Polyamines are required for microtubule formation during
gastric mucosal healing
Ali
Banan,
Shirley A.
McCormack, and
Leonard R.
Johnson
Department of Physiology and Biophysics, The University of
Tennessee College of Medicine, Memphis, Tennessee 38163
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ABSTRACT |
Polyamines are essential for the repair of
gastric and duodenal erosions. Concentrated NaCl (3.4 M) given
intragastrically damages the oxyntic gland mucosa and increases the
activity of gastric mucosal ornithine decarboxylase (ODC), the first
rate-limiting enzyme in polyamine synthesis. The nature of the process
of restitution of damaged mucosa is not well known, except that cell
migration and the actin cytoskeleton play a prominent role.
Microtubules are cytoskeletal components essential for cell migration.
The present investigation determines the relationship between
polyamines, the distribution of microtubules, and gastric healing in
mucosa damaged with hypertonic NaCl solution. Rats were fasted for 22 h
and then given 1.0 ml of 3.4 M NaCl intragastrically. Animals were
killed 1, 2, 4, 8, and 10 h after 3.4 M NaCl. The oxyntic gland mucosa
was removed, and tubulin was visualized by immunofluorescence. Microtubule density was increased around and below the damaged mucosa
in the upper one-third of the glandular epithelium at 2 and 4 h and
returned to near control levels by 10 h. In rats damaged with 3.4 M
NaCl and pretreated intraperitoneally with
-difluoromethylornithine (DFMO), a specific inhibitor of ODC, microtubule content was reduced significantly at all time points after NaCl treatment. Addition of
spermidine after pretreatment with DFMO and 3.4 M NaCl significantly prevented the effects of DFMO. Colchicine, a potent
microtubule-disrupting drug, significantly delayed normal gastric
mucosal healing with no effect on ODC activity. These data show that
polyamines influence the distribution of microtubules during damage in
vivo and indicate a partial mechanism for the dependency of mucosal
healing on polyamines.
colchicine; gastric ulcers; hypertonic NaCl;
-difluoromethylornithine; spermidine; ornithine decarboxylase
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INTRODUCTION |
FULL REPAIR OF MUCOSA damaged by stress or hypertonic
NaCl occurs by at least two different mechanisms. One mechanism, the rapid process of mucosal restitution or reepithelialization, takes place by migration of remaining viable cells from areas adjacent to or
just beneath the injured surface to cover the areas of partial or
complete surface desquamation (19, 20). The other mechanism, the
replacement of lost cells by cell division whereby mucosal thickness
returns to normal, is much slower and usually does not begin until
12-16 h after the insult. The process of restitution is
independent from cell division, depends on an intact basal lamina, may
take place in the absence of blood flow, and may not involve an
inflammatory response (20).
The mechanisms involved in the early rapid restoration of surface
epithelial continuity after mucosal damage are poorly understood. Mucosal reepithelialization occurs before the appearance of an inflammatory response and thus may prevent deeper mucosal damage after
injury. A series of studies indicates that polyamines, either synthesized endogenously or supplied luminally, are required for healing of gastric and duodenal mucosal stress erosions and that they
exert effects on both early mucosal restitution and the later phase
dependent on cell division (24, 25, 27). Oral administration of
putrescine, cadaverine, spermidine, or spermine after stress prevents
the delayed mucosal healing resulting from polyamine depletion.
Polyamines are used as substrates by enzyme transglutaminases and may
be involved in subsequent protein cross-linking and normal repair of
damaged mucosa (27). The time course of the repair process suggests
that polyamines are important for early mucosal restitution independent
of cell replacement (25). Furthermore, polyamine depletion causes the
disappearance of actin and microtubules in polyamine auxotrophic
Chinese hamster ovary (CHO) cells (17). When putrescine is added to the
medium after polyamine starvation, the cytoskeleton recovers within a
few hours (17).
Because mucosal restitution occurs by the sloughing of the damaged
epithelial cells and migration of remaining viable cells over the
denuded basal lamina, the microtubular cytoskeleton may be crucial in
reepithelialization. Microtubules provide a system of fibers for
vesicular and other membrane-bound organelle transport (2, 16). They
also regulate cell shape, cell movement, and the plane of cell division
(1, 6, 12). Fibroblasts treated with colchicine or Colcemid,
microtubule disrupting drugs, were unable to move directionally due to
the disappearance of stable zones of pseudopodial activity at the
leading edge (4). The role of the microtubules in the early restitution
of damaged gastric mucosa and their relationship to ornithine
decarboxylase (ODC, the first rate-limiting enzyme in polyamine
synthesis) activity and polyamine levels in vivo are unknown. The
objective of the current study was to investigate the relationship
between microtubule distribution, polyamines, ODC activity, and mucosal
healing in vivo.
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MATERIALS AND METHODS |
Animals and procedures.
Studies were performed using four or five rats per group. They were
conducted under protocol number 549 approved by The University of
Tennessee Health Science Center Animal Care and Use Committee on August
31, 1994. Male Sprague-Dawley rats weighing 125-150 g were housed
in wire-bottomed, raised cages and given water and standard laboratory
rat food ad libitum. All animals were obtained from Harlan
Sprague-Dawley (Indianapolis, IN). Animal quarters were maintained at a
temperature of 22 ± 1°C with a 12:12-h light-dark cycle.
Animals were fasted but allowed free access to tap water for 22 h
before the experiments.
In the studies determining the influence of colchicine, experimental
animals received a 0.2-ml intraperitoneal injection of 3 mg/kg
colchicine (Sigma Chemical) in 0.9% saline 30 min before and 6 h after
1 ml of 3.4 M NaCl given intragastrically. This dose of colchicine
disrupts microtubules in vivo (16). Control animals received 0.2 ml of
0.9% saline intraperitoneally, followed by 1 ml 3.4 M NaCl. Animals
were killed at 8 or 12 h after 3.4 M NaCl (5 rats per group). The
effect of colchicine on ODC activity has also been determined (4 rats
per group). Colchicine was given (3 mg/kg ip) 30 min before the
intragastric administration of 1 ml 3.4 M NaCl or isotonic saline. Rats
were killed 4 h after treatments, and ODC activity was determined.
Controls received 0.2 ml of 0.9% saline intraperitoneally followed by
1 ml 3.4 M NaCl or isotonic saline alone intragastrically.
In experiments with hypertonic NaCl, five rats were given 1 ml of 3.4 M
NaCl intragastrically, as described previously (23). The animals were
killed by anesthesia with excess methoxyflurane 1, 2, 4, 8, and 10 h
after hypertonic NaCl. The stomachs were removed and processed for
microtubule distribution. Control animals were given 1 ml isotonic
saline intragastrically.
In another experiment
-difluoromethylornithine (DFMO) was dissolved
in distilled water and administered intraperitoneally (5,000 mg/kg) 10 min before 3.4 M NaCl was administered intragastrically. This dose of
DFMO significantly delays the normal repair of gastric and duodenal
mucosa after stress (23). Four rats were killed at each time: 1, 2, 4, 8, and 10 h after 3.4 M NaCl administration. In another experiment
related to this series, DFMO was administered intraperitoneally (500 mg/kg), followed in 10 min by intragastric administration of 3.4 M NaCl
(4 rats). One hour later 100 mg/kg spermidine (100 µmol), dissolved
in 1 ml distilled water, was administered intragastrically. Control
rats were given isotonic saline intragastrically. Animals were killed 4 h after 3.4 M NaCl administration, and the distribution of microtubules
was determined.
In the final experiment, the influence of colchicine before the
administration of 1 ml of 3.4 NaCl on the distribution of gastric
mucosal microtubules was determined. Experimental animals received a
0.2-ml intraperitoneal injection of 3 mg/kg colchicine (Sigma Chemical)
in 0.9% saline 30 min before 3.4 M NaCl treatment (intragastric).
Control animals received 0.2 ml of 0.9% saline intraperitoneally,
followed by either 1 ml isotonic saline or 1 ml 3.4 M NaCl
intragastrically. Animals were killed 4, 8, and 12 h after treatments
(4 rats/group). The stomachs were removed and processed for staining.
The experimental protocols are outlined in Fig.
1.
Histology (microtubule staining).
Animals were quickly anesthetized with methoxyflurane at 1, 2, 4, 8, and 10 h after administration of 3.4 M NaCl and opened via a midline
laparotomy. A ligature was tied around the lower esophagus and proximal
duodenum, and 3 ml of 3.7% paraformaldehyde and 0.2% Triton X-100 in
buffer consisting of 10 mM PIPES, 5 mM EGTA, and 2 mM
MgCl2, pH 6.8, were injected into
the gastric lumen. Excised stomachs were placed in this fixative for 5 min, incised along the greater curvature, immersed for 20 min, and
postfixed in 95% ethanol at
20°C for 5 min. Samples of
oxyntic gland mucosa 1-3 mm thick were removed at right angles to
the long axis of the stomach, washed in Dulbecco's phosphate-buffered
saline (DPBS), and embedded in paraffin. Slices 5 µm thick were
obtained, attached to slides, and rehydrated, finally remaining in DPBS
for 30 min at room temperature. The slides were then incubated with
monoclonal anti-
-tubulin (Sigma ImmunoChemicals, St. Louis, MO),
1:200 dilution, for 1 h at room temperature. Slides were washed three
times in DPBS, incubated with tetramethylrhodamine B
isothiocyanate-conjugated goat anti-mouse IgG (Sigma ImmunoChemicals),
1:32 dilution for 1 h at room temperature, again washed in DPBS,
quickly rinsed in H2O, and mounted
with Aquamount (Lerner Laboratories, Pittsburgh, PA). Slides were
examined in a blinded fashion by coding them so that the examiner (A. Banan) had no knowledge of the experimental protocol. Slides were
viewed in a Nikon photomicroscope (Diaphot) and decoded only after
examination was completed. The intensity of microtubule staining per
10,000 square pixels was determined using images collected with a
Photometric CH250 cooled charge-coupled device camera and IPLab
Spectrum software for intensity determination. Gastric mucosa was
considered injured based on the presence of one or more of the
following: necrotic surface epithelium, hyperemic vessels, hemorrhage,
or damage of superficial cells indicated by cytoplasmic vacuolization,
cytoplasmic swelling, nuclear pyknosis, or nuclear swelling with
chromatin margination.
Ulcer index (macroscopic and microscopic).
Animals were quickly anesthetized with methoxyflurane at 4, 8, and 12 h
after treatment, and stomachs were exposed via a midline laparotomy.
The stomachs were removed, opened along the greater curvature, and
rinsed in ice-cold saline. They were spread flat on a petri dish (over
ice) and examined for gross damage (macroscopic). The method described
by Takagi and Okabe (22) was used to determine the severity of lesions.
The incidence of lesions was noted, and the length of the visible
lesions was measured. The macroscopic ulcer index was expressed as
total lesion length in millimeters. Samples of the stomach were cut
from the glandular epithelium in an area along the greater curvature
2-3 mm below the limiting ridge that separates the forestomach
from the glandular area. Two blocks were removed at right angles to the
long axis of the stomach, separated 0.5 cm, and placed in 10% Formalin
in DPBS (total of 5 sections, each 1 × 10 mm, obtained from 2 blocks separated 0.5 cm distally from each other). Four rats were
examined from each group. Routine paraffin-embedded hematoxylin and
eosin-stained sections were used for evaluation under a light
microscope. Slides were coded so that the examiner (A. Banan) had no
knowledge of the experimental protocol used. The slides were decoded
only after examination was complete. Within each tissue block from each
stomach, the overall length of each tissue section was measured using a graded micrometer eyepiece. The corresponding percentage of the mucosal
surface injury (percentage of microscopic lesions) was determined as
the ratio of surface damage to the overall length of each section.
Gastric mucosa was considered injured based on the criteria mentioned
previously.
ODC assay.
The activity of ODC was assayed by a radiometric technique in which the
amount of
14CO2
liberated from
L-[1-14C]ornithine
(New England Nuclear, Boston, MA) was measured (20). Tissue samples
were collected as described above and placed in 1.0 ml 20 mM
Tris · HCl, pH 7.4, 0.05 EDTA, 0.05 mM pyridoxal phosphate, and 5 mM dithiothreitol. ODC activity is
dependent on the presence of pyridoxal phosphate, and DTT stabilizes
enzymatic activity. The mucosa was homogenized, sonicated, and
centrifuged at 30,000 g at 4°C for
30 min. An aliquot from the 30,000 g
supernatant was incubated in stoppered vials in the presence of 6.8 nmol of [14C]ornithine
for 15 min at 37°C. The
14CO2
liberated by the decarboxylation of ornithine was trapped on a piece of
filter paper impregnated with 20 µl of 2 N NaOH, which was suspended
in a center well above the reaction mixture. The reaction was stopped
by the addition of trichloroacetic acid to a final concentration of
10%. The
14CO2
trapped in the filter paper was measured by liquid scintillation spectroscopy at a counting efficiency of 95%. Aliquots of the 30,000 g supernatant were assayed for total
protein, using the method described by Bradford (5). Enzymatic activity
was expressed as picomoles of CO2
per milligram of protein per hour.
Statistics.
Data are presented as means ± SE of four to five rats per group.
Statistical analysis was performed using the two-tailed Dunnett's multiple comparison test (9), and values of
P < 0.05 were considered significant. Analysis of variance and a Duncan's multiple-range test
were used to compare the points in Fig. 2.

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Fig. 2.
Intensity of microtubule luminescence in control gastric mucosa and
mucosa damaged with 3.4 M NaCl 0.5, 1.5, 2, 4, 8, and 10 h after
treatment. Intensity of luminescence per 10,000 square pixels was
determined from gastric pit areas. Data are means ± SE from 5 rats
per group. * P < 0.05 compared
with control group.
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RESULTS |
Microtubular luminescence at 0.5, 1.5, 2, 4, 8, and 10 h after
hypertonic NaCl treatment is shown in Fig. 2. The intensity of
microtubule staining increased significantly 2 and 4 h after 3.4 M
NaCl. Intensity was greatest 4 h after 3.4 M NaCl and decreased to the
control level 10 h after treatment. Microtubule staining 8 and 10 h
after exposure to NaCl was similar to the control.
Mucosa from rats pretreated with DFMO had a nearly complete lack of
microtubule staining at all time points after 3.4 M NaCl administration
(Fig. 3). Polyamine depletion by DFMO
resulted in a distribution of microtubules similar to that in control
rats (Fig. 3a). Polyamine depletion
prevented most of the increase in microtubule polymerization produced
by hypertonic NaCl damage (Fig. 3,
b-e).

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Fig. 3.
Distribution of microtubules in oxyntic gland mucosa in rats treated
with isotonic saline (a) or DFMO
(500 mg/kg ip) + 3.4 M NaCl 2 h (b),
4 h (c), 8 h
(d), and 10 h
(e) after treatment
(paraffin-embedded sections; monoclonal anti- -tubulin antibody and
tetremethylrhodamine B isothiocyanate-conjugated anti-mouse IgG
antibody). Bar, 100 µm.
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Figure 4 shows gastric mucosa from rats
treated with isotonic saline (Fig.
4a) or hypertonic NaCl (Fig.
4b; group
b), pretreated with DFMO before 3.4 M NaCl (Fig.
4c), or given 100 mg spermidine intragastrically 1 h after 3.4 M NaCl damage and pretreated with DFMO
10 min before damage (Fig. 4d; DFMO + NaCl + SPD; group d). Rats in
groups b and
d have the highest intensity of
microtubule staining immediately below the damaged mucosa in the
gastric pit area located adjacent to the lumen. Exogenous spermidine
given to NaCl-damaged rats treated with DFMO increased
(P < 0.05) the polymerization and
organization of microtubules in the mucosa in a manner similar to that
seen after damage of untreated rats, preventing the effect of polyamine
depletion. The values for the intensity of microtuble luminescence for
the critical 4-h time point for the various groups depicted in Figs. 3
and 4 are shown in Table 1.

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Fig. 4.
Distribution of microtubules in oxyntic gland mucosa from rats treated
with isotonic saline (a), 3.4 M NaCl
(b; group
b), DFMO + 3.4 M NaCl
(c), and DFMO + 3.4 M NaCl + 100 mg
spermidine (d; group
d) 4 h after treatment. Rats in
groups b and
d have the highest intensity of
microtubule staining immediately below the damaged mucosa in the
gastric pit area. Bar, 100 µm.
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Intraperitoneal injection of colchicine 30 min before intragastric
administration of 3.4 M NaCl significantly
(P < 0.05) prevented healing (Fig.
5), as indicated by an increase in the
macroscopic ulcer index observed 8 and 12 h after 3.4 M NaCl alone.
Control animals healed normally. Microscopic examination also
demonstrated that colchicine prevented healing, as evidenced by a high
level of lesions 8 and 12 h after hypertonic NaCl (Fig.
6). At 4 h the values for 3.4 M NaCl and
3.4 M NaCl plus colchicine were not significantly different for both
indexes of damage.

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Fig. 5.
Ulcer index in rats treated with 3 mg/kg colchicine. Colchicine was
administered intraperitoneally 30 min before 3.4 M NaCl damage and
repeated at 6-h intervals. Animals were killed 8 and 12 h after 3.4 M
NaCl. Effect of colchicine is compared with normal rate of healing seen
in animals 4, 8, and 12 h after administration of 3.4 M NaCl. Data are
means ± SE from 4 rats per group.
* P < 0.05 compared with 8- and 12-h NaCl groups.
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Fig. 6.
Microscopic lesions in rats treated as in Fig. 5. Microscopic lesion
(%) is ratio of surface damage to overall length of each section. Data
are means ± SE from 4 rats per group.
* P < 0.05 compared with 8- and 12-h NaCl groups.
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Figure 7 shows the distribution of
microtubules in the oxyntic gland mucosa of rats treated with isotonic
saline (Fig. 7a), 3.4 M NaCl (Fig.
7b), and colchicine + 3.4 M NaCl
(Fig. 7c) 4 h after treatment.
Colchicine also totally prevented the increase in luminescence that
normally occurred after damage by 3.4 M NaCl (Table 1). Intraperitoneal
injection of colchicine 30 min before 3.4 M NaCl significantly reduced
the microtubule content of the mucosa, whereas rats treated with
isotonic saline demonstrated a normal distribution of microtubules, and
mucosa damaged with hypertonic NaCl had a significant increase in the
same cytoskeletal parameter. No significant changes in ODC activity
were seen between the controls and rats treated with colchicine or
between those damaged with hypertonic NaCl and those given colchicine
before 3.4 M NaCl treatment (Fig. 8).

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Fig. 7.
Distribution of microtubules in oxyntic gland mucosa of rats treated
with isotonic saline (a), 3.4 M NaCl
(b), and colchicine + 3.4 M NaCl
(c) 4 h after treatment. Colchicine
(3 mg/kg ip) was given 30 min before 3.4 M NaCl treatment. Bar, 100 µm.
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Fig. 8.
Ornithine decarboxylase (ODC) activity in oxyntic gland mucosa from
rats given isotonic saline (control) or damaged with 3.4 M NaCl (NaCl)
with or without 3 mg/kg ip colchicine (Colch.) 30 min earlier. Animals
were killed 4 h after treatment. Values are means ± SE of 4 rats
per group.
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DISCUSSION |
Microtubules are present in nearly all eukaryotic cells, although in
smaller amounts than actin. When a cell migrates, microtubules and the
actin cytoskeleton may act in concert (16). The importance of actin
and/or microtubules in cell migration varies among cell types.
Microtubules seem to be essential for fibroblast migration, since
colchicine treatment, which depolymerizes microtubules, inhibits
fibroblast movement (4). The opposite is true in neutrophils, which are
more dependent on the actin cytoskeleton for migration (11). The growth
cone of nerve cells, however, depends on both microtubules and actin
filaments (16). Maintenance of an elongated shape and polarized
pseudopodic activity of many cultured cell types such as fibroblasts
and neurons depends on intact microtubules. In experiments with
microtubule-depolymerizing agents such as colchicine, Colcemid, or
nocadazole, fibroblasts are unable to move in any direction due to the
disappearance of stable zones of leading-edge pseudopods (4).
Microtubules enhance or localize pseudopodial activity to one part of
the cell, resulting in extension of the cell and directed movement (8).
In another study, microtubule-dependent control of pseudopodial
activity and cell shape was prevented by antikinesin motor domain
antibodies. Kinesin is a motor or translocator protein associated with
microtubules and is responsible for microtubule-dependent transport
toward the cell periphery (18). Thus available information suggests
that microtubules are necessary for cell migration and motility.
Mucosal restitution or reepithelialization establishes surface mucosal
continuity after erosions and before cell division occurs (15, 19, 20).
In one study, restitution of frog gastric mucosa mounted in Ussing
chambers and damaged by exposure to 1 M NaCl for 10 min began 1-2
h later, before incorporation of tritiated thymidine indicated an
increase in DNA synthesis (21). Between 2 and 4 h, the continuity of
the epithelial surface was progressively restored, and the flattened
epithelial cells gradually became more cuboidal. Overall, gastric pits
were shortened, and parietal and mucous neck cells were found closer to
the lumen. In another study in rats, surface mucosal damage induced by
aspirin was covered by an undifferentiated epithelium as early as 30 min after damage (28), and the gastric surface healed within several
hours (10).
Little is known about the nature of the process of early restitution of
damaged mucosa except that cell migration plays an important role. We
have shown that actin polymerization immediately following damage with
hypertonic NaCl appears to be essential to early mucosal restitution
and depends on the induction of ODC (3). DFMO, a potent inhibitor of
ODC activity, not only decreased the amount of F-actin in vivo after
damage but also prevented healing. Prevention of normal actin
polymerization by cytochalasin D also delayed mucosal healing.
Spermidine prevented the effect of DFMO on both actin polymerization
and healing. Finally, the time course for the induction of ODC
paralleled the course of healing of the damaged mucosa, and that in
turn correlated with the time course of F-actin and polymerization.
Overall, these findings suggested that remodeling of the actin
cytoskeleton plays an important part in early mucosal restitution and
that polyamines are essential to this process. The findings reported in
the current study also implicate the microtubular system in the process
of mucosal restitution and present evidence that polyamines are
essential to its involvement.
A few studies have demonstrated that polyamine depletion alters the
microtubular system. Pohjanpelto et al. (17) found that DFMO caused the
disappearance of microtubules as well as of the actin cytoskeleton in
polyamine auxotrophic CHO cells. Addition of putrescine to the medium
resulted within a few hours in the complete recovery of both the
microtubules and actin filaments in the same cells. Kaminska et al.
(13, 14) have reported that inhibitors of polyamine biosynthesis,
methylglyoxal-bis-(guanylhydrazone) and DFMO, inhibited
the mitogen-stimulated increase in mRNA for the cytoskeletal elements
-actin and
-tubulin in mouse splenocytes and T lymphocytes.
We found a significant increase in microtubular formation 2 h after the
exposure of rat gastric mucosa to a damaging concentration of NaCl
(Fig. 2). The amount of microtubules remained significantly elevated
for up to 4 h and returned to near control levels by 8 h. By 8 h the
mucosa was more than 60% restored, and by 12 h healing was near
completion (Fig. 5). Thus the appearance of microtubules correlated
with the period of mucosal restitution. Inhibition of ODC with DFMO
prevented the appearance of microtubules for at least 10 h after damage
(Fig. 3). As we have previously shown, ODC is induced after damage with
hypertonic NaCl or by stress, and DFMO prevents that induction and
prevents healing (23, 26). In the current study, the induction of ODC
by 3.4 M NaCl is shown in Fig. 8. The product of the ODC reaction is
putrescine, which is converted rapidly to spermidine, and addition of
this polyamine to the stomachs of rats treated with DFMO resulted in
the normal appearance of microtubules in response to damage (Fig. 4).
These results, therefore, demonstrate that the appearance of
microtubules after in vivo damage to the gastric mucosa is polyamine
dependent.
Administration of colchicine, a potent microtubule depolymerizing
agent, essentially reproduced the effects of DFMO. Each molecule of
colchicine binds tightly to one tubulin molecule, preventing
polymerization and subsequent microtubule formation (7). Since
microtubules are in equilibrium with tubulin molecules, colchicine also
leads to the disappearance of existing microtubules. As shown in Fig.
7, colchicine prevented the appearance of microtubules in mucosa
damaged with hypertonic NaCl and caused the disappearance of
microtubules from normal control gastric mucosa. These findings are
identical to those produced by DFMO (Fig. 3). Colchicine also delayed
healing, as shown in Figs. 5 and 6. Twelve hours after damage, the
ulcer index from colchicine-treated rats was still 75% of that present
at 4 h, whereas the ulcer index of normally healing stomachs was less
than 10% of the level at 4 h. This finding is also identical to the
effects of DFMO on healing of lesions induced by either hypertonic NaCl
(3, 23) or stress (25, 26). Figure 8 shows that colchicine had no
effect on basal levels of ODC activity and that it did not prevent the
induction of ODC after damage by 3.4 M NaCl. This important control
demonstrates that the effects of cholchicine were due to disruption of
microtubules and to alterations in polyamine metabolism. More
importantly, this is strong evidence that the reverse is true, namely,
that the appearance of microtubules is essential to the normal healing of gastric mucosa.
The molecular basis of the interaction between polyamines and the
microtubular system in cells is unknown. Although polyamine depletion
has been shown to inhibit the transcription of
-tubulin mRNA, the
effect did not occur until 48-72 h later (13), which is well
beyond the time course for mucosal restitution. After damage,
microtubules increased significantly within 2 h. This early time point
suggests that polyamines directly affect the polymerization and
organization of microtubules during the restitution phase, independent
of an effect on tubulin gene expression or protein synthesis.
Polyamines are polycations, since the amine groups are all charged at
physiological pH. Separated by a backbone of carbon atoms, these
positively charged amine groups can bind to negative charges on
macromolecules such as proteins and nucleic acids. Thus polyamines
could link tubulin molecules via electrostatic interactions. Polyamines
also serve as substrates for transglutaminase and cross-link proteins
through normal covalent bonds. We have shown that protein cross-linking
is essential for mucosal healing (27).
In summary, the polymerization and organization of microtubules
immediately after damage with hypertonic (3.4 M) NaCl is essential to
early mucosal restitution in vivo and depends on the presence of
polyamines. DFMO, a potent inhibitor of ODC activity, decreased the
amount of microtubules in vivo after damage. Prevention of normal
microtubule polymerization by colchicine significantly inhibited
mucosal healing. Spermidine prevented the effect of DFMO on the
polymerization and organization of microtubules. These data provide
evidence, for the first time, that the dependence on polyamines for
normal mucosal healing is related to the effects on the assembly of
microtubules.
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ACKNOWLEDGEMENTS |
We thank Danny Morse for photographs and other illustrations.
 |
FOOTNOTES |
This work was supported by National Institute of Diabetes and Digestive
and Kidney Diseases Grant DK-52784.
Address for reprint requests: L. R. Johnson, Dept. of Physiology and
Biophysics, Univ. of Tennessee, Nash Research Bldg., 894 Union Ave.,
Memphis, TN 38163.
Received 23 June 1997; accepted in final form 22 January 1998.
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