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Gastric acid secretion by non-gastrin acid stimulating peptide in islet cell tumor extracts is mediated by release of histamine from gastric ECL cells in rats
A1008
AGA ABSTRACTS
• GASTRIC ACID SECRETION BY NON-GASTRIN ACID STIMULATING PEPTIDE IN ISLET CELL TUMOR EXTRACTS IS MEDIATED BY RELEASE OF HISTAMINE PROM GASTRIC ECL CELLS IN RATS. Y Sonq, T-M Chang, KY Lee, WY Chey. University of Rochester Medical Center, Rochester, NY A non-gastrin acid stimulating peptide (N-GASP) in islet cells tumor extracts (TE) of patients with gastric acid hypersecretion and peptic ulceration without hypergastrinemia, was shown not only tO stimulate acid secretion, but also potentiates pentagastrin-stimulated acid secretion in anesthetized rats (Song et al, Pancreas 9:805,1994). Like in patients with gastnnoma, H2 receptor antagonists suppress profoundly the acid hypersecretion in these patients and that induced by N-GASP in rats. To determine if N-GASP stimulates the release of histamine from ECL cells, we prepared highly purified rat gastric mucosa ECL cells. Rat stomach fundic mucosal cells were dissociated by protease-EDTA digestion and ECL cells were fractionalized by elutdation centrifugation and further enriched by density gradient centdfugation. The purity of ECL cell fraction was as high as 83_-1:8.1%.Histamine-containing cells were confirmed by immunoeytochemistry using antibody against histamine. One ml each of ECL cell suspension (lx106 cells) were incubated at 37~ C for 60 rain in basal or stimulated conditions. Histamine release and content in cells were determined by histamine radioimmunoassay, Both gastrin-17 (10-8M) and TE (10 rag) significantly stimulated histamine release. CCK-B receptor antagonist L365,260 (10-5M) completely abolished G-17 induced histamine release, whereas it failed to prevent that stimulated by TE. It strongly suggests that TE containing N-GASP stimulates ECL cell without acting on gastrin receptors of ECL cells. alone (%) L365,260 (%) 9asal 100.00 100.00 '7 184.39-J:10.69" 104.75+1.60 302.24+33.69" 317,36:tr.26.79" , a, u e are Mean + SE percentage change of histamine release. Conclusion: The acid secretion stimulated by N-GASP in TE of patients with islet ceil tumors appears to b e mediated by the release of histamine from ECL cells via a non-gastdn receptor pathway.
GASTROENTEROLOGY, Vol. 108, No. 4
• CELLULAR SITES OF EXPRESSION OF THE NEUROKININ 1 RECEPTOR (NKIR) IN THE GUINEA PIG ILEUM. C. Sternini. D. Su, N.C. Breeha, N.W. Bunnett. CURE:VA/UCLA Gastroenterie Biology Center and Depts. of Medicine, and Anatomy & Cell Biology, UCLA School of Medicine, and VAMC West-Los Angeles, CA, and Depts. of Surgery and Physiology, UCSF, San Francisco, CA, USA. The guinea pig enteric nervous system contains a large substance P (SP) neuronal cell population, and SP has beenindicated as an excitatory neurolransmitter of myenterie neurons as well as a neuroneuronal and neuromuscular transmitter. SP has high affinity for the NKIR, and lower affinity for NK2R and NK3R. The aim of this study was to identify the cellular sites of expression of the NKIR in the guinea pi~ ileum with an antibody to the intraeeilular portion of this receptor and tmmunohistochemistry. NKIR immunoreaetivity (IR) is present in a large populatiou of myeaterie and submucosalneurons. Two types of neurous were identified. One type is characterized by a smooth cell body with 1 or 2 long and smooth processes which can be followed within and between ganglia. Another type has an ovoid cell body with one long a~ou and many short and broad-base dendritic prueesses. A few NKIR1R processes are seen at the base of the mueosa, whereas the vaseulature is devoid ofNKIR innervatiou. In the inner portion of the circular muscle, there are numerous NKIR-IR non-neuronal cells with large and elongated cell bodies and many long processes. These cells have the appearance of interstitial cells of Cajal (ICC) and are wrapped by a dense network of processes. Double labeling showed that enteric NK~R-IR neurons are wrapped by a dense network of SP-IR fibers. SP-IR processes are also found in close vicinity to the presumed NK]R ICCs. The NKIR expression pattern in the guinea pig differs from that observed in the rat, where NKIR-IR is found only in one type of enteric neurons, i.e. those with smooth cell body and I or 2 long processes. Other differences include the presence of NK IR-IR processes in the mueosa and the morphological appearance of the non-neuronal cells in the cireuler muscle. As for the rat, tbe cellular sites of expressiou of the NKIR in the guinea pig indicates that this receptor is the mediator of SP actions on enteric neurons and smooth muscle. However, it is likely that there are differences in the mechanisms of action through which SP influences neuroneuronal and neuromuscular transmission in these two species as indicated by the different expression pattern of NK1-R. Supportedby the Morphology/ImagingCoreDK41301and DK43207and NS21710. The NK1-R antibodywas Inovidedby S. Vignaof DukeUniversity.
• EVIDENCE FOR A ROLE OF NEUROTROPH1N-3 IN THE ADULT DIGESTIVE SYSTEM. C- Stemini, D. Su, J. Arakawa, R. De Giorgio, N.C. Breeha. CURE:VA/UCLA Gastroenterie Biology Center, and Departments of Medicine and Anatomy & Cell Biology, UCLA and VAMC-West Los Angeles, Los Angeles, CA, USA and Department of Clinical Pharmacology and Therapeutics, University of Bologna, Italy, Neurotmphins are target-derived neumtmphic molecules that.support neuronal survival, differentiation and outgrowth and exert their etteets via high-affinity receptors. The products of ~ e trk pmto-oncogene . . family, trkA trkB and trkc correspond to the lfign-amnity receptors thin mediate the ~ffects of the iieurotrophins, nerve growth factor (NGF), brain derived neurotrophic factor (BDNF) and neum.tro.phin-3.(NT-3). irkA is the preferred receptor for NGF, and trka and tr~care the preferred receptors for BDNF and NT-3, respectively. However, different neurotrophins can interact with the same re;eeptor, but with , varying affinity. Neurotrophins and their receptors nave a wtuespreaa. distribution in the developing and adult nervous system, suggesting mat neurotrophins have other functions in addition to their well established role in neuronal development. As a first step to test the hypothesis that neumtrophins play a role in the adult digestive system, we invesfigatco the cellular localization of neurotrophin receptors in the rat gastrointestinal tract with a) immunohistoehemistry...anda pan-trk antibody that recognizes all Irks and b) in situ hybndizaUou histochemistt7 with RNA probes specific for each trio. trK immnnoreacttvity (IR) is found in a large proportion of myanteric and submucosal neurons and in interconnecting strands. Abundant tr~-IR fibers innervate the muscle, mucosa, and vasculature. IR cell bodies are encircled by dense networks of fibers that obscure their shape, trkc mRNA is expressed in neurons of the myenteric and submueosal.plexus. By contrast, trkA and trkB mRNAs could not be detected m enteric neurons. All three trk mRNAs are expressed in dorsal root ganglia, which were used as positive controls. These findings suggest that 1) neurotrophins play a role in the adult gastrointestinal tract, perhaps being involved in tissue maintenance and differentiation and 2) NT-3, the preferred ligand for trkc, is likely to be the relevant neurotrophin in the gut, even though an involvement of other neurotrophins cannot be completely excluded at this time. Supported by Morphology/Imaging Core DK41301 and VA Research Medical Funds.
IMMUNOHISTOLOGICAL LOCALIZATION OF GASTRIN RECEPTORS IN ANTRAL GASTRIC MUCOSA IN MAN. Oh. Stettler 1, A. Schmassmann 1, N. I. Tarasova 2, L. Varga 1, F. Halter 1. 1 : Gastrointestinal Unit, Univ. Hospital, Inselspital, Bern, Switzerland. 2: ABL-Basic Research Program, National Cancer Institute, FCRDC, Frederick, MD, USA. B a e k a r o u n d : 1) It has been reported, that growth of antral G (gastrin) cells is not only stimulated by gastric hypoa¢idity but also by hypergastrinemia. 2) Experimental ulcer healing studies suggest, that gastrin receptors do not play a relevant role in cell proliferation at the ulcer margin (Schrnassmann A et al 1994, Gut: 35: 896-904). In addition, radio ligand autoradiography revealed a persistent lack of somatostatin receptors in the ulcer margin. Aims: 1) Localization of gastrin receptors on G and D cells. 2) Detection of differences in density of cells expressing gastrin receptors between non-ulcerated and ulcerated mucosa. M e t h o d s : - Specific gastrin receptor antibodies have been recently developed (Gastroenterology 1994; 106: A845). Consecutive paraffin sections (2 t~m thick) of human nonulcerated and ulcerated antral gastric mucosa have been immunohistochemically stained using antibodies against gastrin receptor, gastrin and sornatostatin. Results: 1) In the non-ulcerated mucosa, gastrin receptors were mainly located in ceils of the upper portion of the antral glands. Using antibodies against gastrin and sornatostatin, we could demonstrate in consecutive sections, that gastrin receptors are predominantly located on G cells. 2) In contrast to the nonulcerated area, epithelial cells in the ulcer margin did not show cells expressing gastrin receptors, gastri n or somatostatin. C o n c l u s i o n : 1) Human antral G cells show immunoreactivity to gastrin receptor antibodies. 2) Cells showing immunoreactJvity to gastrin receptor antibodies are absent in the ulcer margin.
Supported by Swiss National Science Foundation (No. 32-040901.94) and National Cancer Institute, DHHS (No. N01-CO-46000 with ABL).
AGA ABSTRACTS
• GASTRIC ACID SECRETION BY NON-GASTRIN ACID STIMULATING PEPTIDE IN ISLET CELL TUMOR EXTRACTS IS MEDIATED BY RELEASE OF HISTAMINE PROM GASTRIC ECL CELLS IN RATS. Y Sonq, T-M Chang, KY Lee, WY Chey. University of Rochester Medical Center, Rochester, NY A non-gastrin acid stimulating peptide (N-GASP) in islet cells tumor extracts (TE) of patients with gastric acid hypersecretion and peptic ulceration without hypergastrinemia, was shown not only tO stimulate acid secretion, but also potentiates pentagastrin-stimulated acid secretion in anesthetized rats (Song et al, Pancreas 9:805,1994). Like in patients with gastnnoma, H2 receptor antagonists suppress profoundly the acid hypersecretion in these patients and that induced by N-GASP in rats. To determine if N-GASP stimulates the release of histamine from ECL cells, we prepared highly purified rat gastric mucosa ECL cells. Rat stomach fundic mucosal cells were dissociated by protease-EDTA digestion and ECL cells were fractionalized by elutdation centrifugation and further enriched by density gradient centdfugation. The purity of ECL cell fraction was as high as 83_-1:8.1%.Histamine-containing cells were confirmed by immunoeytochemistry using antibody against histamine. One ml each of ECL cell suspension (lx106 cells) were incubated at 37~ C for 60 rain in basal or stimulated conditions. Histamine release and content in cells were determined by histamine radioimmunoassay, Both gastrin-17 (10-8M) and TE (10 rag) significantly stimulated histamine release. CCK-B receptor antagonist L365,260 (10-5M) completely abolished G-17 induced histamine release, whereas it failed to prevent that stimulated by TE. It strongly suggests that TE containing N-GASP stimulates ECL cell without acting on gastrin receptors of ECL cells. alone (%) L365,260 (%) 9asal 100.00 100.00 '7 184.39-J:10.69" 104.75+1.60 302.24+33.69" 317,36:tr.26.79" , a, u e are Mean + SE percentage change of histamine release. Conclusion: The acid secretion stimulated by N-GASP in TE of patients with islet ceil tumors appears to b e mediated by the release of histamine from ECL cells via a non-gastdn receptor pathway.
GASTROENTEROLOGY, Vol. 108, No. 4
• CELLULAR SITES OF EXPRESSION OF THE NEUROKININ 1 RECEPTOR (NKIR) IN THE GUINEA PIG ILEUM. C. Sternini. D. Su, N.C. Breeha, N.W. Bunnett. CURE:VA/UCLA Gastroenterie Biology Center and Depts. of Medicine, and Anatomy & Cell Biology, UCLA School of Medicine, and VAMC West-Los Angeles, CA, and Depts. of Surgery and Physiology, UCSF, San Francisco, CA, USA. The guinea pig enteric nervous system contains a large substance P (SP) neuronal cell population, and SP has beenindicated as an excitatory neurolransmitter of myenterie neurons as well as a neuroneuronal and neuromuscular transmitter. SP has high affinity for the NKIR, and lower affinity for NK2R and NK3R. The aim of this study was to identify the cellular sites of expression of the NKIR in the guinea pi~ ileum with an antibody to the intraeeilular portion of this receptor and tmmunohistochemistry. NKIR immunoreaetivity (IR) is present in a large populatiou of myeaterie and submucosalneurons. Two types of neurous were identified. One type is characterized by a smooth cell body with 1 or 2 long and smooth processes which can be followed within and between ganglia. Another type has an ovoid cell body with one long a~ou and many short and broad-base dendritic prueesses. A few NKIR1R processes are seen at the base of the mueosa, whereas the vaseulature is devoid ofNKIR innervatiou. In the inner portion of the circular muscle, there are numerous NKIR-IR non-neuronal cells with large and elongated cell bodies and many long processes. These cells have the appearance of interstitial cells of Cajal (ICC) and are wrapped by a dense network of processes. Double labeling showed that enteric NK~R-IR neurons are wrapped by a dense network of SP-IR fibers. SP-IR processes are also found in close vicinity to the presumed NK]R ICCs. The NKIR expression pattern in the guinea pig differs from that observed in the rat, where NKIR-IR is found only in one type of enteric neurons, i.e. those with smooth cell body and I or 2 long processes. Other differences include the presence of NK IR-IR processes in the mueosa and the morphological appearance of the non-neuronal cells in the cireuler muscle. As for the rat, tbe cellular sites of expressiou of the NKIR in the guinea pig indicates that this receptor is the mediator of SP actions on enteric neurons and smooth muscle. However, it is likely that there are differences in the mechanisms of action through which SP influences neuroneuronal and neuromuscular transmission in these two species as indicated by the different expression pattern of NK1-R. Supportedby the Morphology/ImagingCoreDK41301and DK43207and NS21710. The NK1-R antibodywas Inovidedby S. Vignaof DukeUniversity.
• EVIDENCE FOR A ROLE OF NEUROTROPH1N-3 IN THE ADULT DIGESTIVE SYSTEM. C- Stemini, D. Su, J. Arakawa, R. De Giorgio, N.C. Breeha. CURE:VA/UCLA Gastroenterie Biology Center, and Departments of Medicine and Anatomy & Cell Biology, UCLA and VAMC-West Los Angeles, Los Angeles, CA, USA and Department of Clinical Pharmacology and Therapeutics, University of Bologna, Italy, Neurotmphins are target-derived neumtmphic molecules that.support neuronal survival, differentiation and outgrowth and exert their etteets via high-affinity receptors. The products of ~ e trk pmto-oncogene . . family, trkA trkB and trkc correspond to the lfign-amnity receptors thin mediate the ~ffects of the iieurotrophins, nerve growth factor (NGF), brain derived neurotrophic factor (BDNF) and neum.tro.phin-3.(NT-3). irkA is the preferred receptor for NGF, and trka and tr~care the preferred receptors for BDNF and NT-3, respectively. However, different neurotrophins can interact with the same re;eeptor, but with , varying affinity. Neurotrophins and their receptors nave a wtuespreaa. distribution in the developing and adult nervous system, suggesting mat neurotrophins have other functions in addition to their well established role in neuronal development. As a first step to test the hypothesis that neumtrophins play a role in the adult digestive system, we invesfigatco the cellular localization of neurotrophin receptors in the rat gastrointestinal tract with a) immunohistoehemistry...anda pan-trk antibody that recognizes all Irks and b) in situ hybndizaUou histochemistt7 with RNA probes specific for each trio. trK immnnoreacttvity (IR) is found in a large proportion of myanteric and submucosal neurons and in interconnecting strands. Abundant tr~-IR fibers innervate the muscle, mucosa, and vasculature. IR cell bodies are encircled by dense networks of fibers that obscure their shape, trkc mRNA is expressed in neurons of the myenteric and submueosal.plexus. By contrast, trkA and trkB mRNAs could not be detected m enteric neurons. All three trk mRNAs are expressed in dorsal root ganglia, which were used as positive controls. These findings suggest that 1) neurotrophins play a role in the adult gastrointestinal tract, perhaps being involved in tissue maintenance and differentiation and 2) NT-3, the preferred ligand for trkc, is likely to be the relevant neurotrophin in the gut, even though an involvement of other neurotrophins cannot be completely excluded at this time. Supported by Morphology/Imaging Core DK41301 and VA Research Medical Funds.
IMMUNOHISTOLOGICAL LOCALIZATION OF GASTRIN RECEPTORS IN ANTRAL GASTRIC MUCOSA IN MAN. Oh. Stettler 1, A. Schmassmann 1, N. I. Tarasova 2, L. Varga 1, F. Halter 1. 1 : Gastrointestinal Unit, Univ. Hospital, Inselspital, Bern, Switzerland. 2: ABL-Basic Research Program, National Cancer Institute, FCRDC, Frederick, MD, USA. B a e k a r o u n d : 1) It has been reported, that growth of antral G (gastrin) cells is not only stimulated by gastric hypoa¢idity but also by hypergastrinemia. 2) Experimental ulcer healing studies suggest, that gastrin receptors do not play a relevant role in cell proliferation at the ulcer margin (Schrnassmann A et al 1994, Gut: 35: 896-904). In addition, radio ligand autoradiography revealed a persistent lack of somatostatin receptors in the ulcer margin. Aims: 1) Localization of gastrin receptors on G and D cells. 2) Detection of differences in density of cells expressing gastrin receptors between non-ulcerated and ulcerated mucosa. M e t h o d s : - Specific gastrin receptor antibodies have been recently developed (Gastroenterology 1994; 106: A845). Consecutive paraffin sections (2 t~m thick) of human nonulcerated and ulcerated antral gastric mucosa have been immunohistochemically stained using antibodies against gastrin receptor, gastrin and sornatostatin. Results: 1) In the non-ulcerated mucosa, gastrin receptors were mainly located in ceils of the upper portion of the antral glands. Using antibodies against gastrin and sornatostatin, we could demonstrate in consecutive sections, that gastrin receptors are predominantly located on G cells. 2) In contrast to the nonulcerated area, epithelial cells in the ulcer margin did not show cells expressing gastrin receptors, gastri n or somatostatin. C o n c l u s i o n : 1) Human antral G cells show immunoreactivity to gastrin receptor antibodies. 2) Cells showing immunoreactJvity to gastrin receptor antibodies are absent in the ulcer margin.
Supported by Swiss National Science Foundation (No. 32-040901.94) and National Cancer Institute, DHHS (No. N01-CO-46000 with ABL).