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Departments of Clinical Chemistry and Pharmacology, School of Medicine, University of Crete, Iraklion, GR-711 10, Crete, Greece
Address all correspondence and requests for reprints to: Andrew N. Margioris, Department of Clinical Chemistry, School of Medicine, University of Crete, Iraklion, GR-711 10, Crete, Greece. E-mail: andym{at}med.uch.gr
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Abstract |
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helical CRH; 4) exposure of PC12 cells to NGF (for 1 week) resulted in
their neuronal differentiation and the stimulation of their production
of CRH by 30 times and of dopamine by 10 times, compared with parallel
controls; this effect of NGF was abolished by helical CRH. In
conclusion, our data suggest that the production of CRH by PC12 cells
represents the preservation of a normal chromaffin cell characteristic
rather than a tumor-induced ectopic phenomenon. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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In a pilot experiment, we have found that CRH is present in normal rat
adrenal chromaffin cells and in the PC12 rat pheochromocytoma cell
line, a model for the study of chromaffin cell differentiation and
catecholamine production by pheochromocytomas (20, 21). The aim of the
present work was to test the hypothesis that the production of CRH by
PC12 cells represents the preservation of a normal chromaffin cell
characteristic and not a tumor-induced expression of a previously
unexpressed gene. To prove this hypothesis, we compared dispersed and
cultured normal rat chromaffin cells with PC12 cells in relation to CRH
secretion, regulation of its production, and response to exogenous CRH
and its antagonist. Specifically, we first measured the basal secretion
rate of IR-CRH by both types of chromaffin cells and that after
exposure to K+, nicotine, interleukin (IL)-1ß (7, 22, 23, 24, 25), and nerve growth factor (NGF). Subsequently, we characterized
the IR-CRH in these cells and their culture media by sieve
chromatography and RP-HPLC. Finally, we examined the effect of
exogenous CRH and of its antagonist, helical CRH (hCRH) on basal
and nicotine-induced epinephrine secretion from the chromaffin cells
and on basal, nicotine-, and NGF-induced dopamine secretion from the
PC12 cells. Our results show that CRH is released by both types of
cells, its secretion and synthesis seem to be similarly regulated, and
it exerts a significant and comparable regulatory effect on the
production of catecholamines by both types of chromaffin cells.
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Materials and Methods |
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Culture of PC12 rat pheochromocytoma cells
The PC12 cells were kindly provided by Dr. G. Guroff, Section on
Growth Factors, NICHD, NIH, Bethesda, MD, handled as previously
described (21). Briefly, PC12 cells were cultured on either flat-bottom
wells in 12-well plates of 4.5 cm2 surface area per well
(Costar Europe LTD) or on flasks of 75 cm2 surface area at
an initial concentration of 1 x 105 cells per
cm2. The cells were left to grow in DMEM (GIBCO-BRL Co.),
containing 10 mM L-glutamine, 1.5 mM HEPES, 100
U/ml penicillin, 0.1 mg/ml streptomycin, 7.5% horse serum, and 7.5%
FCS in an incubator (Forma Scientific) at 5% CO2 and 37 C.
Differentiation of PC12 to neuron-like cells followed exposure to 7S
NGF (25 ng/ml for 1 week). The 7S NGF was kindly provided by Dr. G.
Guroff, Section on Growth Factors, NICHD, NIH, Bethesda, MD.
CRH RIA
Peptides in culture media were concentrated by a C-18 reverse
phase column (Sep-Pak, Waters Associates, Milford, MA) after
acidification in 2 vol 0.1 N HCI and centrifuged at 1.5 x
103 rpm for 10 min. The supernatants were extracted by
activated Sep-Pak cartridges, washed with 20 ml 0.1 N HCI eluted with 3
ml acetonitrile 80%-0.01% HCI, then dried under vacuum (Speed-Vac).
The IR-CRH content of the Sep-Pak extracts was assayed by RIA using a
rabbit antiserum (Neosystem Labboratoire, Strasbourg, France) raised
against human/rat CRH; it does not cross-react with ovine CRH, human
ACTH, LHRH, or AVP. The sensitivity of the assay was 1 pg/tube. The
intraassay coefficient of variation was 4.4% and the interassay was
6.6%. Results are expressed as picograms of IR-CRH per mg of total
cellular protein determined on whole cellular homogenates by the
Bradford method (27) using BSA as standard (28).
Sieve chromatography
Pooled culture media and PC12 cell homogenates were acidified in
2 vol 0.1 N HCI, centrifuged at 1 x 105 rpm x
10 min and the supernatant extracted by activated Sep-Pak cartridges,
washed with 20 ml 0.1 N HCI, eluted with 3 ml acetonitrile 80%-0.01%
HCI, and dried under vacuum (Speed-Vac). The sample was reconstituted
in 0.5 ml 10% formic acid containing 0.5% defatted BSA and 6
M urea and chromatographed on Sephadex G-50 (0.9 x 60
cm, bed vol 40 ml, flow rate of 1.5 ml/h, 1-ml fractions collected).
Fractions were dried under vacuum and reconstituted for RIA. The G-50
column was calibrated with blue dextran and synthetic human CRH.
RP-HPLC
PC12 cells were homogenized in 5 vol 0.1% trifluoroacetic acid
(TFA), centrifuged at 10,000 rpm for 20 min, the supernatant dried
under vacuum, reconstituted in 30 µl 25% acetonitrile-0.1% TFA,
centrifuged again for 10 min and one third of its supernatant was
injected into an HPLC connected to a 0.2 x 30 cm C18
µBondapack column (Waters Associates). A linear gradient program was
used, employing a TFA-acetonitrile solvent system (see Fig. 3
). Solvent
A was composed of 20% acetonitrile-0.1% TFA and solvent B of 80%
acetonitrile-0.1% TFA. The flow rate was 1 ml/min. Samples of 1 ml
were collected. The retention time (Rt) of the IR-CRH
extracted from the PC12 cells was determined by RIA after evaporation
of the HPLC fractions and reconstitution in RIA medium. After the
sample runs, 20 ng diluted synthetic r-CRH and a completely oxidized
r-CRH by 5% H2O2 were run and their
Rt determined also by RIA.
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Statistical analysis
The concentration of the IR-CRH in the culture media was
normalized per total cellular protein of the resuspended and
homogenized cells at the end of each experiment. The secretion of
epinephrine and dopamine in the culture media was expressed as either
the mean in ng ± SEM of five or more experiments, per
well per mg of cell protein or as percent change compared with control
wells. Total cellular protein in each well was measured by the Bradford
Coomassie Brilliant Blue G250 method (Serva, GmbH & Co, Germany) using
BSA as standard (27, 28). For the statistical evaluation of our data,
we used ANOVA followed by two multiple comparison tests: the Fisher’s
least significant difference and the Newman-Keuls test. For the data
expressed as percentages of change over controls, we have used the
nonparametric Kruskal-Wallis test for several independent samples.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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depicts the basal concentration of
IR-CRH in the culture media over a period of 48 h. The
determination of IR-CRH was performed in plain test media after removal
of the horse and FCS-enriched culture media. The concentration of
IR-CRH in the media from early passage PC12 cells (5 and 6)
(lower curve) was lower compared with normal chromaffin
cells (upper curve). However, it increased with the
number of passage becoming, in late passages, higher than that of
chromaffin cells. Thus, PC12 cells of passage 17 had two to three times
more CRH compared with normal chromaffin cells (see Fig. 5), an
observation reproduced in multiple experiments. The significance of the
positive correlation between PC12 passage and their capacity to produce
CRH is unclear, but it may suggest that the capacity to produce CRH is
affected by the state of PC12 differentiation, a hypothesis also
supported by the NGF-derived data (see below). Figure 1 also shows that
replacement of the culture media stimulates the secretion of CRH, which
peaks between 6 and 12 h and subsequently either (a) declines
slightly reaching a stable plateaux (in the normal chromaffin cells),
or (b) it declines even further to low levels (in the PC12 cells). The
latter is a well-documented phenomenon, characteristic of several
neoplastic cells, including pheochromocytomas; it results from the
production of large quantities of proteases from the tumoral cells
(29). Incubation of similar concentrations of standard CRH in
PC12-conditioned media produced a similar curve. Based on these data,
for the subsequent experiments testing the effect of exogenous CRH, we
used aprotinin at 100 kU/ml to protect it from these proteases.
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depicts the effect of a depolarizing dose of
KCl on IR-CRH in the PC12 test media (n = 6). The peak CRH
response occurred at 20 min, and it was almost one order of magnitude
higher than that of parallel controls (from 18.6 ± 3 to 138
± 1, n = 12). Similar data were obtained from normal rat
chromaffin cells. On sieve chromatography, the apparent molecular
weight of IR-CRH in the test media was similar or identical to that of
synthetic r-CRH (Fig. 2, insert).
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depicts the RP-HPLC chromatographic profile of
IR-CRH present in PC12 cellular extracts. A gradient of acetonitrile
(CH3CN) was used (indicated by the broken
line) in the presence of 0.1 N TNF. The Rt of
IR-CRH from the PC12 extracts was identical to that of synthetic r-CRH.
On the graph, the first peak represents the oxidized
Met(O) CRH (this oxidation takes place during the extraction
procedure), whereas the second peak is that of the
putative peptide. RP-HPLC chromatography of pooled extracts from 24-h
PC12 culture media exhibited the same IR-CRH profile.
Figure 4 depicts the effect of nicotine on the release
of IR-CRH from normal chromaffin (upper panel) and PC12
cells (lower panel). Nicotine stimulated the release of
IR-CRH from both types of cells in a dose- and time-dependent manner.
The onset of this effect was early, starting within 30 min of the
application of nicotine. This finding suggests that, as expected, the
stimulatory effect of nicotine mainly involves secretion.
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depicts the effect of IL-1ß, at 40 ng/ml for 36
h, on the rate of IR-CRH accumulation in the test media of normal
chromaffin cells (light grey bars) and of PC12 cells
(white bars). The onset of the effect of IL-1ß
required at least 36 h of exposure, suggesting that it involved
mainly synthesis. Indeed, IL-1ß had no statistically significant
acute effects on CRH secretion from either type of chromaffin cell. It
should be noted that the one order of magnitude difference between the
basal (zero exogenous IL-1ß) concentration of IR-CRH in this
experiment (PC12 passage 17) compared with the one depicted in Fig. 1
(PC12 passage 6) illustrates the significance of the number of passage
on CRH production.
As expected, exposure of PC12 to 7S NGF (25 ng/ml for 1 week) induced
their differentiation to neuron-like cells. The thus differentiated
PC12 cells exhibited a highly significant increase in their capacity to
produce CRH (as well as catecholamines). Figure 6
depicts the rate of CRH accumulation in the media of these cells, which
was approximately 30 times higher compared with parallel controls
(i.e. PC12 cells cultured for the same number of days
without NGF). The concentration of IR-CRH in the test media is
normalized per milligram of total cellular protein to eliminate a
possible, although unlikely, proliferative effect of NGF. The secretion
of IR-CRH in terms of cell number is as follows: from normal rat
chromaffin cells in culture: 15 ± 2 at 12 h (mean
IR-CRH ± SE in picograms per million cells, n =
6), 13 ± 1 at 24 h, 12 ± 1 at 48 h. From PC12 of
early passage: 2 ± 0.5 at 12 h, 1 ± 0.1 at 24 h,
1 ± 0.2 at 48 h. From NGF-treated PC12 cells of early
passage: 104 ± 13 at 12 h, 60 ± 10 at 24 h, and
58 ± 24 at 48 h. From PC12 cells of late passage: 44 ±
0.4 at 12 h, 85 ± 20 at 24 h, and 15 ± 2 at
48 h.
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depicts the effect of CRH at 10-6
M over 48 h on epinephrine release from normal
chromaffin cells (upper panel), and on dopamine release from
PC12 cells (lower panel). Figure 8 depicts
the dose-dependent effect of CRH on the concentration of epinephrine in
the test media of normal chromaffin cells (upper panel), and
of dopamine of PC12 cells (lower panel). The stimulatory
effect of CRH was similar between the two types of chromaffin cells.
Indeed, CRH stimulated the secretion of epinephrine from chromaffin
cells (upper curve, upper panel), and dopamine
from PC12 cells (upper curve, lower panel).
hCRH at 10-5 M blocked the stimulatory
effect of CRH on chromaffin cells (lower curve, upper
panel) and partially blocked its effect on PC12 cells (lower
curve, lower panel). hCRH alone did not have any
statistically significant effect on basal or nicotine-induced
catecholamine secretion from either type of chromaffin cell.
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depicts the accumulation of dopamine
into the culture media of the neuronally differentiated cells over
12 h. The differentiated cells produced almost 10 times more
dopamine compared with PC12 cells exposed only to the NGF vehicle
(control cells). The increased rate of dopamine accumulation in the
NGF-treated cells was prevented completely by preincubation with the
CRH antagonist ahCRH at 10-6 M, suggesting
that endogenous CRH may mediate this phenomenon. Synthetic CRH did not
affect the production of catecholamines from NGF-differentiated PC12
cells, suggesting that the local CRH receptors were already
downregulated.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The release of IR-CRH into the culture media paralleled that of catecholamines, suggesting costorage, a property characteristic of most neural crest-derived cells in which neuropeptides and neurotransmitters are packaged into the same secretory granules (31). The role of adrenomedullary CRH and the physiological significance of its simultaneous release with catecholamines is not known.
IL-1ß stimulates the production of CRH from the parvo cells of the paraventricular hypothalamic nucleus (30). We have found that normal chromaffin cells and the PC12 cell line responded to IL-1ß by an increase of CRH release of late onset, suggesting that, in similarity to hypothalamus, this cytokine affects CRH synthesis rather than secretion.
The concentration of CRH in the normal chromaffin and the PC12 media was in the range of 0.2–0.5 x 10-11 M. This secretion rate suggests that chromaffin cell-derived CRH can not have systemic effects. It is more likely that its effects are confined within the adrenals. Indeed, the calculated in vivo concentration of CRH in the interstitial compartment of an intact adrenal medulla may reach the biologically active levels of 1–2 x 10-10 M, i.e. near the Kd of the CRH receptor.
In the central nervous system, there is a close anatomical proximity
between CRH and noradrenergic neurons at the level of locus
ceruleus (32, 33). In the rat, CRH stimulates the release of
catecholamines from locus ceruleus, as well as from hypothalamus,
medial prefrontal cortex, and the hippocampal dentate gyrus in
vivo (34, 35, 36, 37). This effect of CRH is blocked by hCRH (37). This
effect of CRH involves catecholamine synthesis via stimulation of the
activity of tyrosine hydroxylase, the rate limiting enzyme (38, 39).
All available evidence indicates that CRH plays a similar role in the
adrenal medulla because the latter also contains CRH receptors (13, 14). Our data support this hypothesis, demonstrating a dose-depended
stimulatory effect of CRH on the production of catecholamines from both
normal and PC12 tumoral rat adrenal chromaffin cells. The apparent
preservation, by PC12 cells, of the CRH-mediated effect on
catecholamine synthesis may suggest that CRH is an important component
of the adrenomedullary physiology.
A well-documented characteristic of the PC12 line is its differentiation to neuron-like cells after exposure to NGF for 1 week. We have found that the neuron-like PC12 cells secreted 10 times more catecholamines compared with parallel controls and thirty times more CRH. Our finding is in complete agreement with previously published data from primary cultures of human pheochromocytomas, in which NGF increased the concentration of CRH mRNA by 10-fold (19). The stimulatory effect of NGF on the capacity of PC12 cells to produce catecholamines seems to involve CRH because exposure of PC12 cells to the CRH antagonist, ahCRH, during the incubation with NGF completely abolished the NGF-mediated stimulatory effect. It is thus possible that the following sequence of events takes place: the NGF-induced differentiation of PC12 cells causes an increase in their CRH-producing capacity; the high levels of PC12-derived CRH act in a paracrine manner and stimulate the synthesis of tyrosine hydroxylase, which stimulates the synthesis of catecholamines. Blockage of the endogenous CRH paracrine effect prevents the stimulatory effect of NGF.
Exogenous synthetic CRH did not affect the production of catecholamines from the NGF-differentiated PC12 cells, in contrast to the NGF-unexposed PC12 cells. This finding suggests that either the PC12 CRH receptors were already maximally activated and possibly downregulated or the production of catecholamines was already at its highest possible level as a consequence of the NGF-induced differentiation.
In conclusion, our data suggest that the production of CRH by PC12 cells represents the preservation of a normal chromaffin cell characteristic rather than a tumor-induced ectopic phenomenon. From a clinical point of view, our findings may be of value in some inoperable pheochromocytomas in which the production of catecholamines could be inhibited by the new nonpeptide CRH antagonists.
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Acknowledgments |
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Footnotes |
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Received June 10, 1996.
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