From the Department of Biological Sciences, Wayne State University, Detroit, Michigan 48202
Received for publication, May 16, 2000, and in revised form, December 14, 2000
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ABSTRACT |
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Bipolar affective disorder
(manic-depressive illness) is a chronic, severe, debilitating illness
affecting 1-2% of the population. The Food and Drug
Administration-approved drugs lithium and valproate are not completely
effective in the treatment of this disorder, and the mechanisms
underlying their therapeutic effects have not been established. We are
employing genetic and molecular approaches to identify common targets
of lithium and valproate in the yeast Saccharomyces
cerevisiae. We show that both drugs affect molecular targets in
the inositol metabolic pathway. Lithium and valproate cause a decrease
in intracellular myo-inositol mass and an increase in
expression of both a structural (INO1) and a regulatory
(INO2) gene required for inositol biosynthesis. The
opi1 mutant, which exhibits constitutive expression of
INO1, is more resistant to inhibition of growth by lithium
but not by valproate, suggesting that valproate may inhibit the
Ino1p-catalyzed synthesis of inositol 1-phosphate. Consistent
with this possibility, growth in valproate leads to decreased synthesis
of inositol monophosphate. Thus, both lithium and valproate perturb
regulation of the inositol biosynthetic pathway, albeit via different
mechanisms. This is the first demonstration of increased expression of
genes in the inositol biosynthetic pathway by both lithium and
valproate. Because inositol is a key regulator of many
cellular processes, the effects of lithium and valproate on
inositol synthesis have far-reaching implications for predicting
genetic determinants of responsiveness and resistance to these agents.
Bipolar disorder, or manic-depressive illness, is a common
condition with a lifetime prevalence of 1-2% (1). It is characterized by recurring bouts of mania and depression, which have deleterious effects on career and interpersonal relationships. Approximately 15%
of those afflicted commit suicide, and mortality rates because of
physical disorders are also increased (2, 3). For decades, lithium has
been the most effective agent for the treatment of bipolar illness (4).
Despite the marked benefit that many patients obtain from lithium
therapy, 20-40% of patients fail to show a satisfactory antimanic
response to lithium, and many patients suffer significant morbidity
(5). More recently, the branched fatty acid valproate has been used for
treatment of bipolar disorder (6). Like lithium, it is not completely
effective, and the molecular mechanisms underlying its therapeutic
effects have not been elucidated. Lithium and valproate exert a variety
of biochemical effects, only some of which are likely to be related to
their therapeutic mechanisms of action. Identifying common targets of lithium and valproate is an approach that may more directly address the
therapeutic mechanisms underlying their efficacy (7-11).
The inositol depletion hypothesis proposes that lithium acts by
depletion of inositol from the brain. This is based on the observed
uncompetitive inhibition of inositol monophosphatases by lithium,
resulting in decreased inositol, an increase in inositol phosphates,
and subsequent down-regulation of the phosphoinositide cycle (12).
Because the brain obtains inositol primarily from phosphoinositide
turnover and de novo synthesis, it is highly sensitive to
perturbations of the phosphoinositide cycle. Although there is
considerable evidence that lithium affects the phosphoinositide second
messenger system (13-15), a connection between this effect and the
therapeutic mechanism of lithium has not been established. If inositol
depletion formed the basis for the therapeutic effect, then valproate
might be expected to deplete inositol, as well. Previous studies
indicated that valproate does not inhibit bovine brain (16) or yeast
inositol monophosphatase activity (17) and has a minimal effect on
receptor-mediated phosphoinositide turnover (18). In addition,
valproate does not lead to large accumulations of inositol mono- or
bisphosphates, as seen with lithium (19).
We wished to determine whether valproate and lithium affect similar
targets in the inositol metabolic pathway. The yeast
Saccharomyces cerevisiae is an excellent model in
which to address this question. Many of the genes that encode
components of the phosphoinositide pathway have been cloned in yeast,
and regulation of inositol metabolism in yeast is understood at a
molecular level (20, 21). Expression of the structural gene
INO1, coding for
Ins-1-P1 synthase, is
repressed in the presence of inositol. The products of the positive
transcriptional regulators INO2 and INO4 form a
heterodimer that leads to derepressed expression of INO1 in the absence of inositol. The negative regulator Opi1p is required for
repression of INO1 in the presence of inositol, and defects in this gene result in constitutive expression of INO1 and
an inositol excretion phenotype. The regulatory gene INO2 is
controlled in a similar manner, i.e. its
expression is increased in the absence and repressed in the presence of
inositol. Inositol is a key metabolic sensor, and inositol levels play
a major role not only in regulating inositol biosynthesis but also in
regulation of phospholipid biosynthesis and the glucose and unfolded
protein response pathways (21).
Because conservation of function has been demonstrated from yeast to
humans, an understanding of the molecular targets of lithium and
valproate in yeast may have far-reaching implications for understanding
the mechanisms of action of these drugs. In this study, we demonstrated
that both lithium and valproate affect common targets in the inositol
metabolic pathway and deplete inositol by different mechanisms.
Yeast Strains, Media, and Reagents--
The S. cerevisiae strains used in this study include SMY15
(derivative of D273-10B/A1, met6,
ura3-52, MATa), SH302 (derivative of PMY168,
his3
DEAE dextran, NAD, myo-inositol dehydrogenase,
myo-inositol, myo-inositol monophosphate,
resazurin, TEAB buffer, and valproic acid were purchased from
Sigma. Alkaline phosphatase and hexokinase were purchased from Roche
Molecular Biochemicals. AG1-X8 (200-400; formate form) was
purchased from Bio-Rad. Sep-Pak Accell Plus QMA cartridges were from
Waters Corp. (Milford, MA). Lithium chloride was obtained from Fisher.
Growth of Yeast Cells--
Liquid cultures supplemented with the
indicated concentrations of lithium, valproate, or inositol were
inoculated to an A550 of 0.07. To determine
lithium sensitivity on plates, cells from an overnight culture were
washed four times in water, cell number was determined by microscopic
counting using a hemocytometer, and the desired number of cells were
spotted on plates containing the drugs. All growth was monitored at
30 °C.
Measurement of Intracellular Lithium--
Cells were grown to
stationary phase in minimal synthetic medium in the presence of the
indicated concentrations of lithium. Cells were centrifuged at 4 °C
for 5 min and washed two times in osmotic buffer (20 mM
magnesium and appropriate sorbitol to achieve the same osmolarity as
media containing lithium). Cells were disrupted, and cytosolic
and vacuolar compartments were obtained by differential centrifugation
(22). Intracellular lithium was determined by atomic absorption
spectrophotometry and calculated by the method of Welihinda
et al. (23).
Measurement of Intracellular Inositol and Inositol
Monophosphate--
Cells were grown in minimal synthetic medium at
30 °C in the presence or absence of lithium or valproate. Aliquots
were harvested by centrifugation. Cells were washed three times and
resuspended in water (~1 ml/g cells), and glass beads were added to
~50% of the volume of the suspension. Each sample was vortexed for
10 min at 2-min intervals, alternating with 2-min incubations on ice.
The cell extracts were clarified by centrifugation for 2 min at
2,000 × g, and the supernatants were transferred to
Eppendorf tubes and centrifuged for 15 min at 14,000 × g. The supernatants were collected and frozen at Measurement of INO1 and INO2 Expression--
Cells were grown in
minimal synthetic medium in the presence of the indicated
concentrations of lithium, valproate, or inositol and harvested in the
late logarithmic phase of growth. Cells were disrupted with glass
beads. RNA was isolated by hot phenol extraction (26), fractionated on
an agarose gel, and transferred to nitrocellulose. The blots were
hybridized with 32P-labeled INO1 and
INO2 riboprobes, followed by a riboprobe for the
constitutively expressed ribosomal protein gene TCM1 to
normalize for total RNA. RNA probes for Northern analysis were
synthesized using the Gemini II core system from plasmids linearized
with a restriction enzyme as follows (plasmid, restriction enzyme, RNA
polymerase): pJH310, HindIII, T7 (INO1);
pGEM-INO2, SalI, T7 (INO2); pAB309,
EcoRI, SP6 (TCM1) (27). The results of Northern blot hybridization were visualized by autoradiography, and relative levels of INO1, INO2, and TCM1 were
quantitated by phosphorimaging.
Intracellular Concentration of Lithium--
We wished to determine
the effects of lithium and valproate in concentrations that were within
the therapeutic range achieved in patients (0.6-1.2 mM
lithium and 0.6 mM valproate). Because yeast is a
free-living unicellular organism, it is susceptible to toxic
concentrations of deleterious cations in the environment. Yeast cells
possess two mechanisms to counteract lithium toxicity, exclusion of
lithium from the cell (28), and compartmentalization of lithium within
the vacuole where it is unable to affect cell functions (29). As a
result of differences in these mechanisms, yeast strains differ widely
in their degrees of sensitivity to lithium. In two wild type strains
used in this study, SH302 and SMY15, 7.5 mM extracellular
lithium led to a cytosolic concentration of less than 1 mM
(Fig. 1). Although cytosolic and vacuolar
lithium concentrations increased with increased extracellular lithium, the majority of lithium internalized was sequestered to the vacuoles. In 100 mM extracellular lithium, the cytoplasmic
concentration in SMY15 was still only 2.6 mM, whereas the
cytoplasmic lithium in SH302 was almost 10 mM.
In contrast to lithium, there is no evidence to suggest that valproate
uptake is different in yeast and mammalian cells. Indeed, the effects
we observed with this drug on intracellular inositol and expression of
INO1 and INO2 were apparent with 0.6 mM valproate, the therapeutic dose, as discussed below.
Growth in lithium or valproate leads to decreased intracellular
inositol mass. Growth in the presence of lithium for several generations led to a decrease in inositol mass in both SH302 and SMY15
(Fig. 2). A greater decrease in response
to increasing lithium was observed in SH302 than in SMY15. This may
reflect the greater cytoplasmic lithium concentration in SH302 (Fig.
1). Although steady-state intracellular inositol did not appear to
decrease greatly in SMY15, this strain exhibited a transient decrease
in inositol mass, even in response to low concentrations of lithium. As
seen in Fig. 3, the addition of 15 mM (extracellular) lithium to mid-log phase cells resulted
in a 24% decrease in inositol mass by 6 h. At this time,
intracellular inositol levels began to increase until wild type levels
were restored (within 24 h). A decrease in inositol mass was also
seen during growth in the presence of valproate (Fig. 2). In contrast
to the effects of lithium, the valproate-induced decrease in inositol
was not significantly different in the two strains. These data indicate
that both lithium and valproate cause decreased intracellular
inositol.
Expression of INO1 and INO2 Is Increased during Growth in Lithium
or Valproate--
Transcription of the structural gene
INO1, as well as the positive transcriptional regulator
INO2, is increased when inositol is limiting (20).
Therefore, agents that cause a decrease in intracellular inositol would
be expected to increase expression of these genes. The effects of
lithium and valproate on expression of INO1 and
INO2 were analyzed in Northern blots. As shown in Fig.
4, INO1 expression in SH302 is
increased 5.6- and 13.5-fold in the presence of 7.5 and 15 mM lithium, respectively (Fig. 4A). The
increased expression of INO1 is attenuated during growth in the presence of inositol (Fig. 4C). Thus, cells grown in 40 µM inositol show only very slightly increased
INO1 expression in 7.5 mM lithium and less than
a 6-fold increase in expression in 15 mM lithium.
Growth in the presence of 0.1 and 0.6 mM valproate in the
absence of inositol resulted in a 5.2- and 34.7-fold increase in INO1 mRNA, respectively (Fig.
5A). In the presence of 40 µM inositol, even 0.6 mM valproate did not
cause an increase in INO1.
Expression of INO2 in cells grown without inositol is about
twice as high as in cells grown in 75 µM inositol (see
Fig. 4, B and D and Fig. 5D). Growth
in 7.5 mM lithium caused a 2.4-fold increase in
INO2 expression (Fig. 4B). Similarly, valproate
caused a 1.5- to 2.5-fold increase in INO2 expression (Fig.
5). INO1 expression in SMY15 is also increased in response
to lithium and valproate, although to a lesser extent than in SH302
(data not shown). In summary, lithium and valproate cause an increase
in expression of both INO1 and INO2, genes that
are derepressed in response to decreased intracellular inositol.
The opi1 Null Mutant Exhibits Increased Intracellular Inositol and
Increased Resistance to Lithium but Not Valproate--
Opi1p is a
negative regulator that represses transcription of INO1 in
the presence of inositol (20). Opi1 mutant cells express INO1 constitutively, leading to increased Ins-1-P synthase
and the inositol excretion phenotype (30). We observed that the opi1 null mutant has dramatically increased levels of
intracellular inositol (Fig.
6A). To determine whether the
opi1 mutant has altered sensitivity to lithium or valproate,
equal amounts of mutant and isogenic wild type cells were seeded on
plates containing high concentrations of drug. Interestingly, growth of
the opi1 mutant was less sensitive to lithium (Fig.
6B) but did not show altered sensitivity to valproate (data
not shown).
Inositol Monophosphate Levels Are Decreased during Growth in
Valproate and Increased during Growth in Lithium--
The inability of
the opi1 mutant to overcome valproate inhibition despite
higher levels of Ins-1-P synthase suggested that this reaction might be
inhibited by valproate. Inhibition of this reaction by valproate would
lead to decreased levels of Ins-1-P. Therefore, inositol monophosphate
levels were measured in wild type and opi1 mutant cells
grown in the presence or absence of valproate. As seen in Fig.
7, inositol monophosphate levels in the
wild type strain grown in 0.6 and 2.5 mM valproate were
reduced to 30 and 16%, respectively, of the control (growth in the
absence of drug). Levels of inositol monophosphate in the
opi1 mutant were about 10-fold higher than in the wild type
(consistent with the 10-fold increase in intracellular inositol
observed in the mutant; see Fig. 6). As observed with wild type cells,
growth of mutant cells in the presence of valproate also resulted in a
decrease in inositol monophosphate.
Because lithium inhibits the inositol monophosphatase, inositol
monophosphate levels are expected to increase during growth in the
presence of this drug. Fig. 8 depicts
inositol monophosphate levels in extracts of cells grown in the
presence of lithium. In extracts of lithium-grown cells of both wild
type and opi1 mutant, inositol monophosphate was
increased.
In this report, we show that both lithium and valproate have a
profound effect on inositol metabolism in the eukaryote S. cerevisiae. Both drugs, in therapeutically relevant
concentrations, cause a decrease in intracellular inositol mass and an
increase in expression of a structural (INO1) and a
regulatory (INO2) gene required for inositol synthesis. The
mechanism of inositol depletion by lithium is most likely by inhibition
of inositol monophosphatase, as previous studies have shown that
lithium inhibits yeast inositol monophosphatase activity and reduces
expression of the INM1 gene (17, 31). We propose that the
mechanism of inositol depletion by valproate is most likely via
inhibition of Ins-1-P synthase.
Because yeast strains vary widely in their sensitivities to cations
(32), we measured cytosolic lithium and intracellular inositol levels
in two different wild type strains, SH302 and SMY15. SH302, which had
higher cytosolic lithium than SMY15 (Fig. 1), exhibited a more
pronounced decrease in intracellular inositol (Fig. 2). Lithium caused
increased expression of INO1 and INO2 in both
strains (see Fig. 4 and data not shown). Although the magnitude of
increase in INO1 was greater in SH302, this is not due
simply to the differences in intracellular inositol. The increase in
INO1 expression was also greater in SH302 than in SMY15 in response to valproate, although the intracellular inositol was similar
in the two strains. One possibility is that the biosynthesis of
inositol is more efficient in SMY15 than SH302. This is consistent with
the observation that the dramatic increase in INO1
expression in the presence of 15 mM lithium did not restore
intracellular inositol levels in SH302 (Figs. 2 and 4). This could be
because of differences in levels of inositol monophosphatase in the two strains. An alternative possibility is that higher intracellular lithium in SH302 leads to greater inhibition of inositol
monophosphatase, leading to increased accumulation of the substrate
Ins-1-P. Because inhibition of the monophosphatase is uncompetitive, an
increase in the enzyme-substrate complex would lead to further inhibition.
We observed that 40 µM inositol completely reversed the
35-fold valproate-mediated increase in INO1 expression (Fig.
5) but only partly reverses a smaller lithium-induced increase in
INO1 (Fig. 4). Interestingly, both lithium and valproate
down-regulate the level of a sodium-dependent high affinity
myo-inositol transporter in astrocyte-like cells (33). The
effects of these drugs on inositol uptake and transport in yeast
have not been determined, although preliminary evidence suggests that
they do not cause decreased inositol
uptake.2
How do lithium and valproate decrease intracellular inositol? A likely
mechanism for the lithium-induced decrease is via inhibition of
inositol monophosphatase activity (17, 31, 34). We propose that
valproate leads to decreased inositol by inhibition of the Ins-1-P
synthase reaction. The experiments depicted in Figs. 7 and 8
demonstrate that inositol monophosphate levels are reduced by valproate
but not by lithium. Inhibition of this reaction by valproate is further
supported by the observation that the opi1 mutant does not
exhibit increased resistance to valproate, despite constitutive
expression of INO1 and increased levels of Ins-1-P synthase
(30). Previous findings that valproate does not inhibit inositol
monophosphatase or cause an accumulation of inositol phosphates (16,
19) have been cited as evidence against the inositol-depletion
hypothesis. The experiments shown in this report indicate that
valproate does indeed cause inositol depletion. However, the mechanism
of inositol depletion by valproate is not by inhibition of inositol
monophosphatase but most likely by inhibition of the rate-limiting step
in the de novo synthesis of inositol, i.e. the synthesis of Ins-1-P.
We speculate that, in addition to inhibition of de novo
synthesis of Ins-1-P, another possible mechanism for the
valproate-induced decrease in inositol is by down-regulating expression
or activity of protein kinase C. Although there is no evidence
for this in yeast, valproate does decrease mammalian protein kinase C
expression (8). Protein kinase C appears to be required for inositol
synthesis, as yeast protein kinase C mutants are inositol
auxotrophs.3 Therefore, a
valproate-mediated decrease in protein kinase C would result in
decreased intracellular inositol, reduced expression of INM1
(which is down-regulated at low inositol levels) (17), and thus a
further reduction in synthesis of inositol.
In summary, we have shown that therapeutically relevant concentrations
of both lithium and valproate cause inositol depletion and affect
expression of structural and regulatory genes in the inositol
biosynthetic pathway. The increase in INO2 is especially significant, because this transcriptional activator regulates expression of more than 20 genes, in addition to INO1 (20). These include genes for phospholipid synthesis, as well as genes with
no direct link to lipid synthesis. In addition to its role in
phosphoinositide signaling, inositol is a key metabolic sensor involved
in the regulation of numerous cellular processes including phospholipid
metabolism, the unfolded protein response, and the glucose response
(21). Therefore, perturbation of inositol metabolism by these drugs
will no doubt have far-reaching implications for cellular function and
for responsiveness and resistance to the drugs. Experiments to
characterize the effects of lithium and valproate on these genes are in progress.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
200, leu2
1,
trp1
63, ura3-52), and SH304 (same as SH302 except opi1::LEU2). Minimal
synthetic defined medium contained glucose (2% w/v), necessary amino
acids (histidine; 10 mg/l), leucine (60 mg/l), methionine (10 mg/l),
tryptophan (10 mg/l), and uracil (10 mg/ml), and the salts and vitamin
components of Difco Vitamin Free Yeast Base. Complex medium (YPD)
contained glucose (2% w/v), bacto-peptone (2% w/v), agarose (2%w/v),
and yeast extract (1% w/v).
80 °C.
The protein was quantified by the Bradford assay with bovine serum
albumin as a standard (24). Intracellular inositol mass per 100 µg of
protein was determined by the enzyme-coupled fluorescent assay of
Maslanski and Busa (25). To measure inositol monophosphate (per 1-4 mg of protein), cell extracts were prepared as described above. Inositol and inositol phosphates were resolved by Sep-Pak Accell Plus QMA cartridges, utilizing an Amersham Pharmacia Biotech variable
flow pump. The cartridges were prepared for inositol and inositol
monophosphate loading by attachment to 10-ml syringes and washed in
H2O and 1.0 M TEAB (flow rate less than 0.5 ml/min). Cell extracts were diluted in 10 ml of H2O and
applied to the syringe connected to the Sep-Pak Accell Plus QMA
cartridge. Inositol was eluted in 10 ml of H2O followed by
4 ml of 0.02 M TEAB, and inositol monophosphate was eluted
with 4 ml of 0.1 M TEAB at a flow rate less than 2 ml/min. Inositol monophosphate was dephosphorylated by incubation in
the presence of alkaline phosphatase for 2 h at 37 °C, and the
reaction was terminated by incubation at 100 °C for 3 min. Extracts
were incubated in hexokinase for 1 h at 37 °C, and the reaction
was terminated by incubation at 100 °C for 3 min. Inositol mass was
determined by the enzyme-coupled fluorescent assay of Maslanski and
Busa (25).
RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Intracellular lithium in S. cerevisiae. Wild type strains SH302 and SMY15 were
grown in inositol-free medium containing the indicated extracellular
concentration of lithium for 24 h. Cell extracts were prepared,
and the concentrations of lithium in cytosolic and vacuolar
compartments were determined as described under "Materials and
Methods."
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Fig. 2.
The effects of lithium and valproate on
intracellular inositol mass. Wild type strains SH302 and SMY15
were grown in inositol-free medium containing the indicated
extracellular concentration of lithium or valproate (VPA)
for 24 h. Cell extracts were prepared, and the concentrations of
myo-inositol per 100 µg of protein were determined by the
method of Maslanski and Busa (25) as described under "Materials and
Methods."
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Fig. 3.
The short term effect of lithium on
intracellular inositol mass in SMY15. SMY15 cells were grown in
inositol-free medium in the presence or absence of 15 mM
lithium. Aliquots were taken at intervals following the addition of
lithium, and the concentrations of myo-inositol per 100 µg
of protein were determined as described under "Materials and
Methods."
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Fig. 4.
Effect of lithium on expression of
INO1 and INO2. SH302
cells were grown in the presence of the indicated concentrations of
inositol and lithium. Total RNA was isolated, fractionated on a 1.0%
formaldehyde-agarose gel, transferred to nitrocellulose, and hybridized
with 32P-labeled TCM1 and INO1
(A and C) or INO2 (B and
D) riboprobes. 32P was quantified by
phosphorimaging analysis using ImageQuant software.
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Fig. 5.
Effect of valproate on expression of
INO1 and INO2. SH302 cells were
grown in the presence of the indicated concentrations of inositol and
valproate. Total RNA was isolated, fractionated on a 1.0%
formaldehyde-agarose gel, transferred to nitrocellulose, and hybridized
with 32P-labeled TCM1 and INO1
(A and C) or INO2 (B and
D) riboprobes. 32P was quantified by
phosphorimaging analysis using ImageQuant software.
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Fig. 6.
The opi1 mutant has
increased intracellular inositol and increased resistance to
lithium. A, cells from opi1 mutant and
isogenic wild type (WT) strains were grown in synthetic
inositol-free medium for 24 h. Intracellular inositol was
determined as described in the legend to Fig. 2. B, equal
amounts of opi1 mutant and isogenic wild type cells were
spotted on plates containing 0-600 mM lithium. Plates were
incubated at 30 °C for 5-6 days.
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Fig. 7.
Valproate causes a decrease in inositol
monophosphate. Wild type (WT) and opi1
mutant cells were grown in synthetic inositol-free medium in the
presence of the indicated concentrations of valproate (VPA).
Cell extracts were prepared, and inositol monophosphate levels were
measured per 2 mg of protein by the method of Maslanski and Busa (25)
as described under "Materials and Methods."
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Fig. 8.
Lithium causes an increase in inositol
monophosphate. Wild type (WT) and opi1
mutant cells were grown in synthetic inositol-free medium in the
presence of the indicated concentrations of lithium. Inositol
monophosphate levels were measured per 2 mg of protein.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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ACKNOWLEDGEMENTS |
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We thank John Lopes for helpful discussions and for providing the riboprobes and Husseini Manji for helpful discussions.
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FOOTNOTES |
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* This work was supported by National Institutes of Health Grant MH56220 (to M. L. G.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Contributed equally to this work.
§ To whom correspondence should be addressed. Tel.: 313-577-5202; Fax: 313-577-6891; E mail: MLGREEN@sun.science.wayne.edu.
Published, JBC Papers in Press, January 22, 2001, DOI 10.1074/jbc.M004179200
2 D. Ding, unpublished observations.
3 S. Ju and M. L. Greenberg, unpublished data.
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ABBREVIATIONS |
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The abbreviations used are: Ins-1-P, inositol 1-phosphate; TEAB, triethylammonium bicarbonate.
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REFERENCES |
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