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Colocalization of vasoactive intestinal polypeptide (VIP)and nitric oxide synthase (NOS) in the human antrum
Al182 AGA ABSTRACTS • G4832 PROTON PUMP INHIBITORS ENHANCE PROGRESSION THROUGH THE ADENOMA CARCINOMA SEQUENCE AND DECREASES SURVIVAL IN THE M I N MOUSE MODEL OF FAP. A.M. Smith. S.A. Watson. The Academic Unit of Cancer Studies, Dept of Surgery, Nottingham University, UK.
Introduction. Gastrin is a potent mitogen for colorectal epithelium and tumours. Over-expression of the gastrin/CCKB receptor has been shown in hyperproliferative colonic epithelium and all grades of adenomas. Thus a hyper-gastrinaemic state may promote progression through the adenoma -carcinoma sequence. Aim. To determine the effect of omeprazole-induced hypergastrinaemia on the progression of intestinal neoplasia in the Min (multiple intestinal neoplasia) mouse model of polyposis coil Methods. Mice were entered into the study when PCR analysis confirmed the Min+ genotype. Min mice were randomised to 4 groups to receive: omeprazole (75mg/kg, daily oral treatmen0; omeprazole + Gastrimmune (a gastrin immunogen inducing the in situ formation of anti-gastrin antibodies, 100lag/mouse, day 0 and every 3 weeks). Control mice received oral vehicle control +[- a control immunogen. Serum gastrin levels were measured by radioimmunoassay, Prior to termination, bromodeoxyuridine was administered to generate a proliferative index. Results. Treatment with omeprazole resulted in serum gastrin levels of 236pg/ml compared to 67pg/ml in the untreated group. Hypergastrinaemia significantly decreased survival compared to the appropriate control (p=0.0001, Log rank test) with mice in the control group having a 50% survival of 9.5 weeks compared to 6 weeks in the omeprazole treated group. Gastrimmune induced the formation of serum antibodies with an antigen binding capacity of >20ng/ml resulting in effective neutraiisation of the hypergastrinaemic state. Gastrin neutralisation resulted in a reversal of the survival disadvantage induced by omeprazole (p---0.0017). The hypergastrinaemic mice had enhanced proliferation of normal colonic mucosa (9.46% proliferating cells increased to 20.1%, p<0.05, Mann Whitney) and colonic neoplasia (22.3% increased to 35.0%, p<0.01). Conclusions. The level of hypergastrinaemia was in the range attained in humans on a maintenance dose of omeprazole and resulted in enhanced progression through the adenoma - carcinoma sequence. Gastrin was confirmed as the mediator inducing a state of hyper-proliferation within both normal and neoplastic colonic epithelium. This study highlights the need to assess the safety of hypergastrinaemia on the pre-malignant colon. This research was funded by Aphton Corporation, California, USA. G4833 SERUM GASTRIN LEVELS ARE NORMAL IN PATIENTS WITH COLORECTAL POLYPS. AM Smith 1, A Varro2, SA Watson 1. 1The Academic Unit of Cancer Studies, Department of Surgery, University of Nottingham, UK. 2Department of Physiology, Liverpool University, UK.
Introduction. Autocrine gastrin is produced by the majority of colorectal carcinomas. This local production of gastrin causes elevation of nonamidated gastrin in the circulation of patients with colorectal cancer, when confounding factors are controlled for. Recently gastrin has been demonstrated by RT-PCR and immunocytochemistry to be present in most colorectal polyps, irrespective of size and histological type. Aim. To determine whether serum gastrin levels are elevated in patients with colorectal polyps. Methods. After an overnight fast blood samples were obtained from 23 patients with polyps and 20 Helicobacter pylori negative control subjects, and 12 Helicobacter pylori positive asymptomatic control subjects. Subjects with a history of gastric or duodenal disease were excluded. Helicobacter pylori status was assessed by ELISA. Region specific antisera were used to measure progastrin, glycine-extended gastrin-17 and amidated gastrin-17 by radioimmunoassay. Results, 3/23 patients with colorectal polyps were Helicobacterpylori positive.
i No.
Progastrin Gly- G17 I G17-NH2 (lamol/1) (pmol/l) (pmol/l) Control HP+ 12 0 0 0.029 (0.014, 0.037) Polyp HP+ 3 0.02 0 [ 0.022 Control HP- I 20 0.021 (0, 0.035) 0 I 0.007 (0.004, 0.008) Polyp HP20 0.018 (0, 0.025) 0 0.025 (0.012, 0.048) Median (inter-quartile range). Using Mann-Whitney u-test there was no significant difference between the groups. Conclusions. Despite the production of autocrine gastrin in polyps, there is not reflected by an elevation in circulating gastrin. It is likely the amount of gastrin produced by a small polyp is negligible when secreted into the systemic circulation. This research was funded by Aphton Corporation, California, USA.
GASTROENTEROLOGYVol. 114, No. 4 G4834 COLOCALIZATION OF VASOACTIVE INTESTINAL POLYPEPITDE (VIP)AND NITRIC OXIDE SYNTHASE (NOS) IN THE HUMAN ANTRUM. V,C. Smith and A.M.J. Buchan, Department of Physiology, University of British Columbia, Vancouver, Canada.
Nitric oxide has been shown to mediate relaxation in rat gastric fundus and corpus smooth muscle and to influence endocrine and exocrine secretions. Recent studies have demonstrated a co-localization of VIP and NO in enteric inhibitory neurons and both are co-released resulting in smooth muscle relaxation. Although VIP and NO are known to be colocalized in the innervation of the muscle; secretomotor neurons supplying the mucosa are thought to be VIP-immunoreactive (VIP-IR) only. In human antrum, the distribution of VIP and NO has not been established. The distribution of the enzyme NOS is commonly used to define NO producing neural elements. The present study uses immunocytochemical techniques, confocal microscopy and three-dimensional reconstructions to establish the relationship between VIP-NOS- and gastrin-immunoreactive (IR) structures in human antrum, Antral tissue from transplanl donors (N=5) were fixed in 4% paraformaldehyde for 2h. Cryostat sections (40/aM) were immunostained with a rabbit anti-brain nitric oxide synthase (Transduction Laboratories) and monoclonal antibodies to either VIP (UBC) or gastrin (CURE) for 48h at 4°C. The bound antibodies were localized using either FITC anti mouse IgG or a goat anti-rabbit IgG conjugated to Cy3. The single immunostains demonstrated the presence of V/P- and NOSimmunoreactive nerves in all layers of the antrum. Positive fibres were most abundant in the pyloric sphincter region, although, a significant innervation of the mucosa, muscularis mucosae and smooth muscle layers was observed, Double irnmunostaining demonstrated that NOS-IR neurons in the myenteric plexus were not VIP-IR although the neurons were surrounded by VIP-IR fibres, few VIP-IR cell bodies were observed in either myenteric or submucous plexi. In the mucosa, muscularis rnucosae and muscle layers NOSand VIP-IR were colocalized in fibres. Mucosal fibres were concentrated around the base of the gastric glands with few fibres detected in the upper regions of the mucosa. 3-D reconstructions demonstrated that NOS-IR fibres were not in close association with gastrin-IR endocrine ceils. These data demonstrate for the first time the presence of NOS-IR fibres in the mucosal region of the stomach. Funded by NSERC. G4835 CHANGES IN EXTRACELLULAR Ca-~-SENSING RECEPTOR (CAR) EXPRESSION IN COLON EPITHELIAL PROLIFERATION. SJ. Soot, M.J. Rutten, K.D. Bacon, C.L. Meichsner, B.C. Sheppard, R.A. Crass, K.E. Deveney, F.P. Lee, S,A. Hobson, C.W. Deveney, and K.D. Rodland. VA Med. Center, Portland, OR; Depts. of Cell Biology and Surgery, Oregon Health Sciences University, Portland, OR, 97201.
The CaR has been identified in human and rodent intestinal tissue. Besides its possible role in intestinal Ca2÷-absorption, other roles for the CaR have yet to be defined. It is known that Ca2÷ ions can affect colonic epithelial proliferation. However, it is unknown if the CaR is involved in this colonic proliferative process. The aim of the present study was to determine if CaR expression is altered in normal and cancerous colonic cell proliferation. Normal, polyposis, and cancerous colonic ceils were isolated and cultured from surgical tissues. Caco-2, T-84, LoVo cell lines (ATCC) were cultured in DMEM media. Normal, polyposis, and cancerous colonic tissues were obtained from surgical specimens and paraffin-embedded. Cell growth was measured by MTr-assays. Immunohistochemistry, Western-blotting, and confocal microscopy were used for CaR detection. Immunohistochemistry of normal colonic tissue showed light CaR staining in colonic villus ceils, with moderate and heavy CaR immunostaining in polyposis and cancerous colonic cells, respectively. Positive staining was eliminated by antibody preabsorption with CaR protein. Confocal microscopy of cultured normal and Caco-2 colonic cells for CaR showed light irnmunofluorescence at the apical membrane with heavier fluorescence at the basolateral membrane. Cultured polyposis and LoVo cells showed positive fluorescence around the plasma membrane, with the T-84 cell line showing weak CaR immunofluorescence. Western-blotting of cell cultures detected two bands at 120kDa and 135kDa. Preconfluent cultures of normal colonic cells in serum-free media had low CaR expression with little change of CaR in AGS cells. The mitogenic response of the cultured cells to extracellular Ca2÷ ([Ca2+]o 0-5mM) was greatest with normal colon cells and smallest with the LoVo cell line. Except for T-84 cells, there was a direct relationship between [Ca2÷]o-mitogenic responsiveness and CaR expression. These data suggest the CaR may have an important role in colonic epithelial growth. • G4836 SUBSTANCE P INDUCES NEUROKININ I RECEPTOR ENDOCYTOSIS IN INTERSTITIAL CELLS OF CAJAL IN GUINEAPIG SMALL INTESTINE. B.R. Southwell, S.T. Lavin, and J.B. Furness. Department of Anatomy and Cell Biology, University of Melbourne, Parkville, 3052, Australia.
Interstitial cell of Cajal (ICC) are fusiforrn cells forming self connecting plexuses and tight junctions with muscle ceils. They are thought to generate or coordinate the rhythmic activity of muscle contraction and to form an
Introduction. Gastrin is a potent mitogen for colorectal epithelium and tumours. Over-expression of the gastrin/CCKB receptor has been shown in hyperproliferative colonic epithelium and all grades of adenomas. Thus a hyper-gastrinaemic state may promote progression through the adenoma -carcinoma sequence. Aim. To determine the effect of omeprazole-induced hypergastrinaemia on the progression of intestinal neoplasia in the Min (multiple intestinal neoplasia) mouse model of polyposis coil Methods. Mice were entered into the study when PCR analysis confirmed the Min+ genotype. Min mice were randomised to 4 groups to receive: omeprazole (75mg/kg, daily oral treatmen0; omeprazole + Gastrimmune (a gastrin immunogen inducing the in situ formation of anti-gastrin antibodies, 100lag/mouse, day 0 and every 3 weeks). Control mice received oral vehicle control +[- a control immunogen. Serum gastrin levels were measured by radioimmunoassay, Prior to termination, bromodeoxyuridine was administered to generate a proliferative index. Results. Treatment with omeprazole resulted in serum gastrin levels of 236pg/ml compared to 67pg/ml in the untreated group. Hypergastrinaemia significantly decreased survival compared to the appropriate control (p=0.0001, Log rank test) with mice in the control group having a 50% survival of 9.5 weeks compared to 6 weeks in the omeprazole treated group. Gastrimmune induced the formation of serum antibodies with an antigen binding capacity of >20ng/ml resulting in effective neutraiisation of the hypergastrinaemic state. Gastrin neutralisation resulted in a reversal of the survival disadvantage induced by omeprazole (p---0.0017). The hypergastrinaemic mice had enhanced proliferation of normal colonic mucosa (9.46% proliferating cells increased to 20.1%, p<0.05, Mann Whitney) and colonic neoplasia (22.3% increased to 35.0%, p<0.01). Conclusions. The level of hypergastrinaemia was in the range attained in humans on a maintenance dose of omeprazole and resulted in enhanced progression through the adenoma - carcinoma sequence. Gastrin was confirmed as the mediator inducing a state of hyper-proliferation within both normal and neoplastic colonic epithelium. This study highlights the need to assess the safety of hypergastrinaemia on the pre-malignant colon. This research was funded by Aphton Corporation, California, USA. G4833 SERUM GASTRIN LEVELS ARE NORMAL IN PATIENTS WITH COLORECTAL POLYPS. AM Smith 1, A Varro2, SA Watson 1. 1The Academic Unit of Cancer Studies, Department of Surgery, University of Nottingham, UK. 2Department of Physiology, Liverpool University, UK.
Introduction. Autocrine gastrin is produced by the majority of colorectal carcinomas. This local production of gastrin causes elevation of nonamidated gastrin in the circulation of patients with colorectal cancer, when confounding factors are controlled for. Recently gastrin has been demonstrated by RT-PCR and immunocytochemistry to be present in most colorectal polyps, irrespective of size and histological type. Aim. To determine whether serum gastrin levels are elevated in patients with colorectal polyps. Methods. After an overnight fast blood samples were obtained from 23 patients with polyps and 20 Helicobacter pylori negative control subjects, and 12 Helicobacter pylori positive asymptomatic control subjects. Subjects with a history of gastric or duodenal disease were excluded. Helicobacter pylori status was assessed by ELISA. Region specific antisera were used to measure progastrin, glycine-extended gastrin-17 and amidated gastrin-17 by radioimmunoassay. Results, 3/23 patients with colorectal polyps were Helicobacterpylori positive.
i No.
Progastrin Gly- G17 I G17-NH2 (lamol/1) (pmol/l) (pmol/l) Control HP+ 12 0 0 0.029 (0.014, 0.037) Polyp HP+ 3 0.02 0 [ 0.022 Control HP- I 20 0.021 (0, 0.035) 0 I 0.007 (0.004, 0.008) Polyp HP20 0.018 (0, 0.025) 0 0.025 (0.012, 0.048) Median (inter-quartile range). Using Mann-Whitney u-test there was no significant difference between the groups. Conclusions. Despite the production of autocrine gastrin in polyps, there is not reflected by an elevation in circulating gastrin. It is likely the amount of gastrin produced by a small polyp is negligible when secreted into the systemic circulation. This research was funded by Aphton Corporation, California, USA.
GASTROENTEROLOGYVol. 114, No. 4 G4834 COLOCALIZATION OF VASOACTIVE INTESTINAL POLYPEPITDE (VIP)AND NITRIC OXIDE SYNTHASE (NOS) IN THE HUMAN ANTRUM. V,C. Smith and A.M.J. Buchan, Department of Physiology, University of British Columbia, Vancouver, Canada.
Nitric oxide has been shown to mediate relaxation in rat gastric fundus and corpus smooth muscle and to influence endocrine and exocrine secretions. Recent studies have demonstrated a co-localization of VIP and NO in enteric inhibitory neurons and both are co-released resulting in smooth muscle relaxation. Although VIP and NO are known to be colocalized in the innervation of the muscle; secretomotor neurons supplying the mucosa are thought to be VIP-immunoreactive (VIP-IR) only. In human antrum, the distribution of VIP and NO has not been established. The distribution of the enzyme NOS is commonly used to define NO producing neural elements. The present study uses immunocytochemical techniques, confocal microscopy and three-dimensional reconstructions to establish the relationship between VIP-NOS- and gastrin-immunoreactive (IR) structures in human antrum, Antral tissue from transplanl donors (N=5) were fixed in 4% paraformaldehyde for 2h. Cryostat sections (40/aM) were immunostained with a rabbit anti-brain nitric oxide synthase (Transduction Laboratories) and monoclonal antibodies to either VIP (UBC) or gastrin (CURE) for 48h at 4°C. The bound antibodies were localized using either FITC anti mouse IgG or a goat anti-rabbit IgG conjugated to Cy3. The single immunostains demonstrated the presence of V/P- and NOSimmunoreactive nerves in all layers of the antrum. Positive fibres were most abundant in the pyloric sphincter region, although, a significant innervation of the mucosa, muscularis mucosae and smooth muscle layers was observed, Double irnmunostaining demonstrated that NOS-IR neurons in the myenteric plexus were not VIP-IR although the neurons were surrounded by VIP-IR fibres, few VIP-IR cell bodies were observed in either myenteric or submucous plexi. In the mucosa, muscularis rnucosae and muscle layers NOSand VIP-IR were colocalized in fibres. Mucosal fibres were concentrated around the base of the gastric glands with few fibres detected in the upper regions of the mucosa. 3-D reconstructions demonstrated that NOS-IR fibres were not in close association with gastrin-IR endocrine ceils. These data demonstrate for the first time the presence of NOS-IR fibres in the mucosal region of the stomach. Funded by NSERC. G4835 CHANGES IN EXTRACELLULAR Ca-~-SENSING RECEPTOR (CAR) EXPRESSION IN COLON EPITHELIAL PROLIFERATION. SJ. Soot, M.J. Rutten, K.D. Bacon, C.L. Meichsner, B.C. Sheppard, R.A. Crass, K.E. Deveney, F.P. Lee, S,A. Hobson, C.W. Deveney, and K.D. Rodland. VA Med. Center, Portland, OR; Depts. of Cell Biology and Surgery, Oregon Health Sciences University, Portland, OR, 97201.
The CaR has been identified in human and rodent intestinal tissue. Besides its possible role in intestinal Ca2÷-absorption, other roles for the CaR have yet to be defined. It is known that Ca2÷ ions can affect colonic epithelial proliferation. However, it is unknown if the CaR is involved in this colonic proliferative process. The aim of the present study was to determine if CaR expression is altered in normal and cancerous colonic cell proliferation. Normal, polyposis, and cancerous colonic ceils were isolated and cultured from surgical tissues. Caco-2, T-84, LoVo cell lines (ATCC) were cultured in DMEM media. Normal, polyposis, and cancerous colonic tissues were obtained from surgical specimens and paraffin-embedded. Cell growth was measured by MTr-assays. Immunohistochemistry, Western-blotting, and confocal microscopy were used for CaR detection. Immunohistochemistry of normal colonic tissue showed light CaR staining in colonic villus ceils, with moderate and heavy CaR immunostaining in polyposis and cancerous colonic cells, respectively. Positive staining was eliminated by antibody preabsorption with CaR protein. Confocal microscopy of cultured normal and Caco-2 colonic cells for CaR showed light irnmunofluorescence at the apical membrane with heavier fluorescence at the basolateral membrane. Cultured polyposis and LoVo cells showed positive fluorescence around the plasma membrane, with the T-84 cell line showing weak CaR immunofluorescence. Western-blotting of cell cultures detected two bands at 120kDa and 135kDa. Preconfluent cultures of normal colonic cells in serum-free media had low CaR expression with little change of CaR in AGS cells. The mitogenic response of the cultured cells to extracellular Ca2÷ ([Ca2+]o 0-5mM) was greatest with normal colon cells and smallest with the LoVo cell line. Except for T-84 cells, there was a direct relationship between [Ca2÷]o-mitogenic responsiveness and CaR expression. These data suggest the CaR may have an important role in colonic epithelial growth. • G4836 SUBSTANCE P INDUCES NEUROKININ I RECEPTOR ENDOCYTOSIS IN INTERSTITIAL CELLS OF CAJAL IN GUINEAPIG SMALL INTESTINE. B.R. Southwell, S.T. Lavin, and J.B. Furness. Department of Anatomy and Cell Biology, University of Melbourne, Parkville, 3052, Australia.
Interstitial cell of Cajal (ICC) are fusiforrn cells forming self connecting plexuses and tight junctions with muscle ceils. They are thought to generate or coordinate the rhythmic activity of muscle contraction and to form an