Biomedical subjects

E R Seidel

Publications and source records attributed to E R Seidel.

34 records · Page 2Linked to original sources

PubMed1990

Apparent post-transcriptional modification of ornithine decarboxylase accounts for its induction in IEC-6 cells in culture.

Putrescine, the product of the ornithine decarboxylase (ODC)-catalyzed reaction, stimulates macromolecular synthesis in a duodenal crypt cell line, IEC-6 cells, grown in culture. In addition, supplementation of medium with putrescine alone reverses the inhibition of proliferation produced by inhibition of ODC with difluoromethylornithine (DFMO). A series of experiments was initiated in the IEC-6 cell line to study the regulation of induction of ODC, as this enzyme is rate-limiting in putrescine synthesis. Five percent fetal bovine serum (FBS) and 10 nM IGF-II stimulated a 21-fold and 6-fold induction of ODC activity, respectively. Kinetic analysis indicated that the effect was on the Vmax of the reaction and not on the Km, suggesting an increase in total ODC protein. This was verified by measuring [3H]DFMO binding; serum-stimulated induction of activity was accompanied by a corresponding 20-fold increase in the specific binding of DFMO to ODC. In contrast, Northern analysis demonstrated only a two-fold change in ODC mRNA level during induction. Measurement of enzyme stability showed that the half-life of the ODC protein was increased three-fold above basal level in the induced state. Inhibition of induction was produced by pretreatment with either the calmodulin antagonist, W-7, or the product of the ODC-catalyzed reaction, putrescine. Further analysis illustrated that the inhibition produced by these agents was partly the result of destabilization of the enzyme and not a decrease in message level. These results demonstrate that the induction of ODC by trophic agents is the result of post-transcriptional events rather than at the level of RNA synthesis.

Animals↗

PubMed1989

Pentagastrin induction of spermine/spermidine N1-acetyltransferase and mucosal polyamines.

The trophic response of the gastrointestinal mucosa to treatment with the hormone gastrin includes a polyamine-dependent step. Because gastrin does not induce ornithine decarboxylase, experiments were designed to determine whether pentagastrin induced the polyamine interconverting enzyme, spermine/spermidine N1-acetyltransferase (SAT). Eight hours after intraperitoneal treatment of rats with either spermidine (0.8 mmol/kg) or pentagastrin (250 micrograms/kg) oxyntic gland mucosal SAT activity was increased from roughly 400 to 800 pmol [14C]acetate.mg protein-1.h-1. In contrast, colonic mucosa was not sensitive to pentagastrin even though spermidine treatment induced nearly a 400% increase in SAT activity. Measurement of both oxyntic gland and colonic mucosal polyamine concentrations showed that by 16 h after pentagastrin (250 micrograms/kg ip) putrescine, acetylspermidine, and spermidine levels all were increased to a level approximately 200% of that observed in NaCl-treated rats. By 24 h mucosal polyamine content of pentagastrin-treated rats was not different from control. Essentially the same results were found in animals treated with difluoromethylornithine, thus demonstrating that the increase in mucosal polyamine concentration was not related to the induction of ornithine decarboxylase. The results of these experiments demonstrate that unlike most hormones, the hormone gastrin induces the polyamine converting enzyme, SAT, rather than ornithine decarboxylase during stimulation of polyamine-dependent cell growth and/or division.

Acetyltransferases↗

PubMed1989

Polyamine-dependent growth and calmodulin-regulated induction of ornithine decarboxylase.

The proliferation of cultured gastrointestinal crypt epithelial cells (IEC-6) and the role of calcium in polyamine biosynthesis were examined after serum stimulation of quiescent cells. Ornithine decarboxylase (ODC) activity was high when cells grew in 5% fetal calf serum (FCS) and dropped to nearly nondetectable levels when cells reached contact inhibition of growth. Polyamines appeared to be necessary for the proliferation of these cells, as growth was completely inhibited by the addition of 5 mM difluoromethylornithine, a specific inhibitor of ODC, to the media. This effect was reversed by 10 microM putrescine. Serum deprivation of preconfluent cells resulted in a fall in ODC activity. Readdition of serum led to an increase in ODC activity, which peaked at 4 h after addition and preceded both putrescine accumulation and [3H]thymidine incorporation into acid-precipitable material. Furthermore, readdition of serum to serum-deprived cells resulted in an approximately twofold increase in the level of free, ionized, intracellular Ca2+ as measured spectrophotometrically by monitoring fura-2 fluorescence. Inhibition of calmodulin-mediated processes with N-(6-aminohexyl)-5-chloro-1-naphthalelesulfonamide (W-7), a calmodulin antagonist, inhibited the serum-stimulated induction of ODC in a dose-dependent manner, with an IC50 of approximately 10 microM. Similar results were obtained with trifluoperazine. Lastly, 30 microM W-7 completely inhibited serum-stimulated [3H]thymidine incorporation into acid-precipitable material. These data demonstrate that polyamine biosynthesis and subsequent DNA synthesis after serum refeeding of cells is regulated by a Ca2+ activated, calmodulin-dependent process. Furthermore, the production of polyamines is essential for normal proliferation of these epithelial cells in culture.

Animals↗

PubMed1989

Microflora-derived polyamines modulate obstruction-induced colonic mucosal hypertrophy.

Experiments were designed to determine the role of microflora-derived intraluminal polyamines in the colonic mucosal response to obstruction. Sprague-Dawley rats were treated per os with 0.9% NaCl or a combination of nonabsorbable antibiotics prior to the placement of either a sham or complete colonic obstruction. Sixty-six hours after surgery, wet tissue weight, DNA, RNA, and protein content were all increased in the mucosa proximal to the obstruction in NaCl-treated animals; however, DNA content was the only parameter increased after antibiotics. This induction was a purely local effect as neither hyperplasia nor hypertrophy was observed in the ileum or colon distal to the obstruction. In the NaCl-treated animals, mucosal ornithine decarboxylase activity was not induced until 48 h postsurgery, yet mucosal spermidine concentrations were significantly higher as early as 24 h. Intraluminal bacterial lysine, ornithine, and arginine decarboxylase activities were induced by obstruction but were reduced by antibiotic treatment. [14C]putrescine uptake by intestinal epithelial cells (IEC-6) in culture was blocked by the antibiotics employed in this study, but [14C]-lysine transport was relatively unaffected. These data demonstrate that intraluminal polyamines modulate the trophic response of the colonic mucosa after colonic obstruction.

Animals↗

PubMed1989

Putrescine stimulates DNA synthesis in intestinal epithelial cells.

Experiments were designed to examine the effects of exogenously supplied putrescine on the synthesis of DNA, RNA, and protein in cultured epithelial cells (IEC-6). Putrescine increased aphidicolin-sensitive DNA synthesis at concentrations as low as 0.3 microM putrescine with maximal stimulation (267% control) at 10 microM. This response appeared to be an effect of increases in the intracellular concentration of putrescine as the intracellular levels of spermidine and spermine did not change over the time period examined. Furthermore, pulse-chase experiments revealed that putrescine that entered the cell was not metabolized to another polyamine or degraded. In addition, 10 microM putrescine enhanced both cycloheximide-sensitive lysine incorporation and actinomycin D-sensitive uridine incorporation, indexes of protein and RNA synthesis, respectively. Incorporation of both lysine and uridine was maximal 12 h after the addition of putrescine, whereas thymidine incorporation was still increasing at 24 h, the longest time point examined. These data suggest that putrescine synthesis and/or transport during mucosal proliferation is directly involved in the stimulation of epithelial DNA, RNA, and protein synthesis.

Animals↗

PubMed1986

Hormonal regulation of postprandial induction of gastrointestinal ornithine decarboxylase activity.

The growth of gastrointestinal mucosa can be related to ingestion and digestion of diet, with fasting producing mucosal hypoplasia and hyperphagia producing mucosal hyperplasia. Experiments were designed to determine whether induction of polyamine metabolism following ingestion of a meal was related to mucosal growth. Activity of the enzyme ornithine decarboxylase (ODC) in both jejunum and ileum but not in duodenum was dependent on the presence of food in the gut; ODC activity was more than 200-fold greater in mucosa of fed rats than in fasted rats. Inhibition of ODC with difluoromethylornithine lead to mucosal atrophy in ileum but not in duodenum. Refeeding of fasted rats resulted in significant induction of ODC in duodenal, ileal, and colonic, but not fundic, mucosa. In addition, two hormones, epidermal growth factor and glucagon, were effective inducers of ileal ODC activity. Direct evidence for hormonal involvement in the postprandial rise in mucosal ODC activity was provided by experiments in rats that had undergone ileal bypass surgery. After refeeding of fasted rats mucosal ODC activity was induced in both ileum left in continuity and in the bypassed segment. Refeeding of elemental diets demonstrated that ingestion of carbohydrate alone was sufficient for maximal enzyme induction. Mixed amino acids or glyceryl trioleate were no more effective inducers than nonnutritive solutions of cellulose or saccharin. These data demonstrate that hormones which are released during ingestion and digestion of a meal are the stimuli for induction of mucosal polyamine metabolism, suggesting that food-induced mucosal growth is hormonally mediated.

Animals↗

PubMed1985

Induction in vitro of a specific pattern of jejunal propulsive behavior by cholecystokinin.

Experiments were conducted in vitro to determine whether arterial infusion of the C-terminal octapeptide of cholecystokinin (CCK-8) into cat jejunal segments could elicit propulsive behavior when a segment must do hydrostatic work to expel fluid. Oral and aboral ends of jejunal segments, 17 cm in length, were connected to a propulsion evaluation system that imposed input-output conditions of constant capacitance and negligible resistance. Infusion of 1 X 10(-10) to 3 X 10(-9) M CCK-8 produced an initial simultaneous ejection of approximately equal volumes of fluid from both ends of the segments. This reduction in luminal volume lasted for approximately 4 min. Concurrent with this initial reduction in luminal volume were phasic ejections of fluid that often did not occur simultaneously or with equal magnitude from both ends of a segment. This pattern persisted throughout the period of CCK-8 infusion. Arterial infusion of atropine sulfate (10(-6) M) or hexamethonium sulfate (2.8 X 10(-7) M) prevented the induction of propulsive behavior by CCK-8. These results indicate that arterial infusion of CCK-8 can induce a specific pattern of jejunal propulsive behavior by triggering neural activity in the enteric nervous system and that CCK-8 may have a physiological role in regulating propulsion.

Animals↗

PubMed1985

Attenuation of trophic response to gastrin after inhibition of ornithine decarboxylase.

The mechanism by which the hormone gastrin induces growth of the gastrointestinal mucosa is unknown. Many hormones are inducers of ornithine decarboxylase (ODC), the rate-limiting enzyme in the synthesis of the polyamines putrescine, spermidine, and spermine. Although the exact biochemical function of the polyamines is not completely understood, they appear to be required for normal cell growth and differentiation. Rats were maintained on a liquid diet for 5 days and treated with difluoromethylornithine (DFMO, 200 mg/kg ip, 3 times/day), a selective, irreversible inhibitor of ODC, for 5 days. Half of these animals also received pentagastrin (250 mg/kg ip) during the final 3 days of the treatment schedule. DFMO alone had no effect on body weight or mucosal growth. Pentagastrin increased total RNA, DNA, and protein in the oxyntic gland and duodenal and colonic mucosa. Concurrent treatment with DFMO completely inhibited the trophic response to pentagastrin in the oxyntic gland area of the stomach and the duodenal mucosa. In contrast, DFMO was without effect on response of the colonic mucosa to pentagastrin. Pentagastrin treatment did not induce the mucosal ODC activity of the oxyntic gland area of the stomach, the duodenum, the ileum, or the proximal colon of either animals fasted for 48 h or animals maintained on a liquid diet. ODC activity was measured at 4, 8, 12, and 24 h after administration of hormone. These results suggest that, in at least the proximal bowel, the stimulation by gastrin of nucleic acid and protein synthesis requires intact polyamine synthesis but that gastrin itself does not induce ODC.

Adenosylmethionine Decarboxylase↗

PubMed1985

Ileal mucosal growth during intraluminal infusion of ethylamine or putrescine.

Either ethylamine or the diamine putrescine was infused at the rate of 1 mumol/h for 66 h into the ileal lumen of rats. Total mucosal RNA, DNA, and protein content was greater in amine-treated rats than in rats receiving 0.9% NaCl. Growth was greatest in the mucosa surrounding the tip of the infusion catheter but was also observed 9 cm proximal and distal to the catheter tip. Infusion of these amines induced the activity of the enzymes ornithine and S-adenosylmethionine decarboxylase. Ornithine decarboxylase activity was increased 2- and 6-fold and S-adenosylmethionine decarboxylase activity 10- and 5-fold by putrescine and ethylamine, respectively. Induction of the polyamine biosynthetic enzymes was not accompanied by increases in the tissue content of polyamines. Putrescine, spermidine, and spermine content of the ileal mucosa surrounding the catheter tip was the same in 0.9% NaCl-, ethylamine-, and putrescine-treated animals. Finally, ethylamine was without effect on serum gastrin concentration in these experiments. The results suggest that amines regulate mucosal growth and may do so by modulating the activity of the enzymes involved in the synthesis of the polyamines.

Adenosylmethionine Decarboxylase↗

PubMed1984

Ontogeny of gastric mucosal muscarinic receptor and sensitivity to carbachol.

Development of the muscarinic cholinergic receptor and sensitivity of oxyntic gland mucosa to a muscarinic agonist were studied in rats of various ages. The gastric lumen of the fetal rat at the 20th day of gestation contained a statistically significant amount of basal pepsin, which increased log linearly over the first 30 days of life. Carbachol was effective in stimulating the secretion of pepsin as early as 12 h after birth. Basal acid could be measured in the gastric lumen 12 h after birth. The secretion of basal acid increased log linearly over the first 30 days of life. Carbachol was an effective secretagogue even in the fetal rat. The density of the muscarinic cholinergic receptor of the adult rat oxyntic gland mucosa was 99.3 fmol/mg protein with an apparent equilibrium dissociation constant for quinuclidinyl benzilate of 0.40 nM. The receptor was well developed even in the fetal rat, which bound 79.6 fmol/mg protein with an apparent equilibrium dissociation constant of 0.26 nM. Except for immediately after birth, receptor density was maintained between 70 and 90% of the adult level over the first 30 days of life. These results suggest that cholinergic regulation of gastric acid and pepsin secretion is probably functional by either late gestation or at least immediately after birth.

Aging↗

PubMed1984

Polyamines in the response to intestinal obstruction.

The gastrointestinal mucosa immediately proximal to an intestinal obstruction becomes hyperplastic. Since mucosa that is distal to an obstruction atrophies, it appears that the adaptational response to obstruction is regulated by local factors. The hypothesis tested in these studies is that increased polyamine metabolism in the gut proximal to an obstruction is a required local event in the hyperplastic process. Ligation of either rat ileum or colon resulted within 66 h in a doubling of total RNA, DNA, and protein content in the 2 cm of mucosa immediately proximal to the tie. The trophic response was accompanied by an increase in primary amine content of the intestinal chyme in the segment of gut under investigation. These amines were not removed from intestinal chyme by 24 h of lyophilization, suggesting that the more volatile short-carbon-chain aliphatic amines were of limited importance. Subsequent studies focused on polyamine metabolism. Ornithine decarboxylase (ODC) activity was increased in the mucosa proximal to obstruction. In the ileum, ODC activity was increased 10-fold over control values and in the colon about 2-fold. Increased ODC activity was accompanied by corresponding increases in mucosal polyamine content. Finally, treatment of rats with difluoromethylornithine, a selective, irreversible inhibitor of ODC, partially prevented the trophic response to intestinal obstruction.

Animals↗

PubMed1984

Influence of vagotomy on mucosal protection against alcohol-induced gastric damage in the rat.

We examined the role of the vagus nerve in mediating the protective effects of subcutaneous 16,16-dimethyl prostaglandin E2 (16,16-dmPGE2) or of the mild irritant 30% ethanol (topically) against gastric mucosal injury induced by concentrated solutions of ethanol. Anesthetized rats underwent either truncal vagotomy or sham truncal vagotomy and were studied acutely or 7 days later. Under acute conditions, in rats with intact vagi, oral saline followed by 100% ethanol produced severe gastric hemorrhagic and necrotic lesions throughout the glandular gastric mucosa. Oral 30% ethanol or pretreatment with 16,16-dmPGE2 (5, 10, or 25 micrograms/kg) before giving oral saline significantly reduced the magnitude of injury when mucosa was subsequently exposed to 100% ethanol. In animals with truncal vagotomy, the protective effect of 16,16-dmPGE2 or 30% ethanol was not observed. Similar results were noted when animals were studied 7 days after sham or truncal vagotomy. In other studies, the ability of 16,16-dmPGE2 to prevent gastric injury induced by 50% ethanol or 80 mM aspirin in acid solution (160 mM HCl) with and without prior vagotomy was compared. Although 16,16-dmPGE2 (5 or 25 micrograms/kg) pretreatment significantly reduced the degree of gastric damage induced by both agents in the nonvagotomized state, and by aspirin under vagotomized conditions, only partial protection by 16,16-dmPGE2 against ethanol injury was observed in the vagotomized state. These results suggest that the mechanisms whereby prostaglandins mediate their protective effects against aspirin and ethanol may be different and that the vagus nerve influences the ability of 16,16-dmPGE2 and of the mild irritant 30% ethanol to prevent alcohol-induced gastric injury in the rat stomach.

16,16-Dimethylprostaglandin E2↗

PubMed1983

Contraction and [3H]QNB binding in collagenase isolated fundic smooth muscle cells.

Smooth muscle cells from the guinea pig gastric fundus were isolated by successive collagenase digestions. Tritiated quinuclidinyl benzilate [( 3H]QNB) was used to study the binding characteristics of the muscarinic cholinergic receptors on these cells. Each cell bound 8.3 X 10(-19) mol of QNB, and a concentration of QNB of 0.19 nM was required to label one-half of the binding sites. This suggests a concentration of about 500,000 muscarinic cholinergic receptors per smooth muscle cell. The muscarinic cholinergic receptor antagonists atropine and scopolamine inhibited QNB binding with a 50% inhibiting concentration (IC50) in the nanomolar range, whereas the agonists acetylcholine (ACh), oxotremorine, and carbamylcholine had IC50S in the micromolar range. Hill coefficients (nH) for antagonists approached unity, but agonists displayed fractional nH. Exposure of cells to cholinergic muscarinic agonists resulted in dose-dependent decreases in cell length. The concentration of agonist required to induce half-maximal contractions (ED50) was 8.3 X 10(-12) M for ACh and 6.3 X 10(-13) M for oxotremorine. Atropine (10(-9) M) decreased the sensitivity to ACh, increasing the ED50 for ACh-induced contractions to 1.2 X 10(-10) M. These results suggest the existence of muscarinic receptor heterogeneity for cholinergic agonists but not for antagonists.

Acetylcholine↗

PubMed1983

Muscarinic cholinergic receptors in the piebald mouse model for Hirschsprung's disease.

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.

Animals↗

PubMed1982

Distribution of (3H)QNB binding in dog gastric smooth muscle pre- and post vagotomy.

Tritiated quinuclidinyl benzilate [(3H) QNB] was used to characterize muscarinic cholinergic receptors in membrane fragments prepared from the circular smooth muscle of the dog stomach. In preliminary experiments the effect of protein, incubation time, temperature and pH on QNB binding were evaluated. Muscarinic cholinergic antagonists and agonists inhibited QNB binding in a concentration-dependent manner, but the nicotinic antagonist hexamethonium and adrenergic compounds were not effective in displacing QNB from binding sites. Scatchard plot analysis of binding data showed an asymmetric receptor distribution in the stomach. The cardia bound 425fmol of QNB/mg protein with a Kd of 0.05nM, the fundus 267fmol/mg protein with a Kd of 0.09nM and the antrum 147 fmol/mg protein with a Kd of 0.14nM. In a second series of experiments, binding of QNB was measured in dogs which had been vagotomized three weeks earlier. Vagotomy had no effect on the apparent Kd but disrupted the asymmetric receptor distribution seen in the normal dog such that the Bmax of the cardia fell to a value of 222fmol/mg protein.

Animals↗

PubMed1979

The effects of cholinergic agents on morphine-induced circling behavior in the intact mouse.

The effects of morphine on locomotor activity in mice was studied. It was shown that morphine in this species induces circling, a dose-dependent, stereotyped behavior. At high doses of morphine, mice engaged virtually exclusively in circling uninterrupted by other activities. The effect was modified by muscarinic agents. Blockade of muscarinic receptors with atropine potentiated circling while the muscarinic agonist, oxotremorine, attenuated it. The mixed muscarinic/nicotinic cholinergic agonist, physostigmine, had no effect on this behavior.

Animals↗