RESUMEN
Background: Neuromedin B (Nmb) is implicated in the regulation of nociception of sensory neurons. However, the underlying cellular and molecular mechanisms remain unknown. Methods: Using patch clamp recording, western blot analysis, immunofluorescent labelling, enzyme-linked immunosorbent assays, adenovirus-mediated shRNA knockdown and animal behaviour tests, we studied the effects of Nmb on the sensory neuronal excitability and peripheral pain sensitivity mediated by Cav3.2 T-type channels. Results: Nmb reversibly and concentration-dependently increased T-type channel currents (IT) in small-sized trigeminal ganglion (TG) neurons through the activation of neuromedin B receptor (NmbR). This NmbR-mediated IT response was Gq protein-coupled, but independent of protein kinase C activity. Either intracellular application of the QEHA peptide or shRNA-mediated knockdown of Gß abolished the NmbR-induced IT response. Inhibition of protein kinase A (PKA) or AMP-activated protein kinase (AMPK) completely abolished the Nmb-induced IT response. Analysis of phospho-AMPK (p-AMPK) revealed that Nmb significantly activated AMPK, while AMPK inhibition prevented the Nmb-induced increase in PKA activity. In a heterologous expression system, activation of NmbR significantly enhanced the Cav3.2 channel currents, while the Cav3.1 and Cav3.3 channel currents remained unaffected. Nmb induced TG neuronal hyperexcitability and concomitantly induced mechanical and thermal hypersensitivity, both of which were attenuated by T-type channel blockade. Moreover, blockade of NmbR signalling prevented mechanical hypersensitivity in a mouse model of complete Freund's adjuvant-induced inflammatory pain, and this effect was attenuated by siRNA knockdown of Cav3.2. Conclusions: Our study reveals a novel mechanism by which NmbR stimulates Cav3.2 channels through a Gßγ-dependent AMPK/PKA pathway. In mouse models, this mechanism appears to drive the hyperexcitability of TG neurons and induce pain hypersensitivity.
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Canales de Calcio Tipo T/metabolismo , Dolor/metabolismo , Receptores de Bombesina/metabolismo , Potenciales de Acción , Animales , Canales de Calcio Tipo T/fisiología , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Femenino , Adyuvante de Freund/farmacología , Ganglios Espinales/metabolismo , Masculino , Ratones , Ratones Endogámicos ICR , Neuroquinina B/análogos & derivados , Neuroquinina B/metabolismo , Dolor/fisiopatología , Receptores de Bombesina/fisiología , Receptores Acoplados a Proteínas G/metabolismo , Células Receptoras Sensoriales/metabolismo , Células Receptoras Sensoriales/fisiología , Transducción de Señal/efectos de los fármacos , Ganglio del Trigémino/citología , Ganglio del Trigémino/metabolismoRESUMEN
AIMS: Bombesin receptors (BB receptors) and bombesin related peptides are expressed in the lower urinary tract of rodents. Here we investigated whether in vivo activation of BB receptors can contract the urinary bladder and facilitate micturition in sham rats and in a diabetic rat model of voiding dysfunction. MATERIAL AND METHODS: In vivo cystometry experiments were performed in adult female Sprague-Dawley rats under urethane anesthesia. Diabetes was induced by streptozotocin (STZ; 65mg/kg, i.p.) injection. Experiments were performed 9 and 20weeks post STZ-treatment. Drugs included neuromedin B (NMB; BB1 receptor preferring agonist), and gastrin-releasing peptide (GRP; BB2 receptor preferring agonist). KEY FINDINGS: NMB and GRP (0.01-100µg/kg in sham rats; 0.1-300µg/kg in STZ-treated rats, i.v.) increased micturition frequency, bladder contraction amplitude and area under the curve dose dependently in both sham and STZ-treated rats. In addition, NMB (3, 10µg/kg i.v.) triggered voiding in >80% of STZ-treated rats when the bladder was filled to a sub-threshold voiding volume. NMB and GRP increased mean arterial pressure and heart rate at the highest doses, 100 and 300µg/kg. SIGNIFICANCE: Activation of bombesin receptors facilitated neurogenic bladder contractions in vivo. Single applications of agonists enhanced or triggered voiding in sham rats as well as in the STZ-treated rat model of diabetic voiding dysfunction. These results suggest that BB receptors may be targeted for drug development for conditions associated with poor detrusor contraction such as an underactive bladder condition.
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Diabetes Mellitus Experimental/fisiopatología , Receptores de Bombesina/fisiología , Vejiga Urinaria/efectos de los fármacos , Animales , Diabetes Mellitus Experimental/metabolismo , Evaluación Preclínica de Medicamentos , Femenino , Péptido Liberador de Gastrina/farmacología , Contracción Muscular , Neuroquinina B/análogos & derivados , Neuroquinina B/farmacología , Ratas , Ratas Sprague-Dawley , Receptores de Bombesina/agonistas , Vejiga Urinaria/metabolismo , Vejiga Urinaria/fisiopatología , Micción/efectos de los fármacosRESUMEN
OBJECTIVE: To evaluate whether gastrin-releasing peptide (GRP) and GRP receptor (GRP-R) expression correlate with tumor behavior and to examine the mitogenic actions of GRP on neuroblastomas. SUMMARY BACKGROUND DATA: Neuroblastoma is the most common solid tumor of infants and children. Despite recent advances in multimodality treatment regimens, the survival for advanced-stage tumors remains dismal. Neuroblastomas are known to produce GRP; however, the proliferative effects of GRP on neuroblastomas have not been elucidated. METHODS: Sections of paraffin-embedded neuroblastomas from 33 patients were analyzed for GRP and GRP-R protein expression by immunohistochemistry. Functional binding of GRP-R to the Ca2+ signaling pathway was examined. In addition, the proliferative effect of GRP on neuroblastoma cells (SK-N-SH, IMR-32, SH-SY5Y, LAN-1) was determined. RESULTS: Immunohistochemical analysis showed GRP and GRP-R protein expression in neuroblastomas; an increased expression of GRP-R was noted in a higher percentage of undifferentiated tumors compared with tumors that were benign. GRP-R mRNA was confirmed in neuroblastoma cell lines. GRP treatment resulted in intracellular calcium [Ca2+]i mobilization in two cell lines (SK-N-SH, LAN-1). GRP treatment stimulated growth of all four neuroblastoma cell lines; this effect was inhibited in SK-N-SH cells by pretreatment with GRP antibody. CONCLUSIONS: These findings show increased GRP-R expression in the more aggressive and undifferentiated neuroblastomas. The synchronous expression of GRP and its receptor, GRP-R, suggests a role for these proteins in tumor growth. Moreover, these findings show enhanced proliferation of neuroblastoma cells in vitro after GRP treatment, suggesting that GRP may act as an autocrine and/or paracrine growth factor for neuroblastomas. Treatment with specific GRP-R antagonists may provide novel adjuvant therapy for neuroblastomas in children.
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Péptido Liberador de Gastrina/fisiología , Sustancias de Crecimiento/fisiología , Neuroblastoma/metabolismo , Receptores de Bombesina/fisiología , Calcio/metabolismo , División Celular , Línea Celular , Preescolar , Péptido Liberador de Gastrina/metabolismo , Humanos , Inmunohistoquímica , Lactante , ARN Mensajero/biosíntesis , Receptores de Bombesina/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Células Tumorales CultivadasRESUMEN
BACKGROUND/AIMS: This study investigated the correlation between glucocorticoid-regulated gene expression of the bombesin receptor (BR) and cellular sensitivity to bombesin stimulation in the rat pancreatic acinar cell line AR42J. METHODS: BR gene expression was assessed using a cloned complementary DNA probe and radioligand binding assays. Intracellular Ca2+ mobilization was assessed by dual wavelength spectrophotometry using fura-2 in single cells. RESULTS: Dexamethasone resulted in a rapid dose- and time-dependent decrease of BR messenger RNA levels with maximal inhibition to 25% +/- 2% of controls (n = 4) after 6 hours of hormone treatment. BR messenger RNA half-life was approximately 120 minutes and was not affected by dexamethasone pretreatment; nuclear run-on analysis showed a decreased transcription rate of the BR to approximately 25% of control after hormonal treatment. Radioligand binding studies showed a time-dependent decrease of specific bombesin binding to 25% +/- 8% of control after 48 hours of hormone treatment. Down-regulation of BR gene expression by dexamethasone resulted in a time- and dose-dependent decrease of intracellular Ca2+ mobilization after bombesin stimulation compared with untreated controls. CONCLUSIONS: Glucocorticoids decrease BR gene transcription. The subsequent decrease in cellular BR number renders AR42J cells less sensitive for bombesin-stimulated intracellular Ca2+ mobilization.
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Regulación de la Expresión Génica/efectos de los fármacos , Glucocorticoides/farmacología , Páncreas/química , Páncreas/citología , Receptores de Bombesina/análisis , Receptores de Bombesina/genética , Transcripción Genética/efectos de los fármacos , Animales , Bombesina/farmacología , Calcio/metabolismo , Línea Celular , Dexametasona/farmacología , Relación Dosis-Respuesta a Droga , Regulación hacia Abajo/fisiología , Radioisótopos de Yodo , Páncreas/ultraestructura , ARN Mensajero/análisis , ARN Mensajero/genética , Ratas , Receptores de Bombesina/fisiología , Receptores de Glucocorticoides/análisis , Receptores de Glucocorticoides/fisiología , Factores de TiempoRESUMEN
Mitogen-activated protein kinases (MAPKs) are activated by a variety of extracellular stimuli, including agonists for G protein-coupled receptors. Using transient transfection of COS-7 cells, we have studied the stimulation of a hemagglutinin-tagged p44mapk (p44HA-mapk) by receptors coupled to Gs, Gq, and Gi. Agonists that act via all three G proteins stimulated p44HA-mapk activity. A constitutively activated alpha s mutant, forskolin, and a cAMP analog also increased p44HA-mapk activity, indicating that cAMP in COS-7 cells, in contrast to other cell types, activates the MAPK pathway. Similarly, a constitutively activated alpha q mutant, overexpression of phospholipase C-beta 2, and a phorbol ester also stimulated p44HA-mapk, suggesting that Gq-coupled receptors stimulate the MAPK pathway by increasing phosphatidylinositol turnover and probably stimulating protein kinase C. In COS-7 cells, in contrast to Rat-1 cells, mutationally activated alpha i did not stimulate the MAPK pathway. G protein beta and gamma subunits, overexpressed together, did activate p44HA-mapk; this finding suggests that in COS-7 cells Gi-coupled receptors may stimulate the MAPK pathway through beta gamma. These unexpected results in COS-7 cells show that G proteins and second messengers regulate the MAPK pathway differently in different cell types.