Polyamine metabolism of rat gastric mucosa after oral
administration of hypertonic sodium chloride solution
Kenjiro
Otani1,
Yoshihisa
Yano1,
Tadayoshi
Hasuma1,
Tetsuo
Arakawa2,
Kenzo
Kobayashi2,
Isao
Matsui-Yuasa3, and
Shuzo
Otani1
1 Department of Biochemistry
and 2 Third Department of Internal
Medicine, Osaka City University Medical School; and
3 Department of Food and
Nutrition, Faculty of Human Life Science, Osaka City University,
Osaka 545, Japan
 |
ABSTRACT |
Oral administration of 1 ml of 3.42 M
NaCl solution to rats induced spermidine/spermine
N1-acetyltransferase
(SSAT) activity in gastric mucosa as well as ornithine decarboxylase
(ODC) activity. SSAT activity increased and peaked at 5 h and again at
7 h, whereas ODC activity peaked at 6 h. SSAT mRNA also increased after
3.42 M NaCl administration to an extent similar to the increase in SSAT
activity at 5 h. Intracellular putrescine level and DNA synthesis were
increased by NaCl administration. A polyamine oxidase inhibitor,
N,N'-bis(2,3-butadienyl)-1,4-butanediamine (MDL-72527), but not an ODC inhibitor,
-difluoromethylornithine (DFMO), inhibited the increases in putrescine level and DNA synthesis at 5 h. The inhibition of DNA synthesis by MDL-72527 was reversed by
putrescine administration. In contrast, both MDL-72527 and DFMO
inhibited the increase in putrescine level and DNA synthesis at 16.5 h.
These findings suggest that putrescine produced from preexistent
spermidine by SSAT is responsible for the initial DNA synthesis after
mucosal injury induced by NaCl and that both SSAT and ODC are involved
in formation of putrescine, which is required for subsequent DNA
synthesis.
spermidine/spermine
N1-acetyltransferase; putrescine; ornithine decarboxylase
 |
INTRODUCTION |
THE GASTROINTESTINAL MUCOSA has one of the most rapid
turnover rates of any tissue in the body and correspondingly high rates of cell growth and differentiation. It has been reported that polyamines are involved in growth and differentiation of
gastrointestinal and other types of cells (13, 16, 19). In the stomach
luminal polyamines stimulate repair of gastric mucosal injury (36), and
putrescine injection stimulates oxyntic mucosal growth (20). Oral
administration of hypertonic NaCl solution causes gastric mucosal
damage, followed by induction of ornithine decarboxylase (ODC), the
rate-limiting enzyme of polyamine biosynthesis, mucosal restitution,
and cell growth (1, 11, 35). The processes of mucosal restitution and
cell growth require polyamines.
Monoacetylspermidine is known to be present in
Escherichia coli. Tabor (31) showed
that monoacetylspermidine accumulated in bacterial cells when they were
harvested at 4°C or when large amounts of endogenous polyamines
were formed (32). Heat shock and certain types of chemical stress have
also been shown to induce the accumulation of monoacetylspermidine (6).
However, the physiological function of the acetylation of polyamine is
not well understood. Recently, evidence has been obtained that
spermidine/spermine N1-acetyltransferase
(SSAT) and ODC play important roles in the regulation of intracellular
polyamine levels.
N1-acetylpolyamines
produced by SSAT are quite readily excreted from cells, resulting in a
decrease in spermidine and spermine levels (8). In addition,
N1-acetyl derivatives
of spermine and spermidine are good substrates of polyamine oxidase and
are converted to spermidine and putrescine, respectively (3, 22). SSAT
thus participates in the production of putrescine from preexistent
spermidine or spermine. In the liver, conversion of spermidine to
putrescine occurs after treatment with carbon tetrachloride (14) and
after treatment with growth hormone and thioacetamide or partial
hepatectomy (21). SSAT plays a more important role than ODC in
increasing the putrescine level of duodenal mucosa in chicks with
vitamin D deficiency induced by administration of
1
,25-dihydroxyvitamin D3, and
this increase in putrescine level may affect calcium absorption (30).
However, the physiological role of SSAT in the stomach is still
unclear. In this study, we attempted to determine whether
administration of hypertonic NaCl solution induces SSAT activity and
examined the relationship between SSAT activity and DNA synthesis in
rat gastric mucosa. We found that oral administration of hypertonic NaCl caused a biphasic elevation of SSAT activity and a peak in ODC
activity, which was located between the first and second peaks of SSAT
activity. We also found that elevation of SSAT activity rather than ODC
activity is required for early DNA synthesis after gastric mucosal
damage.
 |
MATERIALS AND METHODS |
Materials.
L-[1-14C]ornithine
(2.07 GBq/mmol),
[acetyl-1-14C]acetyl
CoA (2.00 GBq/mmol), and
[methyl-3H]thymidine
(2.22-3.33 TBq/mmol) were obtained from Moravek Biochemicals (Brea, CA), and
[
-32P]UTP (29.6 TBq/mmol) was from DuPont-NEN (Boston MA). Putrescine, pepstatin A,
bovine serum albumin, and calf thymus DNA were obtained from Sigma
Chemical (St. Louis, MO).
N,N'-bis(2,3-butadienyl)-1,4-butanediamine (MDL-72527) and
-difluoromethylornithine (DFMO) were kindly supplied by Marion Merrell Dow Research Institute (Cincinnati, OH).
The other reagents used were products of special grade from Wako
Chemicals (Osaka, Japan).
Animals and experimental procedure.
Male Wistar rats weighing 200-220 g were purchased from CLEA Japan
(Tokyo, Japan) and were fed standard chow and tap water ad libitum. The
study protocol was approved by the Animal Research Committee of Osaka
City University, and care of animals was in accordance with the
standards of this institution (Guide for Animal Experimentation, Osaka
City University). The animals were fasted for 24 h before the
experiments but had free access to drinking water. They were given 1.0 ml of NaCl solution by gastric tube and killed by exsanguination via
the abdominal aorta under ether anesthesia at various times after NaCl
administration. The stomach was excised open along the great curvature
and washed thoroughly in ice-cold phosphate-buffered saline (PBS). The
antrum was removed, and the oxyntic gland mucosa of the stomach was
scraped away from the underlying smooth muscle with a glass slide. The
collected tissues were divided into three portions: the first was used
for examination of polyamine levels, the second for assays of SSAT and
ODC activities, and the third for analysis of SSAT mRNA.
MDL-72527 (1 mg/100 g body wt) dissolved in 0.2 ml of 0.15 M
saline and DFMO (50 mg/100 g body wt) dissolved in 0.4 ml of 0.15 M
saline were administered intraperitoneally 1 h and 15 min respectively, before intragastric 3.42 M NaCl administration. Putrescine (10 µmol/100 g body wt) dissolved in 0.5 ml of distilled water was administered intragastrically 3 h after NaCl administration. Control rats were given 0.15 and 3.42 M NaCl intragastrically, respectively, with intraperitoneal administration of the vehicle. Animals were killed 5 h after NaCl administration, and DNA synthesis and intracellular putrescine level were measured. When the effects of
MDL-72527 and DFMO on putrescine level and DNA synthesis 16.5 h after
NaCl administration were examined, the inhibitors were supplemented
with the same doses 7.5 h after NaCl administration.
Assay of ODC activity.
ODC activity was assayed by the release of
14CO2
from
L-[1-14C]ornithine
as previously described (25). In brief, tissues were washed
twice with ice-cold PBS and suspended in 400 µl of 500 mM
tris(hydroxymethyl)aminomethane (Tris)-HCl (pH 7.5) containing 250 µM
pyridoxal phosphate, 0.1 mM EDTA, 2.5 mM dithiothreitol, 1.5 mM
pepstatin A, and 292 mM phenylmethylsulfonyl fluoride. The tissues were
disrupted by three cycles of freezing and thawing and centrifuged at
30,000 g for 20 min at 4°C. Then
90 µl of the supernatant were added to a glass tube containing 9.25 GBq of L-[1-14C]ornithine
(5 µl) and 80 nmol of
L-ornithine (5 µl). The test tube was sealed with a rubber stopper equipped with a disposable syringe needle, on which a paper disk (8 mm in diam) containing 50 µl
Soluene-350 (Packard Instruments, Downers Grove, IL) was impaled, and
was incubated at 37°C for 1 h. The
CO2 released by the addition of 1 ml of 2 N citrate through the rubber stopper was trapped by the
Soluene-350 absorbed on the paper disk, and the paper disk was then
transferred to a vial containing 5 ml toluene scintillation fluid
(Omnifluor; Daiichi Chemicals, Tokyo, Japan). Radioactivity was
measured in a Beckman liquid scintillation counter (LS-5801).
Assay of SSAT activity.
Cell extracts were prepared as described previously. Aliquots of the
supernatant of 30,000 g centrifugation
previously described were diluted with the buffer previously described
to a protein concentration under 100 mg protein in 50 µl. SSAT
activity was assayed by measurement of the incorporation of
[acetyl-1-14C]acetyl
CoA into monoacetylspermidine (22). In brief, 50-µl aliquots of the
diluted solution were incubated with 0.3 µmol of spermine, 10 µmol
Tris-HCl (pH 7.8), and 1.48 GBq of
[acetyl-1-14C]acetyl
CoA in a final volume of 100 µl at 37°C for 10 min. The reaction
was terminated by chilling and the addition of 20 µl of 1 M
NH2OH, and the reaction mixture
was placed in boiling water for 3 min. Aliquots of 50 µl of the
reaction mixture were then spotted onto a Whatman P81 paper disk (2.4 cm in diam). The paper disk was washed with tap water and then five
times with 1 ml of ethanol on a filter, dried, and transferred to a
vial containing 5 ml of toluene scintillation fluid, and radioactivity
was measured with a Beckman liquid scintillation counter.
Isolation of RNA.
Total RNA was extracted from scraped gastric mucosal specimens by the
acid guanidinium thiocyanate-phenol-chloroform method (9).
RNase protection assay.
Portions of SSAT cDNA and 18S rDNA were obtained by reverse
transcriptase-polymerase chain reaction (PCR) amplification using total
RNA extracted from rat liver as a template. Reverse transcription was
primed using random hexanucleotides as described previously (34). The
primers 5'-CTGAACGTCTCTGCCCTATCA-3' and
5'-CTTGGATGTGGTGCCGTT-3' were used for amplifying the 83-bp
18S rDNA. The primers 5'-GAAGATGGTTTTGGAGAGCA-3' and
5'-TCTGATCCTATGCCAAAGCC-3' were used for amplifying the
198-bp SSAT cDNA. The thermal cycle profile was as follows:
denaturation for 1 min at 94°C, annealing of primers for 1 min at
55°C, and extension of primers for 1 min at 72°C. The number of
PCR cycles was 25. These PCR products were cloned into the Srf I site
of pCR-Script SK(+) plasmid vector (Stratagene, La Jolla, CA),
sequenced, and used as templates for production of antisense RNA probe
with the ribonuclease (RNase) protection assay. The antisense RNA probe for SSAT mRNA and 18S rRNA were synthesized according to the
manufacturer's instructions (MAXI script T7 in vitro transcription kit
and MEGA script T7 in vitro transcription kit; Ambion, Austin, TX). The specific activities of
[
-32P]UTP used to
label the RNA probe for SSAT mRNA and 18S rRNA were 5.92 GBq/mmol and
88.8 kBq/mmol, respectively. RNase protection assay (17) was performed
according to the manufacturer's instructions (RNase protection kit;
Boehringer Mannheim, Mannheim, Germany). The RNase digestion products
were subjected to denaturing polyacrylamide gel electrophoresis on
4.5% polyacrylamide-7 M urea gel. The amounts of protected RNA probes
were measured using a bioimaging analyzer (BAS 2000II; Fujix, Tokyo,
Japan). To normalize the amounts of RNA applied to the gel, the amounts
of SSAT mRNA relative to those of 18S rRNA were calculated.
Measurement of polyamine contents.
The tissues were washed with ice-cold PBS and disrupted by
ultrasonication in 750 µl of 0.4 N perchloric acid. After
centrifugation at 18,000 g
for 20 min, the supernatant was stored at
20°C until assay
for polyamines. The pellet was used for measurement of DNA content. The
concentration of polyamine in the acid extract was determined by
high-performance liquid chromatography (Shimadzu LC-6A; Shimadzu,
Kyoto, Japan) equipped with a fluorescence detector. Polyamines were
separated on an STR ODS-II column (4.6 × 150 mm, particle size 5 µm, Shimadzu Techno-Research) as described previously (23). Polyamine
contents were expressed as picomoles per milligram of DNA.
Assay of DNA content.
DNA precipitated with 0.4 N perchloric acid was washed and solubilized
by heating at 75°C for 20 min in 0.4 N perchloric acid. After
centrifugation at 18,000 g for 20 min,
DNA of the supernatant was measured by diphenylamine reaction (5) using
calf thymus DNA as a standard.
Assay of DNA synthesis.
DNA synthesis was assayed by the incorporation of
[3H]thymidine into the
acid-insoluble fraction using the method of Furihata et al. (12) with
some modifications. Briefly, five specimens weighing about 20 mg each
were obtained from the oxyntic gland mucosa with a 3-mm dermatological
punch. These specimens were then incubated for 1 h at 37°C in 2 ml
of Eagle's minimum essential medium containing
[3H]thymidine (370 kBq/ml) with gentle shaking. DNA was precipitated with 0.4 N perchloric
acid and centrifuged at 18,000 g for
20 min. The precipitate was washed with 0.4 N perchloric acid, and DNA
was solubilized from the precipitate as previously described. The
radioactivity of the supernatant was measured with a liquid scintillation counter, and the amount of DNA was measured as previously described. Incorporation of
[3H]thymidine into DNA
was expressed as counts per hour per microgram of DNA.
Measurement of protein content.
Protein content was assayed by the method of Bradford (4) using reagent
from Bio-Rad Laboratories (Richmond, CA) and bovine serum albumin as a
standard.
Statistical analysis.
Results obtained are expressed as means ± SD. The significance of
differences between mean values was determined by one- or two-way
analysis of variance (ANOVA). Differences between means were evaluated
by ANOVA, with specific differences tested using Scheffé's
F-test as a post hoc test. Differences
with P < 0.05 were considered
significant.
 |
RESULTS |
Effects of oral administration of NaCl solution on gastric SSAT and
ODC activity.
Effects of the administration of various concentrations of NaCl on
gastric SSAT are shown in Fig. 1. Compared
with physiological saline solution, 3.42 M of NaCl solution increased
SSAT activity significantly, whereas concentrations of NaCl higher and
lower than 3.42 M had less effect on SSAT activity. The changes in SSAT activity after oral administration of 3.42 M NaCl solution are shown in
Fig. 2. SSAT activity increased
biphasically, with a first peak at 5 h and a second at 7 h after NaCl
administration. ODC activity was also increased by NaCl solution and
peaked at 6 h, when the first peak of SSAT activity had already
declined (Fig. 3).

View larger version (17K):
[in this window]
[in a new window]
|
Fig. 1.
Spermidine/spermine
N1-acetyltransferase
(SSAT) activities of oxyntic gland mucosa after 5 h exposure to various
concentrations of NaCl. One milliliter of various concentrations of
NaCl solution was administered intragastrically. Rats were then killed,
and the stomach was removed. SSAT activity of gastric mucosa was
measured as described in MATERIALS AND
METHODS. Each value represents mean ± SD for
3-4 rats. ** P < 0.01 compared with 0.15 M NaCl-treated group.
|
|

View larger version (22K):
[in this window]
[in a new window]
|
Fig. 2.
Changes in SSAT activities of oxyntic gland mucosa after 3.42 M NaCl
administration. Rats were administered 3.42 M NaCl solution and were
killed at times indicated. SSAT activity of gastric mucosa was assayed
as described in MATERIALS AND METHODS.
, 0.15 M NaCl-treated group; , 3.42 M NaCl-treated group. Each
value and bar represent mean ± SD for 4-7 rats.
** P < 0.01 compared with 0.15 M NaCl-treated group;  P < 0.01 compared with 0.15 M NaCl-treated and immediately killed group.
|
|

View larger version (21K):
[in this window]
[in a new window]
|
Fig. 3.
Changes in ornithine decarboxylase (ODC) activities of oxyntic gland
mucosa after 3.42 M NaCl administration. , 0.15 M NaCl-treated
group; , 3.42 M NaCl-treated group. ODC activity was measured as
described in MATERIALS AND METHODS.
Each value and bar represent mean ± SD for 4-7 rats.
** P < 0.01 compared with 0.15 M NaCl-treated group; 
P < 0.01 compared with 0.15 M
NaCl-treated and immediately killed group.
|
|
Effect of oral administration of NaCl solution on gastric SSAT mRNA.
SSAT mRNA level was measured at the times indicated after 3.42 M NaCl
administration. It increased about threefold by 3 h after NaCl
administration and peaked at 7 h (Fig. 4).

View larger version (21K):
[in this window]
[in a new window]
|
Fig. 4.
Changes in oxyntic gland mucosal SSAT mRNA level after 3.42 M NaCl
administration. Total RNA was extracted, and mRNA level was measured by
RNase protection assay as described in MATERIALS AND
METHODS. A:
representative autoradiogram obtained by RNase protection assay.
B: quantification of SSAT mRNA level.
Amounts of protected RNA probes were measured using bioimaging
analyzer. To normalize amounts of RNA applied to gel, amounts of SSAT
mRNA relative to those of 18S rRNA were calculated and expressed as
ratio to that at 0 h (= 1). , 0.15 M NaCl-treated group; , 3.42 M
NaCl-treated group. Each value and bar represent mean ± SD for 3 rats. ** P < 0.01 compared
with 0.15 M NaCl-treated group;
 P < 0.01 compared with
0.15 M NaCl-treated and immediately killed group.
|
|
Effects of oral administration of NaCl on gastric polyamine levels.
Polyamine levels of gastric mucosa were measured 5, 6, and 7 h after
NaCl administration (Fig. 5). Putrescine
level was increased and spermidine and spermine levels were decreased
by administration of solutions with higher NaCl concentrations.

View larger version (22K):
[in this window]
[in a new window]
|
Fig. 5.
Changes in oxyntic gland mucosal contents of putrescine
(A), spermidine
(B), and spermine
(C) after 3.42 M NaCl
administration. Polyamine contents were assayed by high-performance
liquid chromatography (HPLC) as described in MATERIALS
AND METHODS. Each value and bar represent mean ± SD
for 6 rats. * P < 0.05, ** P < 0.01 compared with 0.15 M NaCl-treated group; P < 0.05,  P < 0.01 compared with 0.15 M NaCl-treated and immediately killed. Open bars,
0.15 M NaCl-treated group; hatched bars, 3.42 M NaCl-treated group.
|
|
Effects of oral administration of NaCl solution on gastric DNA
synthesis.
The changes in incorporation of
[3H]thymidine into the
acid-insoluble fraction after NaCl administration are shown in Fig. 6. Incorporation was increased at 5 h after
higher NaCl administration and continued to increase until 23 h. The
increase in the incorporation 5 h after administration of solutions
with higher NaCl concentrations was inhibited by MDL-72527, a specific
inhibitor of polyamine oxidase, and this inhibition was reversed by
putrescine administration (Fig. 7). The ODC
inhibitor DFMO had no significant effect on DNA synthesis at 5 h. DNA
synthesis at 16.5 h was inhibited by both MDL-72527 and DFMO, and the
inhibition caused by the combination of these two agents was greater
than that caused by either alone (Fig. 8).

View larger version (15K):
[in this window]
[in a new window]
|
Fig. 6.
Changes in DNA synthesis in oxyntic gland mucosa after 3.42 M NaCl
administration. DNA synthesis was assayed by incorporation of
[3H]thymidine into
acid-insoluble fraction as described in MATERIALS AND
METHODS. Each value and bar represent mean ± SD for
6 rats. * P < 0.05, ** P < 0.01 compared with 0.15 M NaCl-treated and immediately killed group.
|
|

View larger version (30K):
[in this window]
[in a new window]
|
Fig. 7.
Effects of
N,N'-bis(2,3-butadienyl)-1,4-butanediamine
(MDL-72527), -difluoromethylornithine (DFMO), and exogenous
putrescine on oxyntic gland mucosal DNA synthesis 5 h after 3.42 M NaCl
administration. MDL-72527 (1 mg/100 g body wt) was administered
intraperitoneally 1 h before intragastric NaCl administration. DFMO (50 mg/100 g body wt) was administered intraperitoneally 15 min before NaCl
administration. Putrescine (10 µmol/100 g body wt), dissolved in 0.5 ml of distilled water, was administered intragastrically 3 h after NaCl
administration. Animals not treated with putrescine were given 0.5 ml
of distilled water as a vehicle. DNA synthesis was assayed by
incorporation of
[3H]thymidine into the
acid-insoluble fraction as described in MATERIALS AND
METHODS. Each value and bar represent mean ± SD for
6-8 rats. Open bar, 0.15 M NaCl-treated group; hatched bars, 3.42 M NaCl-treated groups. * P < 0.05, ** P < 0.01.
|
|

View larger version (28K):
[in this window]
[in a new window]
|
Fig. 8.
Effects of MDL-72527 and DFMO on oxyntic gland mucosal DNA synthesis
16.5 h after 0.15 M or 3.42 M NaCl administration. MDL-72527 (1 mg/100
g body wt) was administered intraperitoneally 1 h before and 7.5 h
after intragastric NaCl administration. DFMO (50 mg/100 g body wt) was
administered intraperitoneally 15 min before and 7.5 h after NaCl
administration. DNA synthesis was assayed by incorporation of
[3H]thymidine into
acid-insoluble fraction as described in MATERIALS AND
METHODS. Each value and bar represent mean ± SD for
4-7 rats. Open bar, 0.15 M NaCl-treated group; hatched bars, 3.42 M NaCl-treated groups. * P < 0.05, ** P < 0.01.
|
|
Effects of inhibitors of ODC and polyamine oxidase on putrescine
level.
Putrescine level was increased 5 h after administration of solutions
with higher NaCl concentrations, and this increase was inhibited by
MDL-72527 but not by DFMO (Fig. 9).
However, the increase in putrescine level at 16.5 h was inhibited by
both MDL-72527 and DFMO, and the combination of these two reduced the
putrescine level more than did either of the inhibitors alone (Fig.
10).

View larger version (32K):
[in this window]
[in a new window]
|
Fig. 9.
Effects of MDL-72527, DFMO, and exogenous putrescine on oxyntic gland
mucosal putrescine content 5 h after 0.15 or 3.42 M NaCl
administration. MDL-72527, DFMO, and putrescine were administered as
described in legend for Fig. 7. Putrescine content was assayed by HPLC
as described in MATERIALS AND METHODS.
Each value and bar represent mean ± SD for 6 rats. Open bar, 0.15 M
NaCl-treated group; hatched bars, 3.42 M NaCl-treated groups.
** P < 0.01.
|
|

View larger version (26K):
[in this window]
[in a new window]
|
Fig. 10.
Effects of MDL-72527 and DFMO on oxyntic gland mucosal putrescine
content 16.5 h after 0.15 or 3.42 M NaCl administration. MDL-72527 and
DFMO were administered as described in legend for Fig. 8. Putrescine
content was assayed by HPLC as described in MATERIALS
AND METHODS. Each value and bar represent mean ± SD
for 4 or 5 rats. Open bar, 0.15 M NaCl-treated group; hatched bars,
3.42 M NaCl-treated groups. * P < 0.05, ** P < 0.01.
|
|
 |
DISCUSSION |
It has been reported that oral administration of hypertonic NaCl
solution increases ODC activity in the gastric mucosa (11, 35). We
found that administration of solutions with higher NaCl concentrations
caused biphasic induction of SSAT activity in addition to induction of
ODC activity, with the first peak of SSAT activity occurring earlier
than that of ODC activity. Intracellular putrescine level increased and
spermidine and spermine levels decreased after administration
of solutions with higher NaCl concentrations. Furthermore, a polyamine
oxidase inhibitor, but not an ODC inhibitor, inhibited the increase in
putrescine level 5 h after NaCl administration, indicating that SSAT
and polyamine oxidase participate in putrescine formation from
preexistent spermidine and spermine early after NaCl administration.
Banan et al. (1) recently reported that DFMO inhibited the increase in
gastric mucosal putrescine level 4 h after 3.4 M NaCl treatment. Their
results are not consistent with our own, even though the dose and
method of DFMO administration they used were the same as in our study.
The reason for these differences in findings is unclear at present.
DNA synthesis induced by mucosal injury began 5 h after NaCl
administration and continued until 16.5 h with administration of
solutions with higher NaCl concentrations. This finding is consistent
with those reported previously by Furihata et al. (10), who showed that
the proportion of S-phase cells detected with 5-bromo-2'-deoxyuridine staining in oxyntic gland mucosa of rat stomach increased over 6-24 h after 2.6 M NaCl administration. Our
results showed that the effects of MDL-72527 and DFMO on DNA synthesis
5 h (Fig. 7) and 16.5 h (Fig. 8) after NaCl administration were very
similar to those of the inhibitors on increase in putrescine level at
the same times (Figs. 9 and 10). These findings suggest that putrescine
plays a very important role in DNA synthesis and that SSAT rather than
ODC is involved in putrescine formation during the initial phase after
NaCl administration but that both enzymes are involved in this
formation in the later phase.
SSAT mRNA level was elevated 3 h after NaCl administration and
peaked at 7 h. Treatment with
-amanitin, an inhibitor of RNA polymerase II, inhibited the increase in SSAT activity and SSAT mRNA levels at 3, 5, and 7 h after NaCl administration (data not shown), suggesting that NaCl administration stimulates transcription of
the SSAT gene. However, the increase in SSAT enzymatic activity at the
second peak was less than that in SSAT mRNA level, suggesting that in
addition to transcriptional regulation, posttranscriptional regulation
is involved in the control of SSAT enzymatic activity, as reported
previously (26).
In addition to ODC activity, SSAT activity has been found to be
elevated in tumors (24, 33) and proliferating cells stimulated by
various types of growth factors (21). These findings suggest that both
SSAT and ODC are involved in cell growth. However, Pegg and Erwin (28)
found that SSAT activity was increased after ODC induction and that
intracellular accumulation of spermidine and spermine induced SSAT
activity, suggesting that SSAT plays a role in the control of
intracellular spermidine and spermine levels by conversion of these
polyamines to
N1-acetylated
derivatives, which were excreted extracellularly, resulting in a
decrease in spermidine and spermine levels (8). Basu et al. (2)
and Casero et al. (7) reported that polyamine analogs that induced SSAT
activity decreased intracellular polyamine levels and inhibited cell
growth. Ignatenko and Gerner (15) also showed that SSAT mRNA level
increased as human colon tumor-derived HCT1H cells traversed the log
phase and entered the plateau phase. The overexpression of SSAT
activity in E. coli transfected with a
plasmid containing SSAT gene reduced cell growth (27). These findings
suggest that SSAT causes growth arrest by decreasing spermidine and
spermine levels. On the other hand, it has been reported that SSAT
activity is increased before ODC induction. Höltta et al. (14)
reported that putrescine level increased in association with a decrease
in spermidine level and that this was followed by ODC induction; this
finding is consistent with that of Matsui et al. (22) that treatment of
rats with carbon tetrachloride increased hepatic SSAT activity. These
findings also suggest that SSAT has a physiological role other than
preventing overaccumulation of polyamines. However, the role of the
SSAT activity induced earlier than ODC activity has not been
elucidated. Seidel and Snyder (29) reported that the trophic response
of gastrointestinal mucosa to treatment with pentagastrin was
associated with SSAT induction but not with ODC induction, suggesting
that the polyamine interconversion pathway plays a role in DNA
synthesis. Löser and Fölsch (18) reported that simultaneous
administration of DFMO and MDL-72527 resulted in significant inhibition
of camostate-induced increases in rat pancreatic putrescine and DNA.
However, because they did not administer camostate plus MDL-72527
alone, it was not proved that putrescine formed by SSAT and polyamine
oxidase was responsible for DNA synthesis. Our findings suggest that
SSAT plays an important role in putrescine formation early after NaCl treatment and triggers DNA synthesis and that both SSAT and ODC are
required for the continuation of DNA synthesis in repair after gastric
mucosal damage.
 |
FOOTNOTES |
Address for reprint requests: S. Otani, Dept. of Biochemistry,
Osaka City Univ. Medical School, 1-4-54 Asahimachi, Abeno-ku,
Osaka 545, Japan.
Received 3 March 1997; accepted in final form 14 November 1997.
 |
REFERENCES |
1.
Banan, A.,
J.-Y. Wang,
S. A. McCormack,
and
L. R. Johnson.
Relationship between polyamines, actin distribution, and gastric healing in rats.
Am. J. Physiol.
271 (Gastrointest. Liver Physiol. 34):
G893-G903,
1996[Abstract/Free Full Text].
2.
Basu, H. S.,
M. Pellarin,
B. G. Feuerstein,
D. F. Deen,
R. J. Bergeron,
and
L. J. Marton.
Effect of N1, N14-bis(ethyl)homospermine on the growth of U-87 MG and SF-126 human brain tumor cells.
Cancer Res.
50:
3137-3140,
1990[Abstract].
3.
Bolkenius, F. N.,
and
N. Seiler.
Acetyl derivatives as intermediates in polyamine catabolism.
Int. J. Biochem.
13:
287-292,
1981[Medline].
4.
Bradford, M. M.
A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding.
Anal. Biochem.
72:
248-254,
1976[Medline].
5.
Burton, K.
A study of the conditions and mechanism of diphenylamine reaction for colorimetric estimation of deoxyribonucleic acid.
Biochem. J.
62:
315-323,
1956.
6.
Carper, S. W.,
D. G. Willis,
K. A. Manning,
and
E. W. Gerner.
Spermidine acetylation in response to a variety of stress in E. coli.
J. Biol. Chem.
266:
12439-12441,
1991[Abstract/Free Full Text].
7.
Casero, R. A., Jr.,
P. Celano,
S. J. Ervin,
C. W. Porter,
R. J. Bergeron,
and
P. R. Libby.
Differential induction of spermidine/spermine N1-acetyltransferase in human lung cancer cells by the bis(ethyl)polyamine analogues.
Cancer Res.
49:
3829-3833,
1989[Abstract].
8.
Casero, R. A., Jr.,
and
A. E. Pegg.
Spermidine/spermine N1-acetyltransferase. The turning point in polyamine metabolism.
FASEB J.
7:
653-661,
1993[Abstract/Free Full Text].
9.
Chomczynski, P.,
and
N. Sacchi.
Single-step method of RNA isolation by acid guanidium thiocyanate-phenol-chloroform extraction.
Anal. Biochem.
162:
156-159,
1987[Medline].
10.
Furihata, C.,
H. Ohta,
and
T. Katsuyama.
Cause and effect between concentration-dependent tissue damage and temporary cell proliferation in rat stomach mucosa by NaCl, a stomach tumor promoter.
Carcinogenesis
17:
401-406,
1996[Abstract].
11.
Furihata, C.,
Y. Sato,
M. Hosaka,
T. Matsushima,
F. Furukawa,
and
M. Takahashi.
NaCl-induced ornithine decarboxylase and DNA synthesis in rat stomach mucosa.
Biochem. Biophys. Res. Commun.
121:
1027-1032,
1984[Medline].
12.
Furihata, C.,
Y. Yamawaki,
S. S. Jin,
H. Moriya,
K. Kodama,
T. Matsushima,
T. Ishikawa,
S. Takayama,
and
M. Nakadate.
Induction of unscheduled DNA synthesis in rat stomach mucosa by glandular stomach carcinogens.
J. Natl. Cancer Inst.
72:
1327-1334,
1984[Medline].
13.
Heby, O.
Role of polyamines in the control of cell proliferation and differentiation.
Differentiation
19:
1-20,
1981[Medline].
14.
Höltta, E.,
R. Sinervirta,
and
J. Jänne.
Synthesis and accumulation of polyamines in rat liver regenerating after treatment with carbon tetrachloride.
Biochem. Biophys. Res. Commun.
54:
350-357,
1973[Medline].
15.
Ignatenko, N. A.,
and
E. W. Gerner.
Growth arrest- and polyamine-dependent expression of spermidine/spermine N1-acetyltransferase in human tumor cells.
Cell Growth Differ.
7:
481-486,
1996[Abstract].
16.
Johnson, L. R.
Regulation of gastrointestinal mucosal growth.
Physiol. Rev.
68:
456-502,
1988[Free Full Text].
17.
Lee, J. J.,
and
N. A. Costlow.
A molecular titration assay to measure transcript prevalence levels.
In: Methods Enzymology, edited by S. L. Berger,
and S. L. Kimmel. New York: Academic, 1987, p. 633-648.
18.
Löser, C.,
and
U. R. Fölsch.
Importance of various intracellular regulatory mechanisms of polyamine metabolism in camostate-induced pancreatic growth in rats.
Digestion
54:
213-223,
1993[Medline].
19.
Luk, G. D.,
L. J. Marton,
and
S. B. Baylin.
Ornithine decarboxylase is important in intestinal mucosal maturation and recovery from injury in rats.
Science
210:
195-198,
1988.
20.
Majumdar, A. P.,
and
L. R. Johnson.
Effect of putrescine on oxyntic gland and colonic mucosal growth in rats.
Life Sci.
41:
961-966,
1987[Medline].
21.
Matsui, I.,
and
A. E. Pegg.
Effect of thioacetamide, growth hormone or partial hepatectomy on spermidine acetylase activity of rat liver cytosol.
Biochim. Biophys. Acta
633:
87-94,
1980[Medline].
22.
Matsui, I.,
L. Wiegand,
and
A. E. Pegg.
Properties of spermidine N-acetyltransferase from livers of rats treated with carbon tetrachloride and its role in the conversion of spermidine into putrescine.
J. Biol. Chem.
256:
2454-2459,
1981[Free Full Text].
23.
Matsui-Yuasa, I.,
M. Obayashi,
T. Hasuma,
and
S. Otani.
Enhancement of spermidine/spermine N1-acetyltransferase activity by treatment with lithium chloride in Ehrlich ascites tumor cells.
Chem.-Biol. Interact.
81:
233-242,
1992[Medline].
24.
Matsui-Yuasa, I.,
S. Otani,
Y. Yano,
N. Takada,
M. A. Shibata,
and
S. Fukushima.
Spermidine/spermine N1-acetyltransferase, a new biochemical marker for epithelial proliferation in rat bladder.
Jpn. J. Cancer Res.
83:
1037-1040,
1992[Medline].
25.
Otani, S.,
I. Matsui,
S. Nakajima,
M. Masutani,
Y. Mizoguchi,
and
S. Morisawa.
Induction of ornithine decarboxylase in guinea pig lymphocytes by the divalent cation ionophore A23187 and phytohemagglutinin.
J. Biochem.
88:
77-85,
1980[Medline].
26.
Parry, L.,
R. Balaña Fouce,
and
A. E. Pegg.
Posttranslational regulation of the content of spermidine/spermine N1-acetyltransferase by N1,N12-bis(ethyl)spermine.
Biochem. J.
305:
451-458,
1995[Medline].
27.
Parry, L.,
J. Lopez-Ballester,
L. Wiest,
and
A. E. Pegg.
Effect of expression of human spermidine/spermine N1-acetyltransferase in Escherichia coli.
Biochemistry
34:
2701-2709,
1995[Medline].
28.
Pegg, A. E.,
and
B. G. Erwin.
Induction of spermidine/spermine N1-acetyltransferase in rat tissues by polyamines.
Biochem. J.
231:
285-289,
1985[Medline].
29.
Seidel, E. R.,
and
R. G. Snyder.
Pentagastrin induction of spermidine/spermine N1-acetyltransferase and mucosal polyamines.
Am. J. Physiol.
256 (Gastrointest. Liver Physiol. 19):
G16-G21,
1989[Abstract/Free Full Text].
30.
Shinki, T.,
T. Kadofuku,
T. Sato,
and
T. Suda.
Spermidine N1-acetyltransferase has a larger role than ornithine decarboxylase in 1
, 25-dihydroxyvitamin D3-induced putrescine synthesis.
J. Biol. Chem.
261:
11712-11716,
1986[Abstract/Free Full Text].
31.
Tabor, C. W.
The effect of temperature on the acetylation of spermidine.
Biochem. Biophys. Res. Commun.
30:
339-342,
1968[Medline].
32.
Tabor, C. W.,
and
L. G. Dobbs.
Metabolism of 1,4-diaminobutane and spermidine in E. coli. The effect of low temperature during storage and harvesting of cultures.
J. Biol. Chem.
245:
2086-2091,
1970[Abstract/Free Full Text].
33.
Takenoshita, S.,
S. Matsuzaki,
G. Nakano,
H. Kimura,
H. Hoshi,
H. Shoda,
and
T. Nakamura.
Selective elevation of the N1-acetylspermidine level in human colorectal adenocarcinomas.
Cancer Res.
44:
845-847,
1984[Abstract].
34.
Tamori, A.,
S. Nishiguchi,
T. Kuroki,
N. Koh,
K. Kobayashi,
Y. Yano,
and
S. Otani.
Point mutation of ornithine decarboxylase gene in human hepatocellular carcinoma.
Cancer Res.
55:
3500-3503,
1995[Abstract].
35.
Thirumalai, C. H.,
C. C. Tseng,
K. Tabata,
L. R. Fitzpatrick,
and
L. R. Johnson.
Relationship between ornithine decarboxylase activity and gastric damage.
Am. J. Physiol.
253 (Gastrointest. Liver Physiol. 16):
G1-G6,
1987[Abstract/Free Full Text].
36.
Wang, J. Y.,
and
L. R. Johnson.
Luminal polyamines stimulate repair of gastric mucosal stress ulcers.
Am. J. Physiol.
259 (Gastrointest. Liver Physiol. 22):
G584-G592,
1990[Abstract/Free Full Text].
AJP Gastroint Liver Physiol 274(2):G299-G305
0193-1857/98 $5.00
Copyright © 1998 the American Physiological Society