of Environmental Medicine, Norwegian Institute of Public
Health, Geitmyrsvn. 75, PO Box 4404 Nydalen, N-0403 Oslo, Norway.
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.
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
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
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
XXVth ISFR Conference
between guanosine diphosphate (GDP) and aluminum
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
(3) Dept. of Physical Chemistry, Technical Univ., Szczecin,
For Correspondence: Machoy Z, Dept. of Biochemistry and Chemistry,
PAM, Al. Powstancow Wlkp. 72, 70-111 Szczecin, Poland. Email:
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
J Gen Virol 2002
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
Biol Chem 2000
- As cited and abstracted
in Fluoride 2000; 33(2):96-97
mechanisms for activation of cyclic PIP synthase: by a G
protein or by protein tyrosine
HK, Gebel M, Hucken S, Schaefer M, Kincses M
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
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
- As cited and abstracted
in Fluoride 2000; 33(2):97-98
contribution of the G protein-mediated
mechanism to NaF-induced vascular contractions in diabetic rats:
association with an increased level of G(qalpha) expression
Y, Matsuda N, Sato A, Watanuki S, Tomioka H, Kawasaki H, Kanno
Department of Pharmacology,
Hokkaido University School of Medicine, Sapporo, Japan. email@example.com
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
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
depletion exacerbates impairment by oxidative stress of phosphoinositide
hydrolysis, AP-1, and NF-kappaB activation by cholinergic stimulation.
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
signaling in human brain.
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
in phosphoinositide signaling and G-protein
levels in depressed suicide brain.
MA, Stockmeier C, Meltzer HY, Overholser JC, Dilley GE, Jope
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
stimulation of AP-1 and NF kappa B transcription factors is
differentially sensitive to oxidative stress in SH-SY5Y neuroblastoma:
relationship to phosphoinositide hydrolysis.
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
Platelet phosphoinositide signaling
system: an overstimulated pathway in depression.
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
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
of G-protein activator fluorides,
protein kinase C activator phorbol ester and protein kinase
inhibitor on insulin binding and hormonal imprinting of Tetrahymena.
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
Mol Cell Biochem
1994 Nov 23;140(2):163-70
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,
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.
Appl Pharmacol 1994 Dec;129(2):223-34
receptor overexpression in the fetal rat:
distribution, receptor subtypes, and coupling to adenylate cyclase
activity via G-proteins.
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
infection of myocardial cells induces an enhanced sensitivity
to beta-adrenergic agonists by increasing the concentration
of the stimulatory G-protein.
J, Gustafson B, Kvapil P, Ransnas LA.
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.