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Introduction
Opiates, such as morphine and heroin, exhibit a variety
of physiological activities and have been utilized
extensively in medicine, most prominently in the treatment of
pain. However, at analgesic doses, opioid receptor
agonists or partial agonists can induce unwanted side effects
such as ventilatory depression[1,2], constipation, and the
development of physical tolerance and
dependence[3,4]. It is very important to investigate the biochemical mechanism and
the basis of tolerance and dependence of morphine and heroin.
There were several reports on the relationship between
opiate administration and changes in nucleotide metabolism.
We have demonstrated that heroin could enhance the
catabolism of purine nucleotides in
vivo[5]. Heroin is a semisynthetic morphine ester, with 2 acetyl groups coupled to
the 3- and 6-hydroxyl groups of morphine. After absorption,
heroin is rapidly hydrolyzed to 6-monoacetylmorphine and
morphine by serum and liver esterases. Our previous
experiments in rat C6 glioma cells[6] found that the expression of
these key enzymes, hypoxanthine guanine phosphoribosyl
transferase (HGPRT) and adenosine kinase (AK), in the
purine nucleotide salvage pathway were downregulated by
morphine, which suggested that morphine inhibits purine
nucleotide anabolism in cells. Other studies on primary cell
cultures and cell lines from the brain in
vitro demonstrated that morphine and other opioids often inhibit the synthesis
of DNA and RNA[7_11].
Experiments have revealed that the metabolic problem of
nucleic acid and nucleotides induced by morphine
administration is an important subject. In order to clarify the effects
and possible mechanism of morphine and heroin in
nucleotide catabolism in vivo and in
vitro, and to explore the biochemical pharmacological mechanism of morphine
dependence, tolerance, and withdrawal, we sought to
determine whether morphine exposure affects the
metabolism of purine nucleotide in vivo and
in vitro. In this study, the rat model of morphine dependence and
withdrawal was established and rat C6 glioma cells were used. As
adenosine is not only the intermediate of purine nucleotide
metabolism, but also an important neuromodulator involved
in both acute and chronic effect of
opiates[12_14], we focused on AK and adenosine deaminase (ADA), 2 key enzymes of
anabolism and catabolism of adenosine, respectively.
However, ADA might be the predominant pathway for
adenosine metabolism[15]. ADA and xanthine oxidase (XO)
are 2 key enzymes regulating the catabolism of purine
nucleotides. Therefore those 2 enzymes were chosen for
the investigation of the biochemical pharmacology
mechanism of morphine function.
Materials and methods
Animals and drug treatment Adult, female Wistar rats
(n=50, Grade II, initial weight: 180±50 g, Jilin University
Animal Laboratories, Jilin, China) were chosen for the
study. The certificate number of the breeder was 3020086.
The rats were housed under controlled environmental
conditions with free access to food and water, and were
randomly divided into 5 groups for the study. Each group of
rats included 10 animals and was administered the drug by
injection twice a day at 12 h intervals. The first group of
rats was the control group, in which each rat was
administered saline (ip) at the same dose as the daily morphine
administration for 7 d. The second group was the 3 d
morphine-administered group, in which each rat was
administered morphine (ip) for 3 d with increasing daily doses; 20
mg/kg on d 1, 30 mg/kg on d 2, and 45 mg/kg on d 3. The
third group was the 7 d morphine-administered group, in
which each rat was administered morphine (ip) for 7 d with
daily doses of 20, 30, 45, 55, 65, 85, and 95 mg/kg,
respec-tively. The fourth and fifth groups of rats were for the
morphine natural withdrawal investigation. Both groups of rats
received the same dose of drugs as the third group. The
fourth group of rats were euthanatized on the 11th day (3 d
after morphine withdrawal), while the fifth group of rats were
euthanatized on the 15th day (7 d after morphine withdrawal).
Cell culture C6 glioma cells were provided by the
Shanghai Institute of Cell Research (China). The cells were
maintained in monolayer culture at 37 °C and 5%
CO2 in Dulbecco's modified Eagle's medium, plus 10% fetal calf serum
(heat-inactivated). For all the experiments, the cells were treated
with 0.25% trypsin on plastic culture flasks
(5×105 cells/mL). After overnight incubation, the culture medium was changed;
the cells were replenished with fresh media and treated with
opioid receptor agonist morphine (10 µg/mL) for a selected
time course from 6 to 72 h in the first group. In the second
group, the cells were pretreated with the opioid receptor
antagonist naloxone (0.4 µg/mL diluted with the culture
medium) for 1 h and then treated with naloxone plus
morphine (10 µg/mL diluted with the culture medium) for 24 h. In
the third group, the cells were pretreated with morphine (10
µg/mL diluted with the culture medium) for 1 h and then
treated with naloxone plus morphine (10 µg/mL diluted with
the culture medium) for 24 h. The cells were then washed
with Phosphate-Buffered Salines (PBS), harvested and
processed for RNA isolation with Trizol.
Reagents Morphine hydrochloride was provided by the
First Pharmacy of Shenyang (Shenyang, China). Trizol was
provided by Gibco BRL (Grand Island, NY, USA ). Reverse
transcriptase (MMLV-RT) and RNAsin were provided by
Promega (Shanghai, China). Oligo-dT, dNTP, and
Taq polymerase were provided by Dingguo (Beijing, China). The ADA
and XO detection kits were provided by Nanjing Jiancheng
Biotechnology (Nanjing, China). The uric acid detection
kits, creatinine detection kits, and urea nitrogen detection
kits were bought from Kehua Dongling Diagnostic Products
(Shanghai, China). The specific primers used for RT-PCR
are listed in Table 1. The pGEM-T easy vector and
Prime-a-Gene labeling system were provided by Promega (Shanghai,
China).
Uric acid detection The concentrations of plasma uric
acid were measured by the uricase-rap method with uric acid
detection kits as noted earlier.
Plasma creatinine detection The concentrations of
plasma creatinine were measured by the sarcosine
oxidase-rap method with creatinine detection kits as noted earlier.
Plasma urea nitrogen detection The concentrations of
plasma urea nitrogen were measured by the UV-GLDH method
with the urea nitrogen detection kits as noted earlier.
ADA detection Ten percent homogenates of the parietal
lobe, liver, small intestine, and skeletal muscles were prepared,
and concentrations of ADA in the plasma and tissues were
measured by the ADA detection kits as noted earlier.
XO detection Ten percent homogenates of the parietal
lobe, liver, small intestine, and skeletal muscles were prepared,
and concentrations of XO in the plasma and tissues were
measured by the XO detection kits as noted earlier.
RT-PCR for the evaluation of ADA mRNA transcripts
Two µg total RNA isolated with Trizol from tissues was
reverse transcribed in a volume of 15 µL. PCR was
performed with 1 µL cDNA solution (representing about 0.1 µg
cDNA). The PCR mixture contained PCR buffer (10 mmol/L
Tris-HCl, pH 8.3; 50 mmol/L KCl, 1.5 mmol/L
MgCl2, 0.001% [w/v] gelatin), 0.2 mmol/L dNTP, 50 pmol of each of the 5' and
3' primers for ADA, 25 pmol of the 5' and 3' β-actin 1 primers,
and 2.5 units Taq polymerase in a total volume of 50 µL. The
PCR reaction was carried out in a DNA thermal cycler. The
amplification cycle for ADA denaturation was 95 °C for 35 s,
annealing at 62 °C for 1 min, and extension at 72 °C for 1 min,
and repeated for 35 cycles. The band intensity was
quantified with a Kodak electrophoresis documentation analysis
system (EDAS; New Haven, Connecticut , USA). The ratios
of the intensity of bands of ADA/b-actin 1 were calculated
to estimate the level of ADA mRNA transcripts.
RT-PCR-Southern hybridization for the evaluation of
the XO mRNA transcript level RT-PCR was performed in
the same way as mentioned earlier, except that 50 pmol of
each of the 5' and 3' XO primers and 5' and 3' of the
b-actin 2 primers were used. The amplification cycle for XO was 94 °C
for 35 s, 60 °C for 1 min, and 72 °C for 1 min, with 30 repeated
cycles.
Southern blot hybridization When 15 µL XO and
b-actin 2 PCR products were electrophoresed on 1.5% agarose gel
stained with ethidium bromide, only b-actin 2 products were
observed; therefore, Southern hybridization was performed.
The DNA was transferred to a nylon membrane. The blots
were prehybridized in 5×SSPE (1×SSPE contains 0.15 mol/L
NaCl, 0.01 mol/L NaH2PO4, 0.001 mol/L EDTA), 1% SDS and
10×Denhardt's solution (1×Denhardt's solution contains
0.02% polyvinylpyrrolidine, 0.02% ficoll 400, and 0.02%
bovine serum albumin at 61 °C for 1 h. Internal XO cDNA
obtained from previously made recombinant pGEM-T Easy
vector was labeled with [α-32P]-dCTP according to the
instructions included with the Prime-a-Gene labeling system,
and was then added to the prehybridization buffer and
hybridized at 65 °C for 16 h. The blots were washed twice at
room temperature in 2×SSC (0.3 mol/L sodium chloride and
0.03 mol/L sodium citrate) with 0.1% SDS, then washed at
65 °C in 0.1×SSC with 0.1% SDS, and finally washed at room
temperature in 0.1×SSC.
Statistical analysis Data were presented as mean±SD.
Statistical significance was assessed by the paired
t-test with significance assumed when
P£0.05.
Results
Plasma biochemical detection
Uric acid detection There were significant increases in
the concentrations of plasma uric acid in the rats
administered with morphine for 3 and 7 d, as compared to the control
group (P<0.05). During natural withdrawal, the
concentrations of plasma uric acid in the rats exhibited a decreased
tendency compared to the 7 d morphine-administered group,
but were still higher than that of the control group (Table 2).
Urea nitrogen detection There was no increase of plasma
urea nitrogen in the 3 d morphine-administered group when
compared to the control, while the plasma urea nitrogen in
the 7 d morphine-administered group and 2 withdrawal
groups were significantly higher than that of the control
(P<0.05; Table 2).
Creatinine detection Compared to the control group,
there was no significant plasma creatinine enhancement in
the morphine-administered and withdrawal groups (Table 2).
ADA and XO detection During morphine
administration and natural withdrawal, plasma ADA and XO levels were
all significantly higher than that of the control
(P<0.05; Tables 3, 4).
Tissue ADA detection The activity detection of ADA in
tissues found that morphine administration significantly
increased the parietal lobe ADA in the 7 d
morphine-administered group and in the 3 d natural withdrawal group when
compared to the control (P<0.05). There was a significant
increase of liver ADA in the morphine-administered rats when
compared to the control (P<0.05), while the liver ADA in the
withdrawal group recovered to the control level gradually.
The level of small intestine ADA was higher in the 3 and 7 d
morphine-administered groups and the natural withdrawal
7 d group than in the control group, but with no significant
changes. There was a significant increase of skeletal muscle
ADA in the morphine-administered groups compared to the
control group (P<0.05), while the ADA levels were still higher
in the natural withdrawal groups than in the control, but with
no significant changes. Whereas, the ADA level in the
natural withdrawal 7 d group was significantly lower than in the
7 d morphine-administered group (Table 3).
ADA mRNA detection showed a significant increase of
the parietal lobe ADA mRNA levels in the 3 d
morphine-administered group; however, there was no significant
change of the parietal lobe ADA mRNA in the 7 d
morphine-administered and natural withdrawal groups, compared to
the control groups (Figure 1A, 1B). There was a significant
increase of the liver ADA transcription level in the 3 and 7 d
morphine-administered groups. Compared to the
morphine-administered groups, the effect of morphine decreased
gradually and the liver ADA mRNA levels recovered to the control
level in the withdrawal groups (Figure 2A, 2B). There was
significant upregulated transcription of small intestine ADA
in the rats after treatment with morphine for 7 d. Compared
to the controls, there were no significant changes of small
intestine ADA mRNA in the natural withdrawal groups (Figure
3A, 3B). The ADA mRNA levels of the skeletal muscles were
upregulated both in the morphine-administered groups and
the natural withdrawal groups when compared to the control
group (Figure 4A, 4B).
Tissue XO detection Activity detection of XO in tissues
showed that during morphine administration and natural
withdrawal, the amounts of parietal lobe XO were
significantly higher than the control level
(P<0.05). There was a significant increase of liver XO in the morphine-administered
rats (P<0.05). Compared to the morphine-administered groups,
the effect of morphine gradually decreased and the amount
of liver XO decreased to the control level in the withdrawal
groups (P<0.05). The amounts of small intestine XO in the 3
and 7 d morphine-administered groups were significantly
higher than in the control group (P<0.05), while compared to
the morphine-administered groups, the amounts of XO in
the withdrawal group recovered to the control level
gradually (P<0.05). There was a significant increase of skeletal
muscle XO in the 7 day morphine-administered group as
compared to the corresponding control group
(P<0.05), while the amounts of XO in the withdrawal group gradually
recovered to that of the control level (Table 4).
XO mRNA detection showed a significant increase of the
parietal lobe XO mRNA in the 7 d morphine-administered
group, but there was no significant change of the parietal
lobe XO mRNA in the natural withdrawal groups, compared
to the corresponding control group (Figure 5A_5C). There
was a significant increase of liver XO mRNA levels in the 3
and 7 d morphine-administered groups. Compared to the
morphine-administered groups, the effect of morphine
gradually decreased and the liver XO mRNA levels recovered to
the control level in the withdrawal groups (Figure 6A_6C).
There were significant upregulation of small intestine XO
mRNA in the morphine-administered and natural withdrawal
groups compared to the control group (Figure 7A_7C). The
amounts of skeletal muscle XO mRNA were upregulated in
the 7 d morphine-administered and natural withdrawal groups
compared to the control group (Figure 8A_8C).
Detection of ADA mRNA in C6 cells Compared to the
corresponding control, morphine treatment upregulated ADA
mRNA in C6 cells. ADA mRNA was increased at all of the
selected time courses after exposure to morphine (Figure
9A, 9B). When the cells were pretreated with naloxone for
1 h and then treated with morphine for 24 h, or pretreated
with morphine for 1 h and then treated with naloxone for
24 h, the upregulation of ADA mRNA was prevented (Figure
10A, 10B).
Detection of XO mRNA in C6 cells The
[α-32P]-labeled specific XO cDNA probe hybridized with the RT-PCR
product of the cells. The hybridizing signals were compared to
the total intensity of b-actin 2 products stained on agarose
by ethidium bromide. Compared with the corresponding
period control cells, morphine upregulated XO mRNA in C6
cells. The XO mRNA increased at all selected time periods
after exposure of the cells to morphine, (Figure 11A_11C).
When the cells were pretreated with naloxone for 1 h and
then treated with morphine for 24 h, or pretreated with
morphine for 1 h and then treated with naloxone for 24 h, the
upregulated transcription of XO was inhibited (Figure
12A_12C).
Discussion
In addition to their roles as the subunits of nucleic acids,
purine nucleotides have a variety of other functions in every
cell such as being energy carries, components of enzyme
cofactors, and chemical messengers. Many experiments have
showed that intracellular cAMP and cGMP regulation
systems play an important role in the mechanisms of morphine
dependence and withdrawal[16,17]. Recent papers have
reported that tetrahydrobiopterin (BH4) is an essential
cofactor for tyrosine hydroxylase, tryptophan hydroxylase,
and nitric oxide (NO) synthase[18]. Dopamine and NO are
important neurotransmitters. Many previous studies
showed that their abnormality was concerned with morphine
dependence and tolerance[19_22]. As GTP is the precursor of
BH4, excessive catabolism and insufficient anabolism of
purine nucleotides by morphine administration may cause
serious subsequent effects in vivo.
Previous experiments have demonstrated that morphine
administered intravenously increases the rate of efflux of
purines from the intact rat cerebral cortex, and naloxone
antagonized morphine's action[23]. In addition, recent
ex vivo and in vivo findings have shown that morphine increases
uric acid concentrations in the rat
striatum[24_26]. The reasons for uric acid concentration increases have not been
explained. Uric acid is the final product of purine nucleotide
catabolism in primates. While in some other species,
including rodents, uric acid is the near terminal product which can
be converted to allantoin[27]. The determination of plasma
uric acid concentration may reflect the catabolism state of
purine nucleotides. In this study, we found that there was a
significant increase in plasma uric acid in
morphine-administered rats, compared to the controls. This preliminary data
suggest that morphine exposure may cause the
enhancement of purine nucleotide catabolism. Our further study
showed that the increase of uric acid concentration highly
correlated with ADA and XO, 2 key enzymes of purine
nucleotide catabolism. During morphine administration, the
levels of plasma ADA and XO increased significantly when
compared to the control (P<0.05), and after morphine withdrawal,
the concentration of ADA and XO was still high. The high
concentration of plasma ADA and XO might have resulted
from the release of intracellular enzymes from 1 or more
tissues into the blood; therefore, we determined the ADA and
XO levels in several important tissues, including the brain,
liver, small intestine, and skeletal muscles. The results
indicated that morphine can increase the amounts of ADA and
XO in these tissues, which is believed to contribute to the
enhancement of purine nucleotide catabolism and the high
level of plasma uric acid.
In order to investigate one of the possible mechanisms
of morphine's effect on ADA and XO, RT-PCR, and
RT-PCR-Southern blot were used to examine the gene transcripts of
ADA and XO in these tissues. Compared to the control
group, the transcripts of ADA and XO were significantly
higher in rats administered with morphine, while ADA and
XO mRNA levels in most tissues gradually recovered to the
normal during morphine abstinence. The changes of ADA
and XO mRNA are paralleled with their enzymes, respectively.
Rat C6 glioma cells have been demonstrated to exhibit
stable expression of the μ receptor and other opioid receptors,
such as delta and kappa receptors. The μ receptor mainly
mediates morphine dependence and
tolerance[28_31]. We choose rat C6 glioma cells to try and clarify whether morphine
affects the catabolism of purine nucleotides in
vitro and to study the relationship between the transcripts of ADA and
XO and the opioid receptors. The results suggested that
ADA and XO gene expression were upregulated during
morphine administration, whereas the upregulated expression
of ADA and XO could be reversed by naloxone 1 h before or
after morphine administration. As it is well known that
naloxone is a pure, competitive antagonist of opioid
receptors[32], the up-regulation of ADA and XO gene expression were
probably mediated by opioid receptors.
Creatinine and urea nitrogen, sensitive indices reflecting
the renal clearance rate, were detected to assess the impact
of morphine on renal function. The results showed that the
concentrations of plasma creatinine were not significantly
changed in both the 3 and 7 d morphine-administered groups.
Meanwhile, the concentrations of plasma urea nitrogen
increased in the 7 d morphine-administered group, but the
concentration of the plasma uric acid started to increase in
the 3 d morphine-administered group, so the early
enhancement of plasma uric acid might mainly result from the
increase of purine nucleotides catabolism. Renal function
might be affected by long-term morphine administration which
may partly contribute to the late enhancement of plasma uric
acid.
In our previous experiments we found that gene
expression of the key enzymes, HGPRT and AK in the purine
nucleotide salvage pathway are downregulated by morphine
in vivo and in vitro, which is consistent with the findings that
heroin treatment of cells inhibits the anabolism of purine
nucleotides[6,33]. It is well known that there is no
5-phos-phoribosyl-1 pyrophosphate (PRPP) aminotransferase in the
brain, and the anabolism of nucleotides mainly depends on
the salvage pathway, and therefore, the ability of nucleotide
anabolism is slower in the brain. This experiment
demonstrated that morphine caused the increase of brain ADA and
XO concentrations; the reinforcement of purine nucleotides
catabolism induced by morphine did not disappear quickly,
therefore, the effect of morphine on purine nucleotides
catabolism in the brain is more serious.
In summary, acute and chronic morphine administration
can increase the catabolism of purine nucleotide in the brain
and extraneuronal tissues, and the effect of morphine on
nucleotide catabolism remains for a period after withdrawal.
The inhibitory effects of morphine on the anabolism of
purine nucleotides in previous works and the promotive
effects of morphine on the catabolism of purine nucleotide
in this report imply that morphine may affect the nucleotide
metabolism seriously. It is already known that purine
products affect the normal development of neuron cells and that
long-term administration of opioids may produce toxic
effects on the central and peripheral nervous system,
causing irreversible pathological changes in cells. Therefore, we
propose that the effects of morphine on purine nucleotide
metabolism may be an important, new biochemical
pharmacological mechanism of morphine action, and the
relationship between purine nucleotide metabolism and opioid
addiction is worth further study.
Acknowledgement
This work was supported by Dr Jennifer STEWART of
the Department of Biology, Virginia Commonwealth University, Richmond, VA , USA.
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