1 Department of Basic Science and Oral Research, School of Dentistry, University of Colorado, Health Sciences Center, Denver, Colorado 80262; and 2 Department of Pharmacological Sciences, State University of New York at Stony Brook, Stony Brook, New York 11794
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ABSTRACT |
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We have previously shown that protein kinase C (PKC) suppresses
steroidogenesis in Y-1 adrenocortical cells. To ask
directly if the PKC isoform mediates this suppression, we have
developed Y-1 cell lines in which PKC
is expressed from a
tetracycline-regulated promoter. Induction of PKC
expression in
these cell lines results in decreased P450 cholesterol side-chain
cleavage enzyme (P450-SCC) activity as judged by the conversion of
hydroxycholesterol to pregnenolone. Transcription of a P450-SCC
promoter-luciferase construct is also reduced when PKC
expression is
increased. However, expression of PKC
has no effect on 8-bromo-cAMP
induction of steroidogenesis, indicating that these pathways function
independently to regulate steroidogenesis. To determine the
relationship between endogenous PKC activity and steroidogenesis, we
examined 12 Y-1 subclones that were isolated by limited dilution
cloning. In each of these subclones, steroid production correlates
inversely with total PKC activity and with the expression of PKC
but
not PKC
or PKC
. These studies define for the first time the role
of a specific PKC isoform (PKC
) in regulating steroidogenesis and P450-SCC activity in adrenocortical cells.
cholesterol side-chain cleavage; transcription; adenosine 3',5'-cyclic monophosphate regulation
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INTRODUCTION |
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PREVIOUS WORK FROM OUR laboratory and others suggests that the steady-state level of steroid hormone production in adrenocortical cells reflects a balance between stimulation by the cAMP-dependent protein kinase (PKA) pathway and suppression by the protein kinase C (PKC) pathway (11, 16, 17, 23, 24, 30, 36, 37, 39, 43). Activation of the PKA pathway by adrenocorticotropic hormone (ACTH) results in an acute increase in steroid output due to an increased availability of free cholesterol and a sustained increase due to increased expression of the steroid hydroxylase genes, such as P450 cholesterol side-chain cleavage (P450-SCC) (36, 41). In contrast, the primary effect of PKC on steroidogenesis appears to be suppressive. This is supported by two lines of evidence. First, in ovarian granulosa, testicular leydig, and adrenocortical cells, 12-O-tetradecanoylphorbol-13-acetate (TPA) suppresses the induction of steroidogenesis by trophic hormones and cAMP (16, 23, 24, 30). Second, studies using inhibitors of PKC show that PKC acts as a tonic suppressor of basal steroidogenesis in Y-1 adrenocortical cells and MA-10 leydig cells (22, 37, 39).
We have previously reported that treatment of Y-1 cells with inhibitors
of PKC results in a dose-dependent increase in steroid production (37).
This is accompanied by a comparable increase in the expression of mRNAs
for the steroid synthetic enzymes P450-SCC, P450-11-hydroxylase
(P450-11
-OH), and 3
-hydroxysteroid dehydrogenase (3
-HSD).
Furthermore, in Y-1 cells in which the human apolipoprotein E gene is
overexpressed, PKC
expression is increased, whereas steroid
hydroxylase gene expression and steroidogenesis are suppressed. Treatment of these cells with inhibitors of PKC results in recovery of
steroidogenesis and steroid hydroxylase gene expression (38, 39). These
results suggest that PKC may regulate basal steroidogenesis primarily
by determining the steady-state levels of expression of the steroid
hydroxylase genes.
The PKC isoform family consists of at least 11 members that differ in
their sensitivity to Ca2+ and in
their activation by lipid cofactors (32, 33). The subset of PKC
isoforms expressed varies with cell type. In Y-1 cells, we have
detected PKC, PKC
, and PKC
(Reyland and White, unpublished
data), whereas PKC
and PKC
are the only isoforms detected in rat
adrenal glomerulosa cells (31). To demonstrate definitively that PKC
regulates adrenal steroidogenesis, we have created Y-1 cell lines in
which the expression of PKC
is driven by a tetracycline
(tet)-controlled promoter (14). When PKC
expression is induced in
these cell lines, their ability to synthesize steroids is impaired.
These studies thus identify a specific PKC isoform, PKC
, as an
important regulator of steroidogenesis in adrenocortical cells.
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MATERIALS AND METHODS |
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Generation of Tetracycline-Regulated Y1PKC Cell
Lines
Cell culture. The Y-1 adrenal cell line was obtained from American Type Culture Collection. Cells were maintained in Ham's F-10 medium supplemented with penicillin (100 U/ml), streptomycin (100 µg/ml), 2 mM L-glutamine, 12.5% (vol/vol) heat-inactivated horse serum, and 2.5% (vol/vol) heat-inactivated FCS ("complete medium"). Tet and hygromycin B were obtained from Calbiochem. All additional cell culture reagents were obtained from GIBCO.
Plasmid constructs.
pUHD15-1, pUHD10-3, and pUHD13-3 were a generous gift of H. Bujard
(14). The expression vector, pUHD/PKC, was made by cloning the mouse
PKC
cDNA from pLTRPKC
(26) into the
EcoR I site of pUHD10-3, which places
it under the control of the tet-regulated promoter.
Transfection.
Generation of stable lines using the tet-regulated system requires two
rounds of transfection. First, Y-1 cells were cotransfected with
pUHD15-1, which encodes the tet transactivator
(tTA) gene, and pSV2neo, which
encodes resistance to G418 sulfate, at a ratio of 9:1, by calcium
phosphate-mediated gene transfer essentially as described (37). Cell
clones were selected in complete medium containing 200 µg/ml G418
sulfate (Geneticin, GIBCO) and screened for expression of the
tTA gene by transient transfection of
a tet-responsive promoter linked to a luciferase reporter gene. Using
this approach, we selected four clonal Y-1 cell lines that express the
transfected tTA gene. One of these,
Y1UHD/7, was then secondarily transfected with pUHD/PKC together
with pCMV hygromycin at a ratio of 9:1. To make control cell lines,
Y1UHD/7 cells were transfected with the empty pUHD10-3 vector together
with pCMV hygromycin. Hygromycin-resistant cell clones (Y1PKC
or
Y1Con cells) were selected in 200 µg/ml hygromycin B (active form). Tet (2 µg/ml) was included during selection to suppress expression of
PKC
. The Y1PKC
and Y1Con cell lines were maintained in complete medium containing 100 µg/ml each of G418 sulfate and hygromycin B and
2 µg/ml tet. For most experiments, cells were treated as follows. On
day 0, the medium was replaced with
complete medium containing either 0 or 0.2 µg/ml (or 2 µg/ml in
some cases) tet. The medium was replaced with fresh medium of the same
composition on days 2 and
4, and cells were typically used on
day 6.
Analysis of Steroid Production
We discovered after the subclones were made that the Y1UHD/7 clone selected for the second stage of transfection lacks expression of the 3PKC Activity in Cell Homogenates
DEAE-cellulose-purified whole cell, cytosol, and particulate fraction homogenates were prepared as previously described (39). PKC activity in vitro was assayed as described using a kit purchased from BRL-Life Technologies (39). Cell protein was determined by the Bradford method using a kit purchased from Bio-Rad (8).Immunoblot Analysis of PKC Isoforms
Detection of PKC protein was done as previously described (39). DEAE-cellulose-purified cell homogenates (50 µg for PKCTransient Expression From the P450-SCC Promoter
To make the plasmid P450-SCCluc, the mouse 1.5-kb P450-SCC promoter from p-1500P450-SCChGH (40) was PCR amplified and subcloned into the Bgl II, Sal I sites of pXP2 (34) upstream of the luciferase reporter gene. Transient transfections were done as previously described using the calcium phosphate method (37). The DNA-calcium phosphate mixture was left on overnight. The following morning, the cells were washed with PBS and the medium was replaced with complete medium with or without tet. Cells were harvested for luciferase, protein, andNorthern Blot Analysis
RNA was harvested using RNA Stat-60 (Tel Test) and prepared according to the manufacturer's directions. Ten micrograms of total RNA were separated on a 1.2% agarose gel containing 2.2 M formaldehyde, transferred to Nytran (Schleicher & Schuell), cross-linked with ultraviolet light (Stratalinker), and hybridized to the indicated cDNA probe. cDNA probes were prepared by random priming in the presence of [ ![]() |
RESULTS |
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Regulation of PKC Expression From a
Tetracycline-Responsive Promoter
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With the use of the tet-regulated expression system, it should be
possible to modulate expression of the transfected gene by titrating
the amount of tet included in the culture medium. Figure
3A shows
that the level of PKC protein expression in PKC4/12 cells increases
as the tet concentration in the culture medium decreases. In the
presence of 2.0 or 0.2 µg/ml tet, very little PKC
expression is
detected, indicating nearly complete suppression of the transfected
gene. However, as the tet concentration is decreased, expression of
PKC
protein is increased. When PKC
protein abundance was
quantified by densitometry, PKC
protein was shown to increase 4-fold
when cells were maintained in 0.02 µg/ml tet, 11-fold in 0.002 µg/ml tet, 23-fold in 0.0002 µg/ml tet, and a maximum of 27-fold in
the absence of tet.
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The kinetics of PKC induction following withdrawal of tet from
PKC4/12 cells is shown in Fig. 3B.
PKC
protein is maximally induced 4 days after withdrawal of tet, and
expression is stable at least through day
10. Measurements of PKC activity in vitro indicate that
PKC activity increases as early as 24 h, is maximal by 4 days, and
remains increased at least through day
14 (data not shown).
We have previously shown that, in Y-1 cells, a fraction of the
endogenous PKC pool is in an activated form and that this pool functions to tonically suppress steroidogenesis (37). However, increased PKC protein expression in the Y1PKC
cell lines may not
necessarily correlate with an increase in the abundance of activated
enzyme in vivo, since activators such as diacylglycerol could be
limiting. In vivo activation of PKC typically involves translocation of
the cytosolic protein to the membrane, where the intracellular pool of
activated PKC is thought to be located (6). Thus measurement of the
relative distribution of PKC between the membrane and cytosol gives an
indirect measurement of the total amount of activated PKC in the cell.
To determine the subcellular distribution of PKC in the Y1PKC
cell
lines, we analyzed the distribution of PKC activity in the particulate
(membrane) and cytosolic cellular fractions. PKC activity in the
presence of tet presumably reflects primarily endogenous PKC activity.
As can be seen in Table 1, the distribution
of endogenous PKC between the particulate and cytosolic fraction is
characteristic for each cell line, with PKC4/5 cells having only 3%
associated with the particulate fraction, whereas in PKC4/3 and 4/12
cells 17% and 9%, respectively, of the total PKC activity are
associated with the particulate fraction. The range of PKC activity
distribution between these fractions is similar to what we have
observed previously for Y-1 cells (39). When Y1PKC
cells are
withdrawn from tet, the multiples of increase in PKC
activity is similar in both the membrane and cytosolic fractions for
all cell lines, indicating that the relative distribution of PKC
between the two fractions remains fairly constant. However, the total
amount of PKC activity in the particulate fraction increases 3- to
15-fold, suggesting that overexpression of PKC
results in a
proportional increase in the amount of activated PKC in the Y1PKC
cells.
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Increased Expression of PKC Reduces Basal
Steroidogenesis in the Y1PKC
Cell Lines
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To determine if there is a dose-response relationship between the level
of PKC expression and P450-SCC activity in the Y1PKC
cells, the
tet concentration was varied to provide a range of PKC
expression
levels, and pregnenolone synthesis was analyzed under each condition.
As shown in Fig. 5, pregnenolone production showed little dependence on the tet concentration in the Con1/2 line
but showed a clear concentration dependence in the PKC4/12 cell line.
This indicates that the magnitude of suppression of pregnenolone
synthesis in the PKC
cells is dependent on the level of PKC
protein that is expressed.
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PKC Expression Inhibits P450-SCC Transcription
in the Y1PKC
Cell Lines
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We have also examined the effect of PKC expression on endogenous
P450-SCC mRNA levels. Figure 7 shows a
Northern blot of P450-SCC mRNA expression in Con1/2, PKC4/5, and
PKC4/12 cells cultured in F-10 medium containing 1% Nutridoma SP or in
complete medium, with or without the addition of tet. The blot was
stripped and reprobed for expression of GAPDH mRNA as a control. Little or no consistent decrease in P450-SCC mRNA was seen on induction of
PKC
expression when cells were grown under either condition. In
fact, removing tet from the culture medium actually increased P450-SCC
and GAPDH mRNA expression to some extent in all the cell lines.
P450-SCC and GAPDH mRNA expression was quantified by PhosphorImager analysis. After normalization to GAPDH mRNA, the P450-SCC mRNA ratio,
without tet to with tet, is 0.9 for Con1/2 cells, 0.8 for PKC4/5 cells,
and 1.0 for PKC4/12 cells grown in complete medium. We have also
examined changes in P450-SCC protein abundance by Western blot and
likewise saw little or no suppression of P450-SCC protein expression
under conditions in which PKC
expression is induced (data not
shown). Our inability to detect changes in P450-SCC mRNA and protein
expression may be due to technical difficulties inherent in
quantitating such relatively small differences.
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PKC Expression Does Not Inhibit cAMP Induction
of Steroidogenesis in Y-1 Cells
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Elevated PKC Expression In Vivo Correlates With Decreased Steroidogenesis in Y-1 Cells
To determine if PKC negatively correlates with the ability to synthesize steroids in wild-type Y-1 cells, we examined the relationship between PKC expression and steroidogenesis in 12 Y-1 subclonal lines (Y1/DW cell lines) that were isolated from the Y-1 parent cell line by limited dilution cloning. PKC activity in vitro, which measures total cellular activatable PKC, varied up to 9-fold between the subclones, whereas steroid production varied up to 50-fold. Figure 9 plots steroid production against total PKC activity for the Y1/DW subclones and demonstrates that PKC activity is inversely correlated with the ability to synthesize steroids in these cell clones.
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Y-1 cells appear to express a limited set of PKC isoforms; screening in
our laboratory has detected PKC, PKC
, and PKC
expression (data
not shown). To determine if variations in PKC activity in the Y1/DW
cell lines correlate with changes in the expression of specific PKC
isoforms, we analyzed expression of PKC
, PKC
, and PKC
by
immunoblot. As seen in Fig.
10A,
PKC
expression varies dramatically between the subclones. The amount
of PKC
protein expressed in the individual clones is consistent with
the amount of total PKC activity (see legend to Fig. 10); i.e., the
clones that express the most PKC
protein (DW2, DW4, DW15, DW18, and DW25) have the highest level of PKC activity. Conversely, in clones DW6, DW11, DW14, DW19, DW24, and DW31, PKC
protein expression and
PKC activity are both low. Typical PKC activity measurements for the
parental Y-1 cell line range from 60 to 75 pmol · mg
1 · min
1
(39). Figure 10, B and
C, shows immunoblots in which the
Y1/DW subclones were analyzed for expression of PKC
and PKC
,
respectively. As seen in Fig. 10, B
and C, PKC
expression varied
somewhat between the Y1/DW subclones, but there is no apparent
correlation between PKC
expression and total PKC activity or steroid
production. In contrast, PKC
expression varied little between the
cell clones. These data demonstrate that, in vivo, increased PKC
expression correlates with a decreased capacity to synthesize steroids
and suggests that PKC
may function to regulate some aspect of
steroidogenesis in Y-1 cells.
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DISCUSSION |
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Previous reports, which have used chemical modulators to manipulate PKC
activity, suggest that PKC functions as a negative regulator of
steroidogenesis in many cell types (16, 22-24, 30, 37). However,
to date, direct proof of a role for PKC in the regulation of
steroidogenesis and identification of the functional PKC isoform has
been lacking. To address these questions, we have generated novel Y-1
cell lines in which the expression and activity of a specific PKC
isoform (PKC) can be acutely regulated in the absence of exogenous
inhibitors or activators. The use of a regulatable expression system is
particularly desirable in this context, since there is significant
variation between Y-1 clonal cell lines in PKC isoform expression and
basal steroidogenesis. Using this approach, we demonstrate directly
that PKC functions as a suppressor of steroidogenesis and identify
PKC
as the isoform that mediates this suppression.
The PKC isoform family consists of at least 11 members that differ in
their sensitivity to Ca2+ and in
their activation by lipid cofactors (32, 33). Recent studies have
linked extracellular signals in a variety of cell types to the
activation of specific PKC isoforms, e.g., tumor necrosis factor-
and PKC
activation (29), platelet-derived growth factor and PKC
,
PKC
and PKC
activation (28), and nerve growth factor and PKC
activation (15). In addition, in cotransfection experiments, different
PKC isoforms vary in their ability to transactivate TPA-responsive
genes.
The role of PKC in regulating steroidogenic cell functions has been
studied primarily using the PKC activator, TPA. Studies with TPA
suggest that activation of PKC suppresses cAMP-mediated induction of
steroidogenesis and steroid hydroxylase gene expression in
adrenocortical cells (10, 11). Previous work in our laboratory has
extended observations gleaned from studying TPA activation of PKC by
utilizing specific in vivo inhibitors of PKC to investigate the role of
the constitutively activated PKC pool in regulating steroidogenesis in
adrenocortical cells (37). The Y-1 cell line is appropriate to address
this question because Y-1 cells have an appreciable level of basal
steroidogenesis in the absence of exogenous activators of PKA. Our
results showed that treatment of Y-1 cells with either staurosporine or
calphostin C increases both basal steroid production and the level of
expression of mRNAs for P450-SCC, as well as other steroid synthetic
enzymes more distal in the pathway (37). Induction of steroid
production was accompanied by increased P450-SCC transcription as
monitored in transient transfection assays (37). On the basis of these experiments, we hypothesized that a constitutively active pool of PKC
functions to tonically regulate steroidogenesis in Y-1 cells (37). Our
current results show that inducible expression of the PKC isoform
results in an increase in the constitutive pool of active
(membrane-associated) PKC in Y-1 cells, which presumably mediates
suppression of steroidogenesis.
It has long been observed that treatment of steroidogenic cells with
phorbol ester (TPA) blocks stimulation of steroidogenesis by
physiological regulators of cAMP, such as luteinizing
hormone and ACTH (16, 23, 24, 30). In the H295 human
adrenal cell line, activation of PKC via the physiological regulator,
ANG II, can likewise suppress forskolin-induced expression of P450-SCC mRNA (7). However, previous work from our laboratory has shown that the
PKC and PKA pathways function as reciprocal but independent regulators
of steroidogenesis and P450-SCC mRNA expression in Y-1 cells (37).
These studies showed that PKC functions as a suppressor of
steroidogenesis and P450-SCC mRNA expression even in the absence of a
functional PKA pathway, indicating that PKC does not function by
suppressing cAMP induction of steroidogenesis (37). Our current studies
support this hypothesis, since expression of PKC has no effect on
either the acute or chronic stimulation of steroidogenesis by 8-BrcAMP.
Because PKC
expression has no effect on the acute response to cAMP,
it is unlikely that PKC
regulates cholesterol mobilization to the
mitochondria, but of course this does not rule out the possibility that
another PKC isoform may regulate these processes.
The data reported here indicate that increased expression of PKC in
Y-1 cells suppresses transcription from the mouse P450-SCC promoter.
However, decreased transcription of P450-SCC does not appear to account for the entire reduction in P450-SCC activity we
observe. Although our data are consistent with the conclusion that
PKC
decreases P450-SCC expression, it is also possible that P450-SCC
activity is controlled at additional points such as protein turnover or
posttranslational modification of the P450-SCC enzyme. In this regard,
Vilgrain et al. (42) have reported that PKC can phosphorylate P450-SCC
in vitro.
Thus one mechanism by which PKC decreases P450-SCC activity is
transcriptional repression. Although there are many examples of genes
that are positively regulated by PKC, suppression of transcription by
PKC has been reported in only a few instances, such as regulation of
the phosphoenolpyruvate carboxykinase
and gonadotropin-releasing hormone promoters (9, 35). How PKC and other
regulators suppress transcription is not known. Attempts to map the
TPA-responsive region of the P450-SCC promoter have yielded conflicting
results. Lauber et al. (19) have shown that cAMP response elements in
the bovine P450-SCC promoter are sufficient for TPA-mediated repression
of cAMP-induced expression, indicating that the cAMP and PKC regulatory
regions of the P450-SCC promoter colocalize. Moore et al. (27) have
reported that regions of the human P450-P450-SCC promoter responsible
for TPA regulation map to the same area as elements required for basal
regulation. Thus PKC may regulate expression of P450-SCC by
modification of a protein required for basal regulation. In this
regard, Leers-Sucheta et al. (20) have reported that TPA activates
transcription of the 3
-HSD type II promoter via steroidogenic factor
1, a transcription factor that is also required for basal expression of
type II 3
-HSD. Another possibility is that PKC inhibits
transcription via the transactivator complex, activator protein-1
(AP-1), which mediates gene induction by TPA (3, 4). There
is an AP-1 consensus site in the mouse P450-SCC promoter at
319.
AP-1 is composed of homo- and heterodimers of members of the Fos and
Jun families, and overexpression of c-Jun has been shown to inhibit
expression of several tissue-specific genes including creatine kinase
(21), atrial natriuretric factor (25), and prolactin (13), suggesting that c-Jun may inhibit tissue-specific gene expression. The
availability of Y-1 cell lines in which intracellular PKC
activity
can be acutely altered will facilitate studies on the mechanism of PKC suppression of P450-SCC transcription.
In this report, using a system in which we can regulate PKC
expression, we provide direct evidence that PKC
suppresses P450-SCC activity and basal steroidogenesis in Y-1 cells. Furthermore, we show
that in the Y-1 subclonal cell lines isolated from the Y-1 parent cell
line (Y1/DW cell lines), the ability to synthesize steroid hormones is
inversely correlated with expression of PKC
. It is of interest that
the degree of suppression of steroidogenesis in the Y-1 subclones is
larger than would be predicted from the Y1PKC
cell lines. This
suggests that PKC
may function in Y-1 cells to regulate other
aspects of steroidogenesis in addition to P450-SCC activity. Because in
the Y1/DW cell lines we measure steroidogenesis by production of
20
-dihydroprogesterone and its derivatives, the greater decrease in
steroid synthesis in these cells may reflect additional changes in
enzymatic steps downstream of P450-SCC. Finally, it should be noted
that PKC clearly has other regulatory functions in adrenal cells that
are distinct from its role as a chronic regulator of steroidogenesis.
For example, in adrenocortical glomerulosa cells, binding of ANG II to
the ANG II receptor activates PKC and induces mineralocortical hormone synthesis (5). Regulation of steroidogenesis by ACTH has also been
proposed to involve activation of PKC (11). These divergent functions
may be mediated by distinct PKC isoforms and may reflect the multiple
levels of activation and regulation of these signaling pathways by
phospholipids and other metabolic regulators.
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ACKNOWLEDGEMENTS |
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The expert technical assistance of Lisa Huerta, Penny Strockbine, and Wen Zhu is gratefully acknowledged. We are also indebted to Drs. S. Anderson, R. Evans, S. Diamond, and A. Gutierrez-Hartman for helpful discussions throughout the course of this work.
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FOOTNOTES |
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This work was supported by a National American Heart Grant-in-Aid and a Colorado American Heart Affiliate Grant-in-Aid to M. E. Reyland and by National Heart, Lung, and Blood Institute Grant HL-32868 to D. L. Williams.
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. §1734 solely to indicate this fact.
Address for reprint requests: M. E. Reyland, Dept. of Basic Sciences and Oral Research, Box C286, Univ. of Colorado Health Science Center, Denver, CO 80262.
Received 12 January 1998; accepted in final form 27 May 1998.
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