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Human guanylyl cyclase C promoter directs intestinal expression of luciferase reporter gene in transgenic mice
April 1995
• MECHANISM OF Na-ALANINE CO.TRANSPORT INHIBITION IN THE CHRONICALLY INFLAMED RABBIT ILEUM. U. Sundaram and V.M. Rajendren, Department of Medicine, Yale University School of Medicine, New Haven, CT, During chronic ileal inflammation activation of different immuneinflammatory mediator pathways may alter Na-nutrient co-transport (c-t) processes by different mechanisms. Using a rabbit model of chronic ileitis produced by coccidial infection we have previously demonstrated that Na-glucose c-t was inhibited by a decrease in the maximal rate of uptake (Vmax) without a change in the affinity (Km) for glucose (Gastro. 106:A274, 1994). In the present study we have determined the alterations in Na-amino acid c-t during chronic ileitis. aH-alanine uptake was measured in villus cells isolated from normal and inflamed ileum using a calcium chelation technique. Na-dependent alanine uptake was inhibited in villus cells from the inflamed ileum (5.76 _+ 1.18 umol/mg protein • 15 rain in normal and 1.36 _+ 0.48 in inflamed ileum, n = 5, p < 0.005). In intact cells, since Na-K-ATPase provides the favorable Na electrochemical gradient for this cotransporter, it was measured and was found to be inhibited 50% in the inflamed ileum. In order to determine if the Na-alanine co-transporter is directly affected, alanine uptake was determined in brush border membrane vesicles (BBMV). Na-dependent alanine uptake was inhibited in villus ceil BBMV from the inflamed ileum (73.2 -+ 1.2 pmol/mg protein • 10 sec in normal and 21.5 +_ 3.2 in inflamed, n = 3 p < 0.01). Further, kinetic studies demonstrated that the affinity for alanine was decreased in the inflamed ileum (Kin was 8.5 +- 0.65 mM in normal and 21.0 +_ 2.0 in inflamed, n=3, p < 0.01) without a significant change in the maximal rate (Vmax) of alanine uptake. In conclusion, in a rabbit model of chronic ileitis, similar to Naglucose c-t, Na-alanine c-t is also inhibited at the level of the transporter and not, solely secondary to an alteration of Na extrusion by the cells. However, during chronic ileal inflammation, the mechanism of inhibition of Na-alanine e-t differs from that of Naglucose c-t. Thus, these observations suggest that different immuneinflammatory mediators regulate these Na-nutrient c-t pathways in chronic ileitis.
• HUMAN
GUANYLYL CYCLASE C PROMOTER DIRECTS INTESTINAL EXPRESSION OF LUCIFEEASE R E P O R T E R GENE IN TEANSGENIC MICE.
S. Swenson, E. Mann, R . Giannella. +Division of Digestive Diseases, University of Cincinnati and VAMC, Cincinnati, OH. Intestinal ion transport and fluid balance are regulated, at least in part, by guanylyl cyclase C (GC-C). GC-C generates cGMP in response to the endogenous psptides guanylin and uroguanylln, as well as to bacterial heat-stable enterotoxin. Chloride secretion is stimulated by cGMP via the CFTR CI- channel. We assessed the pattern of mouse GC-C expression by Northern blot. GC-C is expressed only in the intestine, with highest levels in cecum. We investigated the transcriptional regulation of GC-C expression using a transgenic mouse model in which a 2.1 kb XbaI fragment of human GC-C promoter (-1973 to +124) was fused to the lucifsrase reporter gene. This construct was previously shown to direct luciferase expression in the Caco2 intestinal cell line, but not in NIH/3T3 fibroblasts. Of 5 potential transgenio founder mice, 4 were found to have undergone DNA rearrangements in the transgene. One mouse had the predicted pattern for an intact, multiple copy transgene. Transgenic offspring of this founder had high levels of luciferase activity only in the intestine. Low levels of activity in non-intestinal tissues may reflect the absence of suppressor elements from the transgenic promoter. Luciferase activity (Rel. light units/m~ Drot) Brain 1051 Stomach 875 Heart 3469 Duodenum 8117 Kidney 0 Proximal Jejunum 17727 Liver 67 Distal Jejunum 37664 Lung 254 Ileum 79799 Skeletal Muscle 6411 Cecum 180407 Spleen 242 Colon 469615 Testis 1939 Identification of ci___~s-acting elements in the human GC-C promoter which direct intestine-specific gene expression will ultimately aid the design of gene transfer vectors targeting the intestine.
Intestinal Disorders
A329
GASTRIC ULCER FORMATION IN SPONTANEOUSLY HYPERTENSIVE RATS SUBJECTED TO ADRENALECTOMY. H, Suzuki. Y. Akiba*, S. Miura*, H. Ishii*, B.W. Zweifach, G.W. SchmidSch6nbein. Inst. for Biomed. Engineer., Univ. of California San Diego, La JoUa, CA & Dept. of Internal Med., Sch. of Med., Keio Univ., Tokyo, Japan* Spontaneously hypertensive rats (SHR) are known to be resistant to gastric ulcers induced by water-immersion restraint (Life Sci. 49:2023, 1991) and leukocyte recruitment to proinflammatory stimuli is attenuated (Hypertension 21:667, 1993). The objective was to investigate in SHR the role of the adrenal gland in leukocyte recruitment as welI as in gastric ulcer formation. SHR and adrenalectomized SHR (Adx.-SHR), which are normotensive, and their normotensive control, Wistar-Kyoto rats (WKY) with and without adrenalectomy were used. Bilateral adrenalectomy as well as sham-operation were carded out at 12 weeks and the observations was made at 13-14 weeks. The mesentery was superfused with histamine (10 -5 M) and the leukocyte adhesion to the postcapillary venular endothelium was counted in 10 min intervals for 60 rain. After the intravital observation, the gastric ulcer index was estimated. In the gastric specimen, the number of myeloperoxidase (MPO) (+) cells (due to leukocyte infiltration), NADPH diaphorase histochemistry (localization of nitric oxide synthase:NOS) and NOS activity were assayed. For NADPH diaphorase histochemistry, the cryostat sections were reacted with B-NADPH (1 mM) and nitroblue tetrazolium (0.5 mM). NOS activity was measured by the formation of L - [ U - l n C ] citrulline from L-[Ut4C] arginine. Venular leukocyte adhesion following histamine was suppressed in SHR, but the number was dramatically increased after adrenalectomy (p<0.05). Only in the Adx.-SHR was a gastric linear ulcer observed. MPO(+) number and NADPH diaphorase activity were significantly lower in sham-SHR compared to sham-WKY, but increased in Adx.-SHR. In contrast, Adx.-WKY developed neither MPO (+) number nor NADPH diaphorase activity compared with sham-WKY. sliam-WKY Adx.-WKY sham-SHR Adx.-SHR MPO(+) No. 54.3+6.2 59.3-'£2.2 33.0+2.1" 120.5+18.01" (No.lfield) (*o<0.01. to<0.01) CaZ+-independent NOS activity showed a higher value only in Adx.SHR. Thus, the present study suggests a significant leukocyte-dependent gastric ulcer formation associated with an activation of Ca2+-independent NOS in Adx.-SHR, but not in WKY. The adrenal gland may protect the gastric mucosa in SHR. (Supportedin part by NIH Grant HL-10881)
• CLONING AND CHARACTERIZATION OF AN IMMEDIATE' EARLY GENIE (PC4/TIS7) THAT IS MARKEDLY INDUCED IN THE ADAFrlNG REMNANT ILEUM FOLLOWING 70% RESECTION OF THE RAT SMALL INTESTINE E, Swietlicki. J.L. Wang, B.D. Dodson, Levin M.S., D.C. Rubin. Dept. of Medicine, WashingtonUniv. Sch of Med, St. Louis, Me. Loss of functional small bowel surface area produces an adaptive response in the remnant gut. Morphological and functional changes occurring in intestinal adaptation have been well studied, but the underlying molecular mechanismsare largely unknown.To study these mechanisms,we have been using a rat resection model to clonegenesthat are differentially expressedin the adapting ileum. Methods A eDNA library constructedfromthe remnantileum48 h following70% proximal small bowel resection was screened using a subtracted eDNA probe to identify genes specifically induced by resection but not transection and reanastomosis (sham reseeted). Cloned eDNAs were sequenced. Temporal and spatial expressionpatterns were assessedwith RNA blot and in situ hybridization. Results One of the isolated clones was identical to the immediate early gene PC4/TIS7. This gene is regulated in marine fibroblasts and rat PC12 cells by peptide growthfactors. It is induced and translocated to the plasma membraneand then to the nucleus in NGF-treated PC12 cells. A single 1.9 kb gut RNA (induced 5.5 fold at 48 h post resection) was detected on Northern blots. Compared to controls, PC4/TIS7 was induced as early as 8 h, maximallyinduced at 16 h and no longer induced by 1 week post resection. By in situ hybridization, PC4/TIS7 was abundantly expressedin villus enterocytesfromresected but not control rats. Fold Imlucflon (RE / SnR)
I
6
il 2 4
16
4S
168
TimePost Surgery0t 0
rime course of PC4/TIS7 inductionin the adaptive ileum. RNA isolated from the residual ileum sJp 70% proximal small bowel resection (RE) or sham-resection [SHR). RNA blots (n=3 rats per timepoim) were probed with PC4/TIS7 eDNA and PC4/TIS7 mRNAwas quantitated by laser densitometry. Conclusions Due to its putative role in regulating cell proliferation and differentiation and the marked and early inductionof PC4/TIS7 mRNAlevels after resection, it is likely to be an importantregulator of the adaptive response.
• MECHANISM OF Na-ALANINE CO.TRANSPORT INHIBITION IN THE CHRONICALLY INFLAMED RABBIT ILEUM. U. Sundaram and V.M. Rajendren, Department of Medicine, Yale University School of Medicine, New Haven, CT, During chronic ileal inflammation activation of different immuneinflammatory mediator pathways may alter Na-nutrient co-transport (c-t) processes by different mechanisms. Using a rabbit model of chronic ileitis produced by coccidial infection we have previously demonstrated that Na-glucose c-t was inhibited by a decrease in the maximal rate of uptake (Vmax) without a change in the affinity (Km) for glucose (Gastro. 106:A274, 1994). In the present study we have determined the alterations in Na-amino acid c-t during chronic ileitis. aH-alanine uptake was measured in villus cells isolated from normal and inflamed ileum using a calcium chelation technique. Na-dependent alanine uptake was inhibited in villus cells from the inflamed ileum (5.76 _+ 1.18 umol/mg protein • 15 rain in normal and 1.36 _+ 0.48 in inflamed ileum, n = 5, p < 0.005). In intact cells, since Na-K-ATPase provides the favorable Na electrochemical gradient for this cotransporter, it was measured and was found to be inhibited 50% in the inflamed ileum. In order to determine if the Na-alanine co-transporter is directly affected, alanine uptake was determined in brush border membrane vesicles (BBMV). Na-dependent alanine uptake was inhibited in villus ceil BBMV from the inflamed ileum (73.2 -+ 1.2 pmol/mg protein • 10 sec in normal and 21.5 +_ 3.2 in inflamed, n = 3 p < 0.01). Further, kinetic studies demonstrated that the affinity for alanine was decreased in the inflamed ileum (Kin was 8.5 +- 0.65 mM in normal and 21.0 +_ 2.0 in inflamed, n=3, p < 0.01) without a significant change in the maximal rate (Vmax) of alanine uptake. In conclusion, in a rabbit model of chronic ileitis, similar to Naglucose c-t, Na-alanine c-t is also inhibited at the level of the transporter and not, solely secondary to an alteration of Na extrusion by the cells. However, during chronic ileal inflammation, the mechanism of inhibition of Na-alanine e-t differs from that of Naglucose c-t. Thus, these observations suggest that different immuneinflammatory mediators regulate these Na-nutrient c-t pathways in chronic ileitis.
• HUMAN
GUANYLYL CYCLASE C PROMOTER DIRECTS INTESTINAL EXPRESSION OF LUCIFEEASE R E P O R T E R GENE IN TEANSGENIC MICE.
S. Swenson, E. Mann, R . Giannella. +Division of Digestive Diseases, University of Cincinnati and VAMC, Cincinnati, OH. Intestinal ion transport and fluid balance are regulated, at least in part, by guanylyl cyclase C (GC-C). GC-C generates cGMP in response to the endogenous psptides guanylin and uroguanylln, as well as to bacterial heat-stable enterotoxin. Chloride secretion is stimulated by cGMP via the CFTR CI- channel. We assessed the pattern of mouse GC-C expression by Northern blot. GC-C is expressed only in the intestine, with highest levels in cecum. We investigated the transcriptional regulation of GC-C expression using a transgenic mouse model in which a 2.1 kb XbaI fragment of human GC-C promoter (-1973 to +124) was fused to the lucifsrase reporter gene. This construct was previously shown to direct luciferase expression in the Caco2 intestinal cell line, but not in NIH/3T3 fibroblasts. Of 5 potential transgenio founder mice, 4 were found to have undergone DNA rearrangements in the transgene. One mouse had the predicted pattern for an intact, multiple copy transgene. Transgenic offspring of this founder had high levels of luciferase activity only in the intestine. Low levels of activity in non-intestinal tissues may reflect the absence of suppressor elements from the transgenic promoter. Luciferase activity (Rel. light units/m~ Drot) Brain 1051 Stomach 875 Heart 3469 Duodenum 8117 Kidney 0 Proximal Jejunum 17727 Liver 67 Distal Jejunum 37664 Lung 254 Ileum 79799 Skeletal Muscle 6411 Cecum 180407 Spleen 242 Colon 469615 Testis 1939 Identification of ci___~s-acting elements in the human GC-C promoter which direct intestine-specific gene expression will ultimately aid the design of gene transfer vectors targeting the intestine.
Intestinal Disorders
A329
GASTRIC ULCER FORMATION IN SPONTANEOUSLY HYPERTENSIVE RATS SUBJECTED TO ADRENALECTOMY. H, Suzuki. Y. Akiba*, S. Miura*, H. Ishii*, B.W. Zweifach, G.W. SchmidSch6nbein. Inst. for Biomed. Engineer., Univ. of California San Diego, La JoUa, CA & Dept. of Internal Med., Sch. of Med., Keio Univ., Tokyo, Japan* Spontaneously hypertensive rats (SHR) are known to be resistant to gastric ulcers induced by water-immersion restraint (Life Sci. 49:2023, 1991) and leukocyte recruitment to proinflammatory stimuli is attenuated (Hypertension 21:667, 1993). The objective was to investigate in SHR the role of the adrenal gland in leukocyte recruitment as welI as in gastric ulcer formation. SHR and adrenalectomized SHR (Adx.-SHR), which are normotensive, and their normotensive control, Wistar-Kyoto rats (WKY) with and without adrenalectomy were used. Bilateral adrenalectomy as well as sham-operation were carded out at 12 weeks and the observations was made at 13-14 weeks. The mesentery was superfused with histamine (10 -5 M) and the leukocyte adhesion to the postcapillary venular endothelium was counted in 10 min intervals for 60 rain. After the intravital observation, the gastric ulcer index was estimated. In the gastric specimen, the number of myeloperoxidase (MPO) (+) cells (due to leukocyte infiltration), NADPH diaphorase histochemistry (localization of nitric oxide synthase:NOS) and NOS activity were assayed. For NADPH diaphorase histochemistry, the cryostat sections were reacted with B-NADPH (1 mM) and nitroblue tetrazolium (0.5 mM). NOS activity was measured by the formation of L - [ U - l n C ] citrulline from L-[Ut4C] arginine. Venular leukocyte adhesion following histamine was suppressed in SHR, but the number was dramatically increased after adrenalectomy (p<0.05). Only in the Adx.-SHR was a gastric linear ulcer observed. MPO(+) number and NADPH diaphorase activity were significantly lower in sham-SHR compared to sham-WKY, but increased in Adx.-SHR. In contrast, Adx.-WKY developed neither MPO (+) number nor NADPH diaphorase activity compared with sham-WKY. sliam-WKY Adx.-WKY sham-SHR Adx.-SHR MPO(+) No. 54.3+6.2 59.3-'£2.2 33.0+2.1" 120.5+18.01" (No.lfield) (*o<0.01. to<0.01) CaZ+-independent NOS activity showed a higher value only in Adx.SHR. Thus, the present study suggests a significant leukocyte-dependent gastric ulcer formation associated with an activation of Ca2+-independent NOS in Adx.-SHR, but not in WKY. The adrenal gland may protect the gastric mucosa in SHR. (Supportedin part by NIH Grant HL-10881)
• CLONING AND CHARACTERIZATION OF AN IMMEDIATE' EARLY GENIE (PC4/TIS7) THAT IS MARKEDLY INDUCED IN THE ADAFrlNG REMNANT ILEUM FOLLOWING 70% RESECTION OF THE RAT SMALL INTESTINE E, Swietlicki. J.L. Wang, B.D. Dodson, Levin M.S., D.C. Rubin. Dept. of Medicine, WashingtonUniv. Sch of Med, St. Louis, Me. Loss of functional small bowel surface area produces an adaptive response in the remnant gut. Morphological and functional changes occurring in intestinal adaptation have been well studied, but the underlying molecular mechanismsare largely unknown.To study these mechanisms,we have been using a rat resection model to clonegenesthat are differentially expressedin the adapting ileum. Methods A eDNA library constructedfromthe remnantileum48 h following70% proximal small bowel resection was screened using a subtracted eDNA probe to identify genes specifically induced by resection but not transection and reanastomosis (sham reseeted). Cloned eDNAs were sequenced. Temporal and spatial expressionpatterns were assessedwith RNA blot and in situ hybridization. Results One of the isolated clones was identical to the immediate early gene PC4/TIS7. This gene is regulated in marine fibroblasts and rat PC12 cells by peptide growthfactors. It is induced and translocated to the plasma membraneand then to the nucleus in NGF-treated PC12 cells. A single 1.9 kb gut RNA (induced 5.5 fold at 48 h post resection) was detected on Northern blots. Compared to controls, PC4/TIS7 was induced as early as 8 h, maximallyinduced at 16 h and no longer induced by 1 week post resection. By in situ hybridization, PC4/TIS7 was abundantly expressedin villus enterocytesfromresected but not control rats. Fold Imlucflon (RE / SnR)
I
6
il 2 4
16
4S
168
TimePost Surgery0t 0
rime course of PC4/TIS7 inductionin the adaptive ileum. RNA isolated from the residual ileum sJp 70% proximal small bowel resection (RE) or sham-resection [SHR). RNA blots (n=3 rats per timepoim) were probed with PC4/TIS7 eDNA and PC4/TIS7 mRNAwas quantitated by laser densitometry. Conclusions Due to its putative role in regulating cell proliferation and differentiation and the marked and early inductionof PC4/TIS7 mRNAlevels after resection, it is likely to be an importantregulator of the adaptive response.