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Department of Biological Chemistry (S.J.M., D.N.C., A.F.S.) and the Mental Health Research Institute (A.F.S.), The University of Michigan, Ann Arbor, Michigan 48109
Address all correspondence and requests for reprints to: Audrey F. Seasholtz, Ph.D., Mental Health Research Institute, 205 Zina Pitcher Place, Ann Arbor, Michigan 48109-0720. E-mail: aseashol{at}umich.edu
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Introduction |
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CRH, a 41-amino acid peptide, is the primary neuroendocrine mediator of the mammalian stress response (1). CRH is released from the hypothalamus, in response to stressful stimuli, and is carried by the hypophysial portal system to anterior pituitary corticotrophs. There, it binds to the type 1 CRH-receptor (CRH-R1), a seven-transmembrane spanning Gs-protein coupled receptor. CRH receptor activation increases POMC transcription and release of ACTH from the pituitary. ACTH is transported via the blood to the adrenal gland, where it signals for release of glucocorticoids.
The CRH-binding protein (CRH-BP) is a 37-kDa secreted protein that has been colocalized with CRH at several sites in the brain and in the anterior pituitary corticotrophs with CRH-R1 (2). CRH-BP binds to CRH with an affinity higher than that of the CRH receptor [Ki = 0.4 and 1.7 nM, respectively; (3, 4, 5)] and has been shown to block the ACTH-releasing activity of CRH in primary pituitary cultures (5) and in cultured mouse anterior pituitary cells (4). Other in vitro studies have begun to elucidate the molecular mechanisms involved in regulation of CRH-BP gene expression. Transfection experiments with CRH-BP reporter constructs demonstrate positive regulation of the CRH-BP promoter by cAMP and by CRH in cells expressing CRH-R1 (6). Experiments with primary rat astrocyte cultures have also demonstrated positive regulation of endogenous CRH-BP gene expression by cAMP and increased secretion of CRH-BP in response to forskolin and/or phorbol myristate acetate (7, 8).
Together, these in vitro results suggest that CRH-BP plays an inhibitory role in modulation of CRH activity and that the expression and secretion of CRH-BP is regulated by second messengers. However, very little is known about the in vivo role of CRH-BP. Transgenic mice overexpressing CRH-BP in the pituitary provide clues as to the function of CRH-BP in the HPA axis. These mice have elevated levels of both CRH and arginine vasopressin (AVP) messenger RNA (mRNA) in the hypothalamus, while showing normal ACTH and corticosterone levels and a normal stress response. These data suggest that the mice have compensated for the excess CRH-BP by increasing hypothalamic CRH and AVP expression to maintain homeostasis within the HPA axis (9). To gain further insight into the role of CRH-BP in the mammalian stress response, we have examined the effect of acute restraint stress and adrenalectomy on regulation of CRH-BP gene expression in the rat pituitary.
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Materials and Methods |
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Total RNA was prepared from each individual pituitary by homogenization with a Polytron (Kinematica, Inc., Johnson City, TN) in Trizol reagent (Gibco BRL, Bethesda, MD) following manufacturer’s instructions. Generally, 1 ml Trizol was used for each tissue, and 0.2 ml chloroform was added after homogenization. Microfuge tubes were tightly capped and shaken. After 5 min, the samples were centrifuged at 9500 rpm for 10 min. The aqueous phase was transferred to a new Eppendorf tube. RNA was precipitated with 0.5 ml isopropanol at -20 C for 1 h. Samples were centrifuged at 9500 rpm for 15 min, and resulting RNA pellets were resuspended in 100 µl sterile water and reprecipitated with 2 µl of 5 M NaCl and 300 µl of 100% ethanol overnight. Samples were then centrifuged as above and resuspended in 20 µl sterile ribonuclease-free (RNase-free) water for RNase protection experiments. Each individual pituitary yielded approximately 20 µg total RNA.
Corticosterone and ACTH assays
Plasma corticosterone was measured using a corticosterone RIA
kit (Diagnostic Products Corporation, Los Angeles, CA),
carried out following manufacturer’s instructions, using 50 µl of
serum. Plasma ACTH levels were determined by an ACTH immunoassay kit
(Nichols Diagnostic Corp., San Juan Capistrano, CA), performed as
instructed, except that 50 or 100 µl of serum was used instead of 200
µl. The zero calibrator was used to dilute samples to 200 µl. All
samples were assayed in duplicate.
Complementary RNA (cRNA) probe synthesis
To determine levels of rat CRH-BP (rCRH-BP) in each
individual pituitary, a 565-bp PstI fragment
[nucleotides, 707-1271; (6)] from the rCRH-BP complementary
DNA (cDNA) was inserted into the PstI site in the
multiple-cloning site of the pGEM-3Z vector (Promega
Corp., Madison, WI) and linearized with ScaI to
prepare template for riboprobe synthesis. This template produced a
252-base labeled riboprobe which protected 232 bases of the rCRH-BP
mRNA. The rat cyclophilin transcript was used as an internal positive
control in all experiments. A 670-bp fragment of this cDNA was cloned
into pSP65 vector (Promega Corp.) and linearized with
AluI to prepare template for riboprobe synthesis. The
resulting cyclophilin probe (rCyc) was 114 bases and protected 85 bases
of rat cyclophilin mRNA.
One microgram of linearized DNA template was used in an in vitro transcription reaction containing 1x transcription buffer (Epicentre Technologies, Madison, WI); 10 mM dithiothreitol; 0.5 mM each of ATP, GTP, and CTP; 10 µM UTP; and 1 µl RNAsin (28 U/µl, Promega Corp.). 32P-uridine 5'-triphosphate (>3000 µCi/mmol, ICN Pharmaceuticals, Inc., Costa Mesa, CA) was added to each transcription reaction as follows: for rCRH-BP, 10 µl (100 µCi) was dried down and resuspended in 5 µl sterile water; for rCyc, 3 µCi was added. Total reaction vol was 20 µl. For each reaction, 10 U of SP6 RNA polymerase (Epicentre) was added, and the reaction was incubated for 90 min at 40 C. One microliter of RNase-free deoxyribonuclease (10U/µl, Promega Corp.) was then added to each transcription reaction. After incubation for 15 min at 37 C, 30 µl of loading dye (95% formamide, 1 mM EDTA, 0.1% xylene cyanol, 0.1% bromophenol blue) was added to terminate the reaction. Samples were heated 5 min at 70 C. The resulting riboprobe was gel purified by electrophoresis on a short 6% polyacrylamide/7 M urea gel. The RNA was eluted from the gel for 4–5 h in probe elution buffer (0.5 M NH4OAc, 1 mM EDTA, 0.2% SDS).
RNase protection assay
For solution hybridization, 10 µg (or half of each pituitary
sample) of RNA was precipitated with 500,000 cpm of rCRH-BP and 100,000
cpm of rCyc cRNA probes using 0.5 M NH4OAc and
3 vol 100% ethanol. Samples were microfuged for 15 min, ethanol was
removed with a pasteur pipet, and samples were resuspended in 30 µl
of hybridization buffer [80% formamide, 100 mM sodium
citrate (pH 6.4), 300 mM sodium acetate (pH 6.4), and 1
mM EDTA). The samples were then heated to 90 C for 5 min
and immediately hybridized overnight (12–16 h) submerged in a 40 C
water bath.
For RNase digestions, RNase A/RNase T1 mixture (250 U/ml RNase A; 10,000 U/ml RNase T1) was diluted 1:1000 in Digestion Buffer Bx (RPA II kit; Ambion, Inc., Austin, TX), and 200 µl of diluted RNase A/RNase T1 was added to each sample tube. Tubes were incubated at 37 C for 30 min, 300 µl of RNase Inactivation Buffer Dx (Ambion, Inc.) and 200 µl 100% ethanol was added to terminate reactions, and samples were precipitated for 2 h at -20 C. RNA hybrids were pelleted in a microcentrifuge for 15 min, and ethanol was removed carefully. Pellets were resuspended in 6 µl of gel loading buffer (95% formamide, 0.025% xylene cyanol, 0.025% bromophenol blue, 0.5 mM EDTA, 0.025% SDS), then heated to 70 C for 5 min and loaded on a sequencing-length 6% polyacrylamide/7 M urea gel. Gels were run at 60 watts for 2 h, dried, and exposed. The RNase protection assays were repeated with the second half of the pituitary RNA sample, with consistent results. Control assays with varying amounts of RNA showed the RNase protection assays to be linear over 5–20 µg of sample RNA using the amounts (100,000 cpm of rCyc and 500,000 cpm of rCRH-BP) of radioactive cRNA probes prepared as described above. Yeast transfer RNA (10 µg) was used as the negative control for nonspecific hybridization, and rat cerebral cortex RNA (10 µg) was used as the positive control for the RNase protection assays.
Data analysis
Gels were exposed to a PhosphorImager screen (Molecular
Dynamics, Inc., Sunnyvale, CA) and Biomax MS film and
intensifying screens (Eastman Kodak Co., Rochester, NY).
PhosphorImager analysis was carried out using ImageQuant software
(Molecular Dynamics, Inc.). RNase protection assays with
the cyclophilin cRNA probe generate two protected fragments of 84 and
85 bases, most likely caused by breathing of the hybrid; both bands
were included in the quantitation. CRH-BP hybrid densities were divided
by cyclophilin hybrid densities to normalize for variations in RNA
concentrations and recovery. The normalized values are presented as
CRH-BP/cyclophilin mRNA ratios, and results are expressed as the
mean ± SEM. The significance of differences was
assessed by ANOVA with Fisher’s least significant difference
(LSD) post hoc analysis for multiple-time point data
(see Figs. 1
and 2) and Student’s unpaired t test (see
Figs. 3 and 4) using Statview software (Abacus Concepts, Berkeley,
CA).
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Results |
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Figure 1
depicts the levels of
corticosterone and ACTH during the time course of the experiment. In
response to restraint stress, both corticosterone and ACTH increase
significantly at 30 min, to 411 ± 27 ng/ml (mean ±
SEM) and 375 ± 73 pg/ml, respectively. Both
corticosterone and ACTH return to basal levels by 120 min after the
initiation of the restraint period (corticosterone = 28 ± 8
ng/ml; ACTH = 41 ± 9 pg/ml).
The effect of this restraint stress on pituitary CRH-BP mRNA levels is
shown in Fig. 2. Total RNA was isolated
from each pituitary and used to perform RNase protection assays. Figure 2A shows the sizes of the CRH-BP (252 bases) and cyclophilin (internal
control, 114 bases) cRNA probes (lane 1) and their corresponding
protected bands (lane 3; 232 and 85 bases, respectively). Figure 2B
shows a representative RNase protection assay, with the CRH-BP
protected hybrids in the top panels and the cyclophilin
internal control hybrids in the bottom panels. Each lane is
representative of RNA isolated from a single rat pituitary, and the
intensity of the CRH-BP hybrid was normalized to the intensity of the
cyclophilin hybrid. This representative RNase protection assay was
scanned from a 7-day exposure of Biomax-MS film, which was necessary
for publication-quality visualization of the control CRH-BP hybrids.
The actual quantitation of the RNase protection assay was completed
using a Phosphor-Imager and was determined to be in the linear
range of the sensitivity of the instrument.
The quantitation of the RNase protection assay is shown in Fig. 2C.
Immediately after the restraint stress (30-min time point), the
normalized levels of CRH-BP mRNA are 2.1 times higher than basal levels
(control level = 0.69 ± 0.10; 30-min level = 1.45
± 0.28; P = 0.09, compared with control). At the
60-min time point, steady-state CRH-BP mRNA levels are 3.1 times basal
levels (60-min level = 2.16 ± 0.62; P =
0.009, compared with control). At the 120-min time point, CRH-BP mRNA
levels are 2.1 times basal level (120-min level = 1.48 ±
0.43; P = 0.08, compared with control). Although the
30-, 60-, and 120-min time points range between 2–3 times control,
with a visual peak at 60 min, these values are not statistically
distinct from each other. By 240 min after the start of the restraint
period, the levels of CRH-BP steady-state mRNA have returned to basal
levels (240-min level = 0.49 ± 0.12). These results
demonstrate that acute restraint stress significantly increases
pituitary CRH-BP steady-state mRNA levels. Each pituitary RNA sample
was analyzed in two separate RNase protection assays, with consistent
results. The data presented in Fig. 2B is a representative sampling of
the results.
Adrenalectomy decreases pituitary CRH-BP mRNA levels
To examine the effect of glucocorticoids on pituitary CRH-BP mRNA
levels in vivo, we compared steady-state CRH-BP mRNA levels
in unstressed Adx and Sham-Adx rats. Trunk blood was collected for
corticosterone and ACTH assays, and pituitaries were isolated as
described above. The experiment was carried out 7 days post
adrenalectomy. Corticosterone assays demonstrated that the Sham-Adx
animals were unstressed (11.9 ± 0.8 ng/ml) and that the Adx
animals had undetectable levels of corticosterone, as expected. ACTH
assays confirmed that basal ACTH levels were significantly higher in
Adx rats, compared with Sham-Adx rats [1643 ± 167 pg/ml (n
= 6) vs. 23.6 ± 6.7 pg/ml (n = 10),
P < 0.0001; mean ± SEM], as
previously demonstrated by other groups (12).
Figure 3 depicts the effect of adrenalectomy on
CRH-BP steady-state mRNA levels. Figure 3A shows an RNase protection
assay with representative CRH-BP hybrids in the top panels
and the corresponding cyclophilin internal control hybrids in the
bottom panels. As in Fig. 2, each lane is representative of
the RNA isolated from an individual pituitary. To properly visualize
the low intensity of the Adx CRH-BP hybrids, the rest of the hybrids
are overexposed. Quantitations were performed within the sensitivity of
the PhosphorImager.
Figure 3B represents the quantitation of the RNase protection assay
shown in Fig. 3A. The data are presented as CRH-BP/cyclophilin mRNA
ratios. The Adx CRH-BP levels are approximately 8% of the Sham-Adx
control levels (Sham-Adx = 0.28 ± 0.08; Adx =
0.023 ± 0.007; P = 0.01, n = 6),
demonstrating a significant decrease in CRH-BP steady-state mRNA levels
after adrenalectomy in unstressed animals.
Pituitary CRH-BP steady-state mRNA levels in Adx animals after
restraint stress
To examine the effect of acute stress and adrenalectomy on CRH-BP
steady-state mRNA levels, we performed the restraint stress protocol
described above with control, Sham-Adx, and Adx animals and killed
animals at 30-min and 60-min time points. Corticosterone and ACTH
assays confirmed an acute stress response. Corticosterone levels
increased, as expected, for control and Sham-Adx animals (data not
shown). ACTH levels increased for all animals at the 30-min time point
(Sham-Adx, from 23.6 ± 6.7 pg/ml (0 min, n = 10) to 392
± 116 pg/ml (30 min, n = 6), P = 0.0009; Adx,
from 1643 ± 167 pg/ml (0 min, n = 12) to 1998 ± 187
pg/ml (0 min, n = 6), P = 0.18). However, at 60
min after initiation of stress, ACTH levels in both Sham-Adx and
control animals began to return to basal levels, as expected; but ACTH
levels in Adx animals remained elevated (1993 ± 153 pg/ml, n
= 6), presumably because of lack of negative feedback by
glucocorticoids.
RNase protection analysis demonstrated that the stressed Adx
pituitaries had normalized steady-state CRH-BP mRNA levels that were
1.2 times control Adx CRH-BP levels (Fig. 4) at 30 min after initiation of acute
restraint stress (Adx = 0.023 ± 0.007 vs. Adx +
stress = 0.028 ± 0.006). Although these data suggest a small
increase in steady-state CRH-BP mRNA levels after restraint stress in
Adx animals, the statistical analysis shows that the change is not
significant. However, it should be noted that the very low level of
CRH-BP expression in Adx animals greatly increases the variability in
quantitation of the data. As stated above, the representative
autoradiograph has overexposed cyclophilin hybrids to allow
visualization of the corresponding, low-abundance Adx CRH-BP
levels.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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We examined the effect of stress and adrenalectomy on CRH-BP gene
expression using RNase protection assays. Because of the sensitivity of
these assays, we were able to quantitate CRH-BP steady-state mRNA
levels in individual pituitaries. This analysis provides a
determination of CRH-BP mRNA levels in each animal in a group and
demonstrates the variable increases of CRH-BP in individual animals
during the stress response. This variability was most apparent in mRNA
determinations from pituitaries with very low abundance of CRH-BP, such
as the mRNA from Adx rats (see Figs. 3 and 4). It should also be noted
that the nature of these RNase protection assays does not allow
distinction between increased CRH-BP gene transcription and increased
mRNA stability.
Changes in CRH, ACTH, and glucocorticoid secretion, as well as CRH and
POMC gene transcription, have been thoroughly studied in response to
acute stress, but our results are the first describing the effect of
acute restraint stress on CRH-BP. The CRH-BP steady-state mRNA levels
in the pituitary during the stress response increase to two to three
times basal levels (30 and 60 min after initiation of restraint,
respectively), remain elevated until 120 min, and return to basal
levels by 240 min (Fig. 2). During stress, intracellular stores of CRH
and other ACTH secretagogues are rapidly cosecreted into the
hypophysial portal circulation from terminals of parvocellular neurons
of the paraventricular nucleus (PVN) of the hypothalamus. In response
to the activation of pituitary CRH receptors, pituitary ACTH and
adrenal corticosterone are released, with maximal levels detected in
plasma by 30 min after initiation of restraint. Over this same time
course, the transcription of CRH in the PVN has been shown, by intronic
in situ hybridization analysis, to increase by 30 min, with
a rapid decrease to basal levels by 60 min (12). Steady-state CRH mRNA
levels increase slightly, but not dramatically, and remain elevated
during this time course (Ref. 12 ; and McClennen, unpublished
observations). POMC transcription is also increased rapidly in response
to stress (specifically by release of CRH peptide) and peaks at 15 min
(14, 15), but is so abundant in pituitary corticotrophs that the
steady-state mRNA levels do not seem to increase with stress (16).
These time course profiles suggest that both the immediate release of
CRH and the resultant increases in glucocorticoids may play important
roles in the observed rapid increase in pituitary steady-state CRH-BP
mRNA levels in response to acute restraint stress.
Glucocorticoids are the main homeostatic switch in the HPA axis and have previously been shown to down-regulate the stress response at many levels. Glucocorticoids have two modes of feedback: fast and slow (10). The fast-feedback mechanism decreases secretion of CRH and ACTH within minutes of a stress initiation. The delayed-feedback inhibition is responsible for changes in gene transcription involved in stress response. These changes in gene transcription include decreased CRH expression in the hypothalamus (11, 17) and decreased POMC (15) and CRH-R1 expression in the pituitary (18).
To begin to examine the glucocorticoid effect on CRH-BP gene
expression, we used Adx rats, which have no detectable levels of
glucocorticoids, to determine the levels of steady-state CRH-BP gene
expression. In the absence of any stressor, CRH-BP steady-state mRNA
levels in Adx rats decrease significantly, to approximately 8% of
Sham-Adx control levels (Fig. 3). This decrease suggests that
glucocorticoids positively regulate CRH-BP gene expression in the
normal pituitary and confirms that CRH-BP has a role in the mammalian
HPA axis during normal axis activity. This result also demonstrates a
different glucocorticoid-mediated regulation for CRH-BP than for CRH.
As mentioned above, CRH is negatively regulated at two levels by
glucocorticoids in the HPA axis. The fast-feedback inhibition decreases
secretion of CRH, whereas the delayed feedback inhibits CRH gene
transcription (19, 20). Our data suggest an additional role for
glucocorticoids in HPA axis regulation by rapidly increasing the
expression of CRH-BP, which can then bind and sequester free CRH
peptide, thus inhibiting its ACTH-releasing activity. Together, these
studies suggest both a rapid mechanism for down-regulation of CRH by
glucocorticoids and a more sustained response by increasing CRH-BP
levels and, therefore, CRH-BP:CRH protein-protein interactions.
After acute restraint stress, the levels of CRH-BP in Adx rats increase
to 122% of levels in unstressed Adx animals (Fig. 4). Although this
increase is small in comparison with the wild-type stress-induced
increase and does not reach statistical significance, it suggests that
CRH-BP mRNA levels may be increased by stress in the Adx animal.
Because there are no detectable glucocorticoids in this system, CRH may
play a role in this increase in CRH-BP steady-state mRNA levels. The
potential increase in CRH-BP gene expression by CRH would be consistent
with in vitro data, which show an increase in CRH-BP
reporter activity by CRH in transcription regulation studies with
transfected cells (6). However, interpretations from the Adx animal
model are complicated because Adx animals exhibit elevated CRH mRNA
levels in the PVN (21, 22) and decreased CRH-R1 mRNA and receptor
binding levels in the pituitary (18, 23, 24). Restraint stress
significantly increases CRH heteronuclear RNA levels in both Adx
and Sham-Adx animals (12) but does not alter the already reduced
pituitary CRH receptor binding capacity in Adx animals (24). The
decreased CRH receptor binding capacity in the pituitary may,
therefore, impair the pituitary response to elevated CRH levels in the
stressed Adx state. Therefore, it is difficult to clearly assess the
contribution of CRH in CRH-BP mRNA regulation using stressed and
unstressed Adx animals. Additional stress studies in Adx rats with
varying levels of glucocorticoid replacement or in CRH-deficient mice
(25) will allow us to more carefully elucidate the specific roles of
glucocorticoids and CRH on pituitary CRH-BP gene expression. It is
nonetheless clear from our data that glucocorticoids play an important
role in positive regulation of CRH-BP gene expression in both the
nonstressed state and in the stress response.
It is also possible that other hypothalamic or pituitary factors are affecting the levels of CRH-BP, in addition to glucocorticoids and CRH. One other possible regulator of pituitary CRH-BP gene expression is AVP. Although AVP is a weak ACTH secretagogue on its own, it potentiates CRH-mediated ACTH secretion (26, 27). AVP heteronuclear RNA is also elevated in the medial PVN as early as 30 min after restraint stress initiation (28). The immediate release of CRH and AVP, in response to stress, and the elevated expression of AVP in the medial PVN after acute stress suggest that there could be a relationship between AVP and pituitary CRH-BP gene expression that has not yet been determined. Another potential mediator is urocortin, a novel CRH-like neuropeptide (29). Recent data suggest that urocortin is expressed in the pituitary (30, 31), and it has been previously established that urocortin can bind CRH-BP with an affinity comparable with that of CRH (29, 32). Urocortin can also mediate ACTH release in anterior pituitary cultures and after iv injection in the unanesthetized rat (29). Although no clear in vivo role for urocortin has been established in the HPA axis, both of these observations suggest that it could have an effect on CRH-BP expression and activity.
Although individual components of the HPA axis have been extensively examined, the molecular control mechanisms of the mammalian endocrine stress response are not yet well defined. In general, glucocorticoids increase in response to stress. The corticosteroids then feed back on the HPA axis at a number of levels: 1) to quickly decrease secretion of CRH and ACTH; 2) to decrease CRH mRNA levels in the hypothalamus (17); 3) to decrease CRH-R1 mRNA in the pituitary (18); 4) to decrease POMC expression in the pituitary (15); and 5) to increase CRH-BP mRNA in the pituitary. These responses suggest that the overall effect is to sequester CRH from its receptors on target cells, decrease bioactive ACTH, and thus attenuate the stress response. The results in this paper present the first evidence that CRH-BP steady-state mRNA levels are significantly increased by stress and that glucocorticoids clearly play a significant role in the positive regulation in both unstressed and stressed states. This positive glucocorticoid regulation is most clearly shown as a dramatic decrease in CRH-BP steady-state mRNA levels after adrenalectomy. These results suggest that CRH-BP is directly involved in the mammalian stress response and may be important in HPA axis homeostasis. Additional studies on the impact of stress and/or glucocorticoid levels on CRH-BP protein expression and binding activity will further address the in vivo role of CRH-BP in HPA axis regulation.
There is extensive clinical relevance for defining a regulatory role for CRH-BP in the HPA axis. It is believed that hyperactivity of the HPA axis, and specifically hypersecretion of CRH, may play a role in a number of psychiatric disorders, including depression and anorexia (1). The data presented in this paper show that CRH-BP is involved in regulation of HPA axis homeostasis and thus could potentially be modulated in these disease states.
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Acknowledgments |
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Footnotes |
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2 D.N.C. was supported, in part, by National Institutes of
Health Genetics Training Grant T32-GM-07544.
Received May 12, 1998.
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) is
expressed in ng/ml, and ACTH (•) is expressed in pg/ml. Statistical
analysis consisted of ANOVA with Fisher’s LSD post
hoc analysis. **, P < 0.05
vs. all other time points for ACTH; #,
P < 0.05 vs. all other time points
for CORT. These results are representative of n = 8 (60- and
240-min time points), n = 15 or 16 (control, ACTH and CORT,
respectively), and n = 14 (30- and 120-min time points).
Error bars for 120- and 240-min points are insignificant
on this scale.
