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Association of cholecystokinin-a receptor gene polymorphisms and panic disorder in japanese
The differential- and tissue-specific expression of RGSs make these potent regulatory proteins attractive targets for therapeutic interventions.
suggested that naturally occurring alterations in CCK systems could contribute to the development of PD. However, among recent investigations of the polymorphisms of CCK, CCKAR, and CCK-BR in patients with PD, the results were inconclusive. We recently cloned the genomic structures of human CCK-AR, and determined the transcriptional start site of the human CCK-AR gene. Two sequence changes were detected in the promoter region: a G to T change in nucleotide -128 and an A to G change in nucleotide -81 (FEBS Lett, 2000). In this study, we investigated a possible association between the CCK-AR gene and PD by evaluating the distribution of not only the genotypes but also the haplotypes of the two polymorphisms. Methods: The subjects consisted of 109 Japanese patients with PD (64 males, 18-63 years old; 45 females, 21-71 years old), all of whom met DSM-II1-R critena for PD. The age- and gentler-matched control group consisted of 400 unrelated Japanese (234 males, 20-62 years old; 166 females, 21-71 years old). Examination of the polymorphism in the promoter region of the CCK-AR gene was accomplished using a mismatch PCR-RFLP method (Funakoshi et al 2000). Results: The variant genotype frequency was significantly higher in the patients with PD than in the controls (P(0.005)(OR = 1.87, 95% CI = 1.22-2.86). Because of their proximity to each other, the two polymorphisms are in linkage disequilibrium, and the haplotype -81A/-128T was not present. The statistical differences between the haplotype distributions in the PD and control groups were highly significant (p(0.001): The frequency of variant haplotypes (-81G/-128G and -81G/-128T) was higher in the former group than in the latter. Stratification of the PD samples and controls with respect to age and gender did not alter these relationships. Nor did the age at onset of PD affect the distributions of the CCK-AR gene polymorphisms. Conclusions: The CCK-A receptor gene polymorphism may be involved in the neurobiology of panic disorder.
M1054 Proteinase Activated Receptors (PAR-l, 2) Regulate Intestinal Wound Repair in vitro Chris Armstrong, S. Mokashi, Josee Wong, Sunita Swaminathan, M. Hollenberg, Paul L. Beck Intestinal wound repair following injury plays a key role in normal intestinal homeostasis. Impairment of wound repair has been noted in patients with inflammatory bowel disease and animals with abnormal wound repair are markedly more susceptible intestinal inflammation. PARs are a unique subclass of G-prntein-coupled receptors that have been implicated in modulating the intestinal inflammatory response and pain signaling. Both PAR 1 and 2 are expressed in the human, murine and rat intestine. Both thrombin (which can act via PAR-l) and trypsin (which can act wa PAR-2) are known to enhance wound repair. Aim; To determine if PAR 1,2 are directly important in the regulation of wound repair and determine the cellular pathways involved in these responses. Methods; IEC-6 cells were chosen for this model system in that they are a rat intestinal cell line that has been well characterized in this wounding model. In short, confluent IECo6 cells were wounded with a razor blade; either agonists of PAR-1 or 2, thrombin or trypsin were added. Migration cross the wound edge was assessed at 24h. To assess the role of MAPK, SrcK and P38 in these responses specific agonists of these pathways were used. Results; In our model system both trypsin, and thrombin were found to enhance wound repair (p<0.01). Both the PARi and PAR-2 agonists also promoted wound repair (p
M1057 Glucagon-like Petide-2 (GLP-2) Ameliorates Intestinal Pathophysiology Induced by Acute or Chronic Stress in the Mouse Heather Cameron, Ping-Chang Yang, Mary H. Perdue GLP-2, an intestinotrophic growth hormone, has been shown to enhance intestinal epithelia] barrier function, reducing permeability of both paracellular and transcellular pathways. In rats, acute psychological stress causes changes in epithelial physiology, including permeability defects, and chronic stress both sustains pathophysiology and initiates mucosal inflammation. Here, our aim was to define the effects of psychological stress on intestinal function in mice, and to determine if GLP-2 treatment could ameliorate gut pathology/pathophysiology. Methods: Mice were injected se with 5~g GLP-2 4h before exposure to water avoidance stress (WAS, mice on platform surrounded by water) for lh. For acute stress, studies were conducted immediately following WAS; for chronic stress, GLP-2 injection and WAS were repeated for 10 days and studies were conducted following the final stress period. Segments of small and large intestine were mounted in Ussing chambers and conductanc~ (G), and the flux of horseradish peroxidase (HRP) were determined. Adjacent segments were processed for histological assessment and electron microscopy. Results: Acute stress increased G and the flux of HRP across both jejunal and colonic segments, while pretreatment with GLP-2 attenuated the defects. Chronically stressed mice showed a significant reduction in weight gain over the 10d period compared to controls. G and HRP flux increased in both the jejunum and colon of stressed mice, and an increase in inflammatory cells was documented in the colon. GLP-2 treatment improved weight gain, and restored normal conductance and permeability in the jejunum. In the colon, GLP-2 also significantly reduced the uptake of luminal protein and diminished the inflammatory cell infiltration. Conclusions: This study identifies that mouse intestine is adversely affected by both acute and chronic psychological stress. GLP-2 treatment can attenuate stress-induced intestinal pathophysiology and inflammation. Strategies promoting epithelial barrier function may be beneficial for treating stressinduced gut dysfunction.
M1055 Decreased Inflammation Associated with Corticotropin -Releasing Hormone (CRH) Deficiency Does Not Prevent Weight Loss During Acute Tnbs-lnduced Colitis in Mice Jerome Gay, Michael O'Brien, Charalabos Pothoulakis, Katia Karalis Background: CRH, the hypothalamic component of the HPA axis, regulates inflammation through stimulation of glucocorticoid release, while peripherally expressed CRH acts as a proinflammatory factor. Inflammatory bowel dLse.ase(1BD), i.e. ulcerative colitis and Cruhn's dLsease, represent chronic inflammatory disorders involving the mucosal immune system. CRH is expressed in the human and rodent intestine and we have previously shown its proinflammatory effects in acute, enterotoxin-mediated enteritis in rodents. However, the role of CRH in the acute phase of IBD is not known. Aim: To assess the consequence(s) of CRH deficiency in the development and progress of experimental colitis in mice. Methods : Colitis was induced by rectal instillation of triintrobenzene sulfonic acid (TNBS, 250 rag/ kg in 35% ethanol), in CRH deficient (Crh-/-) female mice and their wild type littermates (Crh + / + ) (n = 6-7/group). After 48 hrs inflammation was assessed by histological scoring, myeloperoxidase (MPO) activity, and colonic weight/length ratio. We also measured body weight changes, as well as plasma corticosterone. IL-6 and leptin levels were also measured since they have been suggested to contribute to the anorexia and weight loss associated with acute colitis. We and others have previously reported paradoxically increased IL-6 levels in Crh-A mice subjected to immune activation. Results : see table Conclusions : Our findings support an important proinflammatory role for CRH during acute TNBS colitis, based on the significantly compromised inflammatory responses of the Crh-/- mice. However, the diminished colonic inflammation associated with CRH deficiency did not prevent TNBSinduced weight loss. We speculate that the higher circulating leptin and IL-6 levels of the Crh-/- mice may be the underlying cause(s) of their colitis-induced wasting. Supported by the National Institutes of Health (DK 33506 and DK 47977). MPO actl~ty
Coloelc
Cdt#kdlamN
1.5,1
Cdt,H~ Crk','/~ Inlk=~
9.2~0.9
Gastrin Induces Human Monocyte and Endothelial Cell Activation Angeles Alvarez, Sales Ibiza, Elsa Quintana, Enrique O'Connor, Juan V. Espfugues, Sara Calatayud Hypergastrmemia is a feature of several gastric inflammatory conditions such as Helicobacter pylori infection or chronic treatment with proton pump inhibitors. Experimental studies conducted in our laboratory indicate that gastrin induces leukocyte / endothelial cell interactions in rat mesenteric venules by activating its CCK-B receptor. The aim of the present study was to analyze whether this effect of gastrin is due to a direct action on leukocytes and/or endothehal cells. Methods: The expression of surface adhesion molecules responsible for leukocyte adhesion and emigration has been analyzed in human leukocytes and human umbilical vein endothelial cells (HUVECs). Blood samples from healthy volunteers and HUVECs were treated separately with diflerem concentrations of gastrin and the expression of L-selectin and beta-2 imegrins (CD11/CD18) in leukocytes and E-selectin and immunoglobulins in HUVECs was measured by flow cytometry. Results: Treatment of human blood with gastrin specifically activated monocytes, and not lymphocytes or polymorphonuclear leukocytes. In human monocytes, gastrin induced a concentration-dependent up-regulation of the alpha (CD11a, CDIlb and CD11c) and beta (CD18) subunits of beta-2 integrins. Although a reduction in L-selectin expression was observed after treatment with gastrin, it did not reach statistical significance (Table 1). Treatment of HUVECs with gastrin (10~M, 10"8M and 10-TM) induced significant increases on the expression of ICAM-3 (6.1 _+2. l*, 7.4 -+ 3.6* and 10.4_+ 2.4") and VCAM-1 (12.4 _+4.5, 26.8 - 8.8* and 22.8_+ 4.5")(*P(0.05 vs control, ANOVA + Newman-Keuls test), but no changes were observed on the expression of E-selectin, 1CAM-1 or 1CAM-2. Conclusions: Gastrin activates human monocytes and endothelial cells to express adhesion molecules responsible for the interactions between these two cell types and thus, for the formation of inflammatory foci. These results confirm the pro-inflammatory activity of gastrin and suggest that hypergastrinemia is an ethyopathogenic factor in the development of gastritis in circumstances such as Helicobacter pylori infection or chronic treatment with proton pump inhibitors.
Mou~
r
Cr~) Cdt,./.
M1058
3,33~ 0.40
0.018 ~ 0.0009
2.23,0.26
10127,. 3,41 ~ 1
835.4 46.8 ~
54,46 ~ 23.92
0.037 ~ 0.005
20.4 ~ 6.8
86.74 ~ 2.3
1582, 156
8.79,4.8
0.02~0.002
7.36,2
100%
0.053 , 0.005
43.35~3.2
13.54 2.6
1023,59~ 4.4 0.9 , 84.7~1
5,0.9
508.9~ 73.4 424.5~68
M1056 Association of Cholecystokinin-A Receptor Gene Polymorphisms and Panic Disorder in Japanese Kyoko Miyasaka, Yuki Yoshida, Minoru Ohta, Setsuko Kanai, Hiroko Hosuya, Naomi Yamakawa, Osamu Shirakawa, Sachio Matsushita, Susumu Higuchi, Akihiro Funakoshi Background and Aims: CCK is one of the most abundant neurotransmitter peptides in the central nervous system. Panic disorder (PD) is a common anxiety condition, characterized by unprovoked anxiety attacks distinguished by such symptoms as palpitations, chest pain, dyspnea, choking, tremors, faintness, and sweating, in addition to fear of dying, losing control, or going crazy. CCK-4 induces panic-hke attacks when administered as an intravenous bolus in healthy volunteers, and in patients with panic disorder. Several lines of evidence have
AGA
Abstracts
A-300
suggested that naturally occurring alterations in CCK systems could contribute to the development of PD. However, among recent investigations of the polymorphisms of CCK, CCKAR, and CCK-BR in patients with PD, the results were inconclusive. We recently cloned the genomic structures of human CCK-AR, and determined the transcriptional start site of the human CCK-AR gene. Two sequence changes were detected in the promoter region: a G to T change in nucleotide -128 and an A to G change in nucleotide -81 (FEBS Lett, 2000). In this study, we investigated a possible association between the CCK-AR gene and PD by evaluating the distribution of not only the genotypes but also the haplotypes of the two polymorphisms. Methods: The subjects consisted of 109 Japanese patients with PD (64 males, 18-63 years old; 45 females, 21-71 years old), all of whom met DSM-II1-R critena for PD. The age- and gentler-matched control group consisted of 400 unrelated Japanese (234 males, 20-62 years old; 166 females, 21-71 years old). Examination of the polymorphism in the promoter region of the CCK-AR gene was accomplished using a mismatch PCR-RFLP method (Funakoshi et al 2000). Results: The variant genotype frequency was significantly higher in the patients with PD than in the controls (P(0.005)(OR = 1.87, 95% CI = 1.22-2.86). Because of their proximity to each other, the two polymorphisms are in linkage disequilibrium, and the haplotype -81A/-128T was not present. The statistical differences between the haplotype distributions in the PD and control groups were highly significant (p(0.001): The frequency of variant haplotypes (-81G/-128G and -81G/-128T) was higher in the former group than in the latter. Stratification of the PD samples and controls with respect to age and gender did not alter these relationships. Nor did the age at onset of PD affect the distributions of the CCK-AR gene polymorphisms. Conclusions: The CCK-A receptor gene polymorphism may be involved in the neurobiology of panic disorder.
M1054 Proteinase Activated Receptors (PAR-l, 2) Regulate Intestinal Wound Repair in vitro Chris Armstrong, S. Mokashi, Josee Wong, Sunita Swaminathan, M. Hollenberg, Paul L. Beck Intestinal wound repair following injury plays a key role in normal intestinal homeostasis. Impairment of wound repair has been noted in patients with inflammatory bowel disease and animals with abnormal wound repair are markedly more susceptible intestinal inflammation. PARs are a unique subclass of G-prntein-coupled receptors that have been implicated in modulating the intestinal inflammatory response and pain signaling. Both PAR 1 and 2 are expressed in the human, murine and rat intestine. Both thrombin (which can act via PAR-l) and trypsin (which can act wa PAR-2) are known to enhance wound repair. Aim; To determine if PAR 1,2 are directly important in the regulation of wound repair and determine the cellular pathways involved in these responses. Methods; IEC-6 cells were chosen for this model system in that they are a rat intestinal cell line that has been well characterized in this wounding model. In short, confluent IECo6 cells were wounded with a razor blade; either agonists of PAR-1 or 2, thrombin or trypsin were added. Migration cross the wound edge was assessed at 24h. To assess the role of MAPK, SrcK and P38 in these responses specific agonists of these pathways were used. Results; In our model system both trypsin, and thrombin were found to enhance wound repair (p<0.01). Both the PARi and PAR-2 agonists also promoted wound repair (p
M1057 Glucagon-like Petide-2 (GLP-2) Ameliorates Intestinal Pathophysiology Induced by Acute or Chronic Stress in the Mouse Heather Cameron, Ping-Chang Yang, Mary H. Perdue GLP-2, an intestinotrophic growth hormone, has been shown to enhance intestinal epithelia] barrier function, reducing permeability of both paracellular and transcellular pathways. In rats, acute psychological stress causes changes in epithelial physiology, including permeability defects, and chronic stress both sustains pathophysiology and initiates mucosal inflammation. Here, our aim was to define the effects of psychological stress on intestinal function in mice, and to determine if GLP-2 treatment could ameliorate gut pathology/pathophysiology. Methods: Mice were injected se with 5~g GLP-2 4h before exposure to water avoidance stress (WAS, mice on platform surrounded by water) for lh. For acute stress, studies were conducted immediately following WAS; for chronic stress, GLP-2 injection and WAS were repeated for 10 days and studies were conducted following the final stress period. Segments of small and large intestine were mounted in Ussing chambers and conductanc~ (G), and the flux of horseradish peroxidase (HRP) were determined. Adjacent segments were processed for histological assessment and electron microscopy. Results: Acute stress increased G and the flux of HRP across both jejunal and colonic segments, while pretreatment with GLP-2 attenuated the defects. Chronically stressed mice showed a significant reduction in weight gain over the 10d period compared to controls. G and HRP flux increased in both the jejunum and colon of stressed mice, and an increase in inflammatory cells was documented in the colon. GLP-2 treatment improved weight gain, and restored normal conductance and permeability in the jejunum. In the colon, GLP-2 also significantly reduced the uptake of luminal protein and diminished the inflammatory cell infiltration. Conclusions: This study identifies that mouse intestine is adversely affected by both acute and chronic psychological stress. GLP-2 treatment can attenuate stress-induced intestinal pathophysiology and inflammation. Strategies promoting epithelial barrier function may be beneficial for treating stressinduced gut dysfunction.
M1055 Decreased Inflammation Associated with Corticotropin -Releasing Hormone (CRH) Deficiency Does Not Prevent Weight Loss During Acute Tnbs-lnduced Colitis in Mice Jerome Gay, Michael O'Brien, Charalabos Pothoulakis, Katia Karalis Background: CRH, the hypothalamic component of the HPA axis, regulates inflammation through stimulation of glucocorticoid release, while peripherally expressed CRH acts as a proinflammatory factor. Inflammatory bowel dLse.ase(1BD), i.e. ulcerative colitis and Cruhn's dLsease, represent chronic inflammatory disorders involving the mucosal immune system. CRH is expressed in the human and rodent intestine and we have previously shown its proinflammatory effects in acute, enterotoxin-mediated enteritis in rodents. However, the role of CRH in the acute phase of IBD is not known. Aim: To assess the consequence(s) of CRH deficiency in the development and progress of experimental colitis in mice. Methods : Colitis was induced by rectal instillation of triintrobenzene sulfonic acid (TNBS, 250 rag/ kg in 35% ethanol), in CRH deficient (Crh-/-) female mice and their wild type littermates (Crh + / + ) (n = 6-7/group). After 48 hrs inflammation was assessed by histological scoring, myeloperoxidase (MPO) activity, and colonic weight/length ratio. We also measured body weight changes, as well as plasma corticosterone. IL-6 and leptin levels were also measured since they have been suggested to contribute to the anorexia and weight loss associated with acute colitis. We and others have previously reported paradoxically increased IL-6 levels in Crh-A mice subjected to immune activation. Results : see table Conclusions : Our findings support an important proinflammatory role for CRH during acute TNBS colitis, based on the significantly compromised inflammatory responses of the Crh-/- mice. However, the diminished colonic inflammation associated with CRH deficiency did not prevent TNBSinduced weight loss. We speculate that the higher circulating leptin and IL-6 levels of the Crh-/- mice may be the underlying cause(s) of their colitis-induced wasting. Supported by the National Institutes of Health (DK 33506 and DK 47977). MPO actl~ty
Coloelc
Cdt#kdlamN
1.5,1
Cdt,H~ Crk','/~ Inlk=~
9.2~0.9
Gastrin Induces Human Monocyte and Endothelial Cell Activation Angeles Alvarez, Sales Ibiza, Elsa Quintana, Enrique O'Connor, Juan V. Espfugues, Sara Calatayud Hypergastrmemia is a feature of several gastric inflammatory conditions such as Helicobacter pylori infection or chronic treatment with proton pump inhibitors. Experimental studies conducted in our laboratory indicate that gastrin induces leukocyte / endothelial cell interactions in rat mesenteric venules by activating its CCK-B receptor. The aim of the present study was to analyze whether this effect of gastrin is due to a direct action on leukocytes and/or endothehal cells. Methods: The expression of surface adhesion molecules responsible for leukocyte adhesion and emigration has been analyzed in human leukocytes and human umbilical vein endothelial cells (HUVECs). Blood samples from healthy volunteers and HUVECs were treated separately with diflerem concentrations of gastrin and the expression of L-selectin and beta-2 imegrins (CD11/CD18) in leukocytes and E-selectin and immunoglobulins in HUVECs was measured by flow cytometry. Results: Treatment of human blood with gastrin specifically activated monocytes, and not lymphocytes or polymorphonuclear leukocytes. In human monocytes, gastrin induced a concentration-dependent up-regulation of the alpha (CD11a, CDIlb and CD11c) and beta (CD18) subunits of beta-2 integrins. Although a reduction in L-selectin expression was observed after treatment with gastrin, it did not reach statistical significance (Table 1). Treatment of HUVECs with gastrin (10~M, 10"8M and 10-TM) induced significant increases on the expression of ICAM-3 (6.1 _+2. l*, 7.4 -+ 3.6* and 10.4_+ 2.4") and VCAM-1 (12.4 _+4.5, 26.8 - 8.8* and 22.8_+ 4.5")(*P(0.05 vs control, ANOVA + Newman-Keuls test), but no changes were observed on the expression of E-selectin, 1CAM-1 or 1CAM-2. Conclusions: Gastrin activates human monocytes and endothelial cells to express adhesion molecules responsible for the interactions between these two cell types and thus, for the formation of inflammatory foci. These results confirm the pro-inflammatory activity of gastrin and suggest that hypergastrinemia is an ethyopathogenic factor in the development of gastritis in circumstances such as Helicobacter pylori infection or chronic treatment with proton pump inhibitors.
Mou~
r
Cr~) Cdt,./.
M1058
3,33~ 0.40
0.018 ~ 0.0009
2.23,0.26
10127,. 3,41 ~ 1
835.4 46.8 ~
54,46 ~ 23.92
0.037 ~ 0.005
20.4 ~ 6.8
86.74 ~ 2.3
1582, 156
8.79,4.8
0.02~0.002
7.36,2
100%
0.053 , 0.005
43.35~3.2
13.54 2.6
1023,59~ 4.4 0.9 , 84.7~1
5,0.9
508.9~ 73.4 424.5~68
M1056 Association of Cholecystokinin-A Receptor Gene Polymorphisms and Panic Disorder in Japanese Kyoko Miyasaka, Yuki Yoshida, Minoru Ohta, Setsuko Kanai, Hiroko Hosuya, Naomi Yamakawa, Osamu Shirakawa, Sachio Matsushita, Susumu Higuchi, Akihiro Funakoshi Background and Aims: CCK is one of the most abundant neurotransmitter peptides in the central nervous system. Panic disorder (PD) is a common anxiety condition, characterized by unprovoked anxiety attacks distinguished by such symptoms as palpitations, chest pain, dyspnea, choking, tremors, faintness, and sweating, in addition to fear of dying, losing control, or going crazy. CCK-4 induces panic-hke attacks when administered as an intravenous bolus in healthy volunteers, and in patients with panic disorder. Several lines of evidence have
AGA
Abstracts
A-300