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Development of 1,4,5-inositol trisphophate receptors in the feline gastric antrum
April 1995
• EFFECT OF PROTEIN PRE-DIGESTION ON CHOLECYSTOKININ RELEASE IN CELIAC PATIENTS. P. Deprez, V. Szmata, P. Mainguet, A. Geubel. Gastroenterology Unit, Dept. of Internal Medicine, Cliniques Universitaires Saint-Luc, UCL, 1200 Brussels, Belgium. In celiac sprue cholecystokinin (CCK) release is impaired, which contributes to malabsorption. Whether this defect results from CCK cell dysfunction or from a reduced intraluminal stimulus due to a lack of nutrient's hydrolysis in the jejunum remains controversial. In the latter hypothesis, administration of a predigested diet would correct the defect. Our aim was thus to study the effect of protein pre-digestion on CCK release. Methods. On two separate days, 12 patients with celiac disease, 9 women and 3 men, mean age 39, range 30-45, were given a standard (SondalisR , Clintec) and a semi-elemental (Reabilan", Clintec) liquid diet, The patients were divided in two groups: one with a flat jejunal mucosa (destructive type) (n=6) and one with normal architecture but infiltration of intra-epithelial lymphocytes (infiltrative type) (n=6). A control group (n=9) was also given the two liquid meals. CCK plasma levels were determined at 15 min intervals for 120 min by RIA and expressed as integrated release (IR). Results. Celiac patients had lower basal CCK values than controls: 0.64 _+ 0.1 vs 1.06 _+ 0.2 pmole/I, p<0.05. Both groups had diminished CCK responses compared to the control group (IR 55.6 _+ 11.4, 74.5 _+ 18 and 202 _ 24.9 pmole/I/120 min, respectively, p<0.05). No significant effect of pre-digestion of protein was observed in controls or in celiac patients (IR 62.4 +_20.9, 69.8 + 14.7 and 230.9 +- 34.5 pmole/I/120 rain). Conclusion. Protein pre-digestion does not correct impairement of CCK post-prandial release in celiac patients. Moreover, in celiac patients on gluten-free diet impairement of CCK release persists in spite of improvement in mucosal atrophy.
• IMMUNOLOCALIZATION OF ALPHA INTEGRIN SUBUNITS AND EXTRACELLULAR MATRIX (ECM) COMPONENTS DURING HUMAN COLONIC OR.GANOGENESIS. B.K. Dieck~ranfe, S.A. Santoro, D.H. Alpers. Washington University School of Medicine, St. Louis, MO Morphogenesis is a poorly understood process involving cell adhesion and migration. Intagrins are a family of structurally related transmembrane receptors that participate in cell-cell and cell-ECM interactions. In order to investigate potential integrin roles in human colonic organogenesis, we have examined the tx subunit expression of 131 integrins and extracellular matrix ligands. At 9 weeks, the colon consists ofpseudostratified columnar epithelium surrounded by mesenchyme. Collagen type IV and lamlnin were present underlying the epithelium, around early vascular structures, and surrounding inner circular muscle layer fibers. Fibronectin was uniformly expressed by 9 weeks in the mesenehyme, sparing the epithelium and developing Auerbach's plexus. Tenasein distribution was restricted at 9 weeks to the region of the presumptive muscle layer. After the development ofwell-formad villi, tenascin was expressed in the villus core and region of the muscularis mucosee. The 9 week epithelium expressed o.2, e.3, and ~t6, and by 11 weeks expressed ct9. Expression of~x3 and ct6 was accentuated at the basal membrane, suggesting they may play a role in adhesion to the basement membrane. During transition from a pseudostratified epithelium to a simple columnar surface at 13 weeks, a vertical gradient of a2 expression became evident. Crypt cells strongly expressed et2, with decreasing immunoreactivity noted as cells migrated towards the lumenal surface. Mesenchymal cells diffusely expressed ct5 and 0t8 by 9 weeks. Accentuation ofet5 expression was present in muscle, early in the forming museularis proprin, and later with development of the museularis mucosee by 16 weeks. In contrast, ctg expression became localized to the region of the museularis mucosae and extended into the villus core. Vascular elements developing in the meaenehymewere seen as early as 9 weeks, and stained strongly with anti-e.2 and ¢t6. These observations provide evidence both for the complexity and overlap of adhesive receptor expression and ligands during development, and reveal early mesenehymal cell commitment to specific structures.
Growth, Development, and Nutrition A723
ODEVELOPMENT OF 1,4,5-INOSITOL TRISPHOSPHATE RECEPTORS IN THE FELINE GASTRIC ANTRUM. D. Deutsch, K. N. Bitar, C. Hillemeier. Department of Pediatrics University of Michigan, Ann Arbor, MI 48109. We have previously reported that, unlike isolated circular smooth muscle cells from the feline adult gastric antrum, feline newborn antral cells are unable to contract in response to myogenic agonists in the absence of extracellularcalcinm or to exogenous 1,4,5-inositol trisphosphate (IP3) after permeabilization. IP3 binding was determined in gastric antral circular smooth muscle tissue homogenates from adult cats and 10 day old kittens by the evaluating the competitive binding of D-myo-[3H] inositol 1,4,5-trisphosphate and cold 1,4,5-IP3 (0-200 pmoles). Receptor density (Bmax) and binding affinity (Kd) were determined by Scatchard analysis. Bmax was standardized to tissue protein content and expressed as fmol/mg. The binding affinity (Kd) was similar in both age groups at (30.5 nM + 3.5) in the adult and (32.5 nM + 7.6) in the kitten. However the receptor density (Bmax) was markedly decreased in the kitten at (760 fmol/mg + 162.0) compared to the adult (1755 fmol/mg + 251.6). The contractile response to the myogenic agonist cholecystokinin octapeptide (CCK-OP) which is transient in adult cells and sustained in kitten antral cells was used to further examine the functional role of IP3 and it's receptor. We have measured changes in the intracellular levels of 1, 2 diacylglycerol (DAG), an endogenous activator of protein kinase C, and IP3, which is known to release intracellular calcium stores. In adult antral tissue CCKOP causes an early transient increase in the production of both DAG and IP3 while in the newborn antrum CCK-OP causes an early increase in IP3 and a sustained increase in DAG production. U73122, a phospholipase C inhibitor which blocks IP3 production, blocks the initial cona'action in adult antral cells but not the sustained contraction seen in kitten antral cells. We conclude that in feline antral circular smooth muscle ceils the 1,4,5-inositol trisphosphate receptor is characterized by a single binding site in both the kitten and adult cat. The binding affinity (Kd) was similar between the kitten and adult eat, however the receptor density (Bmax) in the kitten is approximately one-half that of the adult cat. The relative inaccessibility of IP3 sensitive intracellular calcium stores is associated with a shift in signal transduction pathways that are followed in response to myogenic agonists such as CCK-OP that results in sustained contraction and sustained production of DAG. We hypothesize that this relative inaccessibility of IP3 sensitive intracellular calcium may be due developmental immaturity of I]?3receptors.
THE DISTRIBUTION OF NITRIC OXIDE (NO) SYNTHESIZING NEURONS IN THE DEVELOPING PIGLET INTESTINE M. Di Lorenzo, and A. Krantis, Digestive Diseases Research Group, Department of Physiology, University of Ottawa, Ottawa (Ontario) K1H 8M5 NO is a putative mediator of intrinsic inhibitory innervation of the gastrointestinal tract. The status of this innervation can profoundly influence gut motility and function. However, little is known about this innervation in the developing gut. We investigated the distribution of NO neurons in myenterie plexus (MP) and in Henle's plexus (HI') of the developing piglet intestine. Method: Laminar preparations of MP and HP of small and large intestine at different stages of development were processed for histoehemical localization of nitrergic neural elements. Piglets 9 days premature, <72 hours old and 2 weeks old were evaluated for the frequency of NO neurons in the duodenum, jejunum, ileum, caecum and colon. Results: In the MP, all 3 stages of development showed an aboral increase in frequency of positive neurons/ganglion from duodenum to colon. However, we observed a significant decrease in frequency of positive neurons with increasing post-conceptional age in all regions of the small bowel. This decrease was also evident for the large bowel, with maximal decrease already evident in the < 72 hour group. HP demonstrated a similar trend towards an increase in frequency in the cranio-eaudal direction. Unlike the MP, premature animals did not have the highest frequency of NO neurons. Rather, this was found in the proximal small bowel of 2 week olds. In the large bowel, though, this number was highest in premature. Conclusion: NO neurons are present in MP and HP of developing piglet intestine. In the MP this frequency increases in the cranio-eaudal direction. There is an overall decrease in these neurons with increasing post-conceptional age. This finding correlates with recent evidence in the developing human gut. However, in HP, there is an opposite trend, with the highest number of NO positive neurons in proximal small bowel of older developing piglets. Our findings likely reflect the developmental variations among the different sources of NO and the change in regulatory potential of the enteric nervous system with age.
• EFFECT OF PROTEIN PRE-DIGESTION ON CHOLECYSTOKININ RELEASE IN CELIAC PATIENTS. P. Deprez, V. Szmata, P. Mainguet, A. Geubel. Gastroenterology Unit, Dept. of Internal Medicine, Cliniques Universitaires Saint-Luc, UCL, 1200 Brussels, Belgium. In celiac sprue cholecystokinin (CCK) release is impaired, which contributes to malabsorption. Whether this defect results from CCK cell dysfunction or from a reduced intraluminal stimulus due to a lack of nutrient's hydrolysis in the jejunum remains controversial. In the latter hypothesis, administration of a predigested diet would correct the defect. Our aim was thus to study the effect of protein pre-digestion on CCK release. Methods. On two separate days, 12 patients with celiac disease, 9 women and 3 men, mean age 39, range 30-45, were given a standard (SondalisR , Clintec) and a semi-elemental (Reabilan", Clintec) liquid diet, The patients were divided in two groups: one with a flat jejunal mucosa (destructive type) (n=6) and one with normal architecture but infiltration of intra-epithelial lymphocytes (infiltrative type) (n=6). A control group (n=9) was also given the two liquid meals. CCK plasma levels were determined at 15 min intervals for 120 min by RIA and expressed as integrated release (IR). Results. Celiac patients had lower basal CCK values than controls: 0.64 _+ 0.1 vs 1.06 _+ 0.2 pmole/I, p<0.05. Both groups had diminished CCK responses compared to the control group (IR 55.6 _+ 11.4, 74.5 _+ 18 and 202 _ 24.9 pmole/I/120 min, respectively, p<0.05). No significant effect of pre-digestion of protein was observed in controls or in celiac patients (IR 62.4 +_20.9, 69.8 + 14.7 and 230.9 +- 34.5 pmole/I/120 rain). Conclusion. Protein pre-digestion does not correct impairement of CCK post-prandial release in celiac patients. Moreover, in celiac patients on gluten-free diet impairement of CCK release persists in spite of improvement in mucosal atrophy.
• IMMUNOLOCALIZATION OF ALPHA INTEGRIN SUBUNITS AND EXTRACELLULAR MATRIX (ECM) COMPONENTS DURING HUMAN COLONIC OR.GANOGENESIS. B.K. Dieck~ranfe, S.A. Santoro, D.H. Alpers. Washington University School of Medicine, St. Louis, MO Morphogenesis is a poorly understood process involving cell adhesion and migration. Intagrins are a family of structurally related transmembrane receptors that participate in cell-cell and cell-ECM interactions. In order to investigate potential integrin roles in human colonic organogenesis, we have examined the tx subunit expression of 131 integrins and extracellular matrix ligands. At 9 weeks, the colon consists ofpseudostratified columnar epithelium surrounded by mesenchyme. Collagen type IV and lamlnin were present underlying the epithelium, around early vascular structures, and surrounding inner circular muscle layer fibers. Fibronectin was uniformly expressed by 9 weeks in the mesenehyme, sparing the epithelium and developing Auerbach's plexus. Tenasein distribution was restricted at 9 weeks to the region of the presumptive muscle layer. After the development ofwell-formad villi, tenascin was expressed in the villus core and region of the muscularis mucosee. The 9 week epithelium expressed o.2, e.3, and ~t6, and by 11 weeks expressed ct9. Expression of~x3 and ct6 was accentuated at the basal membrane, suggesting they may play a role in adhesion to the basement membrane. During transition from a pseudostratified epithelium to a simple columnar surface at 13 weeks, a vertical gradient of a2 expression became evident. Crypt cells strongly expressed et2, with decreasing immunoreactivity noted as cells migrated towards the lumenal surface. Mesenchymal cells diffusely expressed ct5 and 0t8 by 9 weeks. Accentuation ofet5 expression was present in muscle, early in the forming museularis proprin, and later with development of the museularis mucosee by 16 weeks. In contrast, ctg expression became localized to the region of the museularis mucosae and extended into the villus core. Vascular elements developing in the meaenehymewere seen as early as 9 weeks, and stained strongly with anti-e.2 and ¢t6. These observations provide evidence both for the complexity and overlap of adhesive receptor expression and ligands during development, and reveal early mesenehymal cell commitment to specific structures.
Growth, Development, and Nutrition A723
ODEVELOPMENT OF 1,4,5-INOSITOL TRISPHOSPHATE RECEPTORS IN THE FELINE GASTRIC ANTRUM. D. Deutsch, K. N. Bitar, C. Hillemeier. Department of Pediatrics University of Michigan, Ann Arbor, MI 48109. We have previously reported that, unlike isolated circular smooth muscle cells from the feline adult gastric antrum, feline newborn antral cells are unable to contract in response to myogenic agonists in the absence of extracellularcalcinm or to exogenous 1,4,5-inositol trisphosphate (IP3) after permeabilization. IP3 binding was determined in gastric antral circular smooth muscle tissue homogenates from adult cats and 10 day old kittens by the evaluating the competitive binding of D-myo-[3H] inositol 1,4,5-trisphosphate and cold 1,4,5-IP3 (0-200 pmoles). Receptor density (Bmax) and binding affinity (Kd) were determined by Scatchard analysis. Bmax was standardized to tissue protein content and expressed as fmol/mg. The binding affinity (Kd) was similar in both age groups at (30.5 nM + 3.5) in the adult and (32.5 nM + 7.6) in the kitten. However the receptor density (Bmax) was markedly decreased in the kitten at (760 fmol/mg + 162.0) compared to the adult (1755 fmol/mg + 251.6). The contractile response to the myogenic agonist cholecystokinin octapeptide (CCK-OP) which is transient in adult cells and sustained in kitten antral cells was used to further examine the functional role of IP3 and it's receptor. We have measured changes in the intracellular levels of 1, 2 diacylglycerol (DAG), an endogenous activator of protein kinase C, and IP3, which is known to release intracellular calcium stores. In adult antral tissue CCKOP causes an early transient increase in the production of both DAG and IP3 while in the newborn antrum CCK-OP causes an early increase in IP3 and a sustained increase in DAG production. U73122, a phospholipase C inhibitor which blocks IP3 production, blocks the initial cona'action in adult antral cells but not the sustained contraction seen in kitten antral cells. We conclude that in feline antral circular smooth muscle ceils the 1,4,5-inositol trisphosphate receptor is characterized by a single binding site in both the kitten and adult cat. The binding affinity (Kd) was similar between the kitten and adult eat, however the receptor density (Bmax) in the kitten is approximately one-half that of the adult cat. The relative inaccessibility of IP3 sensitive intracellular calcium stores is associated with a shift in signal transduction pathways that are followed in response to myogenic agonists such as CCK-OP that results in sustained contraction and sustained production of DAG. We hypothesize that this relative inaccessibility of IP3 sensitive intracellular calcium may be due developmental immaturity of I]?3receptors.
THE DISTRIBUTION OF NITRIC OXIDE (NO) SYNTHESIZING NEURONS IN THE DEVELOPING PIGLET INTESTINE M. Di Lorenzo, and A. Krantis, Digestive Diseases Research Group, Department of Physiology, University of Ottawa, Ottawa (Ontario) K1H 8M5 NO is a putative mediator of intrinsic inhibitory innervation of the gastrointestinal tract. The status of this innervation can profoundly influence gut motility and function. However, little is known about this innervation in the developing gut. We investigated the distribution of NO neurons in myenterie plexus (MP) and in Henle's plexus (HI') of the developing piglet intestine. Method: Laminar preparations of MP and HP of small and large intestine at different stages of development were processed for histoehemical localization of nitrergic neural elements. Piglets 9 days premature, <72 hours old and 2 weeks old were evaluated for the frequency of NO neurons in the duodenum, jejunum, ileum, caecum and colon. Results: In the MP, all 3 stages of development showed an aboral increase in frequency of positive neurons/ganglion from duodenum to colon. However, we observed a significant decrease in frequency of positive neurons with increasing post-conceptional age in all regions of the small bowel. This decrease was also evident for the large bowel, with maximal decrease already evident in the < 72 hour group. HP demonstrated a similar trend towards an increase in frequency in the cranio-eaudal direction. Unlike the MP, premature animals did not have the highest frequency of NO neurons. Rather, this was found in the proximal small bowel of 2 week olds. In the large bowel, though, this number was highest in premature. Conclusion: NO neurons are present in MP and HP of developing piglet intestine. In the MP this frequency increases in the cranio-eaudal direction. There is an overall decrease in these neurons with increasing post-conceptional age. This finding correlates with recent evidence in the developing human gut. However, in HP, there is an opposite trend, with the highest number of NO positive neurons in proximal small bowel of older developing piglets. Our findings likely reflect the developmental variations among the different sources of NO and the change in regulatory potential of the enteric nervous system with age.