Intestinal ornithine decarboxylase: and regulation by putrescine KIMIKAZU SHIRLEY Department
half-life
IWAMI, JIAN-YING WANG, RAJEEVE JAIN, McCORMACK, AND LEONARD R. JOHNSON of Physiology and Cell Biology, University of Texas Medical School, Houston, Texas 77030
is one of the earliest events associated with cellular proliferation (14, 17). The most striking characteristics of ODC are its rapid turnover rate and changes in activity in response to various stimuli (29). The half-life of ODC in mammalian tissues, where it has been measured, is 15-30 min, which is much shorter than that of any other mammalian enzyme (29). Another important characteristic of this enzyme is that it is regulated by putrescine, its end product, by a mechanism other than end-product inhibition. Kay and Lindsay (10) first demonstrated that micromolar concentrations of exogenous putrescine prevented the increase in ODC activity in lectin-stimulated lymphocytes. Inhibition occurs with concentrations of putrescine considerably below those that directly inhibit purified ODC and has been shown to occur in many different cell types (29). Inhibition is caused by one or more proteins induced by putrescine. These proteins are called antizymes and inhibit ODC by binding noncovalently to it. Proteins with antizyme activity have been isolated and purified from both Escherichia coli and rat, liver (see Ref. 29 for review). The standard technique for measuring ODC activity is to incubate the enzyme preparation with ornithine, labeled in the l-carbon or carboxyl position. The 14C02is then trapped and counted. Numerous studies employing this technique have shown that ODC in the small intespolyamines; crypt cells; villous cells; enzyme regulation; growth; tine has a relatively high basal activity (1) yet increases rat mucosa; cell culture dramatically with feeding (13, 27, 28). In addition to increased food intake, intestinal mucosal ODC levels increase after partial gut, resection (12), during lactation THE POLYAMINES, spermidine and spermine and their (32), and after luminal obstruction (21). Each of these precursor putrescine, are found in virtually all cells of events increases the exposure of the gut mucosa to food higher eukaryotes (14). The exact action of polyamines or luminal contents, and each is associated with inat the molecular level is unknown. It is likely that these creased mucosal growth. Inhibition of ODC activity by substances are involved in a variety of cellular functions, the specific inhibitor difluoromethylornithine (DFMO) including membrane stabilization, translation processes, prevents the growth responsesto refeeding (28), lactation regulation of RNA and DNA polymerase activity, regu- (32), and resection (12). Numerous laboratories have lation of protein kinase activity, and transfer RNA acylashown that virtually all of the ODC activity measured in tion reactions (14, 17). Polyamines are intimately in- the intestinal mucosa by 14C02collection is localized in volved in, and required for, cell growth and differentiathe differentiated villous cells and not, as one would tion, and their concentrations within cells are highly expect, in the proliferative crypt cells (5, 15, 23). Howregulated. Intracellular polyamine levels are highly de- ever, by use of a highly sensitive ODC antibody we have pendent on the activity of ornithine decarboxylase (ODC; been able to demonstrate specific ODC in rat intestinal EC 4.1.1.17), which is the initial rate-controlling enzyme crypt cells and show that its levels are increased by in the polyamine synthetic pathway. In most tissues, trophic substances such as gastrin and epidermal growth factor (EGF) (9). The ODC of the villous cells did not ODC levels are low, and an increase in enzyme activity IWAMI, KIMIKAZU, LEY MCCORMACK,
JIAN-YING AND LEONARD
WANG,
RAJEEVE
JAIN,
SHIR-
R. JOHNSON. Intestinal ornithine decarboxylase: half-life and regulation byputrescine. Am. J. Physiol. 258 (Gastrointest. Liver Physiol. 21): G308-G315, 1990.-Ornithine decarboxylase (ODC) is the primary ratelimiting enzyme for polyamine synthesis. ODC levels are increased in most tissues, including the intestinal mucosa, by growth-promoting agents. This enzyme has a brief half-life of from 5 to 30 min in mammalian tissues and is regulated by its product, putrescine. The current study examines the turnover and regulation of ODC in the mucosa of the small intestine. With the use of scraped intestinal mucosa from cycloheximidetreated rats, the time course of the decline in ODC activity yielded a half-life of -22 min. Labeling enzyme protein with [“Hldifluoromethylornithine (DFMO) resulted in a nearly identical estimation of half-life. ODC activity of mucosa from isolated gut segments stimulated by luminal glycine (0.1-0.4 M) was enhanced 60-100% by 10 mM putrescine administered luminally. Putrescine alone had no effect on ODC. In contrast, lo-' M putrescine prevented 80% of the ODC activity stimulated by asparagine in IEC-6 cells (a rat intestinal crypt cell line). The half-life of ODC in unstimulated IEC-6 cells was 20 min and increased to 35 min in cells exposed to 10 mM asparagine. These data demonstrate that ODC of nonproliferating villous cells is regulated differently from the identical enzyme in proliferating crypt cells. Therefore, conclusions regarding mucosal growth should not be based totally on ODC activity from whole mucosa, since it is essentially a measure of only the enzyme present in the villous cells.
G308
0193-1857/90
$1.50 Copyright
0 1990 the American
Physiological
Society
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REGULATION
OF
respond to gastrin but was increased by luminal nutrients such as glycine. Thus the agents that increase ODC levels in these two cell types are different. The purpose of the current study is to determine the half-life of ODC in villous cells and in IEC-6 cells and to examine the effect of putrescine on the regulation of enzyme activity. The IEC-6 cell line originated from normal rat intestinal crypt cells, as judged by morphological and immunological criteria, and they retain the growth characteristics of undifferentiated crypt cells. METHODS
Studies are divided into those done in rats and in cultured IEC-6 cells. Each of these categories is further divided into the determination of the half-life of ODC and the measurement of the effect of putrescine on ODC activity. Studies in Rats ODC half-life determination. Male Sprague-Dawley rats weighing X0-170 g were housed in wire-bottomed cages in a room with a constant temperature and a 12-h light-dark cycle and were fed ad libitum on Purina Rat Chow. Rats were then fasted 48 h and refed for 3 h. Refeeding for 3 h results in maximal stimulation of intestinal ODC activity (28). At this time animals were injected intraperitoneally with either cycloheximide (25 mg/kg) or [ 3H] DFMO [ DL- [ 3,4-3H] difluoromethylornithine, 20 Ci/mmol, New England Nuclear, Boston, MA). [3H]DFM0 was given at a dose of 20 &i/animal in 0.4 ml saline. Animals treated with cycloheximide were killed at 0, 30, or 60 min after injecting the drug. The proximal third of the small intestine was removed, rinsed in ice-cold saline, and placed in a beaker of ice-cold saline. The mucosa was collected by scraping with a glass slide over an ice-cold glass plate. Mucosal scrapings were weighed and divided into two portions. One was used to measure protein and the other to determine the rate of ornithine decarboxylation. Ornithine decarboxylase activity was assayed by a radiometric method in which the amount of 14C02 liberated from DL-[ l-14C]ornithine (53 mCi/ mmol, New England Nuclear) was estimated (27). Intestinal mucosa was collected and placed in 0.0667 M sodium-potassium phosphate buffer (pH 7.4) containing 0.02% lauryl ether, 5 mM NaF, 10 PM phosphate, and 10 mM EDTA (ODC assay buffer). The tissues were homogenized, sonicated, and centrifuged at 30,000 g for 25 min. An aliquot from the 30,000-g supernatant was incubated in stoppered vials in the presence of 2 nmol of [14C]ornithine for 15 min at 37OC. The 14C02 liberated by the decarboxylation was trapped on a piece of filter paper and impregnated with 20 ~1 of 2 N NaOH, which was suspended in a centerwell above the reaction mixture. The reaction was stopped by the addition of trichloroacetic acid (TCA) to a final concentration of 10%. The 14C02 trapped in the filter paper was measured by liquid scintillation spectrometry at a counting efficiency of 99%. Blanks were run simultaneously by using a vehicle instead of the supernatant. The protein content
INTESTINAL
ODC
G309
of the samples was determined by using the Bradford (3) method, and results are expressed as picomoles CO2 per hour per milligram protein. The decrease in enzyme activity over time after cycloheximide was used to calculate the half-life of ODC activity. Two rats were studied at each time point during each experiment. A total of three experiments were conducted, so n = 6. Data are expressed as means t SE of six observations. Experiments were conducted in animals injected with [3H]DFM0 to determine the half-life of ODC protein itself. DFMO is an enzyme-activated irreversible inhibitor of ODC activity. When exposed to [3H]DFM0, the enzyme protein becomes labeled as activity is lost (20). Thirty minutes after the injection of [3H]DFM0, 0.6 ml saline containing 50 mg/ml of unlabeled DFMO (kindly donated by Merrell Dow Pharmaceuticals, Cincinnati, OH) was injected into the abdominal cavity. The presence of excess unlabeled DFMO halted the binding of [3H]DFM0 to enzyme protein. Rats were killed by decapitation 20, 30, 50, or 60 min after administering unlabeled DFMO and the small intestine was rapidly excised, rinsed, and frozen in liquid nitrogen. Samples were stored at -80°C until assay. Mucosa from the proximal third of the intestine was collected as previously described above, homogenized in 3 vol of the ODC assay buffer, and centrifuged at 30,000 g for 60 min. The extracts were dialyzed for 3 days to remove [3H]DFM0 that was not bound to protein: 24 h against the ODC assay buffer, and twice (24 h each time) against 1 liter of 50 mM pH 6.86 phosphate buffer containing 0.25 mM NaF, 5 PM EDTA, 0.33 mM dithiothreitol, and 0.02 mM pyridoxal phosphate. The dialysates were transferred to scintillation vials and evaporated on a 40-45°C plate. The remaining protein residues were dissolved in 0.5 ml Protosol. Five milliliters Hydrofluor and one drop of concentrated HCl were added. The radioactivity of the samples was determined by scintillation counting and expressed as counts per minute [3H]DFM0 per gram protein. Each data point represents the mean t SE of five separate animals. Effect of putrescine on ODC activity. ODC activity was measured in isolated intestinal segments as described previously (8). In brief, rats weighing 125-150 g were fasted 48 h and anesthetized with ether; the peritoneal cavity was opened via a midline incision, and one or two 5-cm jejunal segments, 20-30 cm distal from the pylorus, were isolated by ligation. In a typical experiment, 0.5 ml saline or a test solution was injected into each segment using a l-ml syringe. The midline incision was closed and the animals were allowed to recover in individual cages without food or water. Two hours after injecting test substances the rats were killed by an overdose of ether followed by cervical dislocation. All animals were killed between noon and 1 P.M. The individual segments were removed, opened, and rinsed in ice-cold saline. The mucosa was isolated by scraping and the ODC activity measured as described above. In the first study, the effect of putrescine on basal ODC activity was determined. With the use of one isolated segment per rat, putrescine was injected at concentrations of 0.08, 0.20, 5.0, and 10.0 mM. Control rats
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G310
REGULATION
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were injected with saline. The experiment was repeated twice with three rats per group so that n = 9 for each dose of putrescine. This experiment was also carried out killing the rats 4 h after injection instead of after 2 h. This subset was repeated only once, so n = 6. In a second study, the effect of 10 mM putrescine on ODC activity stimulated by different concentrations of glycine was determined. Rats were prepared with two isolated segments, and both segments were injected with saline, a given concentration of glycine (either 0.05, 0.1, 0.2, or 0.4 M), or the same concentration of glycine plus 10 mM putrescine. Only one dose of glycine was used in a given experiment, and separate saline controls were carried out in each experiment. The values from the two loops were averaged and at least six rats were used at each dose of glycine. Studies Using IEC-6 Cells IEC-6 cells (a normal rat small intestinal epithelial cell line) were obtained from the American Type Culture Collection (Rockville, MD) at passage 13. The stock was maintained in T-150 flasks in Dulbecco’s minimal essential medium (DMEM, GIBCO, Grand Island, NY) containing 5% dialyzed fetal bovine serum (FBS, GIBCO), 50 pg gentamicin sulfate, and 10 pg insulin/ml (growth medium) in 90% air-lo% CO2 at 37°C. Tests for mycoplasma were always negative, and the stock was fed three times a week and split 120 once a week. IEC-6 cells have the characteristics of normal cells cultured in vitro and show a number of features that suggest they are derived from undifferentiated small intestinal crypt cells (16). Polyamine dependence of IEC-6 cell. The dependence of IEC-6 cells on polyamines for growth was tested by inhibiting ODC with DFMO and then supplying exogenous putrescine to inhibited cultures. Cells were plated at a density of 12 x lo3 cells/cm2 in T-25 flasks in growth medium (control), growth medium plus 5 mM DFMO, and growth medium plus 5 mM DFMO plus 0.01 mM putrescine. The medium was replaced every other day and cells from three flasks from each treatment group were taken up with trypsin/EDTA and counted with a Coulter counter, model 031 ZF. The experiment was continued for 10 days. Rate of ODC turnover. Cells were plated at a density of -0.5 x lo6 cells/plate and grown for 48 h. In the first study 10 mM asparagine (final concentration) was added and cells were harvested hourly for 5 h. Asparagine has been shown to stimulate ODC activity in many systems. It appears to be the most potent type A amino acid for inducing ODC, and the type A amino acids are much more effective than the others (31). ODC activity was measured and determined to be maximal between 2 and 3 h after stimulation by asparagine. In the next set of experiments cycloheximide (to a final concentration of 50 pg/ml) was added to cultures 2.5 h after the addition of 10 mM asparagine. Other cultures received cycloheximide without being stimulated with asparagine. Cultures were harvested 15, 30, 45, 60, 90, and 120 min after the addition of cycloheximide by washing the cells twice in ice-cold 0.1 M tris( hydroxymethyl)aminomethane (Tris) buffer (pH 7.4) containing 0.1 mM EDTA, 50 PM pyri-
INTESTINAL
ODC
doxal 5-phosphate, and 5 mM dithiothreitol and were scraped into 0.5 ml of the same buffer. Cells were then frozen and thawed three times, sonicated, and centrifuged at 2,000 revolutions/min for 10 min. The ODC activity of an aliquot of the supernatant was incubated in a stoppered test tube in the presence of 2.5 mM [‘4C]-l-ornithine for 15 min at 37°C. Liberated 14C02 was trapped and counted, and the ODC activity was calculated and expressed as described above. Effect of putrescine on ODC activity. Cells were cultured as described above and harvested 2.5 h after the addition of different concentrations of either asparagine or putrescine. Asparagine was added to give concentrations of either 0, 2.5, 5, 7.5, 10, 12.5, 15, or 20 mM. Putrescine was added to concentrations of 0, lo-l2 M, and molar concentrations increasing 1 log unit from 10-l’ to 10W3. Finally 10 mM asparagine was added to cultures containing putrescine in molar concentrations increasing 1 log unit from lo-l1 to 10B5. Cells were harvested and ODC activities determined as described above. Statistics All data are expressed as means t SE. The significance of the differences between means was determined by analysis of variance. The level of significance was determined using the Duncan’s multiple range test (25). RESULTS
Studies in Rats ODC half-life determination. Three hours after the start of refeeding, ODC activity had increased -5O-fold to 232 pmol CO2. h-l mg protein? Cycloheximide inhibited de novo ODC synthesis, causing a rapid decrease in enzyme activity (Fig. 1). By 60 min after cycloheximide, activity had decreased to 37. From Fig. 1 the half-life of enzyme activity was calculated to be 20 min. The determination of the half-life of ODC protein itself is shown in Fig. 2. The radioactivity of undialyzable [3H]DFM0 declined log linearly over time yielding a protein half-life of 21.5 min. Although the value obtained for the half-life corresponded well with that obtained in Fig. 1 and the correlation coefficient of the line (r = -0.86) was reasonable, DFMO-labeled ODC is rapidly cleared and there were few counts remaining by the end of 60 min. Effect of putrescine on ODC actiuity. Injection of putrescine by itself into the isolated proximal intestinal loops caused no significant change in ODC activity compared with saline alone (data not shown). This was true for all doses of the diamine. Glycine alone significantly increased ODC activity in doses from 0.1 to 0.4 M (Fig. 3). As is also shown in Fig. 3, 0.05 M glycine did not alter ODC activity compared with saline. The addition of 10 mM putrescine to glycine significantly enhanced ODC activity compared with glycine alone but only when the concentration of glycine itself significantly stimulated enzyme activity. For example, as is shown in Fig. 3, the addition of 10 mM putrescine to 0.05 M glycine had no effect on ODC activity. The enhancement of ODC activity caused by putrescine over the response to glycine l
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REGULATION
OF INTESTINAL
G311
ODC 500 , -
z.3 g
t
400 I .-
E 2 s-G 300 b ,nE
0 F +
200
x 5 2
3
-
.-
.-
100
8
0 4
0.05
0.10
0.20
Glycine [M] I
1
0
30
60
Time after Cycloheximide
(min)
FIG. 1. Decline in ornithine decarboxylase (ODC) activity 30 and 60 min after injection of 25 mg/kg cycloheximide. Data are means z!z SE of 6 observations.
FIG. 3. ODC activity of mucosa from in situ-isolated segments of proximal small bowel. Segments were filled for 2 h with either saline, different concentrations of glycine, or the combination of glycine doses plus 10 mM putrescine. Columns show means + SE from at least 6 rats. * P < 0.05 compared with saline; t P < 0.05 compared with glycine alone.
500 400 II
P
Control
---s
DFMO
P
8100 G-
DFMO + PUT
l-
80-
k f: m
60
-
0
4
6
8
10
Day
t 40 t I
2
4. Growth of IEC-6 cells in control cultures, cultures in which ODC was inhibited with 5 mM DFMO, and cultures inhibited but to which 0.01 mM putrescine had been added. Cells were grown in Dulbecco’s minimal essential medium containing 5% dialyzed fetal bovine serum. Medium was changed every second day when numbers of cells present in 3 flasks from each group were determined. Means t SE from 3 flasks. * P < 0.05 compared with control and DFMO + putrestine. FIG.
I 20
I 40
h 60
Time After Cold DFMO (min) FIG. 2. Decline in radioactivity of undialyzable fraction from proximal small bowel mucosa of rats injected with 20 &i [3H]difluoromethylornithine (DFMO). Thirty milligrams of unlabeled DFMO were injected at time 0, 30 min after 13H]DFM0. Data points represent [3H]DFM0 bound to ODC and are means + SE for 5 animals. Correlation coefficient is -0.86.
alone ranged from 40 to 60%. Since enzyme activity did not increase in response to putrescine alone, this enhancement can be described as potentiation rather than an additive effect. Studies Using the IEC-6 Cells Polyamine dependence of IEC-6 cells. Continuing
mal cell growth was dependent
on putrescine
nor(Fig. 4).
After initial plating, cells began to divide after day 2. By day 4 there were no significant differences in the numbers of cells present in the three different groups. However, the cells treated with 5 mM DFMO stopped dividing at this point. By day 6 control cultures and cultures treated with DFMO that received exogenous 0.01 mM putrescine contained approximately double the number of cells present in cultures treated with DFMO alone. Cells in control cultures and those treated with putrescine continued to divide until the experiment ended on day 10. From days 6 to 10 the cultures treated with DFMO contained sig-
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G312
REGULATION
OF
I
I
I
I
I
I
0
1
2
3
4
5
INTESTINAL
ODC
(Fig. 7A). Enzyme activity increased linearly with concentrations of asparagine ranging from 2.5 to 10 mM. Although doses of asparagine higher than 10 mM increased enzyme activity, none of the values reached was significantly higher than the response to 10 mM. For this reason 10 mM asparagine was used for the remainder of the studies. As shown in the Fig. 7B, putrescine had no effect on basal (unstimulated) ODC activity until it was administered at a concentration of 10m5M. At this and higher doses, putrescine inhibited nearly all enzyme activity. However, when cells were stimulated by 10 mM asparagine, ODC activity was significantly reduced by 10B7M putrescine (Fig. 8). In fact, 10m7M putrescine inhibited the increase in ODC activity stimulated by asparagine by X5%. Lower concentrations of putrescine also decreased ODC activity, but these differences were not statistically significant.
Time (h) 5. ODC activity of control IEC-6 cells and cells exposed to 10 mM asparagine at 0 time. Each point represents mean of 3 values. Vertical lines are SE. * P c 0.05 compared with control at same time FIG.
IO mM Asparagine t 112= 35 min
I
I
I
I
1
0
30
60 Time (min)
90
120
FIG. 6. Percent ODC activity remaining after exposing IEC-6 cells to cycloheximide (50 pg/ml) at time 0. Figure compares decrease in ODC activity in cells stimulated with 10 mM asparagine 2.5 h before being exposed to cycloheximide with decrease in unstimulated, control cultures. Each point represents means t SE of data from 3 culture dishes.
nificantly fewer cells than the controls and those to which putrescine had been added. Rate of ODC turnover. ODC activity of IEC-6 cells increased significantly within 1 h after adding 10 mM asparagine (Fig. 5). Activity peaked at 2 h and remained at the same level through the 3rd h, after which it began to decrease. After the addition of cycloheximide, ODC activity of IEC-6 cells declined rapidly (Fig. 6). In unstimulated (basal ODC activity) cells, enzyme activity decreased with a half-life of 20 min. ODC activity of cells stimulated with 10 mM asparagine decreased at a slower rate, with a half-life of 35 min. Stimulation with asparagine, therefore, caused an increase in the half-life of enzyme activity. This increase was statistically significant at the 0.01 level. Effect of putrescine on ODC activity. ODC activity of IEC-6 cells was significantly increased by exposure to concentrations of asparagine ranging from 5.0 to 20 mM
DISCUSSION
The most significant of the new findings described in this paper is the realization that ODC activity of mature intestinal enterocytes is regulated differently from that of a proliferative intestinal cell line. As already mentioned, the IEC-6 cell is believed to represent the intestinal crypt cell. In fact this cell line is thought by numerous investigators to provide an appropriate model to examine mucosal proliferative responses, for these cells are morphologically and immunologically indistinct from the proliferative crypt cells (16). In addition, under appropriate conditions these cells will differentiate into mature enterocytes (4). Our data clearly show that ODC activity of IEC-6 cells is inhibited by added putrescine and that this inhibition is considerably more effective when putrescine is added to cells in which ODC activity has been stimulated. On the other hand, putrescine enhances ODC activity in villous cells if the enzyme has been stimulated by luminal amino acids. Somewhat similar results have been reported by Bethe11 and Pegg (2) working with 3T3 and transformed SV-3T3 cells. When the two cell lines were stimulated by the addition of fresh serum, there was a rapid increase in ODC activity. The addition of 50 PM putrescine to the culture medium at the time of serum addition prevented the increase in enzyme activity in 3T3 cells, but not in the transformed cell lines. Measurements of intracellular putrescine showed that both cell types had taken up approximately the same amounts of the diamine. Likewise, the lack of an inhibitory effect of putrescine on ODC activity in villous cells is not due to a failure of these cells to take up polyamines. We have previously shown that isolated enterocytes accumulate putrescine to an eightfold concentration gradient (11). Our current results showing an enhancement of glytine-stimulated ODC activity by putrescine in enterocytes are unusual compared with most findings. However, to our knowledge all other data have been collected in studies of growing cells or at least of cells with the potential to divide. Interestingly, a 66-h infusion of putrescine or a dietary amine, ethylamine, at a rate of 1 pmol/h into the rat small intestine stimulated both mucosal ODC activitv and S-adenosvlmethionine decarbox-
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REGULATION
OF
INTESTINAL
G313
ODC
60 E.a 5 50 k 0 E r‘ 40 \ cu
25
20 FIG. 7. Effects of different concentrations of asparagine (A) or putrescine (B) on ODC activity of IEC-6 cells. ODC activity was measured 2.5 h after asparagine or putrescine administration. Each point represents means t SE of values from 6 dishes of cells. * P < 0.05 compared with controls (0 doses).
0 cl 0
30
15
20
10
E Q .-3 .-> c
a 0 ‘O n 0 0
5
I
I
I
0
5
I
I
I
IO
I
I
0
1
15
20
III
11
0 lo-'*
IO-l0
I
ul E
40
0” 0
30
.
.
Asparagme
AA
c)mm
1 u-c M
h I
i
0 5
20
Putrescine
l
l
1o-6
l
11
1o-4
Putrescine [M]
Asparagine [mM]
n
l
1o-8
[M]
FIG. 8. ODC activity of IEC-6 cells stimulated with 10 mM asparagine in presence of different concentrations of putrescine. ODC activity was assayed 2.5 h after cells had been exposed to asparagine alone or asparagine plus putrescine. Each point is means t SE of determinations from 6 dishes of cells. * P < 0.05 compared with control; t P < 0.05 compared with asparagine alone.
ylase activity (22). Since mature enterocytes do not divide, the presence of high levels of ODC after stimulation is interesting in itself. After a meal the ODC activity of intestinal mucosa increases dramatically. Crypt cell ODC activity is stimulated by trophic hormones such as gastrin, and the enzyme activity of the enterocytes is increased by contact with nutrients (9). Furthermore, nutrients do not appear to stimulate the enzyme in crypt cells and gastrin does not increase the activity in villous cells. To be effective, nutrients must be exposed to the apical or luminal surface of the villous cells; they are ineffective when delivered by the blood (30). Nutrients such as glycine do not increase ODC activity in parts of
the gastrointestinal tract where they are not absorbed such-as the stomach and colon (8). Thus the stimulation of ODC in the villous cells appears linked to their major function of absorption. In this regard the absorption of compounds such as dietary amines and polyamines may enhance ODC activity in villous cells via the same mechanism that nutrients such as glycine do. The function of ODC and the polyamines in the villous cells remains to be elucidated. We have speculated that the increased polyamine levels may somehow reach the crypt cells to stimulate division to replace the villous cells lost during normal function (9). To our knowledge this study also contains the first determination of the half-life of ODC in gastrointestinal mucosa. The half-life of ODC has been determined frequently by using cycloheximide to inhibit the synthesis of the enzyme (18, 29). Originally there was some criticism of this technique, since it measures enzyme activity rather than the amount of enzyme protein (26, 29). However, the method has been substantiated by alternative procedures using immunoassays for ODC (6,7) or by labeling the enzyme protein with DFMO (19, 20). Using both cycloheximide and radioactive DFMO techniques, we obtained virtually identical half-life estimations of -21 min for ODC in villous cells from fasted rats that had been refed for 3 h. We did not measure the half-life of ODC in the mucosa of fasted rats, since basal levels are quite variable. However, it is unlikely to be significantly different from the value obtained in the refed animals. The half-life of 20 min in the refed animals is close to the most rapid turnover rate described for the enzyme (29). Therefore, this turnover time cannot be a significant increase from the basal state. On the other hand, stimulation of the IEC-6 cells significantly lengthened the half-life of ODC from -20 to 35 min. This 75% increase in the stability of the protein, however, accounts for only a small part of the
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G314
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increased level of enzyme after stimulation, since 10 mM asparagine increased ODC activity approximately fivefold. This indicates that most of the increase in enzyme activity in the IEC-6 cells is due to the synthesis of new enzyme protein. The failure of the half-life of ODC in the villous cells to increase after stimulation represents yet another difference between the regulation of the enzyme in these mature cells compared with the proliferative IEC-6 cells. In other systems, increases in ODC activity have been shown to be caused by either increases in protein levels or by the combination of increased protein levels and stability. In rat ovaries, human chronic gonadotropin increased ODC activity and protein fivefold with no change in enzyme half-life (24). The time course of ODC activity change in this study was matched by changes in the amount of ODC mRNA, indicating that the stimulation of synthesis of new enzyme protein occurred at the transcriptional level. Androgen-treated BALB/c mice showed an increased half-life of renal ODC from 18 to 80 min compared with controls. This increase, however, only accounted for a portion of the concurrent 60-fold increase in enzyme activity (20). In conclusion, we have previously shown that rat intestinal crypt cell ODC activity is increased by trophic hormones such as gastrin and EGF (9), whereas the ODC of the mature villous cells is stimulated by absorbed nutrients such as glycine. The current study indicates two additional differences between the regulation of ODC in villous cells and a rat intestinal crypt cell line. First, putrescine inhibits stimulated ODC activity in the crypt cell line while potentiating it in the villous cells. Second, stimulation of ODC activity in the cell line is accompanied by a significant increase in the half-life of the enzyme. ODC half-life does not increase after stimulation of enzymatic activity in the villous cells. These results suggest that during migration and differentiation there is a total change in the regulation of ODC in the mucosal cells of the small intestine. On the other hand, it is also possible that a different ODC enzyme becomes expressed in mature villous cells compared with that present in the crypt cells. The results also indicate that studies of mucosal ODC activity, essentially all due to the enzyme in the villous cells, are not necessarily representative of enzyme activity in the proliferative crypt cells. We are grateful to Barbara E. Eikenburg for expert technical assistance. This study was supported by National Institute of Diabetes and Digestive and Kidney Diseases Grant DK-16505. Address for reprint requests: L. R. Johnson, Dept. of Physiology, Univ. of Texas Medical School, P.O. Box. 20708, Houston, TX 77030. Received
8 May
1989; accepted
in final
form
28 September
1989.
REFERENCES 1. BALL, W. J., J. S. SALSER, AND M. E. BALIS. Biomedical changes in preneoplastic rodent intestines. Cancer Res. 36: 2686-2689,1976. 2. BETHELL, D. R., AND A. E. PEGG. Uptake of putrescine by 3T3 and SV3T3 cells and its effect on ornithine decarboxylase activity. J. CelZ. Physiol. 109: 461-468, 1981. 3. 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.
INTESTINAL
ODC
4. CARROL, K. M., T. T. WONG, D. L. DRABIK, AND E. B. CHANG. Differentiation of rat small intestinal epithelial cells by extracellular matrix. Am. J. Physiol. 254 (Gastrointest. Liver Physiol. 17): G355-G360,1988. 5. FITZPATRICK, L. R., P. WANG, B. E. EIKENBURG, M. K. HADDOX, AND L. R. JOHNSON. Effect of refeeding on polyamine biosynthesis in isolated enterocytes. Am. J. Physiol. 250 (Gastrointest. Liver Physiol. 13): G709-G713, 1986. 6. HOLTTA, E. Immunochemical demonstration of increased accumulation of ornithine decarboxylase in rat liver after partial hepatectomy and growth hormone induction. Biochim. Biophys. Acta 399: 420-427, 1975. 7. ISOMAA, V. V., A. E. I. PAJUNEN, C. W. BARDIN, AND 0. A. JANNES. Ornithine decarboxylase in mouse kidney-purification, characterization and radioimmunological determination of the enzyme protein. J. Biol. Chem. 258: 6735-6740, 1983. 8. JAIN, R., B. E. EIKENBURG, AND L. R. JOHNSON. Stimulation of ornithine decarboxylase activity in digestive tract mucosa. Am. J. Physiol. 253 (Gustrointest. Liver Physiol. 16): G303-G307, 1987. 9. JOHNSON, L. R., C.-C. TSENG, P. WANG, U. R. TIPNIS, AND M. K. HADDOX. Mucosal ornithine decarboxylase in the small intestine: localization and stimulation. Am. J. Physiol. 256 (Gustrointest. Liver Physiol. 19): G624-G630, 1989. 10. KAY, J. E., AND V. J. LINDSAY. Control of ornithine decarboxylase activity in stimulated human lymphocytes by putrescine and spermidine. Biochem. J. 132: 791-796, 1973. 11. KUMAGAI, J., AND L. R. JOHNSON. Characteristics of putrescine uptake in isolated rat enterocytes. Am. J. Physiol. 254 (Gastrointest. Liver Physiol. 17): G81-G86, 1988. 12. LUK, G. D., AND S. B. BAYLIN. Polyamines and intestinal growthincreased polyamine biosynthesis after jejunectomy. Am. J. PhysioZ. 245 (Gustrointest. Liver Physiol. 8): G656-G660, 1983. 13. MOORE, P., AND M. E. SWENSEID. Dietary regulation of the activities of intestinal ornithine decarboxylase by single amino acid feeding. J. Biochem. 98: 133-139, 1985. 14. PEGG, A. E., AND P. P. MCCANN. Polyamine metabolism and function. Am. J. Physiol. 243 (Cell Physiol. 12): C212-C221, 1982. 15. PORTER, C. W., C. DWORAXZYK, B. GANIS, AND M. M. WEISER. Polyamines and biosynthetic enzymes in the rat intestinal mucosa and the influence of methylglyoxal-bis(gluanylhydrazone). Cancer Res. 40: 2330-2335,198O. 16. QUARONI, A., J. WANDS, R. L. TRELSTAD, AND K. J. ISSELBACHER. Epithelial cell cultures from rat small intestine. J. CeZl BioZ. 80: 248-265,1979. 17. RUSSELL, D. H. Ornithine decarboxylase may be a multifunctional protein. Adv. Enzyme Regul. 21: 201-222, 1983. 18. RUSSELL, D., AND S. H. SNYDER. Amine synthesis in regenerating rat liver: extremely rapid turnover of ornithine decarboxylase. IMoL. Pharmacol. 5: 253-262, 1969. 19. SEELY, J. E., AND A. E. PEGG. Changes in mouse kidney ornithine decarboxylase activity are brought about by changes in the amount of enzyme protein as measured by radioimmunoassay. J. Biol. Chem. 258: 2496-2500,1983. on 20. SEELY, J. E., H. Poso, AND A. E. PEGG. Effect of androgens turnover of ornithine decarboxylase in mouse kidney. J. Biol. Chem. 257: 7549-7553,1982. 21. SEIDEL, E. R., M. K. HADDOX, AND L. R. JOHNSON. Polyamines in the response to intestinal obstruction. Am. J. Physiol. 246 (Gustrointest. Liver Physiol. 9): G649-G653, 1984. 22. SEIDEL, E. R., M. K. HADDOX, AND L. R. JOHNSON. Ileal mucosal growth during intraluminal infusion of ethylamine or putrescine. Am. J. Physiol. 249 (Gastrointest. Liver Physiol. 12): G434-G438, 1985. 23. SEPULVEDA, F. V., K. A. BURTON, G. M. CLARKSON, AND G. SYRNE. Cell differentiation and L-ornithine decarboxylase activity in the intestine of rats fed low and high protein diets. Biochim. Biophys. Acta 716: 439-442, 1982. 24. SERTICH, G. J., L. PERSSON, AND A. E. PEGG. Regulation of ovarian ornithine decarboxylase by human chorionic gonadotropin. Am. J. Physiol. 253 (Cell Physiol. 22): C687-C692, 1987. 25. STEEL, R. G. D., AND J. H. TORRIE. PrincipZes and Procedures of Statistics. New York: McGraw-Hill, 1960, p. 107-109. 26. STILES, C. D., K. L. LEE, AND F. T. KENNEY. Differential degradation of messenger RNAs in mammalian cells. Proc. Natl. Acad. Sci. USA 73: 2634-2638,1976.
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27. TABATA, K., AND L. R. JOHNSON. Ornithine decarboxylase and mucosal growth in response to feeding. Am. J. Physiol. 251 (Gmtrointest. Liver Physiol. 14): G270-G275, 1986. 28. TABATA, K., AND L. R. JOHNSON. Mechanism of induction of mucosal ornithine decarboxylase by food. Am. J. Physiol. 251 (Gustrointest. Liver Physiol. 14): G370-G374, 1986. 29. TABOR, C. W., AND H. TABOR. Polyamines. Annu. Reu. Biochem. 53: 749-790, 1984. 30. ULRICH-BAKER, M. G., P. WANG, L. FITZPATRICK, AND L. R. JOHNSON. Amiloride inhibits rat mucosal ornithine decarboxylase
INTESTINAL
ODC
G315
activity and DNA synthesis. Am. J. Physiol. 254 (Gastrointest. Liver Physiol. 17): G408-G415, 1988. 31. VICEPS-MADORE, D., K. Y. CHEN, H.-R. Tsou, AND E. S. CANELAKIS. Studies on the role of protein synthesis and of sodium on the regulation of ornithine decarboxylase activity. Biochim. Biophys. Acta 717: 305-315, 1982. 32. YANG, P., S. B. BAYLIN, AND G. D. LUK. Polyamines and intestinal growth: absolute requirement for ODC activity in adaptation and lactation. Am. J. Physiol. 247 (Gastrointest. Liver Physiol. 10): G553-G557,1984.
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half-life
IWAMI, JIAN-YING WANG, RAJEEVE JAIN, McCORMACK, AND LEONARD R. JOHNSON of Physiology and Cell Biology, University of Texas Medical School, Houston, Texas 77030
is one of the earliest events associated with cellular proliferation (14, 17). The most striking characteristics of ODC are its rapid turnover rate and changes in activity in response to various stimuli (29). The half-life of ODC in mammalian tissues, where it has been measured, is 15-30 min, which is much shorter than that of any other mammalian enzyme (29). Another important characteristic of this enzyme is that it is regulated by putrescine, its end product, by a mechanism other than end-product inhibition. Kay and Lindsay (10) first demonstrated that micromolar concentrations of exogenous putrescine prevented the increase in ODC activity in lectin-stimulated lymphocytes. Inhibition occurs with concentrations of putrescine considerably below those that directly inhibit purified ODC and has been shown to occur in many different cell types (29). Inhibition is caused by one or more proteins induced by putrescine. These proteins are called antizymes and inhibit ODC by binding noncovalently to it. Proteins with antizyme activity have been isolated and purified from both Escherichia coli and rat, liver (see Ref. 29 for review). The standard technique for measuring ODC activity is to incubate the enzyme preparation with ornithine, labeled in the l-carbon or carboxyl position. The 14C02is then trapped and counted. Numerous studies employing this technique have shown that ODC in the small intespolyamines; crypt cells; villous cells; enzyme regulation; growth; tine has a relatively high basal activity (1) yet increases rat mucosa; cell culture dramatically with feeding (13, 27, 28). In addition to increased food intake, intestinal mucosal ODC levels increase after partial gut, resection (12), during lactation THE POLYAMINES, spermidine and spermine and their (32), and after luminal obstruction (21). Each of these precursor putrescine, are found in virtually all cells of events increases the exposure of the gut mucosa to food higher eukaryotes (14). The exact action of polyamines or luminal contents, and each is associated with inat the molecular level is unknown. It is likely that these creased mucosal growth. Inhibition of ODC activity by substances are involved in a variety of cellular functions, the specific inhibitor difluoromethylornithine (DFMO) including membrane stabilization, translation processes, prevents the growth responsesto refeeding (28), lactation regulation of RNA and DNA polymerase activity, regu- (32), and resection (12). Numerous laboratories have lation of protein kinase activity, and transfer RNA acylashown that virtually all of the ODC activity measured in tion reactions (14, 17). Polyamines are intimately in- the intestinal mucosa by 14C02collection is localized in volved in, and required for, cell growth and differentiathe differentiated villous cells and not, as one would tion, and their concentrations within cells are highly expect, in the proliferative crypt cells (5, 15, 23). Howregulated. Intracellular polyamine levels are highly de- ever, by use of a highly sensitive ODC antibody we have pendent on the activity of ornithine decarboxylase (ODC; been able to demonstrate specific ODC in rat intestinal EC 4.1.1.17), which is the initial rate-controlling enzyme crypt cells and show that its levels are increased by in the polyamine synthetic pathway. In most tissues, trophic substances such as gastrin and epidermal growth factor (EGF) (9). The ODC of the villous cells did not ODC levels are low, and an increase in enzyme activity IWAMI, KIMIKAZU, LEY MCCORMACK,
JIAN-YING AND LEONARD
WANG,
RAJEEVE
JAIN,
SHIR-
R. JOHNSON. Intestinal ornithine decarboxylase: half-life and regulation byputrescine. Am. J. Physiol. 258 (Gastrointest. Liver Physiol. 21): G308-G315, 1990.-Ornithine decarboxylase (ODC) is the primary ratelimiting enzyme for polyamine synthesis. ODC levels are increased in most tissues, including the intestinal mucosa, by growth-promoting agents. This enzyme has a brief half-life of from 5 to 30 min in mammalian tissues and is regulated by its product, putrescine. The current study examines the turnover and regulation of ODC in the mucosa of the small intestine. With the use of scraped intestinal mucosa from cycloheximidetreated rats, the time course of the decline in ODC activity yielded a half-life of -22 min. Labeling enzyme protein with [“Hldifluoromethylornithine (DFMO) resulted in a nearly identical estimation of half-life. ODC activity of mucosa from isolated gut segments stimulated by luminal glycine (0.1-0.4 M) was enhanced 60-100% by 10 mM putrescine administered luminally. Putrescine alone had no effect on ODC. In contrast, lo-' M putrescine prevented 80% of the ODC activity stimulated by asparagine in IEC-6 cells (a rat intestinal crypt cell line). The half-life of ODC in unstimulated IEC-6 cells was 20 min and increased to 35 min in cells exposed to 10 mM asparagine. These data demonstrate that ODC of nonproliferating villous cells is regulated differently from the identical enzyme in proliferating crypt cells. Therefore, conclusions regarding mucosal growth should not be based totally on ODC activity from whole mucosa, since it is essentially a measure of only the enzyme present in the villous cells.
G308
0193-1857/90
$1.50 Copyright
0 1990 the American
Physiological
Society
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respond to gastrin but was increased by luminal nutrients such as glycine. Thus the agents that increase ODC levels in these two cell types are different. The purpose of the current study is to determine the half-life of ODC in villous cells and in IEC-6 cells and to examine the effect of putrescine on the regulation of enzyme activity. The IEC-6 cell line originated from normal rat intestinal crypt cells, as judged by morphological and immunological criteria, and they retain the growth characteristics of undifferentiated crypt cells. METHODS
Studies are divided into those done in rats and in cultured IEC-6 cells. Each of these categories is further divided into the determination of the half-life of ODC and the measurement of the effect of putrescine on ODC activity. Studies in Rats ODC half-life determination. Male Sprague-Dawley rats weighing X0-170 g were housed in wire-bottomed cages in a room with a constant temperature and a 12-h light-dark cycle and were fed ad libitum on Purina Rat Chow. Rats were then fasted 48 h and refed for 3 h. Refeeding for 3 h results in maximal stimulation of intestinal ODC activity (28). At this time animals were injected intraperitoneally with either cycloheximide (25 mg/kg) or [ 3H] DFMO [ DL- [ 3,4-3H] difluoromethylornithine, 20 Ci/mmol, New England Nuclear, Boston, MA). [3H]DFM0 was given at a dose of 20 &i/animal in 0.4 ml saline. Animals treated with cycloheximide were killed at 0, 30, or 60 min after injecting the drug. The proximal third of the small intestine was removed, rinsed in ice-cold saline, and placed in a beaker of ice-cold saline. The mucosa was collected by scraping with a glass slide over an ice-cold glass plate. Mucosal scrapings were weighed and divided into two portions. One was used to measure protein and the other to determine the rate of ornithine decarboxylation. Ornithine decarboxylase activity was assayed by a radiometric method in which the amount of 14C02 liberated from DL-[ l-14C]ornithine (53 mCi/ mmol, New England Nuclear) was estimated (27). Intestinal mucosa was collected and placed in 0.0667 M sodium-potassium phosphate buffer (pH 7.4) containing 0.02% lauryl ether, 5 mM NaF, 10 PM phosphate, and 10 mM EDTA (ODC assay buffer). The tissues were homogenized, sonicated, and centrifuged at 30,000 g for 25 min. An aliquot from the 30,000-g supernatant was incubated in stoppered vials in the presence of 2 nmol of [14C]ornithine for 15 min at 37OC. The 14C02 liberated by the decarboxylation was trapped on a piece of filter paper and impregnated with 20 ~1 of 2 N NaOH, which was suspended in a centerwell above the reaction mixture. The reaction was stopped by the addition of trichloroacetic acid (TCA) to a final concentration of 10%. The 14C02 trapped in the filter paper was measured by liquid scintillation spectrometry at a counting efficiency of 99%. Blanks were run simultaneously by using a vehicle instead of the supernatant. The protein content
INTESTINAL
ODC
G309
of the samples was determined by using the Bradford (3) method, and results are expressed as picomoles CO2 per hour per milligram protein. The decrease in enzyme activity over time after cycloheximide was used to calculate the half-life of ODC activity. Two rats were studied at each time point during each experiment. A total of three experiments were conducted, so n = 6. Data are expressed as means t SE of six observations. Experiments were conducted in animals injected with [3H]DFM0 to determine the half-life of ODC protein itself. DFMO is an enzyme-activated irreversible inhibitor of ODC activity. When exposed to [3H]DFM0, the enzyme protein becomes labeled as activity is lost (20). Thirty minutes after the injection of [3H]DFM0, 0.6 ml saline containing 50 mg/ml of unlabeled DFMO (kindly donated by Merrell Dow Pharmaceuticals, Cincinnati, OH) was injected into the abdominal cavity. The presence of excess unlabeled DFMO halted the binding of [3H]DFM0 to enzyme protein. Rats were killed by decapitation 20, 30, 50, or 60 min after administering unlabeled DFMO and the small intestine was rapidly excised, rinsed, and frozen in liquid nitrogen. Samples were stored at -80°C until assay. Mucosa from the proximal third of the intestine was collected as previously described above, homogenized in 3 vol of the ODC assay buffer, and centrifuged at 30,000 g for 60 min. The extracts were dialyzed for 3 days to remove [3H]DFM0 that was not bound to protein: 24 h against the ODC assay buffer, and twice (24 h each time) against 1 liter of 50 mM pH 6.86 phosphate buffer containing 0.25 mM NaF, 5 PM EDTA, 0.33 mM dithiothreitol, and 0.02 mM pyridoxal phosphate. The dialysates were transferred to scintillation vials and evaporated on a 40-45°C plate. The remaining protein residues were dissolved in 0.5 ml Protosol. Five milliliters Hydrofluor and one drop of concentrated HCl were added. The radioactivity of the samples was determined by scintillation counting and expressed as counts per minute [3H]DFM0 per gram protein. Each data point represents the mean t SE of five separate animals. Effect of putrescine on ODC activity. ODC activity was measured in isolated intestinal segments as described previously (8). In brief, rats weighing 125-150 g were fasted 48 h and anesthetized with ether; the peritoneal cavity was opened via a midline incision, and one or two 5-cm jejunal segments, 20-30 cm distal from the pylorus, were isolated by ligation. In a typical experiment, 0.5 ml saline or a test solution was injected into each segment using a l-ml syringe. The midline incision was closed and the animals were allowed to recover in individual cages without food or water. Two hours after injecting test substances the rats were killed by an overdose of ether followed by cervical dislocation. All animals were killed between noon and 1 P.M. The individual segments were removed, opened, and rinsed in ice-cold saline. The mucosa was isolated by scraping and the ODC activity measured as described above. In the first study, the effect of putrescine on basal ODC activity was determined. With the use of one isolated segment per rat, putrescine was injected at concentrations of 0.08, 0.20, 5.0, and 10.0 mM. Control rats
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G310
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were injected with saline. The experiment was repeated twice with three rats per group so that n = 9 for each dose of putrescine. This experiment was also carried out killing the rats 4 h after injection instead of after 2 h. This subset was repeated only once, so n = 6. In a second study, the effect of 10 mM putrescine on ODC activity stimulated by different concentrations of glycine was determined. Rats were prepared with two isolated segments, and both segments were injected with saline, a given concentration of glycine (either 0.05, 0.1, 0.2, or 0.4 M), or the same concentration of glycine plus 10 mM putrescine. Only one dose of glycine was used in a given experiment, and separate saline controls were carried out in each experiment. The values from the two loops were averaged and at least six rats were used at each dose of glycine. Studies Using IEC-6 Cells IEC-6 cells (a normal rat small intestinal epithelial cell line) were obtained from the American Type Culture Collection (Rockville, MD) at passage 13. The stock was maintained in T-150 flasks in Dulbecco’s minimal essential medium (DMEM, GIBCO, Grand Island, NY) containing 5% dialyzed fetal bovine serum (FBS, GIBCO), 50 pg gentamicin sulfate, and 10 pg insulin/ml (growth medium) in 90% air-lo% CO2 at 37°C. Tests for mycoplasma were always negative, and the stock was fed three times a week and split 120 once a week. IEC-6 cells have the characteristics of normal cells cultured in vitro and show a number of features that suggest they are derived from undifferentiated small intestinal crypt cells (16). Polyamine dependence of IEC-6 cell. The dependence of IEC-6 cells on polyamines for growth was tested by inhibiting ODC with DFMO and then supplying exogenous putrescine to inhibited cultures. Cells were plated at a density of 12 x lo3 cells/cm2 in T-25 flasks in growth medium (control), growth medium plus 5 mM DFMO, and growth medium plus 5 mM DFMO plus 0.01 mM putrescine. The medium was replaced every other day and cells from three flasks from each treatment group were taken up with trypsin/EDTA and counted with a Coulter counter, model 031 ZF. The experiment was continued for 10 days. Rate of ODC turnover. Cells were plated at a density of -0.5 x lo6 cells/plate and grown for 48 h. In the first study 10 mM asparagine (final concentration) was added and cells were harvested hourly for 5 h. Asparagine has been shown to stimulate ODC activity in many systems. It appears to be the most potent type A amino acid for inducing ODC, and the type A amino acids are much more effective than the others (31). ODC activity was measured and determined to be maximal between 2 and 3 h after stimulation by asparagine. In the next set of experiments cycloheximide (to a final concentration of 50 pg/ml) was added to cultures 2.5 h after the addition of 10 mM asparagine. Other cultures received cycloheximide without being stimulated with asparagine. Cultures were harvested 15, 30, 45, 60, 90, and 120 min after the addition of cycloheximide by washing the cells twice in ice-cold 0.1 M tris( hydroxymethyl)aminomethane (Tris) buffer (pH 7.4) containing 0.1 mM EDTA, 50 PM pyri-
INTESTINAL
ODC
doxal 5-phosphate, and 5 mM dithiothreitol and were scraped into 0.5 ml of the same buffer. Cells were then frozen and thawed three times, sonicated, and centrifuged at 2,000 revolutions/min for 10 min. The ODC activity of an aliquot of the supernatant was incubated in a stoppered test tube in the presence of 2.5 mM [‘4C]-l-ornithine for 15 min at 37°C. Liberated 14C02 was trapped and counted, and the ODC activity was calculated and expressed as described above. Effect of putrescine on ODC activity. Cells were cultured as described above and harvested 2.5 h after the addition of different concentrations of either asparagine or putrescine. Asparagine was added to give concentrations of either 0, 2.5, 5, 7.5, 10, 12.5, 15, or 20 mM. Putrescine was added to concentrations of 0, lo-l2 M, and molar concentrations increasing 1 log unit from 10-l’ to 10W3. Finally 10 mM asparagine was added to cultures containing putrescine in molar concentrations increasing 1 log unit from lo-l1 to 10B5. Cells were harvested and ODC activities determined as described above. Statistics All data are expressed as means t SE. The significance of the differences between means was determined by analysis of variance. The level of significance was determined using the Duncan’s multiple range test (25). RESULTS
Studies in Rats ODC half-life determination. Three hours after the start of refeeding, ODC activity had increased -5O-fold to 232 pmol CO2. h-l mg protein? Cycloheximide inhibited de novo ODC synthesis, causing a rapid decrease in enzyme activity (Fig. 1). By 60 min after cycloheximide, activity had decreased to 37. From Fig. 1 the half-life of enzyme activity was calculated to be 20 min. The determination of the half-life of ODC protein itself is shown in Fig. 2. The radioactivity of undialyzable [3H]DFM0 declined log linearly over time yielding a protein half-life of 21.5 min. Although the value obtained for the half-life corresponded well with that obtained in Fig. 1 and the correlation coefficient of the line (r = -0.86) was reasonable, DFMO-labeled ODC is rapidly cleared and there were few counts remaining by the end of 60 min. Effect of putrescine on ODC actiuity. Injection of putrescine by itself into the isolated proximal intestinal loops caused no significant change in ODC activity compared with saline alone (data not shown). This was true for all doses of the diamine. Glycine alone significantly increased ODC activity in doses from 0.1 to 0.4 M (Fig. 3). As is also shown in Fig. 3, 0.05 M glycine did not alter ODC activity compared with saline. The addition of 10 mM putrescine to glycine significantly enhanced ODC activity compared with glycine alone but only when the concentration of glycine itself significantly stimulated enzyme activity. For example, as is shown in Fig. 3, the addition of 10 mM putrescine to 0.05 M glycine had no effect on ODC activity. The enhancement of ODC activity caused by putrescine over the response to glycine l
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REGULATION
OF INTESTINAL
G311
ODC 500 , -
z.3 g
t
400 I .-
E 2 s-G 300 b ,nE
0 F +
200
x 5 2
3
-
.-
.-
100
8
0 4
0.05
0.10
0.20
Glycine [M] I
1
0
30
60
Time after Cycloheximide
(min)
FIG. 1. Decline in ornithine decarboxylase (ODC) activity 30 and 60 min after injection of 25 mg/kg cycloheximide. Data are means z!z SE of 6 observations.
FIG. 3. ODC activity of mucosa from in situ-isolated segments of proximal small bowel. Segments were filled for 2 h with either saline, different concentrations of glycine, or the combination of glycine doses plus 10 mM putrescine. Columns show means + SE from at least 6 rats. * P < 0.05 compared with saline; t P < 0.05 compared with glycine alone.
500 400 II
P
Control
---s
DFMO
P
8100 G-
DFMO + PUT
l-
80-
k f: m
60
-
0
4
6
8
10
Day
t 40 t I
2
4. Growth of IEC-6 cells in control cultures, cultures in which ODC was inhibited with 5 mM DFMO, and cultures inhibited but to which 0.01 mM putrescine had been added. Cells were grown in Dulbecco’s minimal essential medium containing 5% dialyzed fetal bovine serum. Medium was changed every second day when numbers of cells present in 3 flasks from each group were determined. Means t SE from 3 flasks. * P < 0.05 compared with control and DFMO + putrestine. FIG.
I 20
I 40
h 60
Time After Cold DFMO (min) FIG. 2. Decline in radioactivity of undialyzable fraction from proximal small bowel mucosa of rats injected with 20 &i [3H]difluoromethylornithine (DFMO). Thirty milligrams of unlabeled DFMO were injected at time 0, 30 min after 13H]DFM0. Data points represent [3H]DFM0 bound to ODC and are means + SE for 5 animals. Correlation coefficient is -0.86.
alone ranged from 40 to 60%. Since enzyme activity did not increase in response to putrescine alone, this enhancement can be described as potentiation rather than an additive effect. Studies Using the IEC-6 Cells Polyamine dependence of IEC-6 cells. Continuing
mal cell growth was dependent
on putrescine
nor(Fig. 4).
After initial plating, cells began to divide after day 2. By day 4 there were no significant differences in the numbers of cells present in the three different groups. However, the cells treated with 5 mM DFMO stopped dividing at this point. By day 6 control cultures and cultures treated with DFMO that received exogenous 0.01 mM putrescine contained approximately double the number of cells present in cultures treated with DFMO alone. Cells in control cultures and those treated with putrescine continued to divide until the experiment ended on day 10. From days 6 to 10 the cultures treated with DFMO contained sig-
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G312
REGULATION
OF
I
I
I
I
I
I
0
1
2
3
4
5
INTESTINAL
ODC
(Fig. 7A). Enzyme activity increased linearly with concentrations of asparagine ranging from 2.5 to 10 mM. Although doses of asparagine higher than 10 mM increased enzyme activity, none of the values reached was significantly higher than the response to 10 mM. For this reason 10 mM asparagine was used for the remainder of the studies. As shown in the Fig. 7B, putrescine had no effect on basal (unstimulated) ODC activity until it was administered at a concentration of 10m5M. At this and higher doses, putrescine inhibited nearly all enzyme activity. However, when cells were stimulated by 10 mM asparagine, ODC activity was significantly reduced by 10B7M putrescine (Fig. 8). In fact, 10m7M putrescine inhibited the increase in ODC activity stimulated by asparagine by X5%. Lower concentrations of putrescine also decreased ODC activity, but these differences were not statistically significant.
Time (h) 5. ODC activity of control IEC-6 cells and cells exposed to 10 mM asparagine at 0 time. Each point represents mean of 3 values. Vertical lines are SE. * P c 0.05 compared with control at same time FIG.
IO mM Asparagine t 112= 35 min
I
I
I
I
1
0
30
60 Time (min)
90
120
FIG. 6. Percent ODC activity remaining after exposing IEC-6 cells to cycloheximide (50 pg/ml) at time 0. Figure compares decrease in ODC activity in cells stimulated with 10 mM asparagine 2.5 h before being exposed to cycloheximide with decrease in unstimulated, control cultures. Each point represents means t SE of data from 3 culture dishes.
nificantly fewer cells than the controls and those to which putrescine had been added. Rate of ODC turnover. ODC activity of IEC-6 cells increased significantly within 1 h after adding 10 mM asparagine (Fig. 5). Activity peaked at 2 h and remained at the same level through the 3rd h, after which it began to decrease. After the addition of cycloheximide, ODC activity of IEC-6 cells declined rapidly (Fig. 6). In unstimulated (basal ODC activity) cells, enzyme activity decreased with a half-life of 20 min. ODC activity of cells stimulated with 10 mM asparagine decreased at a slower rate, with a half-life of 35 min. Stimulation with asparagine, therefore, caused an increase in the half-life of enzyme activity. This increase was statistically significant at the 0.01 level. Effect of putrescine on ODC activity. ODC activity of IEC-6 cells was significantly increased by exposure to concentrations of asparagine ranging from 5.0 to 20 mM
DISCUSSION
The most significant of the new findings described in this paper is the realization that ODC activity of mature intestinal enterocytes is regulated differently from that of a proliferative intestinal cell line. As already mentioned, the IEC-6 cell is believed to represent the intestinal crypt cell. In fact this cell line is thought by numerous investigators to provide an appropriate model to examine mucosal proliferative responses, for these cells are morphologically and immunologically indistinct from the proliferative crypt cells (16). In addition, under appropriate conditions these cells will differentiate into mature enterocytes (4). Our data clearly show that ODC activity of IEC-6 cells is inhibited by added putrescine and that this inhibition is considerably more effective when putrescine is added to cells in which ODC activity has been stimulated. On the other hand, putrescine enhances ODC activity in villous cells if the enzyme has been stimulated by luminal amino acids. Somewhat similar results have been reported by Bethe11 and Pegg (2) working with 3T3 and transformed SV-3T3 cells. When the two cell lines were stimulated by the addition of fresh serum, there was a rapid increase in ODC activity. The addition of 50 PM putrescine to the culture medium at the time of serum addition prevented the increase in enzyme activity in 3T3 cells, but not in the transformed cell lines. Measurements of intracellular putrescine showed that both cell types had taken up approximately the same amounts of the diamine. Likewise, the lack of an inhibitory effect of putrescine on ODC activity in villous cells is not due to a failure of these cells to take up polyamines. We have previously shown that isolated enterocytes accumulate putrescine to an eightfold concentration gradient (11). Our current results showing an enhancement of glytine-stimulated ODC activity by putrescine in enterocytes are unusual compared with most findings. However, to our knowledge all other data have been collected in studies of growing cells or at least of cells with the potential to divide. Interestingly, a 66-h infusion of putrescine or a dietary amine, ethylamine, at a rate of 1 pmol/h into the rat small intestine stimulated both mucosal ODC activitv and S-adenosvlmethionine decarbox-
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REGULATION
OF
INTESTINAL
G313
ODC
60 E.a 5 50 k 0 E r‘ 40 \ cu
25
20 FIG. 7. Effects of different concentrations of asparagine (A) or putrescine (B) on ODC activity of IEC-6 cells. ODC activity was measured 2.5 h after asparagine or putrescine administration. Each point represents means t SE of values from 6 dishes of cells. * P < 0.05 compared with controls (0 doses).
0 cl 0
30
15
20
10
E Q .-3 .-> c
a 0 ‘O n 0 0
5
I
I
I
0
5
I
I
I
IO
I
I
0
1
15
20
III
11
0 lo-'*
IO-l0
I
ul E
40
0” 0
30
.
.
Asparagme
AA
c)mm
1 u-c M
h I
i
0 5
20
Putrescine
l
l
1o-6
l
11
1o-4
Putrescine [M]
Asparagine [mM]
n
l
1o-8
[M]
FIG. 8. ODC activity of IEC-6 cells stimulated with 10 mM asparagine in presence of different concentrations of putrescine. ODC activity was assayed 2.5 h after cells had been exposed to asparagine alone or asparagine plus putrescine. Each point is means t SE of determinations from 6 dishes of cells. * P < 0.05 compared with control; t P < 0.05 compared with asparagine alone.
ylase activity (22). Since mature enterocytes do not divide, the presence of high levels of ODC after stimulation is interesting in itself. After a meal the ODC activity of intestinal mucosa increases dramatically. Crypt cell ODC activity is stimulated by trophic hormones such as gastrin, and the enzyme activity of the enterocytes is increased by contact with nutrients (9). Furthermore, nutrients do not appear to stimulate the enzyme in crypt cells and gastrin does not increase the activity in villous cells. To be effective, nutrients must be exposed to the apical or luminal surface of the villous cells; they are ineffective when delivered by the blood (30). Nutrients such as glycine do not increase ODC activity in parts of
the gastrointestinal tract where they are not absorbed such-as the stomach and colon (8). Thus the stimulation of ODC in the villous cells appears linked to their major function of absorption. In this regard the absorption of compounds such as dietary amines and polyamines may enhance ODC activity in villous cells via the same mechanism that nutrients such as glycine do. The function of ODC and the polyamines in the villous cells remains to be elucidated. We have speculated that the increased polyamine levels may somehow reach the crypt cells to stimulate division to replace the villous cells lost during normal function (9). To our knowledge this study also contains the first determination of the half-life of ODC in gastrointestinal mucosa. The half-life of ODC has been determined frequently by using cycloheximide to inhibit the synthesis of the enzyme (18, 29). Originally there was some criticism of this technique, since it measures enzyme activity rather than the amount of enzyme protein (26, 29). However, the method has been substantiated by alternative procedures using immunoassays for ODC (6,7) or by labeling the enzyme protein with DFMO (19, 20). Using both cycloheximide and radioactive DFMO techniques, we obtained virtually identical half-life estimations of -21 min for ODC in villous cells from fasted rats that had been refed for 3 h. We did not measure the half-life of ODC in the mucosa of fasted rats, since basal levels are quite variable. However, it is unlikely to be significantly different from the value obtained in the refed animals. The half-life of 20 min in the refed animals is close to the most rapid turnover rate described for the enzyme (29). Therefore, this turnover time cannot be a significant increase from the basal state. On the other hand, stimulation of the IEC-6 cells significantly lengthened the half-life of ODC from -20 to 35 min. This 75% increase in the stability of the protein, however, accounts for only a small part of the
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G314
REGULATION
OF
increased level of enzyme after stimulation, since 10 mM asparagine increased ODC activity approximately fivefold. This indicates that most of the increase in enzyme activity in the IEC-6 cells is due to the synthesis of new enzyme protein. The failure of the half-life of ODC in the villous cells to increase after stimulation represents yet another difference between the regulation of the enzyme in these mature cells compared with the proliferative IEC-6 cells. In other systems, increases in ODC activity have been shown to be caused by either increases in protein levels or by the combination of increased protein levels and stability. In rat ovaries, human chronic gonadotropin increased ODC activity and protein fivefold with no change in enzyme half-life (24). The time course of ODC activity change in this study was matched by changes in the amount of ODC mRNA, indicating that the stimulation of synthesis of new enzyme protein occurred at the transcriptional level. Androgen-treated BALB/c mice showed an increased half-life of renal ODC from 18 to 80 min compared with controls. This increase, however, only accounted for a portion of the concurrent 60-fold increase in enzyme activity (20). In conclusion, we have previously shown that rat intestinal crypt cell ODC activity is increased by trophic hormones such as gastrin and EGF (9), whereas the ODC of the mature villous cells is stimulated by absorbed nutrients such as glycine. The current study indicates two additional differences between the regulation of ODC in villous cells and a rat intestinal crypt cell line. First, putrescine inhibits stimulated ODC activity in the crypt cell line while potentiating it in the villous cells. Second, stimulation of ODC activity in the cell line is accompanied by a significant increase in the half-life of the enzyme. ODC half-life does not increase after stimulation of enzymatic activity in the villous cells. These results suggest that during migration and differentiation there is a total change in the regulation of ODC in the mucosal cells of the small intestine. On the other hand, it is also possible that a different ODC enzyme becomes expressed in mature villous cells compared with that present in the crypt cells. The results also indicate that studies of mucosal ODC activity, essentially all due to the enzyme in the villous cells, are not necessarily representative of enzyme activity in the proliferative crypt cells. We are grateful to Barbara E. Eikenburg for expert technical assistance. This study was supported by National Institute of Diabetes and Digestive and Kidney Diseases Grant DK-16505. Address for reprint requests: L. R. Johnson, Dept. of Physiology, Univ. of Texas Medical School, P.O. Box. 20708, Houston, TX 77030. Received
8 May
1989; accepted
in final
form
28 September
1989.
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INTESTINAL
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4. CARROL, K. M., T. T. WONG, D. L. DRABIK, AND E. B. CHANG. Differentiation of rat small intestinal epithelial cells by extracellular matrix. Am. J. Physiol. 254 (Gastrointest. Liver Physiol. 17): G355-G360,1988. 5. FITZPATRICK, L. R., P. WANG, B. E. EIKENBURG, M. K. HADDOX, AND L. R. JOHNSON. Effect of refeeding on polyamine biosynthesis in isolated enterocytes. Am. J. Physiol. 250 (Gastrointest. Liver Physiol. 13): G709-G713, 1986. 6. HOLTTA, E. Immunochemical demonstration of increased accumulation of ornithine decarboxylase in rat liver after partial hepatectomy and growth hormone induction. Biochim. Biophys. Acta 399: 420-427, 1975. 7. ISOMAA, V. V., A. E. I. PAJUNEN, C. W. BARDIN, AND 0. A. JANNES. Ornithine decarboxylase in mouse kidney-purification, characterization and radioimmunological determination of the enzyme protein. J. Biol. Chem. 258: 6735-6740, 1983. 8. JAIN, R., B. E. EIKENBURG, AND L. R. JOHNSON. Stimulation of ornithine decarboxylase activity in digestive tract mucosa. Am. J. Physiol. 253 (Gustrointest. Liver Physiol. 16): G303-G307, 1987. 9. JOHNSON, L. R., C.-C. TSENG, P. WANG, U. R. TIPNIS, AND M. K. HADDOX. Mucosal ornithine decarboxylase in the small intestine: localization and stimulation. Am. J. Physiol. 256 (Gustrointest. Liver Physiol. 19): G624-G630, 1989. 10. KAY, J. E., AND V. J. LINDSAY. Control of ornithine decarboxylase activity in stimulated human lymphocytes by putrescine and spermidine. Biochem. J. 132: 791-796, 1973. 11. KUMAGAI, J., AND L. R. JOHNSON. Characteristics of putrescine uptake in isolated rat enterocytes. Am. J. Physiol. 254 (Gastrointest. Liver Physiol. 17): G81-G86, 1988. 12. LUK, G. D., AND S. B. BAYLIN. Polyamines and intestinal growthincreased polyamine biosynthesis after jejunectomy. Am. J. PhysioZ. 245 (Gustrointest. Liver Physiol. 8): G656-G660, 1983. 13. MOORE, P., AND M. E. SWENSEID. Dietary regulation of the activities of intestinal ornithine decarboxylase by single amino acid feeding. J. Biochem. 98: 133-139, 1985. 14. PEGG, A. E., AND P. P. MCCANN. Polyamine metabolism and function. Am. J. Physiol. 243 (Cell Physiol. 12): C212-C221, 1982. 15. PORTER, C. W., C. DWORAXZYK, B. GANIS, AND M. M. WEISER. Polyamines and biosynthetic enzymes in the rat intestinal mucosa and the influence of methylglyoxal-bis(gluanylhydrazone). Cancer Res. 40: 2330-2335,198O. 16. QUARONI, A., J. WANDS, R. L. TRELSTAD, AND K. J. ISSELBACHER. Epithelial cell cultures from rat small intestine. J. CeZl BioZ. 80: 248-265,1979. 17. RUSSELL, D. H. Ornithine decarboxylase may be a multifunctional protein. Adv. Enzyme Regul. 21: 201-222, 1983. 18. RUSSELL, D., AND S. H. SNYDER. Amine synthesis in regenerating rat liver: extremely rapid turnover of ornithine decarboxylase. IMoL. Pharmacol. 5: 253-262, 1969. 19. SEELY, J. E., AND A. E. PEGG. Changes in mouse kidney ornithine decarboxylase activity are brought about by changes in the amount of enzyme protein as measured by radioimmunoassay. J. Biol. Chem. 258: 2496-2500,1983. on 20. SEELY, J. E., H. Poso, AND A. E. PEGG. Effect of androgens turnover of ornithine decarboxylase in mouse kidney. J. Biol. Chem. 257: 7549-7553,1982. 21. SEIDEL, E. R., M. K. HADDOX, AND L. R. JOHNSON. Polyamines in the response to intestinal obstruction. Am. J. Physiol. 246 (Gustrointest. Liver Physiol. 9): G649-G653, 1984. 22. SEIDEL, E. R., M. K. HADDOX, AND L. R. JOHNSON. Ileal mucosal growth during intraluminal infusion of ethylamine or putrescine. Am. J. Physiol. 249 (Gastrointest. Liver Physiol. 12): G434-G438, 1985. 23. SEPULVEDA, F. V., K. A. BURTON, G. M. CLARKSON, AND G. SYRNE. Cell differentiation and L-ornithine decarboxylase activity in the intestine of rats fed low and high protein diets. Biochim. Biophys. Acta 716: 439-442, 1982. 24. SERTICH, G. J., L. PERSSON, AND A. E. PEGG. Regulation of ovarian ornithine decarboxylase by human chorionic gonadotropin. Am. J. Physiol. 253 (Cell Physiol. 22): C687-C692, 1987. 25. STEEL, R. G. D., AND J. H. TORRIE. PrincipZes and Procedures of Statistics. New York: McGraw-Hill, 1960, p. 107-109. 26. STILES, C. D., K. L. LEE, AND F. T. KENNEY. Differential degradation of messenger RNAs in mammalian cells. Proc. Natl. Acad. Sci. USA 73: 2634-2638,1976.
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27. TABATA, K., AND L. R. JOHNSON. Ornithine decarboxylase and mucosal growth in response to feeding. Am. J. Physiol. 251 (Gmtrointest. Liver Physiol. 14): G270-G275, 1986. 28. TABATA, K., AND L. R. JOHNSON. Mechanism of induction of mucosal ornithine decarboxylase by food. Am. J. Physiol. 251 (Gustrointest. Liver Physiol. 14): G370-G374, 1986. 29. TABOR, C. W., AND H. TABOR. Polyamines. Annu. Reu. Biochem. 53: 749-790, 1984. 30. ULRICH-BAKER, M. G., P. WANG, L. FITZPATRICK, AND L. R. JOHNSON. Amiloride inhibits rat mucosal ornithine decarboxylase
INTESTINAL
ODC
G315
activity and DNA synthesis. Am. J. Physiol. 254 (Gastrointest. Liver Physiol. 17): G408-G415, 1988. 31. VICEPS-MADORE, D., K. Y. CHEN, H.-R. Tsou, AND E. S. CANELAKIS. Studies on the role of protein synthesis and of sodium on the regulation of ornithine decarboxylase activity. Biochim. Biophys. Acta 717: 305-315, 1982. 32. YANG, P., S. B. BAYLIN, AND G. D. LUK. Polyamines and intestinal growth: absolute requirement for ODC activity in adaptation and lactation. Am. J. Physiol. 247 (Gastrointest. Liver Physiol. 10): G553-G557,1984.
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