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Brain Research 887 (2000) 16–22
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Research report
Neonatal polyamine depletion by a-difluoromethylornithine: effects on
adenylyl cyclase cell signaling are separable from effects on brain
region growth
T.A. Slotkin a , *, S.A. Ferguson b , A.M. Cada b , E.C. McCook a , F.J. Seidler a
b
a
Department of Pharmacology and Cancer Biology, Box 3813, Duke University Medical Center, Durham, NC 27710, USA
Division of Neurotoxicology, National Center for Toxicological Research, U.S. Food and Drug Administration, Jefferson, AR 72079, USA
Accepted 12 September 2000
Abstract
Ornithine decarboxylase (ODC) and the polyamines play an essential role in brain cell replication and differentiation. We administered
a-difluoromethylornithine (DFMO), an irreversible inhibitor of ODC, to neonatal rats on postnatal days 5–12, during the mitotic peak of
the cerebellum, a treatment regimen that leads to selective growth inhibition and dysmorphology. In adulthood, cell signaling responses
mediated through the adenylyl cyclase pathway were evaluated in order to determine if synaptic dysfunction extends to regions that
appear to be otherwise unaffected by DFMO. Total adenylyl cyclase catalytic activity, evaluated with the direct enzymatic stimulant,
Mn 21 , was significantly elevated in male rats both in the cerebellum and in brain regions showing no growth retardation (cerebral cortex,
brainstem); there were no significant effects in females. In contrast, signaling mediated through the G proteins that couple
neurotransmitter receptors to adenylyl cyclase showed a deficit in the DFMO group, as evaluated with the response to fluoride; in males,
there was no corresponding increase in activity as would have been expected solely from the enhancement of adenylyl cyclase, and in
females, there was actually a significant decrease in the response to fluoride. Again, the deficits were not restricted to the cerebellum.
Stimulation of adenylyl cyclase by isoproterenol, a b-adrenergic receptor agonist that acts through G s , likewise displayed deficits in both
males and females, and without distinction by brain region. These results indicate that the ODC / polyamine pathway plays a role in the
development of cell signaling, and hence in neurotransmission, above and beyond its role in cell replication and differentiation. Given the
fact that numerous drugs and environmental contaminants have been shown to alter ODC and the polyamines in the developing brain, our
findings suggest that changes in brain region growth or structure are inadequate to predict the targeting of specific neurotransmitter or
signaling pathways, and that gender-selective functional defects may be present despite the absence of morphological differences.
2000 Elsevier Science B.V. All rights reserved.
Theme: Development and regeneration
Topic: Neurotrophic factors: receptors and cellular mechanisms
Keywords: Adenylyl cyclase; b-Adrenergic receptor; a-Difluoromethylornithine; G protein; Ornithine decarboxylase
1. Introduction
Ornithine decarboxylase (ODC) catalyzes the initial step
in the formation of polyamines, major intracellular regulators of cell replication and differentiation [9,17]. A
normal ODC developmental pattern is essential for proper
*Corresponding author. Tel.: 11-919-681-8015; fax: 11-919-6848197.
E-mail address: t.slotkin@duke.edu (T.A. Slotkin).
neurogenesis and architectural assembly [1,16,17] and
accordingly, ODC is rapidly responsive to conditions that
change these developmental events [17]. For this reason,
ODC has proven useful to identify adverse developmental
effects of drugs and environmental contaminants that target
the developing brain [4,6,7,9,12,15,17,27,28].
Whereas the roles of ODC and the polyamines in cell
replication and differentiation have been well characterized, it is unclear whether they influence the ontogeny of
specific neurotransmitter or signaling pathways over and
0006-8993 / 00 / $ – see front matter 2000 Elsevier Science B.V. All rights reserved.
PII: S0006-8993( 00 )02961-9
T. A. Slotkin et al. / Brain Research 887 (2000) 16 – 22
above more generalized aspects of cell development. This
question is of critical importance because alterations in
ODC activity have been noted after fetal or neonatal
exposure to drugs or toxic chemicals that do not evoke
gross alterations in brain growth or morphology
[4,6,7,9,12,15,17,27,28]. If changes in polyamine synthesis
influence the development of cellular function, and not just
replication and differentiation, then straightforward measurements of growth and morphology are inadequate
predictors of fetal or neonatal brain injury. To test this
hypothesis, we have utilized a-difluoromethylornithine
(DFMO), a specific and irreversible inhibitor of ODC that
is devoid of systemic toxicity [4,12,17,26]. Daily postnatal
administration of DFMO to neonatal rats results in immediate and persistent deficits of ODC activity, depletion of
putrescine and spermidine, and retardation of brain growth
targeted toward regions undergoing peaks of cell replication and differentiation [1,3,4,16,17,21,25]. With appropriate selection of a treatment window, DFMO causes
selective growth retardation of specific brain regions while
sparing other regions.
In the current study, we have utilized a brief DFMO
treatment in neonatal rats that elicits alterations in growth
and structure of the cerebellum only, including atrophy and
deficits in cell number [1,5,16], and have examined the
effects when the animals reach adulthood. However, there
is some evidence that this treatment alters functional
development of neurotransmitter pathways elsewhere in
the brain, culminating in disruption of complex behavioral
patterns [3,11,18]. Among the pathways showing potential
alterations from neonatal DFMO treatment, catecholamine
systems stand out as prominent candidates [2,11,13,18,21].
Accordingly, we have concentrated on the effects of
DFMO on b-adrenergic receptors and their transduction of
cell signals through the adenylyl cyclase signaling pathway, comparing effects in the cerebellum to structurally
unaffected regions [4,5,17,25].
2. Materials and methods
2.1. Animal treatments
Studies were carried out in accordance with the declaration of Helsinki and with the Guide for the Care and Use
of Laboratory Animals as adopted and promulgated by the
National Institutes of Health. Fifteen litters of Sprague–
Dawley rats were given subcutaneous injections of DFMO
(500 mg / kg) on postnatal days 5–12 while another 15
litters received equivalent volumes of vehicle (isotonic
saline). Animals were weaned, housed separately by
gender, and allowed to reach approximately 6 months of
age before assays were conducted. Animals were selected
such that each litter contributed no more than one male and
one female rat. The DFMO regimen used here has been
shown previously to cause complete suppression of ODC
17
activity in the developing brain, with corresponding depletion of putrescine and spermidine [21]; activities recover
within 1 week post-treatment, with only transient impairment of general growth. DFMO treatment during this
postnatal period impairs cerebellar growth and elicits
reductions in cell number [5] and lasting anomalies of
cerebellar structure [1,16].
Brains were dissected into nine regions [8], weighed,
flash-frozen and stored at 2458C. For this study, the
cerebral cortex (less the frontal cortex), brainstem, and
cerebellum were assayed. Care was taken so that each
researcher dissected an equal number of animals from each
treatment group.
2.2. b -Receptor binding
Receptor binding capabilities were determined by methods described in earlier publications [14,20,24,29]. The
overall strategy was to examine binding at a single,
subsaturating ligand concentration in preparations from
every animal. The selection of a single concentration of
radioligand for the receptor analysis enables the detection
of changes in either Kd or Bmax but does not permit
distinction between the two possible mechanisms; this
strategy was necessitated by the requirement to measure
receptor binding along with several different measures of
adenylyl cyclase activity, across two treatment groups,
both genders and three tissues (over 100 separate membrane preparations).
Tissues were thawed and homogenized (Polytron, Brinkmann Instruments, Westbury, NY) in 39 volumes of icecold buffer containing 145 mM NaCl, 2 mM MgCl 2 , and
20 mM Tris (pH 7.5). Homogenates were sedimented at
40 0003g for 15 min. The pellets were washed twice,
resuspended (Polytron) in homogenization buffer followed
by resedimentation, and were then dispersed with a
homogenizer (smooth glass fitted with a Teflon pestle) to
achieve a final protein concentration of 1.25 mg / ml in a
buffer consisting of 250 mM sucrose, 2 mM MgCl 2 , 50
mM Tris (pH 7.5). [ 125 I]Iodopindolol (final concentration
of 67 pM; specific activity 2200 Ci / mmol; New England
Nuclear Corp., Boston, MA) was incubated with |125 mg
of membrane protein in a medium of 145 mM NaCl, 2 mM
MgCl 2 , 1 mM Na ascorbate, 20 mM Tris (pH 7.5), for 20
min at room temperature in a total volume of 250 ml.
Incubations were stopped by dilution with 3 ml of ice-cold
buffer, and the labeled membranes were trapped by rapid
vacuum filtration onto Whatman GF / C filters, which were
then washed with additional buffer and counted by liquid
scintillation spectrometry. Non-specific binding (displacement by 100 mM isoproterenol; Sigma Chemical Co., St.
Louis, MO) was generally about 10% of total binding.
2.3. Adenylyl cyclase activity
Membrane fractions were prepared as already described
18
T. A. Slotkin et al. / Brain Research 887 (2000) 16 – 22
for b-receptor binding. The membrane preparations were
diluted 20-fold with 250 mM sucrose, 1 mM EGTA and 10
mM Tris (pH 7.4) prior to the assay. Aliquots of membrane preparation containing 30 mg of protein were
incubated for 10 min at 308C with final concentrations of
100 mM Tris–HCl (pH 7.4), 10 mM theophylline, 1 mM
adenosine 59-triphosphate, 10 mM guanosine 59-triphosphate, 10 mM MgCl 2 , 1 mg / ml of bovine serum albumin,
and a creatine phosphokinase-ATP-regenerating system
consisting of 10 mM sodium phosphocreatine and 8 IU
phosphocreatine kinase (all reagents from Sigma) in a total
volume of 250 ml. The enzymatic reaction was stopped by
placing the samples in a 90–1008C water bath for 5 min,
followed by sedimentation at 30003g for 15 min, and the
supernatant solution was assayed for cAMP using radioimmunoassay kits (Amersham Corp., Chicago, IL). Preliminary experiments showed that the enzymatic reaction was
linear well beyond the assay time period and was linear
with membrane protein concentration; concentrations of
cofactors were optimal.
Adenylyl cyclase activity was measured under several
different conditions. First, basal activity was evaluated.
Second, the maximal G-protein-linked response was evaluated in samples containing 10 mM NaF. Third, maximal
total activity of the AC catalytic unit, independent of
receptors or G proteins, was evaluated with 10 mM MnCl 2
[10]. Finally, b-receptor-mediated effects were evaluated
with 100 mM isoproterenol [19]. The concentrations of all
the agents used here have been found previously to be
optimal for effects on AC and were confirmed in preliminary experiments.
2.4. Data analysis
Results are presented as means and standard errors.
Alterations caused by DFMO were first examined with a
global analysis of variance (ANOVA), incorporating all
variables: treatment, brain region and gender, and for
adenylyl cyclase, the four types of activities (a repeated
measure, since for each sample, all four measures were
evaluated in the same membrane preparation). When the
initial test identified interactions of treatment with other
variables, lower-order ANOVAs were then conducted,
followed by post hoc analysis (Fisher’s Protected Least
Significant Difference) to identify individual variables for
which the DFMO group differed significantly from the
controls; however, where interactions were not detected,
main effects are reported without post hoc analyses. For
convenience, results are presented as the percent change
from control values; however, statistical determinations
were performed on the original data. Main effects were
considered significant at the level of P,0.05 and interaction terms at P,0.1 [23]; in the latter case, the significance was verified by identification of main effects at
P,0.05 with lower-order ANOVAs, after separation of the
interactive factors.
3. Results
In control rats, there were substantial regional and
gender-related differences in adenylyl cyclase activity
(Table 1). Overall activity was lower in the brainstem than
in the other two regions; the response to Mn 21 , which
causes maximal, direct activation of the enzyme was 5- to
10-fold in the cerebellum and cerebral cortex, but only
3-fold in the brainstem. Similarly, fluoride, which produces
complete activation of G proteins, evoked only a 25–30%
increase in the brainstem, compared to 30–40% in the
cerebellum and 50–70% in the cerebral cortex. In contrast,
the response to isoproterenol, a b-receptor stimulant, was
largest in the brainstem (25–40% increase, compared to
10–15% in the other regions). Across all three regions,
females showed higher adenylyl cyclase activities than
males (main effect of gender); the effect was most noticeable in the cerebellum (P,0.007), whereas there was no
significant gender difference in the brainstem and a
difference only for the Mn 21 response in the cerebral
Table 1
Values in control rats a
Cerebellum
Adenylyl cyclase (pmol / mg protein / min)
Basal
Mn 21 -Stimulated
Fluoride-stimulated
Isoproterenol-stimulated
b-Receptor binding (fmol / mg protein)
Weight (mg)
a
Cerebral cortex
Brainstem
Male
Female
Male
Female
Male
Female
13064
678655
17069
14166
14866
876631
20569
16065
10164
683661
152610
11262
9864
824621
166612
11364
8664
287632
10965
11866
8762
339626
12063
10863
1561
1861
1761
1861
333613
30868
696622
640622
–
–
453615
412612
Data represent means and standard errors obtained from 9–10 rats in each group. Across the four adenylyl cyclase measures, ANOVA indicates main
effects of region (P,0.0001), gender (P,0.007) and measure (P,0.0001), as well as interactions of region3measure (P,0.0001) and gender3measure
(P,0.0003). For b-receptor binding, there were no significant main effects or interactions. For brain region weights, there were main effects of region
(P,0.0001) and gender (P,0.008).
T. A. Slotkin et al. / Brain Research 887 (2000) 16 – 22
cortex. There was no significant regional or gender difference for b-receptor binding in control rats but main effects
of both variables were present for tissue weights.
Global statistical analyses of the effects of DFMO
identified effects on adenylyl cyclase activity that were
directed toward specific types of activity (treatment3
measure interaction) and that were gender selective (interaction of treatment3gender3measure). Accordingly, results for each type of adenylyl cyclase measurement are
presented, separated by gender. In contrast, b-receptor
binding did not show any significant treatment effects or
interactions of treatment3other variables; brain region
weights showed regionally dependent effects of DFMO
(treatment3region interaction), without any gender selectivity (no interaction of treatment3gender or of
treatment3gender3region).
Across all three brain regions and both genders, basal
adenylyl cyclase showed a trend (P,0.07) toward reduced
activity in the DFMO group (Fig. 1, top panel). However,
total enzymatic activity, evaluated with Mn 21 , indicated
instead a significant overall elevation after neonatal DFMO
treatment (middle panel), with a distinct gender selectivity
(males.females). Although the effect was lower in the
brainstem than in the other regions, it is difficult to ascribe
regional significance, as there was no overall statistical
difference among regions in the global test (Table 2). A
reduction in basal adenylyl cyclase activity in the face of
an increase in total enzymatic activity implies that DFMO
is likely to affect cofactors that modulate the activity.
Accordingly, we also evaluated the adenylyl cyclase
response to fluoride, which evokes maximal association of
the enzyme with G proteins (Fig. 1, bottom panel). DFMO
treatment impaired the fluoride response, with a decided
gender-dependence (significant deficits in females but not
males).
Fluoride acts upon both stimulatory G proteins (G s ) and
inhibitory proteins (G i ), so we next examined the response
to isoproterenol, which activates b-receptors that work
predominantly through G s . DFMO impaired the adenylyl
cyclase response to isoproterenol in both males and
females (Fig. 2, top panel). However, given that DFMO
induced total adenylyl cyclase activity (Mn 21 response)
and altered overall G-protein signaling (fluoride response),
we evaluated the effects on isoproterenol-stimulated activity relative to the other two stimulants. Comparing the
effects on b-receptor-mediated responses to total enzymatic activity revealed profound deficits in the G s -linked
receptor response, with a greater effect on males (middle
panel). The same pattern was seen when the isoproterenol
response was compared to that of fluoride (bottom panel):
significant overall deficits with the greater loss seen in
males.
Deficiencies in b-receptor cell signaling could also
reflect changes at the level of the receptors themselves.
However, we did not observe any significant differences in
b-receptor sites (Fig. 3, top panel). If anything, DFMO
19
Fig. 1. Effects of neonatal DFMO administration on basal, Mn 21 -stimulated and fluoride-stimulated adenylyl cyclase activity in brain regions of
adult rats, presented as the percent change from control values (Table 1).
Data represent means and standard errors obtained from 8–10 animals for
each gender. ANOVA across both genders appears at the top of each
panel and separate evaluations for males and females are shown at the
bottom of the panel. Post hoc tests for each region were not carried out
because of the absence of significant interactions of treatment3region or
of treatment3region3gender. cb, cerebellum; ctx, cerebral cortex; bs,
brainstem.
20
T. A. Slotkin et al. / Brain Research 887 (2000) 16 – 22
Table 2
Global statistical analyses
Adenylyl cyclase activity
Treatment
Region
Gender
Measure
Treatment3Region
Treatment3Gender
Treatment3Measure
Region3Gender
Region3Measure
Gender3Measure
Treatment3Region3Gender
Treatment3Region3Measure
Treatment3Gender3Measure
Region3Gender3Measure
Treatment3Region3Gender3Measure
a
a
NS
P,0.0001
P,0.002
P,0.0001
NS
NS
P,0.0001
NS
P,0.0001
P,0.0006
NS
NS
P,0.09
NS
NS
b-Receptor binding
Brain region weight
NS
NS
P,0.007
–
NS
NS
–
NS
–
–
NS
–
–
–
–
NS
P,0.0001
P,0.0001
–
P,0.03
NS
–
NS
–
–
NS
–
–
–
–
NS, not significant.
treatment increased receptor binding. The alterations in
adenylyl cyclase signaling also did not correspond to
regional growth impairment by DFMO (bottom panel). As
expected from previous studies [1,5,16], growth impairment was limited to the cerebellum and showed no gender
selectivity.
4. Discussion
The present results indicate that the role of polyamines
in brain development extends beyond the control of
general growth, to the programming of expression and / or
function of specific signaling proteins involved in synaptic
transmission. Although neonatal DFMO compromises the
growth and structure of the cerebellum while sparing the
cerebral cortex and brainstem [1,5,16], effects on the
adenylyl cyclase signaling cascade were present in all three
regions. Furthermore, although adverse changes in growth
and morphology are shared by both males and females, the
effects on signaling displayed distinct gender selectivity.
Accordingly, the DFMO-induced alterations in the functioning of proteins in the adenylyl cyclase cascade are not
manifestations of impaired cerebellar growth, nor do they
represent a spreading of adverse effects to other regions
simply because they connect to cerebellar cells or cerebellar function. It is therefore clear that even a brief developmental period of ODC inhibition exerts effects on
cellular signaling that are entirely distinct from those on
cell growth, and in turn, this implies that polyamines play
a more specified role in the development of cell signaling.
We obtained two main characteristics effects on the
adenylyl cyclase signaling cascade: induction of adenylyl
cyclase activity itself, and interference with signaling
mediated through G-protein-coupled receptors. Adenylyl
cyclase induction was evident from the overall increase in
the response to Mn 21 , which binds directly to the Mg 21 -
sensitive site of the enzyme [10]. If this were the only
effect, then all responses mediated through adenylyl
cyclase should be equally enhanced; however, basal activity actually tended to be reduced, indicating that a defect in
signaling exists upstream from adenylyl cyclase. Approximately half of basal activity is actually dependent on
G-protein interactions with the enzyme, even in the
absence of receptor stimulation [19,22]. Accordingly, we
next evaluated the response to fluoride, which stimulates
all G proteins maximally. There was no global increase as
would be expected from the induction of adenylyl cyclase
catalytic activity. Instead, activity was unchanged in males,
and actually obtunded in females, suggesting that additional gender-selective deficits reside in G-protein-coupled
responses.
Because fluoride activates both G s and G i , we also then
evaluated the response to isoproterenol, which activates
b-adrenergic receptors and thus stimulates adenylyl
cyclase primarily through G s . In this case, we identified
clear-cut deficiencies across both genders in the DFMO
group. Taking into account the fact that the loss of
response to isoproterenol occurs in the presence of genderselective induction of adenylyl cyclase, the deficit in the
specific b-receptor mediated component was actually far
more profound in males, as demonstrated by the sharp
decline in the proportion of adenylyl cyclase activated by
b-receptor stimulation (the isoproterenol:Mn 21 activity
ratio) or in the proportion of G proteins activated by the
receptors (isoproterenol:fluoride activity ratio). Indeed, the
deficiencies in G-protein-mediated responses are likely to
involve separate mechanisms in the two genders. In males,
the deficits comprise loss of b-receptor coupling to G s ,
since the response to isoproterenol was reduced relative to
that of fluoride, without a change in b-receptor binding; in
addition, there are defects in G-protein coupling to
adenylyl cyclase, demonstrated by the fact that the fluoride
response was not enhanced despite induction of adenylyl
T. A. Slotkin et al. / Brain Research 887 (2000) 16 – 22
21
Fig. 3. Effects of neonatal DFMO administration on b-receptor binding
and on brain region weights, presented as the percent change from control
values (Table 1). Data represent means and standard errors obtained from
5–10 animals for each gender. ANOVA across both genders appears at
the top of each panel and separate evaluations for males and females are
shown at the bottom of the panel. Post hoc tests for each region were
carried out only for brain region weight, the only variable to show a
significant interaction of treatment3region. cb, cerebellum; ctx, cerebral
cortex; bs, brainstem.
Fig. 2. Effects of neonatal DFMO administration on isoproterenol-stimulated adenylyl cyclase activity, and on isoproterenol stimulation relative
to Mn 21 or fluoride-stimulated activity, presented as the percent change
from control values (Table 1). Data represent means and standard errors
obtained from 8–10 animals for each gender. ANOVA across both
genders appears at the top of each panel and separate evaluations for
males and females are shown at the bottom of the panel. Post hoc tests for
each region were not carried out because of the absence of significant
interactions of treatment3region or of treatment3region3gender. cb,
cerebellum; ctx, cerebral cortex; bs, brainstem.
cyclase. In females, the effects of DFMO are limited to the
G-protein interaction with adenylyl cyclase. The response
to fluoride was actually reduced despite adenylyl cyclase
induction, implying a greater loss of G-protein function
than in males; on the other hand, there was no further
deficit in the response to isoproterenol, which simply
mirrored the overall deficit in G-protein function (no effect
on the isoproterenol:fluoride activity ratio). These results
thus indicate that neonatal polyamine depletion can have
vastly different effects on cell signaling in males and
females. Ultimately, these defects may contribute to gender-specific effects on behavior, and our results indicate
that selectivity may best be revealed by behavioral responses that challenge receptor systems operating through
adenylyl cyclase.
In conclusion, the role of polyamines in brain development extends to expression or function of specific proteins
that control synaptic responses, entirely separately from
T. A. Slotkin et al. / Brain Research 887 (2000) 16 – 22
22
growth. Thus, effects on signaling extend to regions other
than cerebellum, the only region whose growth is affected
by transient neonatal polyamine depletion [1,5,16]. The
significance of these results extends beyond the effects of
DFMO. Perturbations of ODC activity and polyamines
have been identified for numerous suspected neuroteratogens [4,9,12,15,17], suggesting in these cases, too,
the effects on the ODC pathway may be mechanistically
connected to lasting changes in synaptic function, even
when growth and structure are apparently normal. The
ODC / polyamine system thus provides a potential mechanistic link between initial neuroteratogenic insults and the
eventual functional outcome, rather than representing just a
marker for perturbed growth and development.
Acknowledgements
This study was supported by USPHS HD-09713.
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P.A. Trepanier, F.J. Seidler, Role of ornithine decarboxylase and the
polyamines in nervous system development: short-term postnatal
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of ornithine decarboxylase, Int. J. Dev. Neurosci. 1 (1983) 7–16.
[22] T.A. Slotkin, J. Zhang, E.C. McCook, F.J. Seidler, Glucocorticoidtargeting of the adenylyl cyclase signaling pathway in the cerebellum of young vs. aged rats, Brain Res. 800 (1998) 236–244.
[23] G.W. Snedecor, W.G. Cochran, Statistical Methods, Iowa State
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Pharmacol. 145 (1997) 158–174.
[25] M. Sparapani, M. Virgili, M. Caprini, F. Facchinetti, E. Ciani, A.
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in developing rat brain and on adult rat neurochemistry, Exp. Brain
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www.elsevier.com / locate / bres
Research report
Neonatal polyamine depletion by a-difluoromethylornithine: effects on
adenylyl cyclase cell signaling are separable from effects on brain
region growth
T.A. Slotkin a , *, S.A. Ferguson b , A.M. Cada b , E.C. McCook a , F.J. Seidler a
b
a
Department of Pharmacology and Cancer Biology, Box 3813, Duke University Medical Center, Durham, NC 27710, USA
Division of Neurotoxicology, National Center for Toxicological Research, U.S. Food and Drug Administration, Jefferson, AR 72079, USA
Accepted 12 September 2000
Abstract
Ornithine decarboxylase (ODC) and the polyamines play an essential role in brain cell replication and differentiation. We administered
a-difluoromethylornithine (DFMO), an irreversible inhibitor of ODC, to neonatal rats on postnatal days 5–12, during the mitotic peak of
the cerebellum, a treatment regimen that leads to selective growth inhibition and dysmorphology. In adulthood, cell signaling responses
mediated through the adenylyl cyclase pathway were evaluated in order to determine if synaptic dysfunction extends to regions that
appear to be otherwise unaffected by DFMO. Total adenylyl cyclase catalytic activity, evaluated with the direct enzymatic stimulant,
Mn 21 , was significantly elevated in male rats both in the cerebellum and in brain regions showing no growth retardation (cerebral cortex,
brainstem); there were no significant effects in females. In contrast, signaling mediated through the G proteins that couple
neurotransmitter receptors to adenylyl cyclase showed a deficit in the DFMO group, as evaluated with the response to fluoride; in males,
there was no corresponding increase in activity as would have been expected solely from the enhancement of adenylyl cyclase, and in
females, there was actually a significant decrease in the response to fluoride. Again, the deficits were not restricted to the cerebellum.
Stimulation of adenylyl cyclase by isoproterenol, a b-adrenergic receptor agonist that acts through G s , likewise displayed deficits in both
males and females, and without distinction by brain region. These results indicate that the ODC / polyamine pathway plays a role in the
development of cell signaling, and hence in neurotransmission, above and beyond its role in cell replication and differentiation. Given the
fact that numerous drugs and environmental contaminants have been shown to alter ODC and the polyamines in the developing brain, our
findings suggest that changes in brain region growth or structure are inadequate to predict the targeting of specific neurotransmitter or
signaling pathways, and that gender-selective functional defects may be present despite the absence of morphological differences.
2000 Elsevier Science B.V. All rights reserved.
Theme: Development and regeneration
Topic: Neurotrophic factors: receptors and cellular mechanisms
Keywords: Adenylyl cyclase; b-Adrenergic receptor; a-Difluoromethylornithine; G protein; Ornithine decarboxylase
1. Introduction
Ornithine decarboxylase (ODC) catalyzes the initial step
in the formation of polyamines, major intracellular regulators of cell replication and differentiation [9,17]. A
normal ODC developmental pattern is essential for proper
*Corresponding author. Tel.: 11-919-681-8015; fax: 11-919-6848197.
E-mail address: t.slotkin@duke.edu (T.A. Slotkin).
neurogenesis and architectural assembly [1,16,17] and
accordingly, ODC is rapidly responsive to conditions that
change these developmental events [17]. For this reason,
ODC has proven useful to identify adverse developmental
effects of drugs and environmental contaminants that target
the developing brain [4,6,7,9,12,15,17,27,28].
Whereas the roles of ODC and the polyamines in cell
replication and differentiation have been well characterized, it is unclear whether they influence the ontogeny of
specific neurotransmitter or signaling pathways over and
0006-8993 / 00 / $ – see front matter 2000 Elsevier Science B.V. All rights reserved.
PII: S0006-8993( 00 )02961-9
T. A. Slotkin et al. / Brain Research 887 (2000) 16 – 22
above more generalized aspects of cell development. This
question is of critical importance because alterations in
ODC activity have been noted after fetal or neonatal
exposure to drugs or toxic chemicals that do not evoke
gross alterations in brain growth or morphology
[4,6,7,9,12,15,17,27,28]. If changes in polyamine synthesis
influence the development of cellular function, and not just
replication and differentiation, then straightforward measurements of growth and morphology are inadequate
predictors of fetal or neonatal brain injury. To test this
hypothesis, we have utilized a-difluoromethylornithine
(DFMO), a specific and irreversible inhibitor of ODC that
is devoid of systemic toxicity [4,12,17,26]. Daily postnatal
administration of DFMO to neonatal rats results in immediate and persistent deficits of ODC activity, depletion of
putrescine and spermidine, and retardation of brain growth
targeted toward regions undergoing peaks of cell replication and differentiation [1,3,4,16,17,21,25]. With appropriate selection of a treatment window, DFMO causes
selective growth retardation of specific brain regions while
sparing other regions.
In the current study, we have utilized a brief DFMO
treatment in neonatal rats that elicits alterations in growth
and structure of the cerebellum only, including atrophy and
deficits in cell number [1,5,16], and have examined the
effects when the animals reach adulthood. However, there
is some evidence that this treatment alters functional
development of neurotransmitter pathways elsewhere in
the brain, culminating in disruption of complex behavioral
patterns [3,11,18]. Among the pathways showing potential
alterations from neonatal DFMO treatment, catecholamine
systems stand out as prominent candidates [2,11,13,18,21].
Accordingly, we have concentrated on the effects of
DFMO on b-adrenergic receptors and their transduction of
cell signals through the adenylyl cyclase signaling pathway, comparing effects in the cerebellum to structurally
unaffected regions [4,5,17,25].
2. Materials and methods
2.1. Animal treatments
Studies were carried out in accordance with the declaration of Helsinki and with the Guide for the Care and Use
of Laboratory Animals as adopted and promulgated by the
National Institutes of Health. Fifteen litters of Sprague–
Dawley rats were given subcutaneous injections of DFMO
(500 mg / kg) on postnatal days 5–12 while another 15
litters received equivalent volumes of vehicle (isotonic
saline). Animals were weaned, housed separately by
gender, and allowed to reach approximately 6 months of
age before assays were conducted. Animals were selected
such that each litter contributed no more than one male and
one female rat. The DFMO regimen used here has been
shown previously to cause complete suppression of ODC
17
activity in the developing brain, with corresponding depletion of putrescine and spermidine [21]; activities recover
within 1 week post-treatment, with only transient impairment of general growth. DFMO treatment during this
postnatal period impairs cerebellar growth and elicits
reductions in cell number [5] and lasting anomalies of
cerebellar structure [1,16].
Brains were dissected into nine regions [8], weighed,
flash-frozen and stored at 2458C. For this study, the
cerebral cortex (less the frontal cortex), brainstem, and
cerebellum were assayed. Care was taken so that each
researcher dissected an equal number of animals from each
treatment group.
2.2. b -Receptor binding
Receptor binding capabilities were determined by methods described in earlier publications [14,20,24,29]. The
overall strategy was to examine binding at a single,
subsaturating ligand concentration in preparations from
every animal. The selection of a single concentration of
radioligand for the receptor analysis enables the detection
of changes in either Kd or Bmax but does not permit
distinction between the two possible mechanisms; this
strategy was necessitated by the requirement to measure
receptor binding along with several different measures of
adenylyl cyclase activity, across two treatment groups,
both genders and three tissues (over 100 separate membrane preparations).
Tissues were thawed and homogenized (Polytron, Brinkmann Instruments, Westbury, NY) in 39 volumes of icecold buffer containing 145 mM NaCl, 2 mM MgCl 2 , and
20 mM Tris (pH 7.5). Homogenates were sedimented at
40 0003g for 15 min. The pellets were washed twice,
resuspended (Polytron) in homogenization buffer followed
by resedimentation, and were then dispersed with a
homogenizer (smooth glass fitted with a Teflon pestle) to
achieve a final protein concentration of 1.25 mg / ml in a
buffer consisting of 250 mM sucrose, 2 mM MgCl 2 , 50
mM Tris (pH 7.5). [ 125 I]Iodopindolol (final concentration
of 67 pM; specific activity 2200 Ci / mmol; New England
Nuclear Corp., Boston, MA) was incubated with |125 mg
of membrane protein in a medium of 145 mM NaCl, 2 mM
MgCl 2 , 1 mM Na ascorbate, 20 mM Tris (pH 7.5), for 20
min at room temperature in a total volume of 250 ml.
Incubations were stopped by dilution with 3 ml of ice-cold
buffer, and the labeled membranes were trapped by rapid
vacuum filtration onto Whatman GF / C filters, which were
then washed with additional buffer and counted by liquid
scintillation spectrometry. Non-specific binding (displacement by 100 mM isoproterenol; Sigma Chemical Co., St.
Louis, MO) was generally about 10% of total binding.
2.3. Adenylyl cyclase activity
Membrane fractions were prepared as already described
18
T. A. Slotkin et al. / Brain Research 887 (2000) 16 – 22
for b-receptor binding. The membrane preparations were
diluted 20-fold with 250 mM sucrose, 1 mM EGTA and 10
mM Tris (pH 7.4) prior to the assay. Aliquots of membrane preparation containing 30 mg of protein were
incubated for 10 min at 308C with final concentrations of
100 mM Tris–HCl (pH 7.4), 10 mM theophylline, 1 mM
adenosine 59-triphosphate, 10 mM guanosine 59-triphosphate, 10 mM MgCl 2 , 1 mg / ml of bovine serum albumin,
and a creatine phosphokinase-ATP-regenerating system
consisting of 10 mM sodium phosphocreatine and 8 IU
phosphocreatine kinase (all reagents from Sigma) in a total
volume of 250 ml. The enzymatic reaction was stopped by
placing the samples in a 90–1008C water bath for 5 min,
followed by sedimentation at 30003g for 15 min, and the
supernatant solution was assayed for cAMP using radioimmunoassay kits (Amersham Corp., Chicago, IL). Preliminary experiments showed that the enzymatic reaction was
linear well beyond the assay time period and was linear
with membrane protein concentration; concentrations of
cofactors were optimal.
Adenylyl cyclase activity was measured under several
different conditions. First, basal activity was evaluated.
Second, the maximal G-protein-linked response was evaluated in samples containing 10 mM NaF. Third, maximal
total activity of the AC catalytic unit, independent of
receptors or G proteins, was evaluated with 10 mM MnCl 2
[10]. Finally, b-receptor-mediated effects were evaluated
with 100 mM isoproterenol [19]. The concentrations of all
the agents used here have been found previously to be
optimal for effects on AC and were confirmed in preliminary experiments.
2.4. Data analysis
Results are presented as means and standard errors.
Alterations caused by DFMO were first examined with a
global analysis of variance (ANOVA), incorporating all
variables: treatment, brain region and gender, and for
adenylyl cyclase, the four types of activities (a repeated
measure, since for each sample, all four measures were
evaluated in the same membrane preparation). When the
initial test identified interactions of treatment with other
variables, lower-order ANOVAs were then conducted,
followed by post hoc analysis (Fisher’s Protected Least
Significant Difference) to identify individual variables for
which the DFMO group differed significantly from the
controls; however, where interactions were not detected,
main effects are reported without post hoc analyses. For
convenience, results are presented as the percent change
from control values; however, statistical determinations
were performed on the original data. Main effects were
considered significant at the level of P,0.05 and interaction terms at P,0.1 [23]; in the latter case, the significance was verified by identification of main effects at
P,0.05 with lower-order ANOVAs, after separation of the
interactive factors.
3. Results
In control rats, there were substantial regional and
gender-related differences in adenylyl cyclase activity
(Table 1). Overall activity was lower in the brainstem than
in the other two regions; the response to Mn 21 , which
causes maximal, direct activation of the enzyme was 5- to
10-fold in the cerebellum and cerebral cortex, but only
3-fold in the brainstem. Similarly, fluoride, which produces
complete activation of G proteins, evoked only a 25–30%
increase in the brainstem, compared to 30–40% in the
cerebellum and 50–70% in the cerebral cortex. In contrast,
the response to isoproterenol, a b-receptor stimulant, was
largest in the brainstem (25–40% increase, compared to
10–15% in the other regions). Across all three regions,
females showed higher adenylyl cyclase activities than
males (main effect of gender); the effect was most noticeable in the cerebellum (P,0.007), whereas there was no
significant gender difference in the brainstem and a
difference only for the Mn 21 response in the cerebral
Table 1
Values in control rats a
Cerebellum
Adenylyl cyclase (pmol / mg protein / min)
Basal
Mn 21 -Stimulated
Fluoride-stimulated
Isoproterenol-stimulated
b-Receptor binding (fmol / mg protein)
Weight (mg)
a
Cerebral cortex
Brainstem
Male
Female
Male
Female
Male
Female
13064
678655
17069
14166
14866
876631
20569
16065
10164
683661
152610
11262
9864
824621
166612
11364
8664
287632
10965
11866
8762
339626
12063
10863
1561
1861
1761
1861
333613
30868
696622
640622
–
–
453615
412612
Data represent means and standard errors obtained from 9–10 rats in each group. Across the four adenylyl cyclase measures, ANOVA indicates main
effects of region (P,0.0001), gender (P,0.007) and measure (P,0.0001), as well as interactions of region3measure (P,0.0001) and gender3measure
(P,0.0003). For b-receptor binding, there were no significant main effects or interactions. For brain region weights, there were main effects of region
(P,0.0001) and gender (P,0.008).
T. A. Slotkin et al. / Brain Research 887 (2000) 16 – 22
cortex. There was no significant regional or gender difference for b-receptor binding in control rats but main effects
of both variables were present for tissue weights.
Global statistical analyses of the effects of DFMO
identified effects on adenylyl cyclase activity that were
directed toward specific types of activity (treatment3
measure interaction) and that were gender selective (interaction of treatment3gender3measure). Accordingly, results for each type of adenylyl cyclase measurement are
presented, separated by gender. In contrast, b-receptor
binding did not show any significant treatment effects or
interactions of treatment3other variables; brain region
weights showed regionally dependent effects of DFMO
(treatment3region interaction), without any gender selectivity (no interaction of treatment3gender or of
treatment3gender3region).
Across all three brain regions and both genders, basal
adenylyl cyclase showed a trend (P,0.07) toward reduced
activity in the DFMO group (Fig. 1, top panel). However,
total enzymatic activity, evaluated with Mn 21 , indicated
instead a significant overall elevation after neonatal DFMO
treatment (middle panel), with a distinct gender selectivity
(males.females). Although the effect was lower in the
brainstem than in the other regions, it is difficult to ascribe
regional significance, as there was no overall statistical
difference among regions in the global test (Table 2). A
reduction in basal adenylyl cyclase activity in the face of
an increase in total enzymatic activity implies that DFMO
is likely to affect cofactors that modulate the activity.
Accordingly, we also evaluated the adenylyl cyclase
response to fluoride, which evokes maximal association of
the enzyme with G proteins (Fig. 1, bottom panel). DFMO
treatment impaired the fluoride response, with a decided
gender-dependence (significant deficits in females but not
males).
Fluoride acts upon both stimulatory G proteins (G s ) and
inhibitory proteins (G i ), so we next examined the response
to isoproterenol, which activates b-receptors that work
predominantly through G s . DFMO impaired the adenylyl
cyclase response to isoproterenol in both males and
females (Fig. 2, top panel). However, given that DFMO
induced total adenylyl cyclase activity (Mn 21 response)
and altered overall G-protein signaling (fluoride response),
we evaluated the effects on isoproterenol-stimulated activity relative to the other two stimulants. Comparing the
effects on b-receptor-mediated responses to total enzymatic activity revealed profound deficits in the G s -linked
receptor response, with a greater effect on males (middle
panel). The same pattern was seen when the isoproterenol
response was compared to that of fluoride (bottom panel):
significant overall deficits with the greater loss seen in
males.
Deficiencies in b-receptor cell signaling could also
reflect changes at the level of the receptors themselves.
However, we did not observe any significant differences in
b-receptor sites (Fig. 3, top panel). If anything, DFMO
19
Fig. 1. Effects of neonatal DFMO administration on basal, Mn 21 -stimulated and fluoride-stimulated adenylyl cyclase activity in brain regions of
adult rats, presented as the percent change from control values (Table 1).
Data represent means and standard errors obtained from 8–10 animals for
each gender. ANOVA across both genders appears at the top of each
panel and separate evaluations for males and females are shown at the
bottom of the panel. Post hoc tests for each region were not carried out
because of the absence of significant interactions of treatment3region or
of treatment3region3gender. cb, cerebellum; ctx, cerebral cortex; bs,
brainstem.
20
T. A. Slotkin et al. / Brain Research 887 (2000) 16 – 22
Table 2
Global statistical analyses
Adenylyl cyclase activity
Treatment
Region
Gender
Measure
Treatment3Region
Treatment3Gender
Treatment3Measure
Region3Gender
Region3Measure
Gender3Measure
Treatment3Region3Gender
Treatment3Region3Measure
Treatment3Gender3Measure
Region3Gender3Measure
Treatment3Region3Gender3Measure
a
a
NS
P,0.0001
P,0.002
P,0.0001
NS
NS
P,0.0001
NS
P,0.0001
P,0.0006
NS
NS
P,0.09
NS
NS
b-Receptor binding
Brain region weight
NS
NS
P,0.007
–
NS
NS
–
NS
–
–
NS
–
–
–
–
NS
P,0.0001
P,0.0001
–
P,0.03
NS
–
NS
–
–
NS
–
–
–
–
NS, not significant.
treatment increased receptor binding. The alterations in
adenylyl cyclase signaling also did not correspond to
regional growth impairment by DFMO (bottom panel). As
expected from previous studies [1,5,16], growth impairment was limited to the cerebellum and showed no gender
selectivity.
4. Discussion
The present results indicate that the role of polyamines
in brain development extends beyond the control of
general growth, to the programming of expression and / or
function of specific signaling proteins involved in synaptic
transmission. Although neonatal DFMO compromises the
growth and structure of the cerebellum while sparing the
cerebral cortex and brainstem [1,5,16], effects on the
adenylyl cyclase signaling cascade were present in all three
regions. Furthermore, although adverse changes in growth
and morphology are shared by both males and females, the
effects on signaling displayed distinct gender selectivity.
Accordingly, the DFMO-induced alterations in the functioning of proteins in the adenylyl cyclase cascade are not
manifestations of impaired cerebellar growth, nor do they
represent a spreading of adverse effects to other regions
simply because they connect to cerebellar cells or cerebellar function. It is therefore clear that even a brief developmental period of ODC inhibition exerts effects on
cellular signaling that are entirely distinct from those on
cell growth, and in turn, this implies that polyamines play
a more specified role in the development of cell signaling.
We obtained two main characteristics effects on the
adenylyl cyclase signaling cascade: induction of adenylyl
cyclase activity itself, and interference with signaling
mediated through G-protein-coupled receptors. Adenylyl
cyclase induction was evident from the overall increase in
the response to Mn 21 , which binds directly to the Mg 21 -
sensitive site of the enzyme [10]. If this were the only
effect, then all responses mediated through adenylyl
cyclase should be equally enhanced; however, basal activity actually tended to be reduced, indicating that a defect in
signaling exists upstream from adenylyl cyclase. Approximately half of basal activity is actually dependent on
G-protein interactions with the enzyme, even in the
absence of receptor stimulation [19,22]. Accordingly, we
next evaluated the response to fluoride, which stimulates
all G proteins maximally. There was no global increase as
would be expected from the induction of adenylyl cyclase
catalytic activity. Instead, activity was unchanged in males,
and actually obtunded in females, suggesting that additional gender-selective deficits reside in G-protein-coupled
responses.
Because fluoride activates both G s and G i , we also then
evaluated the response to isoproterenol, which activates
b-adrenergic receptors and thus stimulates adenylyl
cyclase primarily through G s . In this case, we identified
clear-cut deficiencies across both genders in the DFMO
group. Taking into account the fact that the loss of
response to isoproterenol occurs in the presence of genderselective induction of adenylyl cyclase, the deficit in the
specific b-receptor mediated component was actually far
more profound in males, as demonstrated by the sharp
decline in the proportion of adenylyl cyclase activated by
b-receptor stimulation (the isoproterenol:Mn 21 activity
ratio) or in the proportion of G proteins activated by the
receptors (isoproterenol:fluoride activity ratio). Indeed, the
deficiencies in G-protein-mediated responses are likely to
involve separate mechanisms in the two genders. In males,
the deficits comprise loss of b-receptor coupling to G s ,
since the response to isoproterenol was reduced relative to
that of fluoride, without a change in b-receptor binding; in
addition, there are defects in G-protein coupling to
adenylyl cyclase, demonstrated by the fact that the fluoride
response was not enhanced despite induction of adenylyl
T. A. Slotkin et al. / Brain Research 887 (2000) 16 – 22
21
Fig. 3. Effects of neonatal DFMO administration on b-receptor binding
and on brain region weights, presented as the percent change from control
values (Table 1). Data represent means and standard errors obtained from
5–10 animals for each gender. ANOVA across both genders appears at
the top of each panel and separate evaluations for males and females are
shown at the bottom of the panel. Post hoc tests for each region were
carried out only for brain region weight, the only variable to show a
significant interaction of treatment3region. cb, cerebellum; ctx, cerebral
cortex; bs, brainstem.
Fig. 2. Effects of neonatal DFMO administration on isoproterenol-stimulated adenylyl cyclase activity, and on isoproterenol stimulation relative
to Mn 21 or fluoride-stimulated activity, presented as the percent change
from control values (Table 1). Data represent means and standard errors
obtained from 8–10 animals for each gender. ANOVA across both
genders appears at the top of each panel and separate evaluations for
males and females are shown at the bottom of the panel. Post hoc tests for
each region were not carried out because of the absence of significant
interactions of treatment3region or of treatment3region3gender. cb,
cerebellum; ctx, cerebral cortex; bs, brainstem.
cyclase. In females, the effects of DFMO are limited to the
G-protein interaction with adenylyl cyclase. The response
to fluoride was actually reduced despite adenylyl cyclase
induction, implying a greater loss of G-protein function
than in males; on the other hand, there was no further
deficit in the response to isoproterenol, which simply
mirrored the overall deficit in G-protein function (no effect
on the isoproterenol:fluoride activity ratio). These results
thus indicate that neonatal polyamine depletion can have
vastly different effects on cell signaling in males and
females. Ultimately, these defects may contribute to gender-specific effects on behavior, and our results indicate
that selectivity may best be revealed by behavioral responses that challenge receptor systems operating through
adenylyl cyclase.
In conclusion, the role of polyamines in brain development extends to expression or function of specific proteins
that control synaptic responses, entirely separately from
T. A. Slotkin et al. / Brain Research 887 (2000) 16 – 22
22
growth. Thus, effects on signaling extend to regions other
than cerebellum, the only region whose growth is affected
by transient neonatal polyamine depletion [1,5,16]. The
significance of these results extends beyond the effects of
DFMO. Perturbations of ODC activity and polyamines
have been identified for numerous suspected neuroteratogens [4,9,12,15,17], suggesting in these cases, too,
the effects on the ODC pathway may be mechanistically
connected to lasting changes in synaptic function, even
when growth and structure are apparently normal. The
ODC / polyamine system thus provides a potential mechanistic link between initial neuroteratogenic insults and the
eventual functional outcome, rather than representing just a
marker for perturbed growth and development.
Acknowledgements
This study was supported by USPHS HD-09713.
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