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Regulation of prostaglandin production in a rat intestinal epithelial cell line, IEC-18
April 1 9 9 5
EFFECT OF FEED I N T E R V A L A N D FEED T Y P E O N S P L A N C H N I C H A E M O D Y N A M I C S IN H U M A N P R E T E R M N E O N A T E S . A.J.P. L a n e * t , R.C. Coombs*, D.H. E v a n s - , R.J. L e v i n t Departments o f Neonatology* and Biomedical Science'~ University o f Sheffield and Department o f Medical Physics ~, University o f Leicester, England. As part o f a longitudinal study we have used pulsed Doppler ultrasound to examine the effect o f feed interval and feed type on the blood flow velocities in the coeliac axis (CA) and superior mesenteric artery (SMA) o f 37 babies (mean birthweight+s.e.rn. 1.5±0. l kgs, mean gestation +s.e.m. 3 l. 14-0.5 weeks) after ethical committee approval and informed parental consent had been obtained. Babies 'never fed' (n=13 babies, s=13 studies) had significantly higher C A peak systolic velocity (PSV, cm sec -1) (704-6 vs. 584-1 & 59~-2, p<0.05) and significantly lower S M A PSV (414-3 vs. 70~1 & 544-2, p
• REGULATION OF PROSTAGLANDIN PRODUCTION IN A RAT INTESTINAL EPITHELIAL CELL LINE, IEC-18. Louise E. LeDuc and Karen Seibert, Department of Pharmacology, Milton S. Hershey College of Medicine, Pennsylvania State University, Hershey, PA 17033 and Monsanto Co., St Louis, MO 63167. The ability of intestinal epithelial cells to regulate their own functions and to participate in inflammatory reactions is under debate. Prostanoid production by intestinal epithelia represents a potential autocrine regulatory pathway, but is poorly defined. In this study we asked whether enterocytes express cyclooxygenase (COX) which converts arachidonic acid (AA) to prostaglandins (PG), and if so, what factors regulate its activity. We used the nontransformed rat cell line IEC-18, derived from distal ileum. IEC-18 cells were subcultured weekly a t 1 : 2 0 and grown in Dulbecco's modified Eagle's medium supplemented with 5% fetal bovine serum. To determine basal COX activity, we incubated confluent monolayers with and without AA. The role of protein kinase C (PKC) in regulating COX activity was assessed using the PKC activator phorbol 12myristate,13-acetate (PMA). COX activity was determined by measuring the amount of 6-keto-PGFl~x produced after addition Of 5 pM AA. PMA stimulated IEC-18 COX activity in a concentration-dependent manner; maximal effects were seen with 10 nM PMA. Increased COX activity was detectable within 2 h, maximal at 4 - 8 h, and returned to basal levels by 24 h. Sphingosine and high concentrations of H8, which inhibit protein kinase C, blocked the PMA effect. Inhibition of protein transcription by cycloheximide also blocked PMA induction of COX activity. Based on the ability of PMA to stimulate and of corticosteroids to inhibit COX activity, we postulated that IEC-18 can express inducible CO×-2. Such increased expression was confirmed by identification of COX-2 mRNA by nuclease protection assays. Thus COX-2 activity is regulated by protein kinase C, results from increased levels of COX-2 mRNA and requires synthesis of new enzyme. Since normal gut mucosa expresses primarily (constitutive) COX-l, altered expression of one isozyme relative to the other may determine whether enterocyte prostanoid responses are primarily protective or pro-inflammatory.
Intestinal Disorders
CYTOKINE MECHANISMS AND INTERACTIONS IN CYCLOOXYGENASE REGULATION IN A RAT INTESTINAL EPITHELIAL CELL LINE, IEC-18. Louise E. LeDuc and Karen Seibert, Department of Pharmacology, Milton S. Hershey College of Medicine, Pennsylvania State University, Hershey, PA and Monsanto Co., St Louis, MO. Increased prostanoid synthesis by inflamed intestinal mucosa has been well-described, but little is known about the regulation of expression and activity of key enzymes of the cascade. We hypothesized that regulatory pathways for cyclooxygenase (COX)-2 expression might be activated by proinflammatory cytokines such as interleukin 11~ (IL-11~) and tumor necrosis factor (TNF(x). Using the normal, nontransformed rat enterocyte line IEC-18, we investigated the effects of prolonged treatment of monolayers with IL-1, TNF(x, and the protein kinase C (PKC) activator phorbol 12-myristate,13-acetate (PMA). COX activity was assessed by radioimmunoassay of 6-keto-PGFlc¢, the stable metabolite of prostacyclin, following addition of exogenous arachidonic acid (AA}. All three agents increased COX activity with time-and concentration-dependence. To determine whether PKC also mediated the effects of IL-1 and TNF, we downregulated PKC activity with PMA, 1 pM, for 24 h. Under these conditions cultures were unable to respond to further challenge by PMA. In marked contrast, PKC-downregulated cultures showed undiminished, or even increased, responses to IL-1 and TNF, indicating that these cytokines do not exert their effects through activation of PKC. Additional experiments ruled out a role for agonists of the sphingomyelin cycle, Using concentrations which individually produce maximal stimulation of COX activity, we compared the COX activity and COX-1 and COX-2 mRNA levels of cultures treated with each agent alone and in combination. Increases in COX activity were paralleled by increases in expression of COX-2 mRNA, while no COX-1 mRNA was detected. COX induction by both IL-1 and TNF was greatly potentiated by concomitant treatment of cultures with PMA. The effects of the two cytokines together were at best additive, suggesting that they may share a common pathway in the regulation of COX-2 expression. Thus at least two pathways for regulation of COX activity exist in enterocytes and may be modulated in inflammation.
The genetic mechanism of r a t i n t e s t i n a l angiotensin c o n v e r t i n g e n z y m e & d i p e p t i d y l a m i n o p e p t i d a s e IV induction by h i g h p r o l i n e d i e t D.H.Lee, S.H.Yim, J.J.Kim, B.C.Yoon, H.C.Jung, l.S.Song, C.Y.Kim. Dept. of Medicine and Liver Research Institute, Seoul National University, College of Medicine, Seoul, Korea It is known that the diet composed of specific substrate induces the increase of enzymatic activity of bmsh border membrance enzyme which is specific for the substrate, but the genetic mechanism of this adaptive regulation is currently poorly understood. ACE{Angiotensln converting enzyme) and DAP-iV(Dipeptidyl anlinopeptidase IV) are well known bttrsh Imrder membrane enzymes which have major role in the digestion of proline. Although high proline diet is known to induce the increase of enzynmtic activity of ACE and DAP-IV, the genetic mechanism of this phenomenon has not been recognized. AIMS : To clarify the genetic mechaniona of ACE & DAP-IV induction by high proline diet. Method : Total 20 wistar rats were divided into four groups with different diet. Control group was fed with normal diet (17% protein), sucrose group with high sucrose diet :(68% sucrose), protein gorup with high protein diet (50% casein), and proline group with high proline diet (50% gelatin). After 3 weeks the snmll intestine was removed mad divided into three exlual lengfll segments, from which homogenate, brush border membrane and total RNA were prepared. ACE & DAP-IV enzyme activity were estimated in both small intestine honmgenate and brush border membrane. The amount of ACE mRNA and DAP-IV mRNA were also estimated by northern analysis of RNA. Result : 1) ACE activity of homgenate ; There was significant increase of ACE activity in praline group(P<0.01) throughout all sites (proximal, middle, distal). 2) ACE activity of brush border membrane ; There was significant increase of ACE acivity in distal site(P<0.01) of gelatin group. 3) DAP-IV activity of homogenate ; There was significant increase of DAP-IV activity in middle(P<0.01) & distal(P<0.05) site of proline group. 4) DAP-IV activity of brush border membrane ; There was slginficant increase of DAP-IV activity in proline group(P<0.Ol) throughout all sites. 5) ACE mRNA amount ; Proline group showed increase of ACE mRNA in proximal site(P<0.05) & total mean value(P<0.05). 0) DAP-IV mRNA amount ; There was no statistically significant change in praline group. Conclusions : 1) High proline diet indoced the increase of enzymatic activity of ACE and DAP-IV. 2) Increase of ACE activity was associated with increase of ACE mRNA in small intestine, but increase of DAP-IV activity was not associated with DAP-IV mRNA. These results suggested that the induction of increased ACE activity by high praline diet is regulated on transcriptional level mad the induction of increased DAP-IV activity may be regualted on posttranscriptianal level.
A299
EFFECT OF FEED I N T E R V A L A N D FEED T Y P E O N S P L A N C H N I C H A E M O D Y N A M I C S IN H U M A N P R E T E R M N E O N A T E S . A.J.P. L a n e * t , R.C. Coombs*, D.H. E v a n s - , R.J. L e v i n t Departments o f Neonatology* and Biomedical Science'~ University o f Sheffield and Department o f Medical Physics ~, University o f Leicester, England. As part o f a longitudinal study we have used pulsed Doppler ultrasound to examine the effect o f feed interval and feed type on the blood flow velocities in the coeliac axis (CA) and superior mesenteric artery (SMA) o f 37 babies (mean birthweight+s.e.rn. 1.5±0. l kgs, mean gestation +s.e.m. 3 l. 14-0.5 weeks) after ethical committee approval and informed parental consent had been obtained. Babies 'never fed' (n=13 babies, s=13 studies) had significantly higher C A peak systolic velocity (PSV, cm sec -1) (704-6 vs. 584-1 & 59~-2, p<0.05) and significantly lower S M A PSV (414-3 vs. 70~1 & 544-2, p
• REGULATION OF PROSTAGLANDIN PRODUCTION IN A RAT INTESTINAL EPITHELIAL CELL LINE, IEC-18. Louise E. LeDuc and Karen Seibert, Department of Pharmacology, Milton S. Hershey College of Medicine, Pennsylvania State University, Hershey, PA 17033 and Monsanto Co., St Louis, MO 63167. The ability of intestinal epithelial cells to regulate their own functions and to participate in inflammatory reactions is under debate. Prostanoid production by intestinal epithelia represents a potential autocrine regulatory pathway, but is poorly defined. In this study we asked whether enterocytes express cyclooxygenase (COX) which converts arachidonic acid (AA) to prostaglandins (PG), and if so, what factors regulate its activity. We used the nontransformed rat cell line IEC-18, derived from distal ileum. IEC-18 cells were subcultured weekly a t 1 : 2 0 and grown in Dulbecco's modified Eagle's medium supplemented with 5% fetal bovine serum. To determine basal COX activity, we incubated confluent monolayers with and without AA. The role of protein kinase C (PKC) in regulating COX activity was assessed using the PKC activator phorbol 12myristate,13-acetate (PMA). COX activity was determined by measuring the amount of 6-keto-PGFl~x produced after addition Of 5 pM AA. PMA stimulated IEC-18 COX activity in a concentration-dependent manner; maximal effects were seen with 10 nM PMA. Increased COX activity was detectable within 2 h, maximal at 4 - 8 h, and returned to basal levels by 24 h. Sphingosine and high concentrations of H8, which inhibit protein kinase C, blocked the PMA effect. Inhibition of protein transcription by cycloheximide also blocked PMA induction of COX activity. Based on the ability of PMA to stimulate and of corticosteroids to inhibit COX activity, we postulated that IEC-18 can express inducible CO×-2. Such increased expression was confirmed by identification of COX-2 mRNA by nuclease protection assays. Thus COX-2 activity is regulated by protein kinase C, results from increased levels of COX-2 mRNA and requires synthesis of new enzyme. Since normal gut mucosa expresses primarily (constitutive) COX-l, altered expression of one isozyme relative to the other may determine whether enterocyte prostanoid responses are primarily protective or pro-inflammatory.
Intestinal Disorders
CYTOKINE MECHANISMS AND INTERACTIONS IN CYCLOOXYGENASE REGULATION IN A RAT INTESTINAL EPITHELIAL CELL LINE, IEC-18. Louise E. LeDuc and Karen Seibert, Department of Pharmacology, Milton S. Hershey College of Medicine, Pennsylvania State University, Hershey, PA and Monsanto Co., St Louis, MO. Increased prostanoid synthesis by inflamed intestinal mucosa has been well-described, but little is known about the regulation of expression and activity of key enzymes of the cascade. We hypothesized that regulatory pathways for cyclooxygenase (COX)-2 expression might be activated by proinflammatory cytokines such as interleukin 11~ (IL-11~) and tumor necrosis factor (TNF(x). Using the normal, nontransformed rat enterocyte line IEC-18, we investigated the effects of prolonged treatment of monolayers with IL-1, TNF(x, and the protein kinase C (PKC) activator phorbol 12-myristate,13-acetate (PMA). COX activity was assessed by radioimmunoassay of 6-keto-PGFlc¢, the stable metabolite of prostacyclin, following addition of exogenous arachidonic acid (AA}. All three agents increased COX activity with time-and concentration-dependence. To determine whether PKC also mediated the effects of IL-1 and TNF, we downregulated PKC activity with PMA, 1 pM, for 24 h. Under these conditions cultures were unable to respond to further challenge by PMA. In marked contrast, PKC-downregulated cultures showed undiminished, or even increased, responses to IL-1 and TNF, indicating that these cytokines do not exert their effects through activation of PKC. Additional experiments ruled out a role for agonists of the sphingomyelin cycle, Using concentrations which individually produce maximal stimulation of COX activity, we compared the COX activity and COX-1 and COX-2 mRNA levels of cultures treated with each agent alone and in combination. Increases in COX activity were paralleled by increases in expression of COX-2 mRNA, while no COX-1 mRNA was detected. COX induction by both IL-1 and TNF was greatly potentiated by concomitant treatment of cultures with PMA. The effects of the two cytokines together were at best additive, suggesting that they may share a common pathway in the regulation of COX-2 expression. Thus at least two pathways for regulation of COX activity exist in enterocytes and may be modulated in inflammation.
The genetic mechanism of r a t i n t e s t i n a l angiotensin c o n v e r t i n g e n z y m e & d i p e p t i d y l a m i n o p e p t i d a s e IV induction by h i g h p r o l i n e d i e t D.H.Lee, S.H.Yim, J.J.Kim, B.C.Yoon, H.C.Jung, l.S.Song, C.Y.Kim. Dept. of Medicine and Liver Research Institute, Seoul National University, College of Medicine, Seoul, Korea It is known that the diet composed of specific substrate induces the increase of enzymatic activity of bmsh border membrance enzyme which is specific for the substrate, but the genetic mechanism of this adaptive regulation is currently poorly understood. ACE{Angiotensln converting enzyme) and DAP-iV(Dipeptidyl anlinopeptidase IV) are well known bttrsh Imrder membrane enzymes which have major role in the digestion of proline. Although high proline diet is known to induce the increase of enzynmtic activity of ACE and DAP-IV, the genetic mechanism of this phenomenon has not been recognized. AIMS : To clarify the genetic mechaniona of ACE & DAP-IV induction by high proline diet. Method : Total 20 wistar rats were divided into four groups with different diet. Control group was fed with normal diet (17% protein), sucrose group with high sucrose diet :(68% sucrose), protein gorup with high protein diet (50% casein), and proline group with high proline diet (50% gelatin). After 3 weeks the snmll intestine was removed mad divided into three exlual lengfll segments, from which homogenate, brush border membrane and total RNA were prepared. ACE & DAP-IV enzyme activity were estimated in both small intestine honmgenate and brush border membrane. The amount of ACE mRNA and DAP-IV mRNA were also estimated by northern analysis of RNA. Result : 1) ACE activity of homgenate ; There was significant increase of ACE activity in praline group(P<0.01) throughout all sites (proximal, middle, distal). 2) ACE activity of brush border membrane ; There was significant increase of ACE acivity in distal site(P<0.01) of gelatin group. 3) DAP-IV activity of homogenate ; There was significant increase of DAP-IV activity in middle(P<0.01) & distal(P<0.05) site of proline group. 4) DAP-IV activity of brush border membrane ; There was slginficant increase of DAP-IV activity in proline group(P<0.Ol) throughout all sites. 5) ACE mRNA amount ; Proline group showed increase of ACE mRNA in proximal site(P<0.05) & total mean value(P<0.05). 0) DAP-IV mRNA amount ; There was no statistically significant change in praline group. Conclusions : 1) High proline diet indoced the increase of enzymatic activity of ACE and DAP-IV. 2) Increase of ACE activity was associated with increase of ACE mRNA in small intestine, but increase of DAP-IV activity was not associated with DAP-IV mRNA. These results suggested that the induction of increased ACE activity by high praline diet is regulated on transcriptional level mad the induction of increased DAP-IV activity may be regualted on posttranscriptianal level.
A299