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AE2a transcription is stimulated by phorbol ester
April 1998
Intestinal Disorders A359
isobutyrate decreased J mCI by 3, 4 and 1.3 laEqocm-2oh-1, respectively, as compared to a decrease of 0.1 laEq°cm-2oh-l for the gluconate control. These results indicate that cGMP (ST toxin)-induced CI- secretion, like cAMPinduced secretion, can be prevented and reversed in vitro by SCFAs. The similar degree and pattern of inhibition suggest that the mechanism of SCFA action in these secretory models may be similar. G1464 INTESTINAL ANTI-INFLAMMATORY ACTIVITY OF TYPE 4 PHOSPHODIESTERASE INHIBITORS (PDE4) IN RATS. E. Chevalier, M. Chovet and A. Langlois Jouveinal, Parke-Davis, Fresnes, France
Intestinal anti-inflammatory activity of Rolipram has recently been demonstrated (1). The aim of this study was to compare its activity with those of new PDE4 inhibitors on Lipopolysaccharide (LPS)-induced intestinal inflammation in rats. Methods: At t=0 min., LPS (E. Coli) was intravenously (40 mg/kg) administered to male Wistar rats (220-260g). At t=+2 hours, the number of diarrheic rats were counted and a distal jejunal segment was removed to measure macroscopic damage (0=no, l=light, 2=moderate and 3=severe) and tissue hemoglobin (Hb) content. Rolipram, RP73401 and CDP840 (3-30 mg/kg) were administered by oral route at t---24 and -3 hours. Results: LPS significantly increased both macroscopic damage (2.5 -+ 0.1 vs 0 ± 0; p<0.05) and Hb content (15.7 + 0.9 vs 8.5 ±0.3 mg Hb/g tissue; p<0.05) and induced diarrhea in 83% of the animals. After oral administration, Rolipram, RP73401 and CDP840 produced a significant doserelated inhibition of jejunal macroscopic damage and hemoglobin content. Only Rolipram decreased the number of diarrheic rats. The EDsovalues of PDE4 inhibitors are summarized in the following table: Treatment ROLIPRAM RP73401 CDP840
ED~o (confidence limits) mg/kg p.o. Macroscopic Hemoglobin content Diarrhea damage 6.5 (1.5-28.5) 7.1 (1.7-29.8) 19.7 (9.0-43) 7.0 (2.1-23.1) 2.4 (0.7-8.6) no effect 13.5 (7.2-25.1) 13.5 (5.3-34.4) no effect
;onclusion: RP73401 and CDP840, were equipotent to Rolipram for reducing LPS-induced jejunal inflammation in rats. However, only Rolipram presented an antidiarrheal activity. This divergence could be explained by their different actions on high affinity Rolipram binding site (HARBS) and catalytic activities. In conclusion, PDE4 inhibitors, active on lung inflammation (2), also reduce visceral inflammation. [1] Cardelus et al. Eur.J. Pharmacol, 229 (1996): 153-59. [2] Hollbrook et al. Br.J.Pharmacol, 118(5) (1996): 1192-1200. G1465 FATTY A C I D TRANSLOCASE (CD36) GENE EXPRESSION AND REGULATION IN TIlE RAT INTESTINE. M Chen, CM Harmon. University of Michigan, Ann Arbor, MI Background: The CD36 homologous rat Fatty Acid Translocase (FAT) is an 88-kDa transmembrane glycoprotein which has been implicated in long chain fatty acid (LCFA) membrane transport in adipocytes as well as oxidized LDL uptake in monocytes. The mechanism(s) of transmembrane permeation of long chain fatty acids (LCFA) through the enterocyte plasma membrane is not known. We hypothesize that FAT may play a role in LCFA uptake in the gut, and that FAT gene expression may be regulated during development and by one of its ligands, oleic acid. Methods: Total RNA was isolated from rat intestine or intestinal mucosa and analyzed by Northern blot utilizing the P32-1abeled rat FAT eDNA. To investigate the effect of LCFA on FAT mRNA expression, in vivo experiments were done in which the rat duodenum was perfused with 10% oleate for 1 hour, followed by intestinal mucosal RNA isolation and Northern blot analysis. FAT mRNA was quantified using scanning densitometry and mRNA for 18S as an internal control. Statistical analysis used a paired t test. Results: FAT mRNA was found differentially expressed along the gut axis, with highest levels of FAT mRNA noted in the jejunum followed by the duodenum and ileum, with low levels of expression in the stomach and colon. In addition, FAT gene expression in the jejunum was found to be developmentally regulated as the amount of FAT mRNA was found to increase in specimens evaluated from 1, 2, and 5 day old and adult rats. After intraduodenal infusion with 10% oleate for 1 hour, the levels of FAT mRNA in the duodenum and jejunum were significantly reduced by 61% and 65% respectively when compared with control (p < 0.05). The level of FAT mRNA in the ileum of oleate infused rats was increased by 120% compared with control, but this change was not statistically significant (p > 0.05). Conclusion: The mRNA levels of the putative fatty acid transport protein, Fatty Acid Translocase, are differentially expressed along the rat gut axis, with highest levels in the proximal small intestine, a distribution similar to the known location of LCFA absorption. FAT mRNA level also increase developmentally in the rat jejunum from newborn to adult animals. Finally, the long chain fatty acid, oleate, rapidly alters FAT mRNA levels in vivo,
strongly supporting a role for FAT in intestinal fatty acid uptake or physiologic fatty acid signaling. • G1466 AE2a AND AE2b EXPRESSIONS HAVE DISTINCT TISSUERELATED AND DEVELOPMENTAL REGULATIONS. Alice Chow, Rongping Zhang, and Tao Pang. Emory University School of Medicine, Atlanta VA Medical Center, Atlanta, GA 30322.
The single rat AE2 gene has several promoters (Wang et al. JBC 271:7835, 1996), which are predicted to generate distinct polypeptides, which include the full-length AE2a and the N-terminally truncated AE2b. To distinguish AE2a and AE2b, oligonucleotides corresponding to transcriptspecific exons were hybridized to 25-100~g of total RNA from various rat tissues. S1 nuclease assays detected AE2a in rat stomachs, proximal and distal small intestines, colons, kidneys and livers. AE2a was also present in isolated enterocytes. Previous report by Wang et al (JBC 271:7835, 1996) demonstrated that AE2b has a more restricted distribution. Our S1 nuclease studies indicate that AE2b transcripts are more abundant in rat stomachs and colons than in small intestines and livers. AE2a mRNA in small intestines of 14-day old rats was 2.8 fold of that in adults (p < .025). AE2b levels in stomachs, colons, and livers of 14-day old rats only amounted to 39%, 35%, and 11%, respectively, of levels in adult organs. AE2b mRNA level was low in small intestines of both age groups. An AE2b promoter construct was created by fusing the 2.0 kb fragment upstream of exon l b (derived from AE2C 1, a generous gift of GE Shull, Univ. Cincinnati) to the firefly luciferase reporter (pGL-Basic). Caco2 (colon adenocarcinoma) and AGS (gastric adenocarcinoma) cells were cotransfected with Renila luciferase reporter as internal controls. To compare transcriptional activities in Caco2 and AGS, firefly luciferase activity (after normalization to Renila luciferase activity) was expressed as a percentage of mean activity achieved in cells transfected with a reference vector (pGL-control driven by SV40 promoter/enhancer). Basal transcriptional activities of the AE2b construct was 6 fold higher in the stomach-derived AGS cells than in the enterocyte-like Caco2 cells (p < 0.01). Conclusions: (1) AE2a is widely distributed and may serve functions common to most cells; (2) AE2b transcripts are only substantially expressed in rat stomachs and colons, and may serve some tissue-specific functions; (3) the higher abundance of AE2b mRNA in stomachs is associated with higher basal transcriptional activities of AE2b regulatory sequences in AGS; (4) the previously reported developmental changes of AE2 is most likely a result of decreases in AE2a. AE2b appears to gain importance in adults as its mRNA increases in adult tissues which express AE2b. This study indicates that AE2a and AE2b have distinct tissue-related and developmental regulations. • G1467 AE2a TRANSCRIPTION IS STIMULATED BY PHORBOL ESTER. Alice Chow, Rongping Zhang, and Tao Peng. Emory University School of Medicine, Atlanta VA Medical Center, Atlanta, GA 30322.
AE2a encodes a CI:HCO 3 exchanger and its mRNA is widely expressed. We examined if phorbol 12-myristate 13-acetate (TPA) affects AE2a transcript level or activity of the AE2a promoter. Methods and Results: IEC-6 (rat intestinal epithelial cells) were seeded at 4.5x 104 cells/cm 2. Forty-eight hrs later, cultures were treated with serum free media with 0 ng/ml or 50 ng/ml TPA for 2 hrs prior to RNA extraction. AE2a mRNA levels were measured by S1 nuclease assays using an exonla probe. Mean AE2a mRNA level was 27.5% higher in TPA treated cultures (n=4, p < 0.05). AE2a/reporter constructs were obtained by fusing fragments of the rat AE2 gene upstream of exon la to the promoterless, enhancerless firefly luciferase plasmid (pGL-Basic). Caco2 (human colon adenocarcinoma) and AGS (human gastric adenocarcinoma) cells were used for transient transfections. Plasmids carrying the Renila luciferase (pRL-SV40) or chloramphenicolacetyltransferase (pCAT-SV40) reporters were cotransfected to provide internal controls. Caco2 and AGS cells were plated at 2.6x104/cm2 and 5.2x104/cm2, respectively. Twenty-four hours after transfection, media were replaced by sernm-free DMEM containing zero or 50 ng/ml TPA. Forty-eight hrs later, cell extracts were harvested for measurement of reporter activities. Firefly luciferase activities were normalized to either renila luciferase activities (Dual Luciferase System, Promega) or CAT expression (ELISA) from the same extracts. The construct (A4) containing the region from -244 to +47 flanking exonla conferred a 7.6 fold (p < 0.05) and a 5.0 fold (19< 0.01) TPA-induced increase in reporter activities in Caco2 and AGS, respectively. The additional 843 bp upstream sequence (A1 construct) did not increase TPA stimulation. Conclusions: TPA treatment led to an increase in AE2a mRNA level in intestinal cells. The -247 to +47 region has no recognizable TPA-responsive elements (TRE) but conferred response to TPA in both Caco2 and AGS. TPA may act through factors that interact with non-TRE sites to transactivate the AE2a promoter and lead to the observed increase of AE2a mRNA in treated cultures.
Intestinal Disorders A359
isobutyrate decreased J mCI by 3, 4 and 1.3 laEqocm-2oh-1, respectively, as compared to a decrease of 0.1 laEq°cm-2oh-l for the gluconate control. These results indicate that cGMP (ST toxin)-induced CI- secretion, like cAMPinduced secretion, can be prevented and reversed in vitro by SCFAs. The similar degree and pattern of inhibition suggest that the mechanism of SCFA action in these secretory models may be similar. G1464 INTESTINAL ANTI-INFLAMMATORY ACTIVITY OF TYPE 4 PHOSPHODIESTERASE INHIBITORS (PDE4) IN RATS. E. Chevalier, M. Chovet and A. Langlois Jouveinal, Parke-Davis, Fresnes, France
Intestinal anti-inflammatory activity of Rolipram has recently been demonstrated (1). The aim of this study was to compare its activity with those of new PDE4 inhibitors on Lipopolysaccharide (LPS)-induced intestinal inflammation in rats. Methods: At t=0 min., LPS (E. Coli) was intravenously (40 mg/kg) administered to male Wistar rats (220-260g). At t=+2 hours, the number of diarrheic rats were counted and a distal jejunal segment was removed to measure macroscopic damage (0=no, l=light, 2=moderate and 3=severe) and tissue hemoglobin (Hb) content. Rolipram, RP73401 and CDP840 (3-30 mg/kg) were administered by oral route at t---24 and -3 hours. Results: LPS significantly increased both macroscopic damage (2.5 -+ 0.1 vs 0 ± 0; p<0.05) and Hb content (15.7 + 0.9 vs 8.5 ±0.3 mg Hb/g tissue; p<0.05) and induced diarrhea in 83% of the animals. After oral administration, Rolipram, RP73401 and CDP840 produced a significant doserelated inhibition of jejunal macroscopic damage and hemoglobin content. Only Rolipram decreased the number of diarrheic rats. The EDsovalues of PDE4 inhibitors are summarized in the following table: Treatment ROLIPRAM RP73401 CDP840
ED~o (confidence limits) mg/kg p.o. Macroscopic Hemoglobin content Diarrhea damage 6.5 (1.5-28.5) 7.1 (1.7-29.8) 19.7 (9.0-43) 7.0 (2.1-23.1) 2.4 (0.7-8.6) no effect 13.5 (7.2-25.1) 13.5 (5.3-34.4) no effect
;onclusion: RP73401 and CDP840, were equipotent to Rolipram for reducing LPS-induced jejunal inflammation in rats. However, only Rolipram presented an antidiarrheal activity. This divergence could be explained by their different actions on high affinity Rolipram binding site (HARBS) and catalytic activities. In conclusion, PDE4 inhibitors, active on lung inflammation (2), also reduce visceral inflammation. [1] Cardelus et al. Eur.J. Pharmacol, 229 (1996): 153-59. [2] Hollbrook et al. Br.J.Pharmacol, 118(5) (1996): 1192-1200. G1465 FATTY A C I D TRANSLOCASE (CD36) GENE EXPRESSION AND REGULATION IN TIlE RAT INTESTINE. M Chen, CM Harmon. University of Michigan, Ann Arbor, MI Background: The CD36 homologous rat Fatty Acid Translocase (FAT) is an 88-kDa transmembrane glycoprotein which has been implicated in long chain fatty acid (LCFA) membrane transport in adipocytes as well as oxidized LDL uptake in monocytes. The mechanism(s) of transmembrane permeation of long chain fatty acids (LCFA) through the enterocyte plasma membrane is not known. We hypothesize that FAT may play a role in LCFA uptake in the gut, and that FAT gene expression may be regulated during development and by one of its ligands, oleic acid. Methods: Total RNA was isolated from rat intestine or intestinal mucosa and analyzed by Northern blot utilizing the P32-1abeled rat FAT eDNA. To investigate the effect of LCFA on FAT mRNA expression, in vivo experiments were done in which the rat duodenum was perfused with 10% oleate for 1 hour, followed by intestinal mucosal RNA isolation and Northern blot analysis. FAT mRNA was quantified using scanning densitometry and mRNA for 18S as an internal control. Statistical analysis used a paired t test. Results: FAT mRNA was found differentially expressed along the gut axis, with highest levels of FAT mRNA noted in the jejunum followed by the duodenum and ileum, with low levels of expression in the stomach and colon. In addition, FAT gene expression in the jejunum was found to be developmentally regulated as the amount of FAT mRNA was found to increase in specimens evaluated from 1, 2, and 5 day old and adult rats. After intraduodenal infusion with 10% oleate for 1 hour, the levels of FAT mRNA in the duodenum and jejunum were significantly reduced by 61% and 65% respectively when compared with control (p < 0.05). The level of FAT mRNA in the ileum of oleate infused rats was increased by 120% compared with control, but this change was not statistically significant (p > 0.05). Conclusion: The mRNA levels of the putative fatty acid transport protein, Fatty Acid Translocase, are differentially expressed along the rat gut axis, with highest levels in the proximal small intestine, a distribution similar to the known location of LCFA absorption. FAT mRNA level also increase developmentally in the rat jejunum from newborn to adult animals. Finally, the long chain fatty acid, oleate, rapidly alters FAT mRNA levels in vivo,
strongly supporting a role for FAT in intestinal fatty acid uptake or physiologic fatty acid signaling. • G1466 AE2a AND AE2b EXPRESSIONS HAVE DISTINCT TISSUERELATED AND DEVELOPMENTAL REGULATIONS. Alice Chow, Rongping Zhang, and Tao Pang. Emory University School of Medicine, Atlanta VA Medical Center, Atlanta, GA 30322.
The single rat AE2 gene has several promoters (Wang et al. JBC 271:7835, 1996), which are predicted to generate distinct polypeptides, which include the full-length AE2a and the N-terminally truncated AE2b. To distinguish AE2a and AE2b, oligonucleotides corresponding to transcriptspecific exons were hybridized to 25-100~g of total RNA from various rat tissues. S1 nuclease assays detected AE2a in rat stomachs, proximal and distal small intestines, colons, kidneys and livers. AE2a was also present in isolated enterocytes. Previous report by Wang et al (JBC 271:7835, 1996) demonstrated that AE2b has a more restricted distribution. Our S1 nuclease studies indicate that AE2b transcripts are more abundant in rat stomachs and colons than in small intestines and livers. AE2a mRNA in small intestines of 14-day old rats was 2.8 fold of that in adults (p < .025). AE2b levels in stomachs, colons, and livers of 14-day old rats only amounted to 39%, 35%, and 11%, respectively, of levels in adult organs. AE2b mRNA level was low in small intestines of both age groups. An AE2b promoter construct was created by fusing the 2.0 kb fragment upstream of exon l b (derived from AE2C 1, a generous gift of GE Shull, Univ. Cincinnati) to the firefly luciferase reporter (pGL-Basic). Caco2 (colon adenocarcinoma) and AGS (gastric adenocarcinoma) cells were cotransfected with Renila luciferase reporter as internal controls. To compare transcriptional activities in Caco2 and AGS, firefly luciferase activity (after normalization to Renila luciferase activity) was expressed as a percentage of mean activity achieved in cells transfected with a reference vector (pGL-control driven by SV40 promoter/enhancer). Basal transcriptional activities of the AE2b construct was 6 fold higher in the stomach-derived AGS cells than in the enterocyte-like Caco2 cells (p < 0.01). Conclusions: (1) AE2a is widely distributed and may serve functions common to most cells; (2) AE2b transcripts are only substantially expressed in rat stomachs and colons, and may serve some tissue-specific functions; (3) the higher abundance of AE2b mRNA in stomachs is associated with higher basal transcriptional activities of AE2b regulatory sequences in AGS; (4) the previously reported developmental changes of AE2 is most likely a result of decreases in AE2a. AE2b appears to gain importance in adults as its mRNA increases in adult tissues which express AE2b. This study indicates that AE2a and AE2b have distinct tissue-related and developmental regulations. • G1467 AE2a TRANSCRIPTION IS STIMULATED BY PHORBOL ESTER. Alice Chow, Rongping Zhang, and Tao Peng. Emory University School of Medicine, Atlanta VA Medical Center, Atlanta, GA 30322.
AE2a encodes a CI:HCO 3 exchanger and its mRNA is widely expressed. We examined if phorbol 12-myristate 13-acetate (TPA) affects AE2a transcript level or activity of the AE2a promoter. Methods and Results: IEC-6 (rat intestinal epithelial cells) were seeded at 4.5x 104 cells/cm 2. Forty-eight hrs later, cultures were treated with serum free media with 0 ng/ml or 50 ng/ml TPA for 2 hrs prior to RNA extraction. AE2a mRNA levels were measured by S1 nuclease assays using an exonla probe. Mean AE2a mRNA level was 27.5% higher in TPA treated cultures (n=4, p < 0.05). AE2a/reporter constructs were obtained by fusing fragments of the rat AE2 gene upstream of exon la to the promoterless, enhancerless firefly luciferase plasmid (pGL-Basic). Caco2 (human colon adenocarcinoma) and AGS (human gastric adenocarcinoma) cells were used for transient transfections. Plasmids carrying the Renila luciferase (pRL-SV40) or chloramphenicolacetyltransferase (pCAT-SV40) reporters were cotransfected to provide internal controls. Caco2 and AGS cells were plated at 2.6x104/cm2 and 5.2x104/cm2, respectively. Twenty-four hours after transfection, media were replaced by sernm-free DMEM containing zero or 50 ng/ml TPA. Forty-eight hrs later, cell extracts were harvested for measurement of reporter activities. Firefly luciferase activities were normalized to either renila luciferase activities (Dual Luciferase System, Promega) or CAT expression (ELISA) from the same extracts. The construct (A4) containing the region from -244 to +47 flanking exonla conferred a 7.6 fold (p < 0.05) and a 5.0 fold (19< 0.01) TPA-induced increase in reporter activities in Caco2 and AGS, respectively. The additional 843 bp upstream sequence (A1 construct) did not increase TPA stimulation. Conclusions: TPA treatment led to an increase in AE2a mRNA level in intestinal cells. The -247 to +47 region has no recognizable TPA-responsive elements (TRE) but conferred response to TPA in both Caco2 and AGS. TPA may act through factors that interact with non-TRE sites to transactivate the AE2a promoter and lead to the observed increase of AE2a mRNA in treated cultures.