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Background & Aims: We recently identified lysophosphatidic acid (LPA) as a potent antiapoptotic agent for the intestinal epithelium. The objective of the present study was to evaluate the effect of octadecenyl thiophosphate (OTP), a novel rationally designed, metabolically stabilized LPA mimic, on radiation-induced apoptosis of intestinal epithelial cells in vitro and in vivo., Methods: The receptors and signaling pathways activated by OTP were examined in IEC-6 and RH7777 cell lines and wild-type and LPA(1) and LPA(2) knockout mice exposed to different apoptotic stimuli., Results: OTP was more efficacious than LPA in reducing gamma irradiation-, camptothecin-, or tumor necrosis factor alpha/cycloheximide-induced apoptosis and caspase-3-8, and caspase-9 activity in the IEC-6 cell line. In RH7777 cells lacking LPA receptors, OTP selectively protected LPA(2) but not LPA(1) and LPA(3) transfectants. In C57BL/6 and LPA(1) knockout mice exposed to 15 Gy gamma irradiation, orally applied OTP reduced the number of apoptotic bodies and activated caspase-3-positive cells but was ineffective in LPA(2) knockout mice. OTP, with higher efficacy than LPA, enhanced intestinal crypt survival in C57BL/6 mice but was without any effect in LPA(2) knockout mice. Intraperitoneally administered OTP reduced death caused by lethal dose (LD)(100/30) radiation by 50%., Conclusions: Our data indicate that OTP is a highly effective antiapoptotic agent that engages similar prosurvival pathways to LPA through the LPA(2) receptor subtype.

Background & Aims: Inhibition of RhoA activity and depletion of polyamines inhibits cell migration and causes changes in the actin cytoskeleton. In this article we have examined the effect of polyamine depletion on RhoA and evaluated these effects on cell migration., Methods: Polyamines were depleted in intestinal epithelial cell (IEC)-6 cells by incubating them for 4 days with 5 mmol/L alpha-difluoromethylornithine (DFMO), which inhibits ornithine decarboxylase, the first rate-limiting enzyme in the synthesis of polyamines. IEC-6 cells were then transfected with vectors containing HA tags and constitutively active (HA-V14) or dominant-negative (HA-N19) RhoA with pcDNA3 (vector)., Results: DFMO caused a significant decrease in Rho levels in the cytoplasm and membranes of IEC-6 cells. This decrease was caused by an approximate 50% inhibition of RhoA protein synthesis. Neither the half-life of RhoA nor the level of RhoA messenger RNA (mRNA) was affected. HA-V14-RhoA cells migrated much more rapidly than vector-transfected cells, and HA-N19-RhoA cells exhibited almost no motility. The migration of HA-V14-RhoA cells, however, was inhibited markedly by polyamine depletion. Polyamine depletion did not affect the activity of RhoA in HA-V14-RhoA cells, but inhibited it dramatically in the vector-transfected cells. In the presence of DFMO, the HA-V14-RhoA cells lost stress fibers and gained the appearance of HA-N19-RhoA cells or wild-type cells treated with DFMO., Conclusions: First, polyamines are essential for the activity and synthesis and, therefore, normal levels of RhoA protein. Second, RhoA does not mediate the inhibitory effects of polyamine depletion on cell migration.

Background & Aims: We have investigated whether the phospholipid growth factor lysophosphatidic acid (LPA) could prevent intestinal epithelial cells-6 (IEC-6) from apoptosis elicited by 4 different mechanisms. The antiapoptotic effect of LPA was also tested in a mouse model of radiation-induced apoptosis., Methods: Apoptosis was elicited by serum withdrawal, exposure to camptothecin, gamma-irradiation, or rat tumor necrosis factor alpha and evaluated by DNA fragmentation enzyme-linked immunosorbent assay (ELISA) and annexin V staining. Caspase-3/CPP32 activity and activation was measured by ELISA and Western blotting, respectively. Reverse-transcription polymerase chain reaction (RT-PCR) was applied to examine the expression of LPA-receptor transcripts. Mice were treated with 250 microL of 1 mmol/L LPA and exposed to whole-body gamma-irradiation with a dose of 12 or 15 Gy and the number and localization of apoptotic bodies along the crypt were recorded., Results: LPA pretreatment reduced DNA fragmentation induced in all models of apoptosis. LPA rescued cells from apoptosis when applied up to 1 hour after camptothecin treatment or 2 hours after irradiation. LPA inhibited the activation of caspase-3/CPP32 and attenuated its activity. Blocking LPA1 receptors by pertussis toxin and the inhibition of epithelial growth factor receptor tyrosine kinase significantly attenuated the protective effect. In irradiated mice, oral LPA significantly reduced the number of apoptotic bodies in the crypt., Conclusions: (1) LPA prevents and rescues IEC-6 from apoptosis elicited by 4 different mechanisms. (2) This antiapoptotic activity is mediated through LPA1 and LPA2 receptors through the inhibition of caspase-3/CPP32 activation. (3) LPA protects enterocytes against radiation-induced apoptosis. This study suggests that in patients undergoing cancer therapy, dietary LPA might have therapeutically useful antiapoptotic capacity in the intestinal epithelium.

Background: Polyamines serve as substitutes for transglutaminase-catalyzed protein cross-linking and are essential to the healing of gastric mucosal lesions. This study determines whether transglutaminase and protein cross-linking have a role in the healing of hypertonic NaCl-induced gastric lesions., Methods: Rats were fasted 22 hours before given 1 mL 3.4 Mol/L NaCl intragastrically. Gastric mucosa was examined histologically and grossly, and transglutaminase activity was measured as the Ca(2+)-dependent covalent incorporation of [3H]putrescine into acid-precipitable protein., Results: Transglutaminase activity increased significantly from 2 to 8 hours, peaking between 4 and 6 hours after NaCl administration. Lesions were significantly produced after 2 hours, and damage paralleled transglutaminase activity. Dansylcadaverine (200 mg/kg orally), a specific inhibitor of protein cross-linking, prevented the increases in transglutaminase activity and significantly delayed healing but had no effect on lesion formation., Conclusions: These results indicate that (1) hypertonic NaCl-induced gastric mucosal damage is associated with a significant increase in transglutaminase activity and (2) increased transglutaminase activity is involved in the mechanism of normal mucosal healing.

The purpose of this study was to examine whether luminal polyamines administered exogenously accelerate the repair of stress-induced intestinal mucosal damage in rats. Rats were fasted for 22 hours, placed in restraint cages, and immersed in water to the xiphoid process for 6 hours. Animals were killed either immediately after the period of stress or at 4, 12, and 24 hours thereafter. Duodenal mucosa was examined histologically, and ornithine decarboxylase activity and polyamine levels were measured. Repair of duodenal mucosa after stress was extensively delayed by administering 500 mg/kg DL-alpha-difluoromethylornithine (DFMO) IP. DFMO also inhibited ornithine decarboxylase activity and prevented increases in duodenal mucosal polyamine content. Intragastric administration of the polyamines, putrescine, spermidine, and spermine (100 mg/kg), immediately after stress significantly prevented the decreased rate of repair caused by DFMO. Spermidine or spermine accelerated healing better than putrescine in the DFMO-treated rats. Spermine also significantly increased the normal rate of repair of stress-induced damage. The delayed recovery of mucosal DNA, RNA, and protein content following stress in the DFMO-treated rats was prevented by exogenous polyamines. The reduced levels of duodenal mucosal spermidine and spermine in stressed rats treated with DFMO returned toward control levels after administration of exogenous spermidine. These results indicate that (a) luminal polyamines effectively substitute for endogenously synthesized polyamines in the repair process of the duodenal mucosa, (b) luminal polyamines can increase the normal healing rate and, (c) polyamines accelerate healing by increasing both an early phase and a later phase dependent on cell renewal.

This investigation shows whether polyamines and ornithine decarboxylase have a role in duodenal mucosal repair following stress-induced microscopic damage. Rats were fasted for 22 hours, placed in restraint cages, and immersed in water to the xiphoid process for 6 hours. Animals were killed either immediately after the period of stress or at 2-hour intervals up to 24 hours thereafter. Duodenal mucosa was examined histologically, and ornithine decarboxylase and polyamine levels were measured. Ornithine decarboxylase activity was increased significantly up to 6 hours following stress, peaking at 4 hours at a level 10 times the prestress control. By 8 hours, enzyme activity had returned to near normal. Increases in mucosal putrescine, spermidine, and spermine content paralleled the changes in ornithine decarboxylase activity and peaked 4 hours after stress. Stress resulted in microscopic damage evidenced by a nearly complete absence of villi. Significant macroscopic lesions were not present following stress. Mucosal repair was evident 12 hours after stress and almost complete by 24 hours, although the restituted villi were short and blunted. The decreases in mucosal DNA, RNA, and protein content caused by stress were restored and reached near-normal levels 12 hours after the period of stress. In animals given the specific inhibitor of ornithine decarboxylase, alpha-difluoromethylornithine, increases in duodenal mucosal ornithine decarboxylase activity and polyamine levels were inhibited and mucosal repair was almost completely prevented following stress. alpha-Difluoromethylornithine also prevented the recovery of DNA, RNA, and protein content of the duodenal mucosa. These results indicate that duodenal mucosal damage following stress is repaired rapidly; the repair process is accompanied by significant increases in ornithine decarboxylase activity and polyamine levels; and the increases in ornithine decarboxylase and polyamines are absolutely required for the normal repair of the mucosa.

In rats fasted 4 days, immunoreactive gastrin concentrations decreased to one-third of the fed levels in antral tissue and to one-eight of the fed levels in serum. The number of antral cells that reacted with fluorescent antigastrin antiserum was also correspondingly decreased. After refeeding, serum gastrin returned to normal levels in 6 days, whereas antral gastrin concentration recovered after 9 days. Normal gastrin levels were maintained in rats fed a nutritious liquid diet over a 6-day period, whereas tissue and serum hormone concentrations decreased to low levels in rats placed on a high bulk non-nutritive diet over the same period. These results suggest that food in the gastrointestinal tract is necessary for the maintenance of normal serum and antral gastrin concentration in rats. The effect of food is most likely attributable to chemical constituents and not distention by bulk.

The contracted segment of terminal large intestine in both Hirschsprung's disease in humans and in the piebald-lethal strain of mice with congenital megacolon may result from muscarinic cholinergic receptor denervation supersensitivity. This hypothesis was tested by direct binding studies using the muscarinic receptor ligand, tritiated quinuclidinyl benzilate. There were no differences in either the receptor density, dissociation constant, or agonist/antagonist Hill coefficients in the proximal, middle, or distal colon of mice with congenital megacolon as compared with normal littermates. These data do not support the concept of denervation supersensitivity. The data support the suggestion that absence of the inhibitory neurons of the enteric nervous system accounts for the hyperexcitability of the musculature of the aganglionic terminal segment and the obstructive constriction of the segment.

In previous communications we have reported using the rat fed by total parenteral nutrition to examine the effects of the absence of food from the gut on functional and structural parameters of the gastrointestinal tract. In the current study three groups of animals were fed parenterally; one received a continuous infusion of pentagastrin equal to about one-half the D50 for acid secretion, another received a comparable infusion of histamine, and a third group was given only the liquid diet. These animals were compared to orally fed sham operated controls. The parenterally fed animals had significantly lower levels of antral and serum gastrin. When compared to whole body weight, the weights of the oxyntic gland area of the stomach, the pancreas, and the small intestine were significantly lower. In addition, the total and specific activities of the disaccharidase enzymes were significantly reduced. Pentagastrin prevented both the decreases in weights of the gastrointestinal tissues and the decreases in dissaccharidase activity. Histamine was without effect. We conclude that pentagastrin prevents the changes in gastrointestinal structure and function caused by the absence of food from the gut and that the trophic action of gastrin is necessary for the maintenance of the functional and structural integrity of the gastrointestinal tract.

Fasted rats were injected six times over a 48-hr period with G-17 I, G-17 II, G-34 II in doses doubling from 3.38 nmoles per kg to 54 nmoles per kg. Twelve rats were studied at each dose. NaCl-injected animals were used as controls. The rats were killed and the in vitro incorporation of [3H]thymidine into DNA as well as the total DNA content of the mucosa of the duodenum and oxyntic gland area of the stomach were determined. All gastrins, as well as pentagastrin, stimulated DNA synthesis. Peak stimulation occurred at 13.5 nmoles per kg for G-17 I and G-17 II and at 6.75 nmoles per kg for G-34 II. Pentagastrin's peak trophic effect was produced by 325 nmoles per kg. In order to compare the efficacies of the various types of gastrins, 60 rats were divided into groups of 12. One group received saline and the other four groups received the maximally effective dose of one of the four types of gastrins. The responses to each of the gastrins were not significantly different, and DNA synthesis was doubled in each tissue. Total DNA content increased slightly, but significantly, in response to each gastrin. Several conclusions can be drawn from these data: (1) G-17 and G-34 possess trophic activity; (2) the efficacies of pentagastrin, G-17 I, G-17 II, and G-34 II for the stimulation of DNA synthesis are not significantly different; (3) sulfation of G-17 has no significant effect on its trophic activity; (4) based on the effects of exogenous molar doses and their respective half-lives, both G-17 and G-34 would be expected to contribute to the trophic effect of endogenous gastrin; and (5) G-17 and G-34 are at least as effective in stimulating DNA synthesis as they are in stimulating gastric acid secretion.

Fasted rats were injected with either cholecystokinin-octopeptide (CCK-OP), 20 mug per kg; 16,16-dimethyl prostaglandin E2 (16,16-dimethyl PGE2), 0.2 mg per kg; pentagastrin, 250 mug per kg, or saline every 8 hr for 48 hr. The rats were killed and the incorporation of [3H]thymidine into DNA as well as the total DNA and RNA content of the mucosa of the oxyntic gland area and the duodenum were determined. Pentagastrin increased DNA synthesis 60% (P less than 0.001) in gastric mucosa and 90% (P less than 0.001) in duodenal mucosa when compared with rates for saline controls. Neither CCK-OP nor 16,16-dimethyl PGE2 altered gastric mucosal DNA synthesis. Pentagastrin significantly increased the DNA and RNA content of both the gastric and duodenal mucosa. CCK-OP and 16,16-dimethyl PGE2 caused a slight but significant increase in duodenal DNA synthesis, CCK-OP did not significantly increase duodenal DNA content, and 16,16-dimethyl PGE 2 increased duodenal RNA but not DNA content. CCK-OP (20 mug per kg) in combination with pentagastrin did not alter the stimulation of gastric DNA synthesis but significantly decreased the effect of pentagastrin on duodenal DNA. A dose of CCK-OP (370 mug per kg) equimolar to 250 mug per kg of pentagastrin did not stimulate DNA synthesis in either tissue and significantly inhibited stimulation by pentagastrin in both tissues. Low doses of CCK-OP (2.5, 5.0, 10.0, 20.0 mug per kg) caused statistically significant increases in DNA synthesis and DNA content of the pancreas, but had no effect on either mucosa of the oxyntic gland area or duodenum. 16,16-Dimethyl PGE2 did not inhibit the stimulation of DNA synthesis or the increases in DNA and RNA content stimulated by pentagastrin. From these results it appears that: (1) moderate doses of CCK have a weak trophic effect in the duodenum but not in the stomach, (2) physiological doses of CCK-OP stimulated pancreatic DNA synthesis and increased pancreatic DNA content without affecting these parameters in the oxyntic gland area or duodenum in the same animals, (3) in the stomach and duodenum CCK is not as potent a trophic hormone as gastrin and inhibits, probably competitively, the trophic effects of gastrin, (4) 16,16-dimethyl PGE2 does not stimulate growth and does not interfere with the trophic response to gastrin even though it inhibits acid secretion, and (5) 16,16-dimethyl PGE2 increased the RNA content of duodenal mucosa indicating that it may stimulate activity resulting in hypertrophy.

This paper deals with several new findings regarding the trophic action of gastrointestinal hormones. Naturally occurring gastrins (G-17 I, G-17 II, G-34 II) stimulated DNA synthesis and increased total DNA content of gastric mucosa. These were several times more potent than pentagastrin on a molar basis. In a survey of various tissues from the gastrointestinal tract, pentagastrin exerted trophic effects on mucosa of the oxyntic gland area, duodenum, and colon; it had no effect on the esophagus, antrum, or diaphragm. Maximal stimulation (200% of control) of colonic DNA synthesis was produced by 250 mug of pentagastrin per kg. Vasoactive intestinal peptide did not stimulate DNA synthesis when given alone and inhibited the trophic effect of pentagastrin when administered simultaneously. Glucagon stimulated DNA synthesis in both colon and oxyntic gland mucosa to 40% of the increase caused by pentagastrin and did not inhibit the effects of pentagastrin when administered concurrently.

The effects of hypophysectomy and subsequent replacement therapy with growth hormone on serum and antral gastrin levels was investigated in both fasted and nonfasted pair-fed rats. Hypophysectomy caused a 57% decrease in serum and 47% decrease in antral gastrin content in 18-hr fasted animals. In nonfasted animals, hypophysectomy resulted in a 42% fall in serum gastrin and a 76% fall in antral gastrin. Animals were given injections of growth hormone over a 10-day period, then fasted 16 to 18 hr before being killed. Doses of 100 to 200 mug per 100 g of body weight were ineffective, but a dose of 500 mug per 100 g was sufficient to restore completely serum gastrin levels to intact control values. In nofasted rats this same dose raised serum gastrin to intact levels and increased antral gastrin significantly over hypophysectomized levels, but did not increase it to control values. When given to pair-fed intact animals, growth hormone caused slight but not significant elevations in serum and antral gastrin. The effect of growth hormone on gastrin secretion and /or synthesis may be a significant physiological factor in the regulation of normal gastrointestinal function and growth.

This study examines the effect of prolonged absence of oral food intake on structural parameters of the gastrointestinal tract in rats maintained nutritionally by intravenous feeding for up to 3 weeks. During this time, their body weights increased by 22%. Controls fed a nearly isocaloric oral diet were sham operated and harnessed in the same manner as their parenterally fed counterparts. Parenteral feeding resulted in a significant decrease in the weights (per 100 g body weight) of the oxyntic gland area of the stomach, small intestine, and pancreas. The weights of the spleen, testes, kidneys, and antral region of the stomach were unaltered. In the small intestine there was a significant loss of DNA and a near doubling of the RNA:DNA ratio in the parenterally fed animals. In the absence of an oral diet antral gastrin levels decreased to one-thirtieth of the control level. The following conclusions are suggested by these results. First, the oral intake and/or physical presence of food within the gastrointestinal tract are necessary for structural maintenance of some tissues of that tract. Second, the disproportionate decrease in weight that occurs in certain tissues is apparently unrelated to the absence of nutrients which might normally be utilized directly from the lumen. Third, maintenance of normal tissue stores of the hormone, gastrin, is dependent on stimuli provided by oral ingestion and the presence of food in the gastrointestinal tract.

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.