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Department of Biochemistry and Molecular Biology, University of Minnesota, Duluth MN 55812
2To whom correspondence should be addressed.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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KEY WORDS: • copper deficiency • rats • mRNA • dopamine ß-monooxygenase
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). When Cu is inadequate
for the cuproenzyme to catalyze its reaction at full capacity, an
altered biochemical phenotype is observed (Prohaska 1990
). For example, decreased melanin formation due to reduced
tyrosinase activity leads to hypopigmentation. Decreased crosslinking
of collagen and elastin due to reduced lysyl oxidase leads to a
connective tissue disorder. For other cuproenzymes, the connection
between lower enzyme activity and altered phenotype is less definitive.
Dopamine ß-monooxygenase
(DBM)3
is a cuproenzyme that catalyzes the final step in the biosynthesis of
norepinephrine (NE) by hydroxylating dopamine (DA) in an ascorbate- and
oxygen-dependent reaction (Friedman and Kaufman 1965
). DBM is located in adrenal medulla, sympathetic neurons,
and noradrenergic and adrenergic neurons of the brain. DBM is essential
for embryonic development of the mouse as demonstrated in recent
studies in which the DBM gene was ablated (Thomas et al. 1995
). In humans, the lack of DBM results in severe hypotension
(Gary and Robertson 1994
). It has not been clearly
established whether the attenuation of DBM activity that occurs when Cu
is limiting results in abnormal physiology. In fact, the Cu story
regarding DBM is quite puzzling.
Shortly after the discovery that DBM was a cuproenzyme, radioisotopic
studies in rats indicated that dietary Cu deficiency decreased the
conversion of cardiac DA to NE (Missala et al. 1967
).
Indeed, the steady-state NE concentration is lower in hearts from
Cu-deficient rats compared with controls (Prohaska and Heller 1982
). Later, it was shown that DA is elevated in hearts
of Cu-deficient rats, supporting the hypothesis that DBM is altered
by Cu deficiency (Prohaska et al. 1990
).
The original Cu-DBM history in the central nervous system (CNS) is
due in large part to the seminal work of Hunt and Johnson (1972)
, who studied mottled mice. Mutations at the mottled
locus in mice are homologous with mutations in humans with Menkes’
syndrome. Before the Cu-mottled mutant story was even known, they
reported that brains of mutant mice converted less
[3H]-tyrosine to NE and more
[3H]-tyrosine to DA than control littermates.
They concluded that the former observation was due to decreased DBM and
the latter to enhanced tyrosine hydroxylase activity, which they
confirmed directly. In addition, they reported lower steady-state
NE levels in brain of the mutant mice compared with littermates
(Hunt and Johnson 1972
). Two years later Hunt (1974)
reported that Cu was lower in brains of the mutant mice,
and the Cu DBM connection in brain was formulated. Concurrently, it was
reported that brain NE was lower in Cu-deficient rats compared with
controls (Prohaska and Wells 1974
). The NE deficit was
also observed in Cu-deficient lambs (O’Dell et al. 1976
). Regional analyses in rodent brain indicated that
Cu-deficient rats and mice have lower NE concentrations in all
regions except hypothalamus (Feller and O’Dell 1980
,
Prohaska and Bailey 1993
and 1994
). Cu-deficient
rodents exhibit elevated DA in brain regions enriched in noradrenergic
neurons (Prohaska and Bailey 1993
and 1994
). These data
provide strong support for limiting DBM activity in brain after Cu
deficiency.
However, direct enzyme assay of brain homogenates demonstrated higher
DBM activity in mutant mouse brain (Hunt 1974
,
Prohaska and Smith 1982
) and higher activity of DBM
after dietary Cu deficiency in mice (Prohaska and Smith 1982
) and rats (Prohaska and Bailey 1994
).
Regional analyses of six rat brain areas confirmed and extended earlier
work that DBM activity, measured in vitro, was higher in brains of
Cu-deficient rats (Prohaska and Bailey 1995
). Thus,
a paradox exists. On the basis of metabolite levels, DBM activity is
lower; using direct assay, however, DBM activity is higher after Cu
deficiency. Assay of DBM requires the addition of an agent, usually
Cu2+ or N-ethylmaleimide (NEM) to
inactivate an endogenous inhibitor. Perhaps there are different levels
of inhibitors or activators after Cu deficiency.
The adrenal gland is a rich source of DBM, and catecholamines have been
studied after Cu deficiency. Hesketh (1981)
reported
lower NE, but not epinephrine levels in adrenals of Cu-deficient
rats and elevated DBM activity. In cattle, the catecholamine data were
confirmed, but not DBM activity (Hesketh 1980
). In
Cu-deficient mutant mice, no change in catecholamine content was
detected, but higher DBM activity in adrenal gland was measured
(Hunt 1977
). After perinatal Cu deficiency in young male
rats, adrenal DBM activity was 44% higher than control values
(Prohaska and Bailey 1994
). Adrenal NE content
was reported to be lower in Cu-deficient mice but not rats; in both
species, DA was markedly higher in adrenal gland of Cu-deficient
rodents (Prohaska et al. 1990
). Epinephrine content was
not altered in either species. These facts suggest phenomena in adrenal
gland similar to those in brain, i.e., a paradox between metabolite and
enzyme assay data.
The purpose of these experiments was to extend earlier observations on brain and adrenal DBM in two models of dietary Cu deficiency, measuring metabolites, enzyme activity and steady-state mRNA levels. A postnatal model studying the effect of postweaning Cu deficiency and a perinatal model studying the effect of gestational-lactational Cu deficiency were compared. DBM mRNA was quantified to test the hypothesis that higher DBM activity is due to increased DBM levels and not the presence of activators or lower inhibitors.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Sperm-positive Sprague-Dawley rats and male weanling Holtzman rats
were purchased commercially (Harlan Sprague Dawley, Indianapolis, IN).
Rats were fed one of two dietary treatments, copper deficient or copper
adequate, consisting of a Cu-deficient purified diet (Teklad
Laboratories, Madison, WI) and either low Cu drinking water or
Cu-supplemented drinking water, respectively. The purified diet was
similar to the AIN-76A diet (AIN 1977
and 1980
) and
contained the following major components (g/kg diet): sucrose, 500;
casein, 200; cornstarch, 150; corn oil, 50; cellulose, 50; modified
AIN-76 mineral mix, 35; AIN-76A vitamin mix, 10;
DL-methionine, 3; choline bitartrate, 2; and ethoxyquin
0.01. Cupric carbonate was omitted from the AIN-76 mineral mix. The
purified diet contained 0.30 mg Cu/kg and 44 mg Fe/kg by chemical
analysis. Holtzman males, Sprague-Dawley offspring and dams
consuming the Cu-deficient treatment drank deionized water, whereas
Cu-adequate treatment groups drank water that contained 20 mg Cu/L
by the addition of CuSO4 to the drinking water. Rats were
given free access to diet and drinking water. All rats were maintained
at 24°C with 55% relative humidity on a 12-h light:dark cycle
(lights on 0700–1900 h). All protocols were approved formally by the
University of Minnesota Animal Care Committee.
In Experiment 1, male weanling Holtzman rats (n = 16) were divided equally and randomly assigned to either Cu-deficient or Cu-adequate treatments. Rats were maintained on their respective treatments for 4 wk in stainless steel cages.
In Experiment 2, Sprague-Dawley pregnant dams were given the
Cu-deficient treatment 7 d after they were identified as
sperm-positive. Two days after parturition, litter size was
adjusted to eight pups. Offspring were weaned when 3 wk old and were
given the same treatment as their respective dams for an additional
24 h. A total of eight litters [four Cu adequate (+Cu) and four
Cu deficient (-Cu)] were studied. This paradigm is similar to that
described previously (Prohaska and Bailey 1994
).
Remaining offspring (both +Cu and -Cu) were offered a nonpurified
commercial diet, Purina LRC 5001 (Ralston Purina, St. Louis, MO), and
tap water. Six weeks after Cu repletion (the LRC diet contained 14 mg
Cu/kg), four rats from each treatment group, Cu-adequate and
Cu-repleted female and male, were sampled to evaluate recovery.
Rats were killed by decapitation. Livers, adrenal glands and brains
were removed, weighed and processed for biochemical analysis. Males and
females were killed on consecutive days in Experiment 2. Brains were
dissected on a chilled glass plate according to established guidelines
(Glowinski and Iversen 1966
). The cerebellum, medulla
oblongata + pons, cerebral cortex (cerebrum) and remainder, referred to
as "midbrain" for these experiments, were dissected and frozen in
liquid nitrogen. Some adrenal glands were homogenized for 30 s in
24 vol of 0.05 mol/L potassium phosphate (pH 7.0) using a Tissumizer
and microprobe (SDT-080 EN, Tekmar, Cincinnati, OH).
Biochemical analyses.
Hemoglobin was determined spectrophotometrically as metcyanhemoglobin.
Adrenal homogenate protein levels were measured by a modified Lowry
procedure using bovine albumin as reference (Markwell et al. 1978
). Adrenal catecholamine pools of epinephrine, NE and DA
were determined by HPLC with electrochemical detection as described
previously (Prohaska et al. 1990
).
Copper analyses.
Portions of liver and diet (~1 g each) and the entire cerebrum were weighed to the nearest milligram and wet-digested with 4 mL concentrated HNO3 (AR select grade, Mallinckrodt, St. Louis, MO); the residue was brought to 4.0 mL with 0.1 mol/L HNO3. Samples were then analyzed for total Cu and Fe by flame atomic absorption spectroscopy (Model 2380, Perkin-Elmer, Norwalk, CT). The method was checked with a certified standard, U.S. National Bureau of Standards 1577 bovine liver (Gaithersburg, MD).
DBM assay.
Activity of adrenal gland DBM (EC 1.14.17.1) was determined
spectrophotometrically as described previously (Prohaska and Smith 1982
). The endogenous inhibitor of DBM activity was
inactivated by 25 mmol/L NEM rather than copper. Homogenates were
diluted in 0.005 mol/L potassium phosphate (pH 7.0) containing 0.2%
Triton X-100 and centrifuged at 6,500 x g for 10
min. This phosphate-Triton buffer was stored in an acid-washed
bottle containing 1 g of Chelex 100 (Bio-Rad Laboratories,
Hercules CA) suspended within dialysis tubing.
Northern blot analysis.
Total adrenal and brain RNA was isolated from quick-frozen samples
using a modified guanidium thiocyanate/phenol/chloroform procedure
(Chomczynski and Sacchi 1987
). Original RNA pellets were
resuspended in 600 µL guanidium thiocyanate buffer,
then reextracted with 60 µL of 2 mol/L sodium acetate
(pH 4.0), 300 µL water saturated phenol and 300
µL chloroform/isoamyl alcohol (49:1). RNA was
precipitated from the aqueous supernate by adding 1 vol cold
isopropanol. RNA concentration and 260/280 ratios were determined by
spectrophotometry (Model DU 640, Beckman Instruments, Palo Alto, CA).
Total RNA, 10–15 µg, was size fractionated on 1.0 or
1.5% agarose gels containing 2 mol/L formaldehyde and 0.02 mol/L
sodium phosphate (pH 7.0). After electrophoresis, gels were stained
with acridine orange (Sigma Chemical, St. Louis, MO) and photographed
to determine RNA integrity. RNA was transferred to Nytran Plus
membranes (Schleicher & Schuell, Keene, NH) by upward capillary
transfer with 20X SSC (3 mol/L NaCl, 0.3 mol/L sodium citrate, pH 7.0).
Positions of 28S and 18S ribosomal RNA were marked after transfer,
membranes were briefly rinsed in 6X SSC and RNA was fixed by baking at
80°C under vacuum for 2 h. Membranes were rinsed in 1X SSC 0.1%
SDS for 30 min and prehybridized in a solution containing 50%
formamide, 5X Denhardt’s, 5X SSC, 0.05 mol/L sodium phosphate (pH
6.5), 0.1% SDS, 0.1% Na4P2O7 and
50 mg/L salmon sperm DNA (Sigma) for 6 h at 42°C. The membranes
were then hybridized overnight with a purified 32P
random-primed 2.2-kb probe for rat DBM (1.7 ng/mL). Membranes were
washed twice with 2X SSC 0.1% SDS for 30 min., once with 0.1X SSC
0.1% SDS for 30 min and once with 0.1X SSC 0.1% SDS at 55°C. Washed
membranes were wrapped in plastic and exposed to film. After
autoradiography, membranes were stripped with near-boiling 0.1X SSC
0.1% SDS and rehybridized with a 32P-labeled 1.2-kb probe
for mouse 18S ribosomal RNA (Ambion, Austin, TX) to verify equal
loading and transfer of RNA. Images of autoradiograms were captured
using Gel Doc 1000 (Bio-Rad) interfaced with a Macintosh PPC computer,
and density of band profiles was integrated using manufacture’s
software Molecular Analyst (Bio-Rad).
DBM probe preparation.
A Bluescript M13-plasmid–containing rat DBM (McMahon et al. 1990
) was expressed in Escherichia coli after
electroporation. Amplified plasmid DNA was purified with Wizard
Megapreps DNA Purification System (Promega, Madison, WI); a 2.2-kb
EcoR1 fragment corresponding to nucleotides 260-2402 of rat DBM was cut
from the plasmid, separated via agarose gel electrophoresis and
purified from the gel using GenElute Agarose Spin Columns (Supelco,
Bellefonte, PA). The purified DBM probe was random-prime labeled
(High Prime Kit, Roche Molecular Biochemicals, Indianapolis, IN) with
[
-32P] dCTP (Amersham, Arlington Heights, IL) and
separated from nucleotides using ProbeQuant G-50 Micro Columns
(Pharmacia Biotech, Piscataway, NJ). Background binding was reduced by
incubating the denatured probe to a blank membrane for 4 h at
42°C before hybridizing to RNA membranes.
Statistics.
Dietary treatment effects were evaluated by Student’s t
test after variance equality was tested, = 0.05. Data were
analyzed using a personal computer and statistical software (Statview
4.5, Abacus Concepts, Berkeley, CA).
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). Compared with +Cu rats, -Cu rats were smaller and had higher
heart/body weight ratios (data not shown). Liver Cu levels in -Cu rats
were 10% of those in +Cu rats, whereas liver Fe levels were 123%
higher than those in +Cu rats. Hemoglobin concentration in -Cu rats
was 69% of that in +Cu rats. These features of -Cu rats indicate a
state of severe Cu deficiency.
|
). The mean value for -Cu rats was 140% higher than the mean +Cu
value. Protein concentration was not altered in adrenal homogenates by
Cu deficiency. A mixing experiment was conducted in which samples from
three +Cu and three -Cu rats were run individually and then mixed in
equal parts to determine whether endogenous inhibitors (+Cu samples) or
activators (-Cu samples) might explain the 1.4-fold difference in
enzyme activity. The value determined was 102 ± 4.5% (mean
± SD) of the theoretical value, consistent with the
lack of any obvious endogenous factors affecting the adrenal DBM
activity in the two sample pools.
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). The -Cu values were 180% higher than those of the +Cu rats, in
agreement with the DBM enzyme activity data. The 18S signal was not
different between groups. The mean density ratio -Cu/+Cu was 1.05.
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)] were extracted and analyzed for catecholamines
(Fig. 3
). Compared with +Cu rats, the -Cu rats had a lower NE content and
higher DA content in adrenal gland (P < 0.05).
Epinephrine content was not affected by dietary Cu deficiency.
|
). Both copper-deficient females (-CuF) and copper-deficient
males (-CuM) were smaller and had enlarged hearts (data not shown)
compared with their sex-matched controls, although body weight
differences in the males were not significant (P < 0.06). Weights of adrenal glands and brains were not affected by
perinatal Cu deficiency (Table 2)
. Anemia was observed in -CuF and
-CuM rats compared with their respective control groups (Table 2)
.
Liver Cu concentration of -CuF and -CuM rats was 3.4 and 5.7%,
respectively, that of +Cu rats. Liver Fe concentration was higher in
-CuM than copper-adequate males (+CuM). The concentration of Cu in
cerebrum of -CuF and -CuM rats was 18 and 20%, respectively, of +Cu
values. Together, these results support the hypothesis that the -CuF
and -CuM rats exhibited signs characteristic of severe Cu deficiency.
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). A robust enhancement of the DBM mRNA steady-state level can be
seen in all three lanes from the -CuM rats compared with the +CuM
samples. On average, the ratio of DBM/18S for -CuM samples was 175%
higher than that of the +Cu ratio, an enhancement similar to that
observed in the adrenal samples of the -Cu rats in the postnatal model
(Fig. 2)
. The 18S signal was not different between -Cu and +Cu lanes.
The mean ratio of -Cu/+Cu was 0.91.
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). In contrast to the -Cu males in the postnatal model (Fig. 3)
, there
was no difference in adrenal NE content in the 22-d-old females (Fig. 5)
. However, in confirmation of the postnatal model data, there was a
significant 3.8-fold elevation in DA content, suggesting DBM
impairment. Adrenal epinephrine content was not altered in the
perinatal model of Cu deficiency, in agreement with the data on older
rats (Fig. 3)
. The absolute levels of the adrenal catecholamines in the
48-d-old Holtzman males and 22-d-old Sprague-Dawley females (Figs. 3
, 5)
seem roughly proportional to adrenal gland size (Tables 1
and 2
).
|
). Panel A shows 8 lanes loaded with 20 µg of
total RNA alternating with four -CuM and four +CuM samples. In only
the first pair of loaded samples did there appear to be an enhancement
of the DBM mRNA signal. The mean DBM/18S area ratio for -CuM samples
was not different than the value for +CuM. However, in panel
B, the mean DBM/18S ratio for -CuF was 52% higher than the
corresponding ratio for copper-adequate females (+CuF),
P < 0.05. The 18S signal for medulla oblongata/pons
for -Cu rats was not different from that of +Cu rats. The mean ratio
for -CuM/+CuM was 1.02 and for -CuF/+CuF was 0.95. Overall, in only
about half of the brain -Cu samples did there appear to be a clear cut
elevation in DBM mRNA after Cu deficiency in contrast to the consistent
and more robust response observed in adrenal gland (Figs. 2
, 4)
. We
attempted to detect DBM mRNA in the "midbrain" samples from male
offspring and found only a weak signal in two +CuM samples and none in
the four -CuM samples (data not shown). We were also unable to detect
a DBM mRNA signal in hypothalamus using RNA from rats of another study
(data not shown) despite the highest DBM enzyme activity in this region
(Prohaska and Bailey 1995
).
|
). Furthermore, adrenal DBM mRNA levels were no longer significantly
elevated, suggesting that the observations made in the 22-d-old -CuM
rats were reversible.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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communicated a similar observation in 7-wk-old -Cu rats in a perinatal
Cu deficiency model. In the current postnatal studies, the mRNA
increase and enzyme activity increase were approximately equal. This
suggests that the mRNA level is reflecting protein levels. In the
perinatal model, the increase in DBM mRNA was much greater than the
44% increase in adrenal enzyme activity that was found in another
group of rats treated similarly (Prohaska and Bailey 1994
). Hesketh (1981)
also reported higher
adrenal DBM activity in a postnatal Cu-deficient rat model. This
rise in adrenal DBM mRNA was eliminated by restoring Cu to the diet of
-CuM rats, suggesting the transitory nature of the induction.
Medulla oblongata/pons was chosen to represent CNS tissue enriched in
cell bodies that contain DBM. The activity of DBM in this region is
~1% that of adrenal gland (Prohaska and Bailey 1994
and 1995
); thus, it was more challenging to detect DBM mRNA levels.
However, results in these studies indicate a modest effect (significant
in females) that supports adrenal data indicating an up-regulation
of DBM mRNA after perinatal Cu deficiency. In -CuF, the average
increase in medulla oblongata/pons DBM mRNA was 52%, similar to the
elevation in DBM enzyme activity measured previously for -CuF (25%)
and -CuM (43%) (Prohaska and Bailey 1995
). The degree
of Cu deficiency in brain in the current model, as assessed by cerebrum
Cu concentration, is similar to that observed previously
(Prohaska and Bailey 1994
), suggesting that the RNA data
are representative of this model. Previous results in the postnatal
model indicated that although brain Cu was lower in -Cu rats by 36%,
there was no change in DBM activity (Prohaska et al. 1995
); thus, RNA analyses were not performed on brains of the
older rats.
What might be the cellular signal responsible for increasing DBM mRNA?
A likely candidate is depletion of NE. Treatment with reserpine, a drug
that depletes catecholamines, results in induction of rat adrenal DBM
mRNA (McMahon et al. 1990
). Reserpine treatment of rats
also elevates DBM activity and protein level in the brain in a time
course that parallels depletion of monoamines (Reis et al. 1975
). In the CNS, there is reproducibly lower NE in the
medulla oblongata/pons of -Cu rodents, supporting the hypothesis that
low NE induces DBM transcription.
However, in the adrenal gland, the data are less clear. In the current
studies, for example, there was evidence of lower NE in the postnatal
model but not the perinatal model, yet both yielded similar mRNA
enhancements. Others have reported varying outcomes of postnatal Cu
deficiency on adrenal NE content. Hesketh (1981)
reported lower NE in -CuM rats. Fields et al. (1991)
reported higher NE in -CuM rats. Prohaska et al. (1990)
reported no changes in NE content in -CuM rats. In those cases in
which DA was measured, a robust increase was detected even though NE
data were not consistent. Others have shown that DBM is
transcriptionally regulated by many factors such as cAMP,
glucocorticoids, bradykinin, nicotine and immobilization stress
(McMahon and Sabban 1992
). Perhaps the rise in DBM mRNA
is due to one or more of these factors. A candidate for further
research is glucocorticoids because it is known that -Cu rats have
elevated levels in the adrenal gland and plasma (Fields et al. 1991
).
Negative results of mixing experiments in the current studies support
the proposal that there is more DBM protein present after Cu deficiency
rather than abnormal levels of endogenous factors. It is possible that
the extra apo-DBM is not active in vivo because of limiting free Cu
ion. Thus far, no specific Cu-chaperon protein for DBM has been
identified; thus, the specific details of how Cu is inserted into
apo-DBM are not known. However, because of the facile and rapid
exchange of free Cu with apo-DBM (Skotland and Flatmark 1983
), it is feasible that extra apo-DBM can be activated
with traces of free Cu when assayed in the laboratory. This would
explain the higher DBM activity detected in the laboratory assay and is
consistent with the higher mRNA levels. Measurement of DBM protein will
be required to confirm this supposition.
Catecholamine data presented in these two experiments and those
discussed previously suggest that DBM activity is impaired in vivo.
Catecholamine data in humans with Cu deficiency due to Menkes’
syndrome also support defective DBM function (Hoeldtke et al. 1988
). It will be more challenging to determine whether any of
the phenotypic and developmental abnormalities associated with Cu
deficiency are due to catecholamine imbalance.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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3 Abbreviations used: CNS, central nervous system; +CuF, copper-adequate female; -CuF, copper-deficient female;
+CuM, copper-adequate male; -CuM, copper-deficient male; DA,
dopamine; DBM, dopamine ß-monooxygenase (EC 1.14.17.1); NE, norepinephrine; NEM, N-ethylmaleimide.
Manuscript received July 8, 1999. Initial review completed August 18, 1999. Revision accepted September 7, 1999.
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