RESUMEN
Cholecystokinin 1 receptor (CCK1R) is activated by singlet oxygen (1O2) generated in photodynamic action with sulphonated aluminum phthalocyanine (SALPC) or genetically encoded protein photosensitizer (GEPP) KillerRed or mini singlet oxygen generator (miniSOG). A large number of GEPP with varied 1O2 quantum yields have appeared recently; therefore, in the present work, the efficacy of different GEPP to photodynamically activate CCK1R was examined, as monitored by Fura-2 calcium imaging. KillerRed, miniSOG, miniSOG2, singlet oxygen protein photosensitizer (SOPP), flavin-binding fluorescent protein from Methylobacterium radiotolerans with point mutation C71G (Mr4511C71G), and flavin-binding fluorescent protein from Dinoroseobacter shibae (DsFbFP) were expressed at the plasma membrane (PM) in AR4-2J cells, which express endogenous CCK1R. Light irradiation (KillerRed: white light 85.3 mWâ§cm-2, 4' and all others: LED 450 nm, 85 mW·cm-2, 1.5') of GEPPPM-expressing AR4-2J was found to all trigger persistent calcium oscillations, a hallmark of permanent photodynamic CCK1R activation; DsFbFP was the least effective, due to poor expression. miniSOG was targeted to PM, mitochondria (MT) or lysosomes (LS) in AR4-2J in parallel experiments; LED light irradiation was found to all induce persistent calcium oscillations. In miniSOGPM-AR4-2J cells, light emitting diode (LED) light irradiation-induced calcium oscillations were readily inhibited by CCK1R antagonist devazepide 2 nM; miniSOGMT-AR4-2J cells were less susceptible, but miniSOGLS-AR4-2J cells were not inhibited. In conclusion, different GEPPPM could all photodynamically activate CCK1R. Intracellular GEPP photodynamic action may prove particularly suited to study intracellular GPCR.
Asunto(s)
Proteínas Bacterianas , Proteínas Luminiscentes , Methylobacterium/genética , Fármacos Fotosensibilizantes/metabolismo , Receptores de Colecistoquinina , Rhodobacteraceae/genética , Proteínas Bacterianas/biosíntesis , Proteínas Bacterianas/genética , Línea Celular , Humanos , Proteínas Luminiscentes/biosíntesis , Proteínas Luminiscentes/genética , Receptores de Colecistoquinina/biosíntesis , Receptores de Colecistoquinina/genética , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/genéticaRESUMEN
Bone morphogenetic protein-2 (BMP2) is a secreted protein that highly expressed in a variety of cancers and contributes to cell proliferation, migration, invasiveness, mobility, metastasis and EMT. However, its clinical significance and biological function in nasopharyngeal carcinoma (NPC) remain unknown up to now. Up-regulation of BMP2 was first observed in NPC cell lines by a genome-wide transcriptome analysis in our previous study. In this study, BMP2 mRNA was detected by qRT-PCR and data showed that it was upregulated in NPC compared with non-cancerous nasopharynx samples. Immunohistochemistry (IHC) analysis in NPC specimens revealed that high BMP2 expression was significantly associated with clinical stage, distant metastasis and shorter survival of NPC patients. Moreover, overexpression of BMP2 in NPC cells promoted cell proliferation, migration, invasiveness and epithelial-mesenchymal transition (EMT). Mechanistically, BMP2 overexpression increase phosphorylated protein level of mTOR, S6K and 4EBP1. Correspondingly, mTORC1 inhibitor rapamycin blocked the effect of BMP2 on NPC cell proliferation and invasion. In conclusion, our results suggest that BMP2 overexpression in NPC enhances proliferation, invasion and EMT of tumor cells through the mTORC1 signaling pathway.
Asunto(s)
Proteína Morfogenética Ósea 2/farmacología , Proliferación Celular/efectos de los fármacos , Diana Mecanicista del Complejo 1 de la Rapamicina/efectos de los fármacos , Neoplasias Nasofaríngeas/patología , Invasividad Neoplásica/patología , Línea Celular Tumoral , Movimiento Celular/efectos de los fármacos , Transición Epitelial-Mesenquimal/efectos de los fármacos , Regulación Neoplásica de la Expresión Génica , Estudio de Asociación del Genoma Completo , Humanos , Diana Mecanicista del Complejo 1 de la Rapamicina/genética , Neoplasias Nasofaríngeas/genética , Metástasis de la Neoplasia , ARN Mensajero/biosíntesis , ARN Mensajero/genética , Receptores de Colecistoquinina/biosíntesis , Transducción de Señal/efectos de los fármacos , Transcriptoma/genética , Regulación hacia Arriba/efectos de los fármacosRESUMEN
AIM: Cholecystokinin (CCK) and gastrin (Gs) are a well known trophic factor for the gastrointestinal tract and their trophic effects are shown mainly toward pancreas and stomach, respectively. Though, the exact characterization of CCK and Gs receptors subtype (cholecystokinin type A receptor [CCKAR] and cholecystokinin type B receptor/gastrin receptor [CCKBR/GR]) in stomach cancer (SC) and pancreatic cancer (PC) is still controversial and necessities further validation. SUBJECTS AND METHODS: CCKAR and CCKBR/GR expression was analyzed by immunohistochemistry in 55 SC, 25 benign gastric diseases (BGDs), 38 PC (including periampullary carcinoma), and 10 normal pancreatic tissue. The results were statistically correlated with the patient's clinical history to observe the prognostic significance if any. RESULT: CCKAR expression was detected in 18.2% of SC, 20% of BGD, 65.8% of PC, and 30.0% of normal pancreas tissue samples. The CCKBR/GR expression was detected in 58.2% of SC, 48.0% of BGD, 18.4% of PC, and 60.0% of normal pancreas tissue samples. CCKBR/GR expression was significantly high in well and moderately differentiated SC samples as compared to poorly differentiated samples. CONCLUSION: Our study showed significantly higher expression of CCKAR and down regulation of CCKBR in PC as compared to control while CCKBR/GR was detected in majority of SC samples. Thus, our study suggests that CCK and Gs receptors may have diagnostic and therapeutic implications. However, study need to be validated in significantly bigger sample size and need to be replicated in different cohorts.
Asunto(s)
Biomarcadores de Tumor/biosíntesis , Neoplasias Pancreáticas/genética , Receptor de Colecistoquinina B/biosíntesis , Receptores de Colecistoquinina/biosíntesis , Neoplasias Gástricas/genética , Adulto , Anciano , Biomarcadores de Tumor/genética , Colecistoquinina/genética , Femenino , Gastrinas/genética , Regulación Neoplásica de la Expresión Génica , Humanos , Masculino , Persona de Mediana Edad , Páncreas/patología , Neoplasias Pancreáticas/patología , Receptor de Colecistoquinina B/genética , Receptores de Colecistoquinina/genética , Estómago/patología , Neoplasias Gástricas/patologíaRESUMEN
We previously found that daidzein decreased food intake in female rats. The present study aimed to elucidate the relationship between dynamics of appetite-mediated neuropeptides and the anorectic effect of daidzein. We examined appetite-mediated gene expression in the hypothalamus and small intestine during the 3 meals per day feeding method. Daidzein had an anorectic effect specifically at the second feeding. Neuropeptide-Y (NPY) and galanin mRNA levels in the hypothalamus were significantly higher after feeding in the control but not in the daidzein group, suggesting that daidzein attenuated the postprandial increase in NPY and galanin expression. The daidzein group had higher corticotrophin-releasing hormone (CRH) mRNA levels in the hypothalamus after feeding, and increased cholelcystokinin (CCK) mRNA levels in the small intestine, suggesting that CCK is involved in the hypothalamic regulation of thisãanorectic effect. Therefore, daidzein may induce anorexia by suppressing expression of NPY and galanin and increasing expression of CRH in the hypothalamus.
Asunto(s)
Anorexia/genética , Apetito/genética , Ingestión de Alimentos/genética , Galanina/biosíntesis , Neuropéptido Y/biosíntesis , Animales , Anorexia/patología , Apetito/fisiología , Peso Corporal , Ingestión de Alimentos/efectos de los fármacos , Métodos de Alimentación , Femenino , Galanina/genética , Regulación de la Expresión Génica/efectos de los fármacos , Humanos , Hipotálamo/metabolismo , Hipotálamo/fisiología , Isoflavonas/administración & dosificación , Neuropéptido Y/genética , ARN Mensajero/biosíntesis , Ratas , Receptores de Colecistoquinina/biosíntesis , Receptores de Hormona Liberadora de Corticotropina/biosíntesisRESUMEN
The cholecystokinin receptor type 1 (CCK1R) is a G protein-coupled receptor (GPCR) that is involved in several biological processes including the regulation of the secretion of digestive enzymes. The peptide hormone cholecystokinin (CCK) binds to CCK1R, which is an important pharmacological target for several diseases, including obesity. Interestingly, nutritional dietary peptides also appear to activate CCK1R, and may play a role in CCK1R signaling in the gut. In this study, a novel technique to screen for CCK1R ligands based on affinity-selection is described. Functional expressed CCK1R is reconstituted into membrane nanoparticles called NABBs (nanoscale apo-lipoprotein bound bilayers). NABBs are native-like bilayer membrane systems for incorporation of GPCRs. CCK1R-NABBs were characterized using a fluorescently labeled CCK analog and can be used as a cutting-edge technology to screen for CCK1R ligands using affinity-selection mass spectrometry.
Asunto(s)
Nanopartículas/química , Receptores de Colecistoquinina/química , Animales , Apolipoproteínas/química , Técnicas Biosensibles , Señalización del Calcio , Evaluación Preclínica de Medicamentos/métodos , Células HEK293 , Humanos , Transporte de Proteínas , Ratas , Receptores de Colecistoquinina/biosíntesis , Receptores de Colecistoquinina/genética , Proteínas de Pez Cebra/químicaRESUMEN
PURPOSE: Cholecystokinin (CCK) is a neuropeptide that has been identified in trigeminal ganglion neurons. Gastrin (GAST) is a related peptide never explored in the cornea. The presence and role of both gastrointestinal peptides in the cornea and corneal sensory neurons remain to be established. We explored here in mice whether CCK, GAST, and their receptors CCK1R and CCK2R are expressed in the corneal epithelium and trigeminal ganglion neurons innervating the cornea. METHODS: We used RT-PCR analysis to detect mRNAs of CCK, GAST, CCK1R, and CCK2R in mouse cornea epithelium, trigeminal ganglia, and primary cultured corneal epithelial cells. Immunofluorescence microscopy was used to localize these peptides and their receptors in the cornea, cultured corneal epithelial cells, and corneal nerves, as well as in the cell bodies of corneal trigeminal ganglion neurons identified by retrograde labeling with Fast Blue. RESULTS: Mouse corneal epithelial cells in the cornea in situ and in cell cultures expressed CCK and GAST. Only the receptor CCK2R was found in the corneal epithelium. In addition, mouse corneal afferent sensory neurons expressed CCK and GAST, and the CCK1R receptors. CONCLUSIONS: The presence of CCK, GAST, and their receptors in the mouse corneal epithelium, and in trigeminal ganglion neurons supplying sensory innervation to the cornea, opens the possibility that these neuropeptides are involved in corneal neurogenic inflammation and in the modulation of repairing/remodeling processes following corneal injury.
Asunto(s)
Colecistoquinina/genética , Córnea/metabolismo , Gastrinas/genética , Nervio Trigémino/metabolismo , Animales , Células Cultivadas , Colecistoquinina/biosíntesis , Córnea/inervación , Epitelio Corneal/citología , Epitelio Corneal/metabolismo , Gastrinas/biosíntesis , Regulación de la Expresión Génica , Masculino , Ratones , Ratones Endogámicos C57BL , ARN/genética , Receptor de Colecistoquinina B/biosíntesis , Receptor de Colecistoquinina B/genética , Receptores de Colecistoquinina/biosíntesis , Receptores de Colecistoquinina/genéticaRESUMEN
In the quest for novel tools for early detection and treatment of cancer, we propose the use of multimers targeting overexpressed receptors at the cancer cell surface. Indeed, multimers are prone to create multivalent interactions, more potent and specific than their corresponding monovalent versions, thus enabling the potential for early detection. There is a lack of tools for early detection of pancreatic cancer, one of the deadliest forms of cancer, but CCK2-R overexpression on pancreatic cancer cells makes CCK based multimers potential markers for these cells. In this Letter, we describe the synthesis and evaluation of CCK trimers targeting overexpressed CCK2-R.
Asunto(s)
Colecistoquinina/análogos & derivados , Colecistoquinina/síntesis química , Neoplasias Pancreáticas/metabolismo , Biomarcadores de Tumor/biosíntesis , Biomarcadores de Tumor/química , Colecistoquinina/química , Humanos , Neoplasias Pancreáticas/diagnóstico , Neoplasias Pancreáticas/tratamiento farmacológico , Receptores de Colecistoquinina/biosíntesis , Receptores de Colecistoquinina/química , Receptores de Colecistoquinina/metabolismoRESUMEN
Obstructive jaundice causes multiple malfunctions in various organs including the pancreas. To establish how such malfunctions occur, we experimentally induced obstructive jaundice through bile duct ligation (BDL) using rats, measured serum bilirubin, amylase and insulin levels, and examined histological, immunohistochemical and cytological changes in the pancreas at 3 days, 1 week, and 4 weeks after the BDL. Morphometrical analysis was also conducted. Serum amylase levels steeply increased at 3 days, and then decreased at 1 and 4 weeks after the BDL to lower than the control level. In contrast, the number of zymogen granules decreased at 3 days after the BDL, then increased and eventually surpassed the control group at 4 weeks after the BDL. On the other hand, serum insulin levels dramatically decreased at 3 days after the BDL but recovered to a level close to that of the control group at 1 week after the BDL. At 4 weeks after the BDL, however, the serum insulin levels again showed a marked decline. Slight decrease in insulin immunoreactivity and number of insulin granules were observed at 4 weeks after the BDL. Cholecystokinin receptors (CCK-R) were expressed in both acinar and islet cells; their immunoreactivity significantly decreased in the acinar cells at 4 weeks after the BDL. Our results suggest that CCK may play a role in regulating changes in the pancreas after obstructive jaundice.
Asunto(s)
Islotes Pancreáticos/patología , Ictericia Obstructiva/patología , Páncreas Exocrino/patología , Amilasas/sangre , Animales , Bilirrubina/sangre , Inmunohistoquímica , Insulina/sangre , Islotes Pancreáticos/enzimología , Islotes Pancreáticos/ultraestructura , Ictericia Obstructiva/sangre , Masculino , Microscopía Electrónica de Transmisión , Páncreas Exocrino/enzimología , Páncreas Exocrino/ultraestructura , Ratas , Ratas Wistar , Receptores de Colecistoquinina/biosíntesisRESUMEN
Cholecystokinin octapeptide (CCK8) can exert the immunoregulatory roles through activating immune cell surface receptors such as T lymphocytes, macrophages, dendritic cells, and so on. In this study, we discussed the effects of CCK8 on lipopolysaccharide (LPS)-activated B cells in terms of the expression of co-stimulatory molecules, and the capacity to activate CD4(+) T cells and cytokines production in vitro. The results revealed that B cells expressed two types of CCK receptors; CCK8 inhibited the expression of co-stimulatory molecules such as CD80 and CD86 on LPS-activated B cells, suppressed the proliferation of allogeneic T cells in a dose-dependent manner, and also reduced the secretion of Th1-type cytokine IFN-γ, whereas enhanced the secretion of Th2-type cytokine IL-4 by LPS-activated B cells. Both CCK1R and CCK2R participated in these effects. Taken together, CCK8 is capable of exerting immunomodulatory functions through B cells.
Asunto(s)
Antígenos CD/biosíntesis , Linfocitos B/efectos de los fármacos , Citocinas/inmunología , Lipopolisacáridos/farmacología , Receptores de Colecistoquinina/biosíntesis , Sincalida/farmacología , Animales , Antígenos CD/inmunología , Linfocitos B/inmunología , Antígeno B7-1/biosíntesis , Antígeno B7-1/inmunología , Antígeno B7-2/biosíntesis , Antígeno B7-2/inmunología , Linfocitos T CD4-Positivos/efectos de los fármacos , Linfocitos T CD4-Positivos/inmunología , Células Cultivadas , Citocinas/biosíntesis , Citometría de Flujo , Activación de Linfocitos/efectos de los fármacos , Activación de Linfocitos/inmunología , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Receptores de Colecistoquinina/inmunologíaRESUMEN
The gastrointestinal hormone cholecystokinin (CCK) can induce acute pancreatitis in rodents through its action on acinar cells. Treatment with CCK, in combination with other agents, represents the most commonly used model to induce experimental chronic pancreatitis. Pancreatic stellate cells (PSC) are responsible for pancreatic fibrosis and therefore play a predominant role in the genesis of chronic pancreatitis. However, it is not known whether PSC express CCK receptors. Using real time PCR techniques, we demonstrate that CCK1 and CCK2 receptors are expressed on rat PSC. Interestingly both CCK and gastrin significantly induced type I collagen synthesis. Moreover, both inhibit proliferation. These effects are comparable with TGF-ß-stimulated PSC. Furthermore, the natural agonists CCK and gastrin induce activation of pro-fibrogenic pathways Akt, ERK, and Src. Using specific CCK1 and CCK2 receptor (CCK2R) inhibitors, we found that Akt activation is mainly mediated by CCK2R. Akt activation by CCK and gastrin could be inhibited by the PI3K inhibitor wortmannin. Activation of ERK and the downstream target Elk-1 could be inhibited by the MEK inhibitor U0126. These data suggest that CCK and gastrin have direct activating effects on PSC, are able to induce collagen synthesis in these cells, and therefore appear to be important regulators of pancreatic fibrogenesis. Furthermore, similar to TGF-ß, both CCK and gastrin inhibit proliferation in PSC.
Asunto(s)
Colágeno/biosíntesis , Regulación de la Expresión Génica , Páncreas/metabolismo , Células Estrelladas Pancreáticas/metabolismo , Receptor de Colecistoquinina B/biosíntesis , Receptores de Colecistoquinina/biosíntesis , Animales , Butadienos/farmacología , Colágeno Tipo I/metabolismo , Inhibidores Enzimáticos/farmacología , Gastrinas/metabolismo , Masculino , Nitrilos/farmacología , Ratas , Ratas Sprague-Dawley , Transducción de Señal , Factor de Crecimiento Transformador beta/metabolismoRESUMEN
Cholecystokinin (CCK) is a gut-brain peptide has been described to be able to induce mitosis according to recent studies. Additionally, conflicting data has been published on whether tumours of the central and peripheral nervous system in general, and gliomas in particular, express CCK receptors. In the present in vitro study we employed reverse transcription followed by the polymerase chain reaction (RT-PCR) to investigate whether mRNA for CCK-A and CCK-B receptors as well as CCK peptide itself is present in primary human gliomas and the U-87 MG GBM cell line. The data show that 14/14 (100%) of the primary gliomas exhibited mRNA expression for the CCK peptide gene and the B receptor including the U-87 MG cells, whereas, only 2/14 (14%) showed presence of the CCK-A receptor. The presence of CCK receptors together with CCK peptide expression itself suggests presence of an autocrine loop controlling glioma cell growth. In support of this conclusion, a neutralizing antibody against the CCK peptide exhibited a dose dependent inhibition of cell growth whereas, antagonists to CCK caused a dose depend inhibition of exogenous stimulated glioma cell growth in vitro, via the CCK-B receptor which is PKC activated. Assessment of apoptosis and proteasome activity were undertaken and we report that treatment with CCK antagonists decreased proteasome and increased caspase-3 activity. These data indicate that CCK peptide and CCK-B are abundant in human gliomas and they act to stimulate cell growth in an autocrine manner, primarily via the high affinity CCK-B receptor, which was blocked by antagonists to CCK, perhaps via apoptosis.
Asunto(s)
Comunicación Autocrina/fisiología , Neoplasias Encefálicas/metabolismo , Neoplasias Encefálicas/fisiopatología , Colecistoquinina/biosíntesis , Glioma/metabolismo , Glioma/fisiopatología , Comunicación Paracrina/fisiología , Receptores de Colecistoquinina/biosíntesis , Caspasa 3/metabolismo , Inhibidores de Caspasas , Línea Celular Tumoral , Proliferación Celular , AMP Cíclico/biosíntesis , Relación Dosis-Respuesta a Droga , Humanos , Hidrólisis , Fosfatidilinositoles/metabolismo , Complejo de la Endopetidasa Proteasomal/metabolismo , ARN Mensajero/biosíntesis , ARN Mensajero/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Timidina/metabolismoRESUMEN
In invertebrates, the biogenic-amine octopamine is an important physiological regulator. It controls and modulates neuronal development, circadian rhythm, locomotion, 'fight or flight' responses, as well as learning and memory. Octopamine mediates its effects by activation of different GTP-binding protein (G protein)-coupled receptor types, which induce either cAMP production or Ca(2+) release. Here we describe the functional characterization of two genes from Drosophila melanogaster that encode three octopamine receptors. The first gene (Dmoa1) codes for two polypeptides that are generated by alternative splicing. When heterologously expressed, both receptors cause oscillatory increases of the intracellular Ca(2+) concentration in response to applying nanomolar concentrations of octopamine. The second gene (Dmoa2) codes for a receptor that specifically activates adenylate cyclase and causes a rise of intracellular cAMP with an EC(50) of approximately 3 x 10(-8) m octopamine. Tyramine, the precursor of octopamine biosynthesis, activates all three receptors at > or = 100-fold higher concentrations, whereas dopamine and serotonin are non-effective. Developmental expression of Dmoa genes was assessed by RT-PCR. Overlapping but not identical expression patterns were observed for the individual transcripts. The genes characterized in this report encode unique receptors that display signature properties of native octopamine receptors.
Asunto(s)
Calcio/metabolismo , AMP Cíclico/biosíntesis , Octopamina/metabolismo , Receptores de Amina Biogénica/fisiología , Receptores de Colecistoquinina/biosíntesis , Secuencia de Aminoácidos , Animales , Línea Celular , Clonación Molecular , AMP Cíclico/genética , Drosophila melanogaster , Regulación del Desarrollo de la Expresión Génica , Humanos , Datos de Secuencia Molecular , Octopamina/farmacología , Receptores de Amina Biogénica/biosíntesis , Receptores de Amina Biogénica/química , Receptores de Colecistoquinina/agonistas , Receptores de Colecistoquinina/genética , Transducción de Señal/genéticaRESUMEN
OBJECTIVES: Pancreatic acini of diabetic rats release amylase less than normal acini on cholecystokinin (CCK) stimulation. Pancreatic enzyme secretion by CCK is closely related to the second messenger inositol 1,4,5-trisphosphate (IP3), which mobilizes intracellular calcium stores via the endoplasmic reticulum-located receptor IP3 (IP3R). Recently, we observed altered intracellular calcium response on CCK-8 stimulation in streptozotocin (STZ)- treated diabetic rat acini. METHODS: To determine whether IP3R is involved in altered calcium response, we measured inositol phosphate (IP) formation and the expression and phosphorylation of type III IP3R protein in diabetic acini. Also, CCK receptor mRNA expression was examined to determine whether the changes in IP formation and IP3R protein phosphorylation in diabetic acini might result from the defect at the postreceptor level. RESULTS: CCK-8-induced IP formation at all concentrations used was significantly reduced in diabetic acini, though IP formation was increased in a concentration-dependent manner. The expression of type III IP3R protein was significantly reduced in diabetic acini. Additionally, CCK-8-stimulated phosphorylation of type III IP3R protein was not observed in diabetic acini. However, the reduction of CCK receptor mRNA expression was not detected in diabetic acini. CONCLUSION: Our results indicate that altered calcium response to CCK-8 in diabetic acini might be associated with a post-CCK receptor defect including the changes in IP formation, type III IP3R protein expression, and phosphorylation of type III IP3R protein.
Asunto(s)
Canales de Calcio/metabolismo , Calcio/metabolismo , Colecistoquinina/metabolismo , Diabetes Mellitus Experimental/metabolismo , Páncreas/metabolismo , Receptores Citoplasmáticos y Nucleares/metabolismo , Sincalida/metabolismo , Animales , Receptores de Inositol 1,4,5-Trifosfato , Fosfatos de Inositol/metabolismo , Masculino , Fosforilación/efectos de los fármacos , ARN Mensajero/biosíntesis , Ratas , Ratas Sprague-Dawley , Receptores de Colecistoquinina/biosíntesis , Estreptozocina , Tritio/metabolismoRESUMEN
Activation of the hypothalamic-pituitary-adrenal axis by fasting seems to involve cholecystokinin (CCK) receptors. This work aims to characterize the role of endogenous CCK in the activity of the paraventricular nucleus (PVN) of the hypothalamus during food withdrawal. We investigated, by c-Fos immunohistochemistry, the effect of CCK1 and CCK2 receptor antagonists (SR-27,897 and L-365,260, respectively) on c-Fos levels expression induced by food deprivation. Under our conditions, the number of cells expressing c-Fos was reduced both by SR-27,897 and L-365,260 in food-deprived rats. To investigate the importance of glucose availability, we studied the effect of CCK receptor antagonists on c-Fos synthesis induced by the glucose antimetabolite 2-deoxyglucose. In these animals, only SR-27,897 decreased c-Fos expression in the PVN. Our results indicate that the effect of CCK antagonists is mainly perceptible when glucose availability decreases, and suggest that CCK-ergic inputs could drive the activity of the PVN under fasting/low glucose conditions.
Asunto(s)
Colecistoquinina/fisiología , Privación de Alimentos/fisiología , Núcleo Hipotalámico Paraventricular/metabolismo , Animales , Genes fos/efectos de los fármacos , Genes fos/fisiología , Ácidos Indolacéticos/farmacología , Masculino , Núcleo Hipotalámico Paraventricular/efectos de los fármacos , Ratas , Ratas Wistar , Receptores de Colecistoquinina/antagonistas & inhibidores , Receptores de Colecistoquinina/biosíntesis , Tiazoles/farmacologíaRESUMEN
The gut-brain peptide cholecystokinin (CCK) has been implicated in the regulation of dopamine (DA) transmission in the brain. CCK agonists have been shown to modify baseline and stimulant-induced DA release in the brain via CCK-A mediated mechanisms. However, the role of endogenous CCK in regulating brain DA via CCK-A receptors has not been fully elucidated. Recently, a strain of rats (Otsuka Long Evans Tokushima Fatty (OLETF)), lacking the CCK-A receptor due to a genetic mutation, was discovered, providing a potentially useful tool to study the DA regulatory role of CCK-A receptors. In order to further clarify the role of CCK-A receptors in the regulation of central DA transmission, extracellular DA levels in the nucleus accumbens (NAC) and the caudate-putamen (CP) of OLETF rats, and their non-mutant counterparts, Long Evans Tokushimo Otsuka rats, was assessed by microdialysis at baseline and in response to cocaine (15 mg/kg) and amphetamine (0.5 mg/kg) administration. Baseline levels of extracellular DA were significantly elevated in the CP but not in the NAC of OLETF rats. In contrast, the NAC exhibited a greater DA response to cocaine (15 mg/kg) and amphetamine (0.5 mg/kg) in OLETF rats. This is the first direct evidence, of which we are aware, supporting altered DA regulation in OLETF rats. These findings suggest that CCK-A receptors play an important role in the regulation of central DA transmission, and support the notion that the OLETF rat is a useful model to study this regulation.
Asunto(s)
Encéfalo/metabolismo , Dopamina/metabolismo , Espacio Extracelular/metabolismo , Receptores de Colecistoquinina/deficiencia , Receptores de Colecistoquinina/genética , Animales , Encéfalo/efectos de los fármacos , Espacio Extracelular/efectos de los fármacos , Ratas , Ratas Endogámicas OLETF , Receptor de Colecistoquinina A , Receptores de Colecistoquinina/biosíntesisRESUMEN
BACKGROUND: Several studies have shown a link between gastrin and gastric cancer, both in humans and animals, especially infected with Helicobacter pylori (H. pylori). However, the exact role of hypergastrinemia in gastric carcinogenesis remains still undetermined. The aim of the present study was to evaluate the interaction between gastrin, cyclooxygenase-2 (COX-2), hepatocyte growth factor (HGF) and apoptosis-related proteins (Bax, Bcl-2, caspase-3, survivin) in cultured gastric epithelial cancer cells. MATERIAL AND METHODS: In the present study, gastric cultured cancer cells (KATO III cells) were exposed to increasing concentrations of gastrin (1-1000 nM). Cells incubated with culture medium alone, without added gastrin, served as controls. Using RT-PCR and Western blot, we examined the mRNA and protein expression for COX-2, HGF and apoptosis-related proteins (Bax, Bcl-2, caspase-3 and survivin). In addition, the gene expression of gastrin and gastrin receptor (CCK-2) as well as the release of gastrin in culture medium in the unstimulated cells were examined by RT-PCR and RIA, respectively. The apoptosis rate in cells was measured by flow cytometric analysis. RESULTS: The present study shows that the gastric cultured epithelial cells exhibit the expression of gastrin and CCK-2 receptors and release of gastrin into the culture medium. The epithelial gastric cancer cells incubated with gastrin showed a concentration-dependent increase of COX-2 and HGF expression. Although no significant changes in apoptosis rate were observed, the exposure of these cells was associated with a dose-dependent increase in the expression of antiapoptotic proteins Bcl-2 and survivin. CONCLUSIONS: This study demonstrates that 1) gastrin stimulates the gene and protein expression of COX-2 and HGF in human cultured gastric cancer cells and 2) gastrin shows antiapoptotic activity through the upregulation of Bcl-2 and survivin.
Asunto(s)
Carcinoma de Células en Anillo de Sello/metabolismo , Gastrinas/fisiología , Factor de Crecimiento de Hepatocito/biosíntesis , Isoenzimas/biosíntesis , Prostaglandina-Endoperóxido Sintasas/biosíntesis , Neoplasias Gástricas/metabolismo , Apoptosis , Carcinoma de Células en Anillo de Sello/genética , Carcinoma de Células en Anillo de Sello/patología , Caspasa 3 , Caspasas/biosíntesis , Ciclooxigenasa 2 , ADN de Neoplasias/biosíntesis , ADN de Neoplasias/genética , Células Epiteliales/efectos de los fármacos , Células Epiteliales/metabolismo , Células Epiteliales/patología , Regulación de la Expresión Génica , Humanos , Técnicas In Vitro , Proteínas Inhibidoras de la Apoptosis , Proteínas de la Membrana , Proteínas Asociadas a Microtúbulos/biosíntesis , Proteínas de Neoplasias , Proteínas Proto-Oncogénicas/biosíntesis , ARN Mensajero/biosíntesis , ARN Mensajero/genética , Receptores de Colecistoquinina/biosíntesis , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Neoplasias Gástricas/genética , Neoplasias Gástricas/patología , Survivin , Células Tumorales Cultivadas , Regulación hacia ArribaRESUMEN
The role of hyper-gastrinaemia in the incidence of colonic cancer remains to be clarified. The aim of this study was to determine whether cholecystokinin-2 (CCK-2) receptor expression predicts the sensitivity of human colonic adenomas to the proliferative effects of serum hyper-gastrinaemia. Gene expression of the classical (74 kDa) CCK-2 receptor in human colonic adenoma specimens and cell lines, was quantified by real-time PCR. Western blotting, using a CCK-2 receptor antiserum, confirmed protein expression. A transformed human colonic adenoma was grown in SCID mice, with hyper-gastrinaemia induced by proton pump inhibitors. CCK-2 receptor blockade was achieved by using neutralising antiserum. Both human colonic adenoma cell lines and biopsies expressed CCK-2 receptor mRNA at levels comparable with CCK-2 receptor transfected fibroblasts and oxyntic mucosa. Western blotting confirmed immunoreactive CCK-2 receptor bands localised to 45, 74 and 82.5 kDa. Omeprazole and lansoprazole-induced hyper-gastrinaemia (resulting in serum gastrin levels of 34.0 and 153.0 pM, respectively) significantly increased the weight of the human adenoma grafts (43% (P=0.016) and 70% (P=0.014), respectively). The effect of hypergastrinaemia on tumour growth was reversed by use of antiserum directed against the CCK-2 receptor. Hyper-gastrinaemia may promote proliferation of human colonic adenomas that express CCK-2 receptor isoforms.
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Adenocarcinoma/patología , Adenoma/patología , Neoplasias del Colon/patología , Gastrinas/fisiología , Proteínas de Neoplasias/fisiología , Receptores de Colecistoquinina/fisiología , Adenocarcinoma/genética , Adenocarcinoma/metabolismo , Adenoma/genética , Adenoma/metabolismo , Animales , Neoplasias del Colon/genética , Neoplasias del Colon/metabolismo , Sistemas de Computación , Inhibidores Enzimáticos/farmacología , Femenino , Fibroblastos/metabolismo , Mucosa Gástrica/efectos de los fármacos , Mucosa Gástrica/metabolismo , Gastrinas/sangre , Gastrinas/metabolismo , Regulación Neoplásica de la Expresión Génica , Humanos , Intrones/genética , Masculino , Ratones , Ratones SCID , Proteínas de Neoplasias/biosíntesis , Proteínas de Neoplasias/genética , Trasplante de Neoplasias , Omeprazol/farmacología , Células Parietales Gástricas/metabolismo , Reacción en Cadena de la Polimerasa , Isoformas de Proteínas/biosíntesis , Isoformas de Proteínas/genética , Isoformas de Proteínas/fisiología , Procesamiento Proteico-Postraduccional , Inhibidores de la Bomba de Protones , ARN Mensajero/biosíntesis , Receptor de Colecistoquinina B , Receptores de Colecistoquinina/biosíntesis , Receptores de Colecistoquinina/genética , Tasa de Secreción/efectos de los fármacos , Células Tumorales Cultivadas/metabolismo , Células Tumorales Cultivadas/patología , Células Tumorales Cultivadas/trasplanteRESUMEN
Abnormal splicing of primary RNA transcripts of normal genes is a recognized mechanism for the production of some abnormal proteins found in cancer cells. A misspliced form of the cholecystokinin-B/gastrin (CCK-B) receptor recently was reported to be present in colon carcinoma, where it was postulated to play a role in stimulating tumor growth (M. R. Hellmich et al., J. Biol. Chem., 275: 32122-32128, 2000). Here, we report the presence of the same abnormal protein in pancreatic carcinoma and explore the molecular basis for this missplicing event. Reverse transcription-PCR and sequencing were used to demonstrate the presence of a misspliced form of the CCK-B receptor having its fourth intron retained in three pancreatic cancer cell lines and in tumor tissue, but not in surrounding healthy pancreas, from two patients with pancreatic carcinoma. A mini-gene construct representing the region of this gene from its third through its fifth exon and containing the two intervening introns was produced and transiently expressed in the MIA PaCa-2 human pancreatic cancer cell line. Specific reverse transcription-PCR reactions with both vector-derived and receptor-specific primers demonstrated the presence of both correctly fully spliced and selectively misspliced forms of this receptor. Mutagenesis of the mini-gene demonstrated that a suboptimal sequence at the 3'-end of intron 4 contributed to this missplicing. This focused attention on the U2 small nuclear ribonucleoprotein particle auxiliary splicing factors (U2AFs) known to interact specifically with this domain. Indeed, quantitative real-time PCR demonstrated a reduced level of expression of one of these factors, U2AF35, in pancreatic cancer cells compared with healthy pancreas. Furthermore, the relative amount of missplicing of the CCK-B receptor mini-gene in the pancreatic cancer cell line was reversed by transfection of the cells with U2AF35 cDNA. This work describes the presence of an additional abnormal protein in pancreatic cancer and describes a new molecular mechanism for its production, providing additional potential therapeutic targets.
Asunto(s)
Intrones , Proteínas Nucleares , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/metabolismo , Receptores de Colecistoquinina/genética , Ribonucleoproteínas/fisiología , Empalme Alternativo , Secuencia de Aminoácidos , Humanos , Datos de Secuencia Molecular , ARN Mensajero/biosíntesis , ARN Mensajero/genética , Receptor de Colecistoquinina B , Receptores de Colecistoquinina/biosíntesis , Ribonucleoproteínas/metabolismo , Factor de Empalme U2AF , Células Tumorales CultivadasRESUMEN
OBJECTIVE: The cholecystokinin(2)-receptor (CCK(2)R) promotes secretion and cell growth induced by its ligands cholecystokinin (CCK) and gastrin. The receptor has recently been shown to be expressed in human medullary thyroid carcinomas (MTCs). The objective of this study was to analyze CCK(2)R expression in MTC samples of different tumor stages as well as in non-malignant thyroid tissues. DESIGN AND METHODS: Using RT-PCR we investigated 19 MTC samples and TT-cells (a human MTC cell line), as well as samples of normal thyroid. In addition, we performed immunohistochemistry using calcitonin- and CCK(2)R-specific antibodies on MTCs and samples of C-cell hyperplasia. RESULTS: We demonstrate for the first time that CCK(2)R is expressed not only in MTCs but in all samples of normal thyroid tissue. Using immunohistochemistry the receptor could be localized on calcitonin-secreting C-cells. The highest incidence of CCK(2)R expression in MTCs was observed in early-tumor stages, whereas CCK(2)R could not be detected in advanced or metastasized tumors. CONCLUSIONS: The expression of CCK(2)R in C-cells suggests a physiological function for gastrin and/or CCK in the regulation of calcitonin release, presumably related to bone and calcium metabolism. Moreover, these ligands might act as growth factors in MTCs. Efforts in the development of CCK(2)R scintigraphy for the detection of MTC lesions might have to consider a lower incidence of the receptor in advanced tumor stages.
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Carcinoma Medular/metabolismo , Receptores de Colecistoquinina/biosíntesis , Glándula Tiroides/metabolismo , Neoplasias de la Tiroides/metabolismo , Adolescente , Adulto , Anciano , Comunicación Autocrina/fisiología , Carcinoma Medular/patología , Colecistoquinina/metabolismo , Femenino , Gastrinas/metabolismo , Humanos , Inmunohistoquímica , Masculino , Persona de Mediana Edad , Mutación/genética , Oncogenes/genética , Pentagastrina , Receptor de Colecistoquinina B , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Estimulación Química , Glándula Tiroides/patología , Neoplasias de la Tiroides/patologíaRESUMEN
The role of the gastric acid secretagogues acetylcholine, gastrin and histamine has been debated for decades. Initially, the mast cell was considered the source of acid stimulatory histamine. Later, Håkanson & Owman (1969) showed that the entero-chromaffinlike (ECL) cell produces and stores histamine in several species, including rat and man. Kahlson et al. (1964) showed that food and gastrin stimulated oxyntic mucosal histamine synthesis and release, Berglindh et at. (1976) that histamine and cholinergics but not gastrin induced acid secretion in isolated oxyntic glands and parietal cells, and Rangachari (1995) that acetylcholine or gastrin released histamine in isolated mucosa. These findings suggested that gastrin stimulates acid secretion through release of ECL cell histamine. Studying simultaneous histamine release and acid secretion in isolated oxyntic mucosal cells, we found that gastrin stimulated acid secretion only in preparations releasing histamine. Moreover, in the isolated rat stomach, gastrin stimulated both histamine release and acid secretion. Maximal acid output was higher with histamine than with gastrin, and augmented by acetylcholine but not by gastrin. These findings strongly suggested that gastrin acts by releasing histamine. Finally, a fluorescein-labelled gastrin analogue bound to the ECL cell, not to the parietal or stem cell regions. This is interesting, recalling that gastrin has a potent and specific trophic effect on the ECL cell and only a general effect on all other oxyntic cell types. In conclusion, physiological observations are best explained by localising the CCK2 receptor only to the ECL cell, the other effects of gastrin on the gastric mucosa being secondary to the release of mediators from the ECL cell.