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The migration of IEC-6 cells is inhibited when the cells are depleted of polyamines by inhibiting ornithine decarboxylase with alpha-difluoromethylornithine (DFMO). Exogenous putrescine, spermidine, and spermine completely restore cell migration inhibited by DFMO. Because polyamines are interconverted during their synthesis and catabolism, the specific role of individual polyamines in intestinal cell migration, as well as growth, remains unclear. In this study, we used an inhibitor of S-adenosylmethionine decarboxylase, diethylglyoxal bis(guanylhydrazone)(DEGBG), to block the synthesis of spermidine and spermine from putrescine. We found that exogenous putrescine does not restore migration and growth of IEC-6 cells treated with DFMO plus DEGBG, whereas exogenous spermine does. In addition, the normal distribution of actin filaments required for migration, which is disrupted in polyamine-deficient cells, could be achieved by adding spermine but not putrescine along with DFMO and DEGBG. These results indicate that putrescine, by itself, is not essential for migration and growth, but that it is effective because it is converted into spermidine and/or spermine.

The current study tests the hypothesis that intracellular polyamines are involved in the regulation of gene expression of transforming growth factor-beta (TGF-beta) during epithelial cell migration after wounding. Administration of alpha-difluoromethylornithine (DFMO), a specific inhibitor of ornithine decarboxylase (the first rate-limiting enzyme for polyamine synthesis), depleted cellular polyamines putrescine, spermidine, and spermine in IEC-6 cells. DFMO also significantly reduced basal levels of TGF-beta mRNA in unwounded cells. Gene expression of TGF-beta was dramatically stimulated after wounding of a monolayer of cells not treated with DFMO. TGF-beta mRNA levels significantly increased from 4 to 12 h after wounding, peaking at 6 h at a level eight times the prewounding control. Increased levels of TGF-beta mRNA in IEC-6 cells after wounding were paralleled by an increase in TGF-beta content. Depletion of intracellular polyamines in DFMO-treated cells significantly inhibited increased expression of the TGF-beta gene in response to wounding. Cell migration also significantly decreased in DFMO-treated cells. In the presence of DFMO, exogenous TGF-beta restored cell migration to normal. These results indicate that 1) polyamine depletion induced by DFMO is associated with decreases in the expression of the TGF-beta gene and cell migration in IEC-6 cells and 2) exogenous TGF-beta reverses the inhibitory effect of polyamine depletion on cell migration. These findings suggest that polyamines are required for epithelial cell migration in association with their ability to regulate TGF-beta gene expression.

The current study tested the hypothesis that the protooncogene c-myc is involved in the mechanism by which gastrin modulates mucosal cell proliferation. Studies were conducted in the IEC-6 cell line, derived from rat small intestinal crypt cells. Administration of gastrin resulted in the rapid appearance of c-myc mRNA in IEC-6 cells. The increased expression of c-myc began 1 h and peaked 4 h after exposure to gastrin. Maximum increase in c-myc mRNA levels was 7.5-fold the normal value. When cellular protein synthesis was inhibited by addition of cycloheximide, gastrin superinduced c-myc mRNA levels. Gastrin also significantly increased the mRNA levels for ornithine decarboxylase (ODC), the rate-limiting enzyme for polyamine biosynthesis, enzyme activity, and intracellular polyamines in IEC-6 cells. Treatment with alpha-difluoromethylornithine (DFMO), a specific inhibitor of ODC, not only completely depleted intracellular polyamines but also significantly prevented the increased expression of c-myc in cells exposed to gastrin. These results show that 1) gastrin stimulates both polyamine biosynthesis and the expression of the c-myc protooncogene, and 2) depletion of intracellular polyamines by DFMO significantly prevented the increased expression of c-myc by gastrin.

Direct exposure of small intestinal mucosal cells to luminal polyamines stimulates proliferation. This study tests the hypothesis that the protooncogenes c-fos, c-myc, c-jun, and junB are involved in the mechanism by which polyamines modulate mucosal growth. Studies were conducted in the IEC-6 cell line, derived from rat small intestinal crypt cells. Cells were grown in Dulbecco's minimal essential medium containing 5% dialyzed fetal bovine serum (dFBS) in the presence of absence of alpha-difluoromethylornithine (DFMO), a specific inhibitor of ornithine decarboxylase, which is the rate-limiting enzyme for polyamine synthesis. Cellular polyamine levels, cell growth, and relative abundance of c-fos, c-myc, c-jun, and junB mRNAs, were measured at 1, 2, 4, 6, 8, and 12 days after initial plating. The intracellular polyamines, spermidine and spermine, and their precursor, putrescine, in DFMO-treated cells decreased significantly at 2 days and remained depleted thereafter. Although DFMO profoundly decreased growth and final cell number, both control and DFMO-treated cells entered a plateau phase by 6 days. In control cells, c-myc and c-jun mRNA levels significantly increased on days 4-6 and then returned to a basal level of expression, which was maintained thereafter. c-fos mRNA in quiescent cells after 24 h serum deprivation was significantly stimulated by 5% dFBS, although a steady-state level of c-fos mRNA was undetectable in control cells. Treatment with DFMO not only prevented increased expression of c-myc and c-jun protooncogenes at 4 days, but also significantly reduced steady-state levels of c-myc and c-jun mRNA between 6 and 12 days.(ABSTRACT TRUNCATED AT 250 WORDS)

Studies from our laboratory have shown that polyamines are essential for the normal repair of duodenal erosions induced in vivo in a rat stress-ulcer model. In that model, the inhibition of ornithine decarboxylase, a rate-limiting enzyme of polyamine biosynthesis, with alpha-difluoromethylornithine (DFMO) almost entirely prevented healing. Healing could be restored by oral polyamines. In this paper, we have investigated whether the polyamines are required for the early stages of epithelial restitution using an IEC-6 cell culture model of cell migration. Treatment of the cells with DFMO for 4 days reduced cell migration 80%. Migration could be restored to normal by concomitant treatment with putrescine (PUT), spermidine (SPD), or spermine (SPM), but not by their addition during the migration period (6 h) only. If DFMO treatment was not begun until the migration period, it still reduced cell migration 20%, and this deficit could not be restored by concomitant addition of the polyamines. Intracellular polyamine levels at these times, i.e., 6 h or 4 days, were an important factor in these results. Only PUT was undetectable after 6 h of DFMO. SPD and SPM were still at normal levels at 6 h. SPD was undetectable at 4 days, but SPM was still at 40% of normal. These data give added importance to PUT because its absence reduced cell migration after only 6 h, while SPD and SPM were still present in normal amounts. Perhaps exogenous SPD and SPM restored cell migration when present with DFMO for 4 days treatment primarily because they contributed to intracellular PUT through the acetyltransferases.(ABSTRACT TRUNCATED AT 250 WORDS)

Two key enzymes in polyamine biosynthesis are ornithine decarboxylase (ODC) and S-adenosylmethionine decarboxylase (SAMDC). SAMDC decarboxylates S-adenosylmethionine, which then donates aminopropyl groups for spermidine and spermine synthesis. The purpose of our study was to determine whether putrescine, taken up from medium or synthesized endogenously by ODC, alters SAMDC activity. Studies were conducted in the IEC-6 cell line derived from rat small intestinal crypt cells. Cells were grown in Dulbecco's minimal essential medium containing 5% dialyzed fetal bovine serum (dFBS). They were deprived of serum for 24 h before experiments. Basal SAMDC activity was increased significantly by > or = 10(-4) M of putrescine. Lower doses had no significant effect. The same doses of putrescine decreased ODC activity to near zero. Asparagine at 10 mM or 5% dFBS not only stimulated ODC activity and the intracellular putrescine levels but also increased significantly SAMDC activity as well. ODC activity peaked at 3 h, and the maximum level of SAMDC occurred 3-4 h after exposure to asparagine or serum. Treatment with DL-alpha-difluoromethylornithine (DFMO), a specific ODC inhibitor, prevented the increases in both cellular putrescine levels and SAMDC activity in asparagine- and serum-treated cells. In the presence of DFMO, exogenous putrescine returned SAMDC activity toward control levels but had no effect on ODC. A very slight increase of SAMDC half-life in IEC-6 cells grown in the presence of putrescine was not statistically significant.(ABSTRACT TRUNCATED AT 250 WORDS)

Cell migration is the principal force behind the early restitution of erosions of the mucosa of the gastrointestinal tract. Despite the importance of cell migration to healing, no attempts to study the process in culture have been reported. We have attempted to standardize conditions for migration and test the migration responses of the small intestinal epithelial crypt cell line IEC-6 in some experimental situations already well known in vivo. We found good correspondence between in culture and in vivo on the following points: 1) migration was independent of DNA synthesis; 2) DNA synthesis was not concentrated at the wound edge; and 3) inhibition of actin polymerization stopped migration altogether. In addition, the presence of an extracellular matrix maximized migration. Protein inhibitors with different modes of action inhibited cell migration to different degrees, not always commensurate with their inhibition of protein synthesis. Cell surface proteoglycans were important; hyaluronic acid had an effect, but the secretion of a migration-stimulating substance by wounded cells was equivocal. Significantly, alpha-difluoromethylornithine (DFMO), which inhibits ornithine decarboxylase and polyamine synthesis, almost totally prevented cell migration. Because DFMO also prevents healing of mucosal erosions in vivo, we believe that this model can be used, keeping in mind its spatial limitations, to study the process of cell migration involved in the early restitution of mucosal erosions.

The aim of this study was to determine whether the circadian changes in ornithine decarboxylase (ODC) activity of different segments of the small intestine were governed by factors other than food intake. First, the effects of fasting on mucosal ODC activity were examined. The results indicate that mucosal ODC activity in 24 hr and 48 hr fasted rats decreased significantly compared with ad libitum-fed rats. Second, the circadian rhythm of mucosal ODC activity was characterized by measuring mucosal ODC activity in fasted rats at four time points (09:00, 15:00, 21:00, and 03:00 hr; light period: 06:00-18:00 hr). The results from this study indicate that there is a detectable baseline ODC activity in different segments of fasting intestine. In duodenum, mucosal ODC activity was highest at 15:00 hr (light period), a time at which the rat was normally not eating. In jejunum and ileum, mucosal ODC activity increased between 21:00 and 03:00 hr (dark period). The observation that small intestine exhibits a distinct circadian rhythm of ODC activity in fasted rats suggests that not only food but also intrinsic factors can modulate physiologic oscillations in mucosal ODC activity.

Ornithine decarboxylase (ODC), through the regulation of polyamine biosynthesis, influences normal mucosal growth and cell proliferation. The purpose of our study was to determine whether mucosal ODC activity and polyamines play a role in the dramatic increase in mucosal mass and crypt elongation associated with parasite-induced inflammation in the small intestine. Rats were inoculated orally with L1 larvae of the parasitic nematode Trichinella spiralis and killed at different times during the enteric phase of infection. Duodenal and jejunal mucosal ODC activities increased significantly from 2 to 14 days, peaking 7 days postinfection (PI). By 21 days PI, enzyme activity had returned to near normal values. In the ileal mucosa, ODC activity was increased only at 7 days PI. Increases in ODC activity were paralleled by increases in mucosal putrescine, spermidine, and spermine content. Infection with T. spiralis induced DNA synthesis and evoked a significant rise in DNA, RNA, and protein content in the mucosa. Increases in nucleic acid and protein levels were most prominent in the proximal half of the small intestine where the majority of worms reside. Treatment of rats with alpha-difluoromethylornithine (DFMO) prevented the infection-induced elevations in mucosal ODC activity, polyamine levels, DNA synthesis, and DNA, RNA, and protein content without influencing the development of inflammation or the parasite's life cycle. These results suggest that mucosal hyperplasia caused by infection may be regulated, in part, by the growth-promoting effects of ODC, presumably through the stimulation of crypt cell proliferation. Thus ODC may be an important determinant of the intestinal response to infection.

Ornithine decarboxylase (ODC) is the primary rate-limiting 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 cycloheximide-treated rats, the time course of the decline in ODC activity yielded a half-life of approximately 22 min. Labeling enzyme protein with [3H]difluoromethylornithine (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, 10(-7) 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.

The importance of the presence of food in the gastointestinal (GI) tract in maintaining normal enzyme activity in the small intestine was investigated in rats that were kept from eating for as long as 2 weeks. During this time animals were sustained on a nutrient solution administered intravenously. Enzyme activity and small bowel mass in these animals and similiar parameters in rats that were administered the same solution orally were compared with control groups fed an essentially isoenergetic diet of stock rat food and water. Disaccharidase and peroxidase levels in mucosal scrapings expressed as activity/milligram protein, activity/intestine, and activity/100 g body weight, were significantly reduced in intravenously nourished rats but not in rats receiving the solution orally. In addition to changes in enzyme activities, parenteral feeding was accompanied by a significant reduction in the small bowel weight:body weight ratio as compared with that of rats fed the stock diet or nutrient solution orally. Results support the conclusion that normal activity of intestinal enzymes spatially distributed with regard to depth within the mucosa depends on the presence of food in theGI tract and cannot be maintained at normal levels by total intravenous nutrition.

The effect of intravenously administered secretin (0.5, 2.0, 6.0 U/kg-h) and intraduodenal acidification (13.2 meq/h HCl) on the electrical activity of the small bowel of three conscious dogs with gastric and duodenal cannulas was observed. Electrical activity was recorded in fasted as well as fed conditions through silver wire electrodes implanted along the entire length of the small bowel. Intravenous infusion of secretin in all dosages and in all dogs delayed the onset of the interdigestive myoelectric complex and reduced the total percentage of slow waves with superimposed spike potentials. Intraduodenal acidification also inhibited the interdigestive myoelectric complex, which developed incompletely with fewer action potentials on slow waves. Secretin did not produce any alteration in the fed pattern of activity, slow-wave frequency, or the caudal migration of the interdigestive myoelectric complex. The present study indicates that the nuerohumoral mechanisms responsible for initiation of the interdigestive myoelectric complex may be different from those responsible for its caudal migration.

The effect of bilateral thoracic vagotomy on the myoelectric activity of the small intestine was determined in conscious dogs. Animals were implanted with electrodes spaced 25 cm apart along the serosal surface of the small intestine, and a cannula was placed in the most dependent portion of the stomach. Recordings were made with dogs in the fasted and fed states. Two distinct patterns of myoelectric activity were recorded: one typical of the fasted state (the interdigestive myoelectric complex) and one typical of the fed state. After completion of the control recording periods, a truncal vagotomy was performed on each animal. Completeness of vagotomy was confirmed by lack of a gastric secretory response to insulin. Gastric stasis occurred after vagotomy; therefore, the animals' stomachs were emptied via the gastric cannula to obtain a fasted condition. Vagotomy had little to no effect on the fasted pattern of myoelectric activity. The fed pattern was significantly altered in two of the three animals. This alteration could be due to the effect of vagotomy on gastrin release. We conclude that nervous pathways within the vagus may exert some influence on intestinal myoelectric activity but that other neural-humoral pathways are probably involved.

In most tissues increases in ornithine decarboxylase (ODC) are associated with growth. Refeeding fasted rats. a potent stimulus for mucosal growth, strongly increases ODC in both small and large intestinal mucosa. In the small bowel, almost all of this increase occurs in the mature villus cells rather than the proliferative crypt cells. Nevertheless, inhibition of ODC with difluoromethylornithine blocks the growth response. Using a highly specific, polyclonal antiserum to ODC, we have determined that in the fasted rat ODC is localized almost exclusively to the villus cells. Using antiserum dilution techniques, we have shown that, within 2 h, refeeding increases the amount of immunoreactive ODC in both villus and crypt cells. Furthermore, the trophic hormone gastrin also increases ODC, but only in the crypt cells. Epidermal growth factor increased ODC to a greater extent than gastrin, but stimulation was more general, including both crypt and villus cells. Perfusing an isolated segment of small bowel in situ with glycine for 2 h also increased immunoreactive ODC but only in the villus cells. Thus in the small intestine the effect of refeeding on ODC activity appears to be mediated by different types of stimuli: luminal nutrients increase enzyme levels in the absorbing villus cells, while trophic peptides stimulate ODC synthesis in the proliferative crypt cells.

Intestinal DNA, RNA, and protein content were decreased to a greater extent than was body weight when rats were starved for 3 days. Specific lactase and maltase activity increased with progressively longer periods of starvation. Antral and serum gastrin concentration significantly decreased during the 3 days of starvation. Pentagastrin (250 mug/kg 3 times daily) was injected into a group of rats for the duration of a 3-day starvation period and caused a small but significant increase in the relative intestinal RNA and protein content and decreased lactase and maltase specific activities in comparison with the levels of 3-day starved controls. Pentagastrin thus partially reversed some of the starvation-induced changes toward fed levels. Thus, a deficiency in the trophic hormone gastrin may be partially responsible for the disproportionate changes in intestinal tissue during starvation.

Ornithine decarboxylase (ODC) activity has been found to be preferentially associated with small intestinal villus cells rather than crypt cells in the rat. In the present study, ODC, S-adenosylmethionine decarboxylase (SAMDC), and polyamines were measured in isolated enterocytes to determine which cell populations increased polyamine biosynthetic activity after refeeding. Two hours following refeeding, significant increases in ODC were observed in villus tip (10 times) and midvillus (20 times) enterocytes. No increase in ODC activity was found in isolated crypt cells. A similar pattern was observed for SAMDC. Enzyme activity increased in villus tip (2 times) and midvillus (27 times) cells but not in crypt enterocytes. Putrescine contents were increased following refeeding in midvillus enterocytes (P less than 0.05) and in crypt cells (P less than 0.05). The accumulation of putrescine in midvillus cells occurs via ODC-induced biosynthesis, whereas in crypt enterocytes it may be due to putrescine uptake. The lack of induction of ODC and SAMDC in crypt enterocytes following acute refeeding suggests these enzymes are apparently not involved in the initiation of cell proliferation known to occur under this condition.

Gastrin is a trophic hormone for the mucosa of the oxyntic gland area of the stomach, the proximal small intestine, and the colon. It also has trophic effects on the pancreas. All these tissues undergo hyperplasia to some extent following distal small bowel resection. This study evaluates the role of gastrin in postresectional hyperplasia by examining the growth of these tissues in antrectomized rats following intestinal resection. Antrectomy itself caused atrophy of the oxyntic gland mucosa, colonic mucosa, and the pancreas but had no effect on ileal mucosa. Resection by itself stimulated growth of all tissues and significantly increased serum gastrin levels. After resection in antrectomized animals, all tissues underwent an adaptational response. The increase in total DNA content after resection in antrectomized rats was as great in all tissues, except the colon, as it was in animals with intact antrums and normal gastrin levels. These results indicate that gastrin plays no role in the postresectional hyperplasia observed in the various tissues of the gastrointestinal tract.

Polyamines are necessary for the growth of eukaryotic cells and are supplied either by new synthesis or cellular uptake. To our knowledge, no information is available on polyamine uptake by gastrointestinal cells. In the current study, isolated villous enterocytes from the rat accumulated putrescine to an eightfold concentration gradient. Uptake was temperature dependent, saturable, and inhibited by 1 mM KCN. Kinetic analysis showed a Km of 1.23 X 10(-5) M and a Vmax of 2.60 X 10(-10) mol.10(6) cells-1.15 min-1. Enterocytes from the distal one-fourth of the gut showed the highest rate of uptake. Putrescine uptake was inhibited by cadaverine and spermine but not by the amino acids asparagine, AIB, or leucine. Sodium replacement by choline, lithium, N-methyl-D-glucamine, or tetramethylammonium significantly inhibited uptake, but replacement of Na+ by sucrose or mannitol was without effect. The inhibition observed was believed to be due to the ability of the cations to interact in some way with the carrier. Neither ouabain nor digitoxigenin had any effect on uptake. These data indicate that putrescine is accumulated by villous enterocytes by a carrier-mediated process that does not appear to involve Na+ contransport.

Feeding an elemental diet (Vivonex) to rats over 9 days causes a decrease in the rate of cell renewal and a reduction in villus size in both the jejunum and ileum, as compared with rats fed regular chow. The addition of bulk to elemental diet cannot prevent the reduction in villous size, but it can cause a small increase in the rate of cell renewal, which is still much lower than that in chow-fed rats. The serum gastrin level of rats fed the elemental diet is about one-third of the level found in chow-fed rats, and it is not changed by the addition of bulk.

A number of peptides having trophic activity on gastrointestinal mucosa and growth factors are known to induce small intestinal ornithine decarboxylase (ODC) activity. The effect of peptides on ODC and S-adenosylmethionine (SAMDC) activities (key enzymes in polyamine biosynthesis) in isolated enterocytes is unknown. Male Sprague-Dawley rats were fasted for 72 h and injected intraperitoneally with epidermal growth factor (EGF), pentagastrin, or glucagon, or intragastrically with EGF. A similar volume of water served as a control. Villus tip, midvillus, and crypt cell fractions were collected and identified. ODC and SAMDC activities were determined in these cells 4 h after peptide injection. EGF given intraperitoneally, but not intragastrically, stimulated ODC activity along the cryptvillus column. Pentagastrin and glucagon did not induce polyamine biosynthetic enzyme activity. ODC and SAMDC activities in intestinal mucosal scrapings from fasted animals also were increased 2-4 h after intraperitoneal EGF treatment. It is possible that EGF binding at the serosal surface of the crypt enterocyte and subsequent ODC induction is important in initiating the cellular proliferation that is known to occur after treatment with this peptide.