RESUMEN
Type 2 diabetes mellitus (T2DM) is characterized by the inability of the pancreatic ß-cells to secrete enough insulin to meet the demands of the body. Therefore, research of potential therapeutic approaches to treat T2DM has focused on increasing insulin output from ß-cells or improving systemic sensitivity to circulating insulin. In this study, we examined the role of the A(1) receptor in glucose homeostasis with the use of A(1) receptor knockout mice (A(1)R(-/-)). A(1)R(-/-) mice exhibited superior glucose tolerance compared with wild-type controls. However, glucose-stimulated insulin release, insulin sensitivity, weight gain, and food intake were comparable between the two genotypes. Following a glucose challenge, plasma glucagon levels in wild-type controls decreased, but this was not observed in A(1)R(-/-) mice. In addition, pancreas perfusion with oscillatory glucose levels of 10-min intervals produced a regular pattern of pulsatile insulin release with a 10-min cycling period in wild-type controls and 5 min in A(1)R(-/-) mice. When the mice were fed a high-fat diet (HFD), both genotypes exhibited impaired glucose tolerance and insulin resistance. Increased insulin release was observed in HFD-fed mice in both genotypes, but increased glucagon release was observed only in HFD-fed A(1)R(-/-) mice. In addition, the regular patterns of insulin release following oscillatory glucose perfusion were abolished in HFD-fed mice in both genotypes. In conclusion, A(1) receptors in the pancreas are involved in regulating the temporal patterns of insulin release, which could have implications in the development of glucose intolerance seen in T2DM.
Asunto(s)
Glucemia/metabolismo , Insulina/metabolismo , Receptor de Adenosina A1/metabolismo , Animales , Dieta Alta en Grasa , Ingestión de Alimentos , Glucagón/sangre , Glucosa/farmacología , Prueba de Tolerancia a la Glucosa , Insulina/sangre , Resistencia a la Insulina , Secreción de Insulina , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Receptor de Adenosina A1/genética , Aumento de PesoRESUMEN
The pancreatic islets of Langerhans are responsible for the regulated release of the endocrine hormones insulin and glucagon that participate in the control of glucose homeostasis. Abnormal regulation of these hormones can result in glucose intolerance and lead to the development of diabetes. Numerous efforts have been made to better understand the physiological regulators of insulin and glucagon secretion. One of these regulators is the purine nucleoside, adenosine. Though exogenous application of adenosine has been demonstrated to stimulate glucagon release and inhibit insulin release, the physiological significance of this pathway has been unclear. We used a novel 7 µm enzyme-coated electrode biosensor to measure adenosine levels in isolated rodent islets. In the mouse islets, basal adenosine levels in the presence of 3 mM glucose were estimated to be 5.7 ± 0.6 µM. As glucose was increased, extracellular adenosine diminished. A 10-fold increase of extracellular KCl increased adenosine levels to 16.4 ± 2.0 µM. This release required extracellular Ca (2+) suggesting that it occurred via an exocytosis-dependent mechanism. We also found that while rat islets were able to convert exogenous ATP into adenosine, mouse islets were unable to do this. Our study demonstrates for the first time the basal levels of adenosine and its inverse relationship to extracellular glucose in pancreatic islets.
Asunto(s)
Adenosina/metabolismo , Líquido Extracelular/metabolismo , Glucosa/farmacología , Islotes Pancreáticos/metabolismo , Cloruro de Potasio/farmacología , Animales , Calcio/metabolismo , Membrana Celular/efectos de los fármacos , Membrana Celular/fisiología , Dipiridamol/farmacología , Exocitosis , Guanosina Monofosfato/farmacología , Islotes Pancreáticos/efectos de los fármacos , Ratones , Proteínas de Transporte de Nucleósidos/antagonistas & inhibidores , Ratas , Tioinosina/análogos & derivados , Tioinosina/farmacologíaRESUMEN
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RESUMEN
Ghrelin, a potent orexigenic hormone released from the stomach, is important in regulating energy metabolism. Abnormal ghrelin levels are associated with eating disorders and metabolic diseases. However, factors involved in the regulation of ghrelin release remain unclear. Here, we examined the involvement of adenosine signaling in the control of ghrelin release from the perfused mouse stomach. Adenosine stimulated ghrelin release concentration-dependently, and the A(2A) receptor-selective antagonists 4-(2-[7-amino-2-(2-furyl)[1,2,4]triazolo[2,3-a][1,3,5]triazin-5-ylamino]ethyl)phenol (ZM 241385) and 2-(2-furanyl)-7-(2-phenylethyl)-7H-pyrazolo[4,3-e][1,2,4]triazolo[1,5-c]pyrimidin-5-amine (SCH 58261) abolished the increased release. The A(2A) receptor-selective agonist 2-p-(2-carboxyethyl)phenethylamino-5-N-ethylcarboxamidoadenosine hydrochloride (CGS 21680) augmented ghrelin release concentration-dependently, whereas the A(1) receptor-selective agonist 2-chloro-N(6)-cyclopentyladenosine inhibited ghrelin release. In A(2A) receptor knockout mice, adenosine inhibited ghrelin release, and the A(1) receptor-selective antagonist 8-cyclopentyl-1,3-dipropylxanthine blocked this inhibition. The adenosine deaminase inhibitor erythro-9-(2-hydroxy-3-nonyl)adenine hydrochloride increased ghrelin release in wild-type and A(1) receptor knockout mice but not in A(2A) receptor knockout mice. Colocalization of ghrelin immunoreactivity with A(1) and A(2A) receptor immunoreactivities in the gastric nerve fibers were observed. Colocalization was also detected for ghrelin and A(1) receptor immunoreactivities in the gastric mucosa. Blockade of neural activities with tetrodotoxin abolished the stimulatory effect of adenosine on ghrelin release. In conclusion, adenosine exerts predominantly a tonic A(2A) receptor-mediated stimulatory action on gastric ghrelin release, whereas an A(1) receptor-mediated inhibitory action is also apparent when the tonic excitatory effect was removed.
Asunto(s)
Adenosina/fisiología , Mucosa Gástrica/metabolismo , Ghrelina/metabolismo , Receptor de Adenosina A1/fisiología , Receptor de Adenosina A2A/fisiología , Transducción de Señal/fisiología , Animales , Relación Dosis-Respuesta a Droga , Mucosa Gástrica/efectos de los fármacos , Masculino , Ratones , Ratones Congénicos , Ratones Endogámicos C57BL , Ratones Noqueados , Perfusión , Receptor de Adenosina A1/deficiencia , Receptor de Adenosina A2A/deficiencia , Transducción de Señal/efectos de los fármacosRESUMEN
AIMS: The gastrointestinal hormone GIP promotes pancreatic islet function and exerts pro-survival actions on cultured beta-cells. However, GIP also promotes lipogenesis, thus potentially restricting its therapeutic use. The current studies evaluated the effects of a truncated GIP analog, D-Ala(2)-GIP(1-30) (D-GIP(1-30)), on glucose homeostasis and beta-cell mass in rat models of diabetes. MATERIALS AND METHODS: The insulinotropic and pro-survival potency of D-GIP(1-30) was evaluated in perfused pancreas preparations and cultured INS-1 beta-cells, respectively, and receptor selectivity evaluated using wild type and GIP receptor knockout mice. Effects of D-GIP(1-30) on beta-cell function and glucose homeostasis, in vivo, were determined using Lean Zucker rats, obese Vancouver diabetic fatty rats, streptozotocin treated rats, and obese Zucker diabetic fatty rats, with effects on beta-cell mass determined in histological studies of pancreatic tissue. Lipogenic effects of D-GIP(1-30) were evaluated on cultured 3T3-L1 adipocytes. RESULTS: Acutely, D-GIP(1-30) improved glucose tolerance and insulin secretion. Chronic treatment with D-GIP(1-30) reduced levels of islet pro-apoptotic proteins in Vancouver diabetic fatty rats and preserved beta-cell mass in streptozotocin treated rats and Zucker diabetic fatty rats, resulting in improved insulin responses and glycemic control in each animal model, with no change in body weight. In in vitro studies, D-GIP(1-30) exhibited equivalent potency to GIP(1-42) on beta-cell function and survival, but greatly reduced action on lipoprotein lipase activity in 3T3-L1 adipocytes. CONCLUSIONS: These findings demonstrate that truncated forms of GIP exhibit potent anti-diabetic actions, without pro-obesity effects, and that the C-terminus contributes to the lipogenic actions of GIP.
Asunto(s)
Proteínas Reguladoras de la Apoptosis/química , Apoptosis , Diabetes Mellitus Experimental/tratamiento farmacológico , Células Secretoras de Insulina/metabolismo , Receptores de la Hormona Gastrointestinal/agonistas , Células 3T3 , Adipocitos/citología , Animales , Glucosa/metabolismo , Prueba de Tolerancia a la Glucosa , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Ratas , Ratas Zucker , Receptores de la Hormona Gastrointestinal/químicaRESUMEN
BACKGROUND & AIMS: Glucose-dependent insulinotropic polypeptide (GIP) and the proglucagon product glucagon-like peptide-1 (GLP-1) are gastrointestinal hormones that are released in response to nutrient intake and promote insulin secretion. Interestingly, a subset of enteroendocrine cells express both GIP and GLP-1. We sought to determine whether GIP also might be co-expressed with proglucagon in pancreatic alpha-cells. METHODS: We assessed GIP expression via reverse-transcription polymerase chain reaction, in situ hybridization, and immunohistochemistry. We developed a novel bioassay to measure GIP release from isolated islets, compared the biological activities of full-length and truncated GIP, and assessed the impact of immunoneutralization of islet GIP on glucose-stimulated insulin secretion in isolated islets. RESULTS: GIP messenger RNA was present in mouse islets; GIP protein localized to islet alpha-cells of mouse, human, and snake pancreas, based on immunohistochemical analyses. However, using a C-terminal GIP antibody, immunoreactivity was detected in islets from prohormone convertase (PC) 2 knockout but not wild-type mice. Bioactive GIP was secreted from mouse and human islets after arginine stimulation. In the perfused mouse pancreas, GIP(1-42) and amidated GIP(1-30) had equipotent insulinotropic actions. Finally, immunoneutralization of GIP secreted by isolated islets decreased glucose-stimulated insulin secretion. CONCLUSIONS: GIP is expressed in and secreted from pancreatic islets; in alpha-cells, PC2 processes proGIP to yield a truncated but bioactive form of GIP that differs from the PC1/3-derived form from K-cells. Islet-derived GIP promotes islet glucose competence and also could support islet development and/or survival.
Asunto(s)
Polipéptido Inhibidor Gástrico/metabolismo , Células Secretoras de Glucagón/metabolismo , Glucosa/metabolismo , Insulina/metabolismo , Islotes Pancreáticos/metabolismo , Animales , Boidae , Línea Celular , Duodeno/metabolismo , Femenino , Polipéptido Inhibidor Gástrico/genética , Péptido 1 Similar al Glucagón/metabolismo , Humanos , Secreción de Insulina , Islotes Pancreáticos/embriología , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Fragmentos de Péptidos/metabolismo , Proglucagón/metabolismo , Proproteína Convertasa 2/deficiencia , Proproteína Convertasa 2/genética , ARN Mensajero/metabolismo , Ratas , Ratas Wistar , Receptores de la Hormona Gastrointestinal/genética , Receptores de la Hormona Gastrointestinal/metabolismo , Factores de Tiempo , Técnicas de Cultivo de Tejidos , TransfecciónRESUMEN
Glucose-dependent insulinotropic polypeptide (GIP) is a hormone released from enteroendocrine K cells in response to meals. Posttranslational processing of the precursor protein pro-GIP at residue 65 by proprotein convertase subtilisin/kexin type 1 (PC1/3) in gut K cells gives rise to the established 42-amino-acid form of GIP (GIP(1-42)). However, the pro-GIP peptide sequence contains a consensus cleavage site for PC2 at residues 52-55 and we identified PC2 immunoreactivity in a subset of K cells, suggesting the potential existence of a COOH-terminal truncated GIP isoform, GIP(1-30). Indeed a subset of mouse and human K cells display GIP immunoreactivity with GIP antibodies directed to the mid portion of the peptide, but not with a COOH-terminal-directed GIP antibody, indicative of the presence of a truncated form of GIP. This population of cells represents approximately 5-15% of the total GIP-immunoreactive cells in mice, depending on the region of intestine, and is virtually absent in mice lacking PC2. Amidated GIP(1-30) and GIP(1-42) have comparable potency at stimulating somatostatin release in the perfused mouse stomach. Therefore, GIP(1-30) represents a naturally occurring, biologically active form of GIP.
Asunto(s)
Células Enteroendocrinas/metabolismo , Polipéptido Inhibidor Gástrico/metabolismo , Fragmentos de Péptidos/metabolismo , Proproteína Convertasa 1/metabolismo , Animales , Furina/biosíntesis , Polipéptido Inhibidor Gástrico/inmunología , Polipéptido Inhibidor Gástrico/fisiología , Mucosa Gástrica/metabolismo , Humanos , Ratones , Fragmentos de Péptidos/fisiología , Proproteína Convertasa 2/metabolismo , Somatostatina/metabolismo , Estómago/efectos de los fármacosRESUMEN
Adenosine inhibits gastric acid secretion, either directly by acting on acid-secreting parietal cells or indirectly by stimulating the release of the acid inhibitor, somatostatin. The present study examined the role of adenosine on somatostatin release in an isolated vascularly perfused mouse stomach model. Concentrations of exogenous adenosine >or= 1.0 microM stimulated gastric release of somatostatin-like immunoreactivity (SLI), and this effect was blocked by the A(2A) receptor antagonist ZM 241385 [4-(2-[7-amino-2-(2-furyl)[1,2,4]triazolo[2,3-a][1,3,5]triazin-5-ylamino]ethyl)phenol]. The A(2A) receptor agonist CGS 21680 [2-p-(2-carboxyethyl)phenethylamino-5'-N-ethylcarboxamidoadenosine hydrochloride] augmented SLI release in a concentration-dependent manner, suggesting that A(2A) receptor activation is involved in the stimulatory effect of adenosine on SLI release. Conversely, SLI release was inhibited by the A(1) receptor agonists N(6)-cyclopentyladenosine and 2-chloro-N(6)-cyclopentyladenosine and lower concentration of adenosine (0.01 microM). The involvement of specific adenosine receptors in controlling the release of gastric SLI was also examined using A(2A) receptor knockout (A(2A)R-KO) mice. In these mice, adenosine (10 microM) inhibited SLI release, and the effect was abolished by the selective A(1) receptor antagonist 8-cyclopentyl-1,3-dipropylxanthine, suggesting a link between the selective A(1) activation and inhibition of SLI release. The adenosine deaminase inhibitor erythro-9-(2-hydroxy-3-nonyl)adenine hydrochloride augmented SLI release in wild-type controls but not in the presence of ZM 241385 or in A(2A)R-KO mice. We conclude that adenosine has dual actions on regulating mouse gastric SLI release: stimulatory at higher concentrations through the A(2A) receptor and inhibitory at lower concentrations through the A(1) receptor, whereas A(2B) and A(3) receptors have a minimal role.
Asunto(s)
Agonistas del Receptor de Adenosina A2 , Antagonistas del Receptor de Adenosina A2 , Adenosina/fisiología , Somatostatina/metabolismo , Estómago/efectos de los fármacos , Adenosina/agonistas , Adenosina/antagonistas & inhibidores , Adenosina/farmacología , Animales , Relación Dosis-Respuesta a Droga , Mucosa Gástrica/metabolismo , Técnicas In Vitro , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Perfusión , Receptor de Adenosina A2A/genética , Estómago/irrigación sanguíneaRESUMEN
Adenosine has been shown to inhibit immunoreactive gastrin (IRG) release and to stimulate somatostatin-like immunoreactivity (SLI) release by activating adenosine A(1) and A(2A) receptors, respectively. Since the synthesis and release of gastrin and somatostatin are regulated by the acid secretory state of the stomach, the effect of achlorhydria on A(1) and A(2A) receptor gene expression and function was examined. Omeprazole-induced achlorhydria was shown to suppress A(1) and A(2A) receptor gene expression in the antrum and corporeal mucosa, but not in the corporeal muscle. Omeprazole treatment produced reciprocal changes in A(1) receptor and gastrin gene expression, and parallel changes in A(2A) receptor and somatostatin gene expression. The localization of A(1) and A(2A) receptors on gastrinsecreting G-cells and somatostatin-secreting D-cells, respectively, suggests that changes in adenosine receptor expression may modulate the synthesis and release of gastrin and somatostatin. Thus, the effect of omeprazole on adenosine receptor-mediated changes in IRG and SLI release was also examined in the vascularly perfused rat stomach. After omeprazole treatment, the A(1) receptor-mediated inhibition of IRG and SLI release induced by N(6)-cyclopentyladenosine (A(1) receptor-selective agonist) was not altered, but the A(2A) receptor-mediated augmentation of SLI release induced by 2-p-(2-carboxyethyl-)phenethylamino-5'-N-ethylcarboxamidoadenosine (A(2A)-selective agonist) was significantly attenuated. These findings agree well with the corresponding omeprazole-induced decrease in antral A(2A) receptor mRNA expression. Overall, the present study suggests that adenosine receptor gene expression and function may be altered by omeprazole treatment. Acid-dependent changes in adenosine receptor expression may represent a novel purinergic regulatory feedback mechanism in controlling gastric acid secretion.
Asunto(s)
Adenosina/análogos & derivados , Expresión Génica/efectos de los fármacos , Omeprazol/farmacología , Receptor de Adenosina A1/metabolismo , Receptor de Adenosina A2A/metabolismo , Adenosina/farmacología , Animales , Interacciones Farmacológicas , Gastrinas/genética , Gastrinas/metabolismo , Gliceraldehído-3-Fosfato Deshidrogenasas/genética , Gliceraldehído-3-Fosfato Deshidrogenasas/metabolismo , Masculino , Fenetilaminas/farmacología , ARN Ribosómico 18S/efectos de los fármacos , Ratas , Ratas Wistar , Receptor de Adenosina A1/genética , Receptor de Adenosina A2A/genética , Somatostatina/genética , Somatostatina/inmunología , Somatostatina/metabolismoRESUMEN
Adenosine has been demonstrated to inhibit gastric acid secretion. In the rat stomach, this inhibitory effect may be mediated indirectly by the inhibition of gastrin release. Results show that the A(1) receptor agonist N(6)-cyclopentyladenosine (CPA) suppressed immunoreactive gastrin (IRG) release in a concentration-dependent manner. CPA significantly inhibited IRG release at 0.001 microM and maximally inhibited IRG release at 1 microM. At concentrations of 0.001 to 0.1 microM, the A(2A) receptor-selective agonist 2-p-(2-carboxyethyl)phenethylamino-5'-N-ethylcarboxamidoadenosine and A(3) receptor-selective agonist 1-deoxy-1-[6-[[(3-iodophenyl)methyl]amino]-9H-purin-9-yl]-N-methyl-beta-d-ribofuranuronamide, had no effect on IRG release, suggesting the involvement of A(1) receptors. In agreement, the A(1) receptor-selective antagonist 8-cyclopentyl-1,3-dipropylxanthine abolished adenosine-induced inhibition of IRG release. Results of immunohistochemistry experiments reveal the presence of A(1) receptor immunoreactivity on mucosal G-cells and D-cells, and the gastric plexi, but not parietal cells, suggesting that adenosine may act directly on G-cells or indirectly on the gastric plexi to modulate IRG release. The structure of the mucosal A(1) receptor was found to be identical to that in the rat brain. Alternative splicing within the coding region of this receptor did not occur. A real-time reverse transcription-polymerase chain reaction assay was developed to measure gastric A(1) receptor gene expression. The highest level of gastric A(1) receptor mRNA was found in the corporeal muscle. However, this level was significantly lower in comparison with the striatum. In conclusion, this study shows that adenosine may suppress IRG release, at least in part, by activating A(1) receptors localized on G-cells and may consequently result in an inhibition of gastric acid secretion.
Asunto(s)
Adenosina/análogos & derivados , Gastrinas/metabolismo , Receptor de Adenosina A1/fisiología , Adenosina/farmacología , Agonistas del Receptor de Adenosina A1 , Animales , Relación Dosis-Respuesta a Droga , Mucosa Gástrica/metabolismo , Masculino , Fenetilaminas/farmacología , Ratas , Ratas WistarRESUMEN
Adenosine has been demonstrated to inhibit gastric acid secretion. In the rat stomach, this inhibitory effect may be mediated indirectly by increasing the release of somatostatin-like immunoreactivity (SLI). Results show that adenosine analogs augmented SLI release in the isolated vascularly perfused rat stomach. The rank order of potency of the analogs in stimulating SLI release was 2-p-(2-carboxyethyl)phenethylamino-5'-N-ethylcarboxamidoadenosine (CGS 21680) approximately 5'-N-ethylcarboxamidoadenosine > 2-chloroadenosine > R-(-)-N(6)-(2-phenylisopropyl)adenosine >1-deoxy-1-[6-[[(3-iodophenyl)methyl]amino]-9H-purin-9-yl]-N-methyl-beta-d-ribofuranuronamide > N(6)-cyclopentyladenosine approximately N(6)-cyclohexyladenosine > S-(+)-N(6)-(2-phenylisopropyl) adenosine, suggesting the involvement of the A(2A) receptor. In agreement, 4-(2-[7-amino-2-(2-furyl)[1,2,4]triazolo[2,3-a] [1,3,5]triazin-5-ylamino]ethyl)phenol (ZM 241385), an A(2A) receptor antagonist, was shown to abolish the adenosine- and CGS 21680-stimulated SLI release. Immunohistochemical studies reveal the presence of A(2A) receptor immunoreactivity on the gastric plexi and mucosal D-cells, but not on parietal cells and G-cells, suggesting that adenosine may act directly on D-cells or indirectly on the gastric plexi to augment SLI release. The present study also demonstrates that the structure of the mucosal A(2A) receptor is identical to that in the rat brain, and that alternative splicing of this gene does not occur. A real-time reverse transcription-polymerase chain reaction assay has also been established to quantify the levels of A(2A) receptor mRNA. Results show that gastric tissues contained significantly lower levels of A(2A) receptor mRNA compared with the striatum. The lowest level was detected in the mucosa. In conclusion, adenosine may act on A(2A) receptors to augment SLI release and consequently control gastric acid secretion.
Asunto(s)
Adenosina/análogos & derivados , Adenosina/metabolismo , Receptor de Adenosina A2A/metabolismo , Somatostatina/metabolismo , Estómago/efectos de los fármacos , Adenosina/farmacología , Agonistas del Receptor de Adenosina A2 , Antagonistas del Receptor de Adenosina A2 , Animales , Mucosa Gástrica/metabolismo , Masculino , Fenetilaminas/farmacología , Ratas , Ratas Wistar , Distribución Tisular , Triazinas/farmacología , Triazoles/farmacología , Xantinas/farmacologíaRESUMEN
In the stomach, the majority of substance P's effects are mediated by the activation of neurokinin-1 (NK1) receptors. The gastric cellular distribution of these receptors in Wistar and Sprague-Dawley rats was determined using immunocytochemistry. The localization of the NK1 receptors with respect to von Willebrand's factor, protein gene product 9.5, substance P, vasoactive intestinal peptide, and calcitonin gene-related peptide was also determined. Results show that NK1 receptor immunoreactivity was dependent on the duration of fixation. In corpus and antrum tissues that were fixed in 4% paraformaldehyde for 30 min, the presence of NK1 receptor immunoreactivity was demonstrated on nerve fibers throughout the stomach, on the surface and in the cytoplasm of myenteric cell bodies, on circular smooth muscle cells, and on vascular endothelial cells. This was observed in tissues from both rodent strains. Overnight fixation in the same fixative, however, demonstrated the presence of NK1 receptor immunoreactivity only on nerve fibers and cell bodies of the myenteric plexus, and on circular smooth muscle cells. In 30-min fixed tissues, the localization of NK1R immunoreactivity on vascular endothelial cells and nerve fibers was confirmed by co-localization with von Willebrand's factor and protein gene product 9.5 immunoreactivity, respectively. In both rodent strains, NK1 receptor immunoreactivity was co-localized with substance P immunoreactivity on nerve fibers of the longitudinal and circular muscle. In the Wistar rat, NK1 receptor immunoreactivity was co-localized with vasoactive intestinal peptide immunoreactivity or calcitonin gene-related peptide immunoreactivity throughout the stomach. However, in the Sprague-Dawley rat, NK1 receptor immunoreactivity was only co-localized with calcitonin gene-related peptide immunoreactivity in a minority of fibers of the circular muscle. The overall results of this study show that the antigenic epitopes of the NK1 receptor are sensitive to overfixation. When tissues were not overfixed, NK1 receptor immunoreactivity was distributed more extensively throughout the rat stomach than has been described previously. The results of this study provide the anatomical basis for many of the actions of substance P in the rat stomach.