Biomedical subjects

E R Seidel

Publications and source records attributed to E R Seidel.

At least 19 recordsLinked to original sources

PubMed2006

Analysis of the interactions of Nrf-2, PMF-1, and CSN-7 with the 5'-flanking sequence of the mouse 4E-BP1 gene.

Nuclear factor erythroid 2-related factor 2 (Nrf-2) binds to a specific polyamine responsive element (PRE) in the promoter region of the spermidine-spermine acetyltransferase (SSAT) gene, a key component of the polyamine catabolic pathway. Regulation of SSAT gene transcription requires the additional interaction of Nrf-2 with polyamine modulated factor 1 (PMF-1). Likewise, transcription of the eukaryotic initiation factor 4E binding protein 1 (4E-BP1) gene is regulated in a polyamine-dependent manner, but the actual mechanism has not previously been determined. Analysis of the 5'-flanking sequence of the murine 4E-BP1 gene indicated the presence of several potential PRE sites, which might be involved in regulating its transcription. Our goal in this research was to determine potential interactions between Nrf-2, PMF-1, the human homologue of the Arabidopsis signalosome complex (CSN-7), and these potential PRE sites. Four PCR fragments containing regions with considerable homology (78%) to the human PRE were generated from the 5'-flanking sequence of the mouse 4E-BP1 gene and the fragments were used in electrophoretic gel mobility shift and supershift assays. Purified Nrf-2 interacted with all four of these fragments, and similar gel shifts were observed with both cytoplasmic and nuclear fractions of NIH-3T3 cells. However, polyamine depletion with difluoromethylornithine (DFMO) eliminated the gel shift. Supershift assays indicated that the shift was due to the binding of Nrf-2, and the binding was competitive with a known Nrf-2 binding sequence. Purified PMF-1 did not bind any of the PCR fragments alone, but when added with Nrf-2, decreased the magnitude of the gel shift for one of the fragments (PRE located at -2060 relative to the transcription start site). CSN-7 did not interact with the sequences, nor did it inhibit protein/DNA interaction. These data indicate a possible mechanism by which polyamines enhance the binding of a Nrf-2/PMF-1 complex to the 5'-flanking region of the 4E-BP1 gene. Since polyamines increase expression of the 4E-BP1 gene, it seems likely that formation of this complex is involved in its transcriptional regulation.

5' Flanking Region↗

PubMed2005

Polyamines are absorbed through a y+ amino acid carrier in rat intestinal epithelial cells.

Due to the similarity in transport characteristics of polyamines and the y+ basic amino acid system, we hypothesized that both substrates could be moving through a common carrier site. Competitive and cross inhibition experiments in intestinal epithelial cells revealed the possibility of a common transport site. N-ethylmalemide (NEM) inhibited both lysine and putrescine transport, confirming that both were carried by a y+ transporter. Overexpressing the y+ transporter CAT-1 in a polyamine transport-deficient cell line, CHO-MG, did not reconstitute polyamine-transport. Thus, polyamines are not traveling through CAT-1. To determine if lysine is carried by a polyamine transport site, an antizyme-overexpressing cell line was used. Antizyme overexpression decreased polyamine uptake by 50%; in contrast, lysine transport was unaffected. Therefore, lysine is not traveling through a polyamine transport site. It appears that polyamines and lysine are likely traveling through a common unknown y+ transport site.

Absorption↗

PubMed2004

Polyamines regulate eukaryotic initiation factor 4E-binding protein 1 gene transcription.

Difluoromethylornithine-induced polyamine depletion produced a significant fall in the rate of 4E-BP1 gene transcription in IEC-6 cells, without a change in stability of the 4E-BP1 message. The effect was reversed by the addition of exogenous putrescine. Decreased 4E-BP1 gene transcription produced a concomitant fall in steady-state concentration of the 4E-BP1 protein. Segments of the 4E-BP1 gene 5' flanking sequence were inserted into a GFP reporter construct. While all the segments containing the first 500 nucleotides 5' to exon 1 were capable of driving GFP expression, two regions (between -2465 and -1965, and between -896 and 511) did so in a polyamine-dependent manner. Steady-state concentration of ornithine decarboxylase (ODC), the first enzyme in the polyamine biosynthetic pathway, was increased in response to polyamine depletion. These data provide a mechanism by which polyamines affect transcription of the 4E-BP1 gene, which in turn affect translation of ODC and perhaps other cap-dependent proteins.

Adaptor Proteins, Signal Transducing↗

PubMed2002

Antizyme mRNA distribution and regulation in rat small intestinal enterocytes.

The protein ornithine decarboxylase antizyme (AZ) is inhibitory to both polyamine transport and synthesis. Experiments were performed to examine the distribution and regulation of AZ mRNA in cells of the small intestinal epithelium, a tissue exposed to high concentrations of extracellular polyamines and high levels of ornithine decarboxylase (ODC) activity. AZ mRNA was expressed in acutely isolated epithelial cells of rat jejunum and ileum; expression was higher in proximal than distal small intestine. In cells isolated from jejunal crypt-villus axis, AZ was expressed to high levels in cells from the small intestinal crypts but the message fell to near undetectable levels in cells of the villus tip. Western blot analysis demonstrated that distribution of AZ protein followed the distribution of AZ message. The distribution of ornithine decarboxylase activity along the crypt-villus axis was also determined. ODC activity and ODC protein were higher in cells from the upper villus than in cells isolated from the crypt. The intestinal lumen contains extremely high concentrations of free polyamines. The effect of depletion of endogenous polyamines or the addition of exogenous polyamines on AZ mRNA was evaluated in IEC-6 cells. Cells were depleted of intracellular polyamines by 72 hr of incubation in difluoromethylornithine. The fall in intracellular polyamine content was accompanied by a corresponding fivefold fall in AZ mRNA. When polyamine-depleted cells were treated with putrescine, the level of the AZ mRNA transcript was increased ninefold. These data demonstrate the expression of AZ gene in the longitudinal and crypt-villus axes of rat small intestine and show that AZ gene transcription is modulated by polyamines, an effect which may be involved in product suppression of polyamine synthesis.

Animals↗

PubMed2001

Peroxynitrite inhibits the activity of ornithine decarboxylase.

Polyamines are required during cell proliferation, whereas NO has anti-proliferative properties. Ornithine decarboxylase (ODC) is a critical enzyme for the synthesis of polyamines. We tested the hypothesis that the modification of ODC by peroxynitrite (OONO-), a short-lived free radical formed from NO and superoxide produces a fall in ODC activity, and therefore polyamine synthesis and cell proliferation. The treatment of a rat recombinant ODC (rODC) with OONO- resulted in a dose-dependent inhibition of rODC activity with an IC50 of approximately 100 microM. A Western blot employing a specific antibody to nitrotyrosine revealed a dose-dependent nitration of rODC tyrosine residues. When intact IEC-6 cells were treated with ONOO-, ODC activity decreased by 49%. These data suggest a correlation between ODC activity and nitration, and a possible mechanism by which NO synthesis may modulate polyamine synthesis.

Animals↗

PubMed1997

Inhibition by rapamycin of ornithine decarboxylase and epithelial cell proliferation in intestinal IEC-6 cells in culture.

1. Induction of the enzyme ornithine decarboxylase (ODC) appears to be controlled primarily at the level of ODC mRNA translation. The immunosuppressant drug, rapamycin, blocked the induction of ODC in response to serum by roughly 50% but was without effect on transport of putrescine into the intracellular space. The effect on ODC was specific for the intracellular signalling pathway leading to activation of p70S6k, as the immunosuppressant FK 506 was without effect on ODC activity. 2. Exposure of IEC-6 duodenal epithelial cells to rapamycin inhibited cellular proliferation. The effect of rapamycin was cytostatic in that removal of the immunosuppressant from the medium resulted in renewed cell division. Conversely, addition of exogenous putrescine, the product of the ODC catalysed reaction, was unable to reverse the cytostatic effects of rapamycin. 3. At a concentration of 10 nM, rapamycin inhibited the induction of ODC by 50%, a level of inhibition which could not be enhanced by exposure cells to 1000 nM rapamycin. This observation suggests that other intracellular signalling pathways, in addition to the p70S6k cascade, might be involved in regulation of translation of ODC mRNA or that rapamycin does not completely inhibit p70S6k.

Cell Division↗

PubMed1996

Insulin-like growth factor I in suckling rat gastric contents.

The small intestinal mucosa of the neonatal rat expresses primarily lactase activity until just prior to weaning when lactase falls to low levels and a full complement of adult digestive enzymes appears. Insulin-like growth factor 1 (IGF-I) is a normal component of maternal milk of humans and experimental animals. Experiments were performed to examine the concentrations of IGF-I in dam milk and the gastric content of suckling pups. Lactase activity in 1-day-old neonates was 0.66 micromol glucose formed/mg protein/hr (unit) and fell progressively until day 25, whereas sucrase activity at day 1 postpartum was 0.07 units and rose progressively to 0.21 units at day 25. The IGF-I content of dam milk was measured at 1, 5, 10, 15, 18, and 20 days postpartum by radioreceptor assay (RRA). Milk contained 1.02 pmol IGF-I/ml milk at one day postpartum, peaked at day 18 with 5.08 pmol IGF-I/ml, and fell to 2.31 pmol/ml at day 20. By day 25, dams were dry. The IGF-I content of the neonate gastric lumen was also measured by RRA. At day 1 the gastric lumen contained 2.63 pmol IGF-I/ml of luminal contents, fell to 1.06 pmol IGF-I/ml at day 5, and then rose again to peak at 3.37 pmol/ml at day 15 just prior to weaning. Two days after weaning, the level of luminal IGF-I had fallen to 1.15 pmol/ml. These data demonstrate the concentration of IGF-I in maternal milk is reflected in the concentration of the peptide in gastric contents of suckling pups and that the concentration in the gastric lumen may be high enough to affect epithelial cell proliferation and differentiation.

Age Factors↗

PubMed1995

Cell spreading and the regulation of ornithine decarboxylase.

The aim of this study was to investigate the effect of cell spreading on the induction of ornithine decarboxylase and the rate of putrescine uptake in anchorage-dependent and anchorage-independent cells. Plating non-transformed IEC-6 epithelial cells at high versus low cell density restricted cell spreading from 900 microns 2 to approximately 140 microns 2, blunted the transient induction of ornithine decarboxylase activity from 202 to 32 pmol 14CO2/mg protein per hour and reduced the rate of [14C] putrescine uptake from 46 to 23 pmol/10(5) cells per hour. The mean spreading area of the cell population was controlled by coating tissue culture dishes with the nonadhesive polymer, polyHEMA. Ornithine decarboxylase activity and putrescine uptake correlated with cell spreading with minimal spreading (263 microns 2) corresponding to an 83% decrease in ornithine decarboxylase activity and 51% decrease in the rate of putrescine uptake. Adding the RGD peptide, Gly-Arg-Gly-Glu-Ser-Pro to the medium of sparsely plated cells resulted in rapid reductions in cell spreading concomitant with dose-dependent decreases in ornithine decarboxylase activity and putrescine uptake. Finally, minimizing cell spreading by depriving cells of substratum contact completely abolished serum-induced increases in ornithine decarboxylase and reduced the rate of putrescine uptake by 47%. In contrast to IEC-6 cells, ornithine decarboxylase of neoplastic HTC-116 cells was constitutively expressed with basal and stimulated activity (193 and 982 pmol 14CO2/mg protein per hour, respectively) completely independent of cell adhesion. Putrescine uptake, however, was abolished in the absence of cell adhesion. These data suggest that the induction of ornithine decarboxylase activity and the rate of putrescine uptake correlate with spreading of anchorage-dependent IEC-6 cells and that ornithine decarboxylase activity but not putrescine uptake, appears to be independent of spreading of neoplastic HTC-116 cells.

Cell Adhesion↗

PubMed1995

Vascular endothelial cell proliferation: regulation of cellular polyamines.

OBJECTIVE: The aims were to investigate the requirement for and regulation of cellular polyamines during vascular endothelial cell proliferation. METHODS: Proliferation of cultured bovine pulmonary artery endothelial cells was determined after cellular polyamine depletion. In addition, polyamine synthesis and uptake mechanisms were examined in the presence and absence of trophic stimuli and density dependent inhibition of cell proliferation. RESULTS: Serum stimulated, subconfluent cells ceased cell division following the inhibition of ornithine decarboxylase (ODC), a rate limiting enzyme for polyamine biosynthesis. The addition of 10 microM putrescine, the product of the enzyme reaction catalysed by ODC, completely reversed the inhibition of cell growth. Serum deprivation reduced cytosolic ODC activity to near non-detectable levels. Readdition of 10% fetal bovine serum (FBS) resulted in transient increases in ODC activity which preceded DNA synthesis and mitosis. Basic fibroblast growth factor also stimulated ODC activity in a dose dependent manner with levels approximating the maximum obtainable with FBS. In contrast, platelet derived growth factor and epidermal growth factor did not stimulate ODC activity. Finally, mitogenic stimuli did not induce ODC activity in density arrested cells. The uptake of radiolabelled putrescine from the cell medium was time dependent and saturable. Kinetic studies from dividing cells revealed a single transport component for putrescine uptake [maximum rate of uptake (Vmax) = 11.2(SEM 2.0) pmol.10(5) cells-1.h-1; Michaelis constant (Km) = 1.1(0.3) microM]. Putrescine uptake by density arrested cells was characterised by a 57% decrease in Vmax with no change in Km. Readdition of FBS to serum deprived subconfluent cells, in the presence of ODC inhibitors, resulted in a rapid increase in the rate of putrescine uptake with Vmax increasing by 533% over FBS alone by 48 h. DISCUSSION: These data suggest that polyamines are essential for endothelial cell proliferation and that synthesis and uptake mechanisms are regulated according to cell growth.

Animals↗

PubMed1994

Insulin-like growth factor I in human gastrointestinal exocrine secretions.

Insulin-like growth factor I (IGF-I) is the mediator of growth hormone dependent growth. The peptide has been identified by radioimmunoassay in a number of human exocrine secretions of the gastrointestinal tract including (nM): saliva 0.9, gastric juice 3.5, jejunal chyme 24.6, pancreatic juice 3.6, and bile 0.9. The identification of IGF-I in pancreatic juice was confirmed by HPLC. The intravenous injection of 1 unit/kg secretin increased pancreatic juice IGF-I content from a basal level of roughly 4 nM to nearly 20 nM. Conversely, the IGF-I content of bile was unaffected by secretin. Radioligand blot analysis of samples of gastric juice, jejunal chyme and pancreatic juice demonstrated that these fluids contained no IGF binding proteins. Thus, unlike IGF-I in serum, IGF-I secreted into the gastrointestinal lumen is not bound to insulin-like growth factor I binding proteins. Since the growth factor is not protein bound, its concentration in the gut lumen may be high enough to exert biological activity.

Adult↗

PubMed1993

Paraquat uptake in the cultured gastrointestinal epithelial cell line, IEC-6.

Although the major target organ of paraquat toxicity is the lung, the primary route of absorption of the herbicide is across the gastrointestinal epithelium. Thus, uptake of paraquat was investigated in the rat duodenal crypt cell line, IEC-6. The herbicide was toxic; as measured by cell number, the LD50 was 75 microM. Using 2 microM [14C]paraquat as tracer, uptake of the herbicide was found to be slow but linear over 24 hr at 37 degrees C. No accumulation was observed at 4 degrees C. Inhibition studies showed paraquat inhibited both putrescine and spermidine uptake after only a 15-min incubation. The Km for putrescine was increased when incubated in the presence of paraquat whereas Vmax was not changed, suggesting a competitive mode of inhibition. In the reverse situation, putrescine also inhibited paraquat uptake (IC50 10 microM). Paraquat uptake was reduced in a dose-dependent manner by the putative calmodulin antagonist W-7, but was not affected by KN-62, a Ca2+/calmodulin kinase II specific inhibitor. These results provide evidence that paraquat uptake occurs through the polyamine transport system in IEC-6 cells. Furthermore, this process is modulated by intracellular events involving a major signaling protein, Ca2+/calmodulin.

1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗

PubMed1993

Characterization of univectorial polyamine transport in duodenal crypt cell line.

High levels of polyamines have been identified in the lumen of the intestines. Luminal polyamines are involved in normal mucosal growth and may be the primary source of extracellular polyamines for tumors grown in animals undergoing polyamine antimetabolite therapy. The vectorial movement of polyamines across an in vitro model of the gut was studied in epithelial cells grown in culture. IEC-6 cells were plated on either plastic or raised inserts. Cells grown on plastic were employed to define the kinetic constants for putrescine and spermidine uptake. Eadie-Hofstee plot analysis of putrescine uptake was characteristic of a single class of transporter with a Michaelis constant (Km) of 4.85 +/- 0.57 microM and a maximal velocity (Vmax) of 627 +/- 85 pmol x 15 min-1 x 10(6) cells-1. The plot for spermidine uptake was curvilinear and representative of the interaction of spermidine with two sites: Km 1 and 2 are 0.26 +/- 0.13 and 2.1 +/- 0.77 microM; Vmax 1 and 2 are 177 +/- 50 and 429.5 +/- 70 pmol x 15 min-1 x 10(6) cells-1, respectively. Calmodulin antagonism blocked the uptake of putrescine and the low-affinity but not high-affinity spermidine uptake system. Seven-day postconfluent cells grown on plastic inserts were used to study the vectorial movement of polyamines in a polarized epithelium. The apical membrane domain expressed two sites with similar kinetic constants to those observed when cells were grown on plastic. In contrast, however, the basolateral membrane did not transport polyamines. Spermidine uptake through this membrane was only a fraction of that in the apical membrane and was completely nonspecific.(ABSTRACT TRUNCATED AT 250 WORDS)

1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗

PubMed1992

Ornithine decarboxylase activity during gastric ulcer healing in dogs.

Ornithine decarboxylase (ODC) activity has been associated with mucosal growth and injury, yet, little information is available on ODC activity during gastric ulcer healing. We measured ODC activity in the ulcer base submucosa and the surrounding mucosa at 1 cm and 2 cm and assessed ulcer surface healing and a histologic score in experimentally induced ulcers (Quinton ulcer-maker) at 0 and 5 hr and at one, two, three, four, and seven days. A total of 26 dogs were studied, eight of which received 2% difluoromethylornithine (DFMO, a specific inhibitor of ODC) in drinking water. Ulcer healing was assessed by digitizing initial (plug size), and final ulcer surface area and was expressed as percent ulcer surface reduction. A histologic score was assessed by two independent pathologists unaware of the treatment. ODC induction was observed in the submucosa of the ulcer base but not in the surrounding mucosa. The baseline submucosal ODC activity was measured at 0.2 +/- 0.1 pmol (14CO2)/mg protein/hr, and at one day the ODC activity increased to 4.0 +/- 0.7, at three days to 15.2 +/- 5.5, and at seven days to 2.6 +/- 1.0 (P less than 0.001). DFMO treatment delayed GU healing significantly up to three days, but no difference was noted at seven days. The assessed histologic parameters did not correlate with ODC activity, and DFMO treatment did not alter the histologic score. These data suggest that polyamine biosynthesis occurs in the ulcer base submucosa during the first seven days of experimentally placed gastric ulcers.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1992

Ca2+/calmodulin regulation of putrescine uptake in cultured gastrointestinal epithelial cells.

Regulation of putrescine uptake in a small intestinal crypt cell line, IEC-6 cells, was examined. Uptake of [14C]putrescine was measured throughout a normal growth curve and was found to be inversely related to growth. Kinetic analysis at low and high cell density revealed the inhibition of uptake in confluent cells was due to a five-fold reduction in Vmax of uptake, 199.5 vs. 43.1 pmol.10(5) cells-1.h-1, respectively. Three gastrointestinal hormones, gastrin, secretin, and cholecystokinin, produced partial inhibition of [14C]putrescine uptake. Conversely, treatment of quiescent cells with 5% fetal bovine serum to stimulate growth did not affect uptake. Influence of putrescine uptake on free ionized intracellular Ca2+ ([Ca2+]i) was measured by microspectrofluorometry using the Ca(2+)-sensitive fluoroprobe fura-2. Basal [Ca2+]i was calculated to be 112 nM and increased rapidly to 313 nM upon addition of 10 microM putrescine. Preventing the rise in [Ca2+]i using an intracellular Ca2+ buffer, 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid acetoxymethyl ester, decreased [14C]putrescine uptake to 29.5 +/- 5.3% of control values. 45Ca2+ flux experiments and measurement of transport in 0 Ca2+ and 0.5 mM EDTA suggested an intracellular source of calcium was mobilized during putrescine uptake. Finally, use of the putative calmodulin antagonist N-(6-aminohexyl)-5-chloro-l-naphthalenesulfonamide caused a dose-dependent inhibition of [14C]putrescine uptake with 50% inhibitory concentration of approximately 7 microM. These data suggest that putrescine uptake in IEC-6 cells may be regulated by a Ca2+/calmodulin-dependent mechanism.

Animals↗

PubMed1992

Trophic action of local intraileal infusion of insulin-like growth factor I: polyamine dependence.

Experiments were performed to determine potential growth-promoting effects of human recombinant insulin-like growth factor I (hrIGF-I) in the gastrointestinal tract. IGF-I and IGF-II, but not insulin, were potent (half-maximal effective concentration 0.3 nM) and efficacious inducers of the growth-related enzyme ornithine decarboxylase (ODC) in the gut-derived cell line IEC-6. Maximal ODC induction was observed after treatment of cells with 10 nM IGF-I. In whole animal experiments, bolus intraileal injection of 10 nM hrIGF-I in anesthetized rats induced a 300% increase in ileal mucosal ODC activity, which was sensitive to inhibition with difluoromethylornithine (DFMO). Rats were implanted intraperitoneally with osmotic minipumps filled with 0.9% NaCl or 10 nM IGF-I that was delivered to the ileal lumen by a short Silastic catheter. Sixty-six hours of 1 microliter/h intraluminal IGF-I infusion produced an approximate doubling of mucosal wet weight (NaCl 50 mg vs. IGF-I 102 micrograms/2 cm mucosa) and total mucosal RNA, DNA, and protein content over that in rats that were infused with NaCl. Intraperitoneal treatment with 200 mg/kg DFMO three times per day had little effect on ileal mucosal mass, but completely inhibited the trophic response to IGF-I infusion. IGF-I infusion had no effect on body weight.

Animals↗

PubMed1992

Protein kinase C regulation of IEC-6 cell ornithine decarboxylase.

Experiments were performed to immunologically identify protein kinase C (PKC) in cultured IEC-6 cells. Polyclonal antibodies specific to PKC revealed an immunoreactive band of approximately 84 kDa in both cytosolic and solubilized particulate fractions. Treatment with phorbol 12-myristate 13-acetate (PMA; 10 nM x 60 min) increased the intensity of the 84-kDa band by 25% in the solubilized particulate fraction while decreasing it by 36% in the cytosolic fraction. Prolonged 24-h treatment with 300 nM PMA completely abolished the 84-kDa band in both fractions. Isoform-specific antisera demonstrated that alpha- and epsilon-isoforms of PKC were expressed in IEC-6 cells. Treatment of quiescent cultures with PMA induced a maximal 400% increase in ornithine decarboxylase (ODC) activity. Similarly, addition of exogenous phospholipase C (PLC) to quiescent cells stimulated ODC activity. Downregulation of PKC with 300 nM PMA x 24 h inhibited basal, serum, and PLC-stimulated ODC activity by 70%. Northern analysis revealed that PKC downregulation was correlated with a marked reduction in ODC mRNA levels, suggesting regulation of ODC enzyme at this level. Despite their ability to modulate ODC activity in quiescent cultures, neither PMA nor PLC induced [3H]thymidine incorporation at 24 h. Furthermore, downregulation of PKC did not attenuate thymidine incorporation. However, chronic PMA treatment caused the cells to contact-inhibit at a 30% lower cell density, 3.16 x 10(6) vs. 2.1 x 10(6) cells/35-mm plate, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)

Blotting, Western↗

PubMed1990

Multiple pathways for the regulation of ornithine decarboxylase in intestinal epithelial cells.

The regulation of ornithine decarboxylase (ODC) was examined in an intestinal epithelial crypt cell line (IEC-6). Addition of fetal bovine serum or growth factors to quiescent preconfluent cells resulted in a 20- to 30-fold increase in the specific activity of ODC, which was maximal at approximately 4 h. In contrast, ODC mRNA levels either did not change or increased only twofold over the time period examined. The increased enzymatic activity was blocked by cycloheximide, putrescine, and the calmodulin antagonist N-(6-aminohexyl)-5-chloro-1-napthalinesulfonamide (W-7). Cycloheximide alone increased mRNA levels and potentiated the induction in response to serum, suggesting that protein synthesis is not required for the increase in mRNA accumulation. In contrast to its effect on ODC activity, W-7 was without effect on the serum-stimulated increase in ODC or c-fos mRNA levels. Putrescine decreased ODC activity, but not mRNA content, in a dose-dependent manner with an IC50 between 0.1 and 1.0 microM. Also, serum stimulation resulted in a threefold increase in the stability of the enzyme in the presence of cycloheximide; this effect was blocked by pretreatment with W-7. Enzymatic activity was paralleled by ODC protein content as determined by [3H] difluoromethylornithine binding. Finally, the induction of enzyme activity was due entirely to an increase in Vmax as no detectable change in Km for ornithine was detected. These results suggest that ODC is regulated at multiple levels by independent signaling pathways in cultured intestinal epithelial cells. Increased levels of active ODC protein and enzymatic activity are sensitive to W-7 and putrescine.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1990

Gastrointestinal luminal polyamines: cellular accumulation and enterohepatic circulation.

The concentration of polyamines contained in the lumen of the gut was quantified. The duodenum and jejunum of the rat contained 2-3 mM putrescine and 1-2 mM cadaverine, whereas in the ileum and colon the concentration of these polyamines was significantly less. In addition, the concentrations of spermine and spermidine in the intestinal lumen were low to undetectable. Putrescine in the lumen of the gut was over 90% free with only 10% or less bound to protein. The activity of the enzymes responsible for the synthesis of polyamines was also measured. In contrast to concentration, enzyme activity was found to be high in the ileum, cecum, and colon and nonexistent in the duodenal and jejunal lumen. This suggested the potential for enterohepatic circulation of polyamines that were synthesized by the colonic microflora and transported to the proximal gut via the portal circulation and biliary tree. Indeed, when [14C]putrescine was instilled into the lumen of the gut, it was secreted in pancreaticobiliary secretions. Upper and lower jejunum and colon all supported enterohepatic circulation of polyamines, whereas it was absent in the ileum. Polyamine accumulation in IEC-6 cells grown under in vitro conditions was also measured. Putrescine was transported under time- and temperature-dependent but sodium-independent conditions. The transporter displayed little selectivity for the various polyamines and compounds with related structures but did not recognize amino acids. The Michaelis constant for putrescine accumulation was 1.26 x 10(-6) M with a maximal velocity of the enzyme reaction of 5,184 pmol putrescine.mg protein-1.h-1.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acids↗