RESUMEN
BACKGROUND: Vasoactive intestinal peptide (VIP) and pituitary adenylate cyclase activating peptide (PACAP)-containing nerves surround cerebral blood vessels. The peptides have potent vasodilator properties via smooth muscle cell receptors and activation of adenylate cyclase. The purpose of this study was to describe the effects of two putative VIP/PACAP receptor antagonists and the distribution of the receptor protein in rat brain vessels. METHODS: The vascular effects of VIP, PACAP-27 and PACAP-38 were investigated in segments of rat middle cerebral artery (MCA) by pressurized arteriography, and in a wire myograph. The antagonistic responses to PACAP6-38 and PG99-465 were evaluated. In addition, the receptor subtypes for VIP and PACAP (VPAC1, VPAC2 and PAC1) were visualized in the rat middle cerebral artery by immunohistochemistry and Western blotting. RESULTS: In the perfusion model, abluminal but not luminal VIP, PACAP-27 and PACAP-38 caused concentration-dependent relaxations of the MCA (27.1±0.2%, 25.2±0.4% and 0.3±0.1%, respectively). In the wire myograph, there was no significant difference in potency of the peptides in the MCA. In both systems, PACAP6-38 and PG99-465 inhibited the VIP induced relaxation. Western blot showed the presence of the receptor proteins in cerebral vasculature and immunohistochemistry showed that all three receptors are present and located in the cytoplasm of smooth muscle cells. CONCLUSION: In both systems, the two blockers antagonized the relaxant VIP effect; the potency order of agonists and the immunohistochemistry suggest the presence of the dilatory VPAC1 and VPAC2 receptors on the smooth muscle cells.
Asunto(s)
Calcio/fisiología , Arterias Cerebrales/metabolismo , Receptores del Polipéptido Activador de la Adenilato-Ciclasa Hipofisaria/fisiología , Receptores de Péptido Intestinal Vasoactivo/fisiología , Ácido 15-Hidroxi-11 alfa,9 alfa-(epoximetano)prosta-5,13-dienoico/farmacología , Angiografía , Animales , Western Blotting , Arterias Cerebrales/efectos de los fármacos , Electromiografía , Técnica del Anticuerpo Fluorescente , Inmunohistoquímica , Contracción Isométrica/efectos de los fármacos , Masculino , Músculo Liso Vascular/fisiología , ARN Mensajero/biosíntesis , ARN Mensajero/genética , Ratas , Ratas Sprague-Dawley , Ratas Wistar , Receptores del Polipéptido Activador de la Adenilato-Ciclasa Hipofisaria/efectos de los fármacos , Receptores del Polipéptido Activador de la Adenilato-Ciclasa Hipofisaria/metabolismo , Receptores de Péptido Intestinal Vasoactivo/efectos de los fármacos , Receptores de Tipo II del Péptido Intestinal Vasoactivo/metabolismo , Vasoconstrictores/farmacologíaRESUMEN
A hallmark in most neurological disorders is a massive neuronal cell death, in which uncontrolled immune response is usually involved, leading to neurodegeneration. The vasoactive intestinal peptide (VIP) is a pleiotropic peptide that combines neuroprotective and immunomodulatory actions. Alterations on VIP/VIP receptors in patients with neurodenegerative diseases, together with its involvement in the development of embryonic nervous tissue, and findings found in VIP-deficient mutant mice, have showed the relevance of this endogenous peptide in normal physiology and in pathologic states of the central nervous system (CNS). In this review, we will summarize the role of VIP in normal CNS and in neurological disorders. The studies carried out with this peptide have demonstrated its therapeutic effect and render it as an attractive candidate to be considered in several neurological disorders linked to neuroinflammation or abnormal neural development.
Asunto(s)
Enfermedades del Sistema Nervioso/tratamiento farmacológico , Fármacos Neuroprotectores/uso terapéutico , Péptido Intestinal Vasoactivo/uso terapéutico , Enfermedad de Alzheimer/fisiopatología , Animales , Trastorno Autístico/fisiopatología , Encéfalo/efectos de los fármacos , Encéfalo/fisiología , Lesiones Encefálicas/fisiopatología , Discapacidades del Desarrollo/tratamiento farmacológico , Síndrome de Down/fisiopatología , Encefalitis/tratamiento farmacológico , Femenino , Trastornos del Espectro Alcohólico Fetal/fisiopatología , Humanos , Ratones , Esclerosis Múltiple/fisiopatología , Fármacos Neuroprotectores/administración & dosificación , Enfermedad de Parkinson/fisiopatología , Embarazo , Receptores de Péptido Intestinal Vasoactivo/efectos de los fármacos , Péptido Intestinal Vasoactivo/administración & dosificación , Péptido Intestinal Vasoactivo/fisiologíaRESUMEN
Vasoactive intestinal peptide (VIP) is a basic 28 amino acid peptide that binds to a member of the class II family of G protein-coupled receptors (GPCRs). It is widely expressed throughout the body and plays an important role in numerous biological functions. VIP acts via three different GPCRs: VPAC1, VPAC2, and PAC1, which have been identified in various tissues, including brain, lung, kidney, gastrointestinal tract, tongue, and also on immunocompetent cells such as macrophages and lymphocytes. There is mounting evidence that VIP expression and signaling is altered in numerous neurological disorders, and it is becoming apparent that VIP and its receptors could be therapeutic loci for the treatment of several pathological conditions of the central nervous system. In this review, we describe the pathology of several major neurological disorders and discuss the potential pharmacotherapeutic role of VIP and its receptors for the treatment of disorders such as Alzheimer's disease, Parkinson's disease, and Autism Spectrum Disorders.
Asunto(s)
Enfermedades del Sistema Nervioso/tratamiento farmacológico , Receptores de Péptido Intestinal Vasoactivo/efectos de los fármacos , Péptido Intestinal Vasoactivo/uso terapéutico , Animales , Sistemas de Liberación de Medicamentos/métodos , Humanos , Modelos Biológicos , Enfermedades del Sistema Nervioso/metabolismo , Enfermedades del Sistema Nervioso/fisiopatología , Receptores de Péptido Intestinal Vasoactivo/metabolismo , Receptores de Péptido Intestinal Vasoactivo/fisiología , Péptido Intestinal Vasoactivo/metabolismo , Péptido Intestinal Vasoactivo/fisiologíaRESUMEN
A novel peptide combination consisting of four synthetic neuropeptide analogs of Vasoactive Intestinal Peptide (VIP), Bombesin, Substance P and Somatostatin has been found to have potent anticancer activity in vitro and in vivo. The receptors of these four neuropeptides are known to be over expressed in various cancers. We have found the presence of native neuropeptides in the culture supernatant of the primary tumor cells of human colon adenocarcinomas. It was further demonstrated by receptor-ligand assays that not only do these tumor cells synthesize and secrete four peptide hormones but also possess specific high affinity receptors on their surface. Screening a large panel of analogs to the four peptide hormones on tumor cell proliferation led to the identification of four cytotoxic analogs, the combination of which was code-named DRF7295. The design and synthesis of the peptide analogs have been described in this paper. In vitro anticancer activity of DRF7295 was studied in a large panel of human tumor cells. Gastrointestinal tumor cells of the colon, pancreas and duodenum were found to be most sensitive to DRF7295 with moderate activity seen in glioblastoma, prostate, leukemia and those of oral cancer cells. Efficacy studies in xenograft models of colon and duodenum resulted in T/C% of less than 40%, which is indicative of strong tumor regressing potential of DRF7295 in gastrointestinal cancers. Acute and long-term toxicity studies as well as safety pharmacology studies conducted indicate the safety of the drug upon systemic administration with no significant adverse pharmacological effects.
Asunto(s)
Antineoplásicos/farmacología , Sistemas de Liberación de Medicamentos , Neoplasias Gastrointestinales/tratamiento farmacológico , Péptidos/farmacología , Animales , Antineoplásicos/efectos adversos , Bombesina/análogos & derivados , Línea Celular Tumoral , Combinación de Medicamentos , Femenino , Neoplasias Gastrointestinales/fisiopatología , Humanos , Masculino , Ratones , Ratones Desnudos , Péptidos/efectos adversos , Ratas , Ratas Wistar , Receptores de Bombesina/efectos de los fármacos , Receptores de Bombesina/metabolismo , Receptores de Neuroquinina-1/efectos de los fármacos , Receptores de Neuroquinina-1/metabolismo , Receptores de Somatostatina/efectos de los fármacos , Receptores de Somatostatina/metabolismo , Receptores de Péptido Intestinal Vasoactivo/efectos de los fármacos , Receptores de Péptido Intestinal Vasoactivo/metabolismo , Somatostatina/análogos & derivados , Sustancia P/análogos & derivados , Pruebas de Toxicidad , Péptido Intestinal Vasoactivo/análogos & derivados , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Our aim was to confirm the role of postganglionic vagal fibres and vasoactive intestinal polypeptide (VIP) in mediating the vagal tachycardia in anaesthetised dogs. Vagal postganglionic stimulation after atenolol (1 mg/kg) and hexamethonium (10 mg/kg) caused a bradycardia (40 beats/min, n = 2), after atropine (0.5 mg/kg i.v.) the resulting tachycardia (37 beats/min) was attenuated by VIP receptor antagonism with VIP (6-28) (100 mug i.c.) by approximately 50%. VIP release from vagal postganglionic fibres mediates the vagal tachycardia.
Asunto(s)
Ganglios/fisiopatología , Taquicardia/fisiopatología , Nervio Vago/fisiopatología , Péptido Intestinal Vasoactivo/fisiología , Antagonistas Adrenérgicos beta/farmacología , Animales , Atenolol/farmacología , Presión Sanguínea/efectos de los fármacos , Cateterismo Cardíaco , Vasos Coronarios/fisiología , Perros , Estimulación Eléctrica , Frecuencia Cardíaca/efectos de los fármacos , Receptores de Péptido Intestinal Vasoactivo/efectos de los fármacos , Receptores de Péptido Intestinal Vasoactivo/metabolismo , Taquicardia/metabolismo , Nervio Vago/metabolismo , Péptido Intestinal Vasoactivo/metabolismoRESUMEN
Chronic inflammatory airway diseases such as bronchial asthma or chronic obstructive pulmonary disease (COPD) are major contributors to the global burden of disease. Although inflammatory cells play the central role in the pathogenesis of the diseases, recent observations indicate that also resident respiratory cells represent important targets for pulmonary drug development. Especially targeting airway neuromediators offers a possible mechanism by which respiratory diseases may be treated in the future. Among numerous peptide mediators such as tachykinins, calcitonin gene-related peptide, neurotrophins or opioids, vasoactive intestinal polypeptide (VIP) is one of the most abundant molecules found in the respiratory tract. In human airways, it influences many respiratory functions via the receptors VPAC1, VPAC2 and PAC1. VIP-expressing nerve fibers are present in the tracheobronchial smooth muscle layer, submucosal glands and in the walls of pulmonary and bronchial arteries and veins. Next to its strong bronchodilator effects, VIP potently relaxes pulmonary vessels, and plays a pivotal role in the mediation of immune mechanisms. A therapy utilizing the respiratory effects of VIP would offer potential benefits in the treatment of obstructive and inflammatory diseases and long acting VIP-based synthetic non-peptide compounds may represent a novel target for drug development.
Asunto(s)
Broncodilatadores/uso terapéutico , Receptores de Péptido Intestinal Vasoactivo/metabolismo , Péptido Intestinal Vasoactivo/uso terapéutico , Vasodilatación , Animales , Asma/tratamiento farmacológico , Asma/metabolismo , Broncodilatadores/metabolismo , Broncodilatadores/farmacología , Humanos , Pulmón/irrigación sanguínea , Pulmón/efectos de los fármacos , Pulmón/inervación , Pulmón/metabolismo , Moco/metabolismo , Neuropéptidos/metabolismo , Neuropéptidos/farmacología , Neuropéptidos/uso terapéutico , Polipéptido Hipofisario Activador de la Adenilato-Ciclasa/metabolismo , Conformación Proteica , Ensayos Clínicos Controlados Aleatorios como Asunto , Receptores de Péptido Intestinal Vasoactivo/química , Receptores de Péptido Intestinal Vasoactivo/efectos de los fármacos , Mucosa Respiratoria/efectos de los fármacos , Mucosa Respiratoria/metabolismo , Linfocitos T/efectos de los fármacos , Linfocitos T/metabolismo , Nervio Vago/metabolismo , Péptido Intestinal Vasoactivo/metabolismo , Péptido Intestinal Vasoactivo/farmacologíaRESUMEN
Prepro-vasoactive intestinal peptide (VIP) mRNA codes for two neuropeptides: VIP and peptide histidine isoleucine (PHI). Two VIP receptors, shared with a similar affinity by pituitary adenylate cyclase-activating polypeptide (PACAP), have been cloned: VPAC(1) and VPAC(2). PHI binds to these receptors with a lower affinity. VPAC receptors are classically associated with a cAMP-dependent pathway, although other pathways, including calcium mobilization and protein kinase C activation have been described. We previously showed that intracerebral administration of the glutamate agonist ibotenate to postnatal day 5 mice induces white matter lesions mimicking human periventricular leukomalacia. In this model, coinjection of VIP protects against white matter lesions. This neuroprotection is independent from cAMP and is mediated by protein kinase C. Using this model, this study aimed to determine the receptor involved in VIP-induced neuroprotection. VIP effects were mimicked with a similar potency by VPAC(2) agonists and PHI but not by VPAC(1) agonists, PACAP 27, or PACAP 38. VIP neuroprotective effects were lost in mice lacking VPAC(2) receptor. In situ hybridization confirmed the presence of VPAC(2) mRNA in the postnatal day 5 white matter. When analyzed between embryonic life and adulthood, VIP-specific binding site density peaked at postnatal day 5. These data suggest that, in this model, VIP-induced neuroprotection is mediated by VPAC(2) receptors. The pharmacology of this VPAC(2) receptor seems unconventional because 1) PACAP does not mimic VIP effects, 2) PHI acts with a comparable potency, and 3) PACAP 27 modestly inhibited the VIP-specific binding, whereas for PHI or VIP, inhibition was complete.
Asunto(s)
Animales Recién Nacidos/fisiología , Fármacos Neuroprotectores/farmacología , Receptores de Péptido Intestinal Vasoactivo/efectos de los fármacos , Péptido Intestinal Vasoactivo/farmacología , Animales , Química Encefálica/efectos de los fármacos , Femenino , Ácido Iboténico/farmacología , Hibridación in Situ , Técnicas In Vitro , Masculino , Membranas/efectos de los fármacos , Membranas/metabolismo , Ratones , Factores de Crecimiento Nervioso/metabolismo , Neuropéptidos/metabolismo , Neurotransmisores/metabolismo , Péptido PHI/metabolismo , Polipéptido Hipofisario Activador de la Adenilato-Ciclasa , Embarazo , Prosencéfalo/efectos de los fármacos , Prosencéfalo/metabolismo , ARN Mensajero/biosíntesis , ARN Mensajero/genética , Receptores de Superficie Celular/efectos de los fármacos , Receptores del Polipéptido Activador de la Adenilato-Ciclasa Hipofisaria , Receptores de Tipo II del Péptido Intestinal VasoactivoRESUMEN
Most drugs target a receptor for a hormone or neurotransmitter. A newer strategy for drug development is to target a downstream signaling element, such as the G protein associated with a receptor. Suramin is considered a lead compound targeting this moiety. It inhibits binding of guanosine 5'-O-(3-thiotriphosphate) (GTPgammaS) to G proteins and reduces agonist binding to G protein-coupled receptors. Suramin is thought to uncouple the G protein from its associated receptor, although there is no direct evidence for this mechanism. We have now examined the effect of suramin on G protein signaling for the vasoactive intestinal peptide (VIP) receptor in lung. The primary experimental strategy was a two-step cross-linking reaction that covalently captures the VIP-receptor-G protein ternary complex. Such cross-linking provided the first direct evidence that suramin physically disrupts receptor-G protein coupling. We investigated how this uncoupling relates to the inhibition of GTPgammaS binding. Suramin indiscriminately hindered the dissociation of various guanine nucleotides from the G protein, implying that its action is not allosteric. Further cross-linking studies suggested that suramin does not obstruct the receptor docking site directly but appears to block the interface between G protein alpha and betagamma subunits. Observations with a purified system of recombinant G protein subunits without a receptor yielded direct evidence that suramin suppresses the association between these subunits. This action can explain how it both disrupts receptor-G protein coupling and inhibits guanine nucleotide release. The improved understanding of suramin's action advances the development of selective inhibitors of G protein signaling.
Asunto(s)
Subunidades alfa de la Proteína de Unión al GTP/antagonistas & inhibidores , Subunidades beta de la Proteína de Unión al GTP/antagonistas & inhibidores , Subunidades gamma de la Proteína de Unión al GTP/antagonistas & inhibidores , Receptores Acoplados a Proteínas G/antagonistas & inhibidores , Suramina/farmacología , Animales , Unión Competitiva/efectos de los fármacos , Reactivos de Enlaces Cruzados , GTP Fosfohidrolasas/metabolismo , Guanosina 5'-O-(3-Tiotrifosfato)/metabolismo , Técnicas In Vitro , Cinética , Pulmón/efectos de los fármacos , Pulmón/metabolismo , Masculino , Ratas , Ratas Sprague-Dawley , Receptores de Péptido Intestinal Vasoactivo/efectos de los fármacos , Proteínas Recombinantes/efectos de los fármacosRESUMEN
Pituitary adenylate cyclase-activating polypeptide (PACAP) potentiates glucose-induced insulin release and increases cytosolic Ca2+ concentration ([Ca2+]i) in islet beta-cells in a concentration-dependent manner with two peaks at 10(-13) and 10(-9) M. PAC1 receptor (PAC1-R) and VPAC2 receptor (VPAC2-R) are expressed in pancreatic beta-cells and thought to be involved in insulin release. We aimed to determine the receptor types involved in the [Ca2+]i responses to 10(-13) and 10(-9) M PACAP. We measured [Ca2+]i in beta-cells and examined comparative effects of PAC1-R-selective agonist maxadilan, its antagonist M65, VPAC2-R-selective agonist Ro25-1553, and native ligands of PACAP and VIP. In the presence of 8.3 mM glucose, maxadilan, Ro25-1553, PACAP, and VIP at 10(-13) and 10(-9) M all increased [Ca2+]i. PACAP and maxadilan elicited greater effects at 10(-9) M than at 10(-13) M both in the incidence and amplitude of [Ca2+]i responses. For VIP and Ro25-1553, in contrast, the effects at 10(-9) and 10(-13) M were comparable. Furthermore, the amplitude of [Ca2+]i responses to 10(-9) M PACAP, but not 10(-13) M PACAP, was suppressed by M65. The results suggest that VPAC2-R and PAC1-R contribute equally to [Ca2+]i responses to sub-picomolar concentrations of PACAP, while PAC1-R has greater contribution to [Ca2+]i responses to nanomolar concentrations of this peptide.
Asunto(s)
Calcio/metabolismo , Islotes Pancreáticos/efectos de los fármacos , Islotes Pancreáticos/metabolismo , Factores de Crecimiento Nervioso/administración & dosificación , Neuropéptidos/administración & dosificación , Neurotransmisores/administración & dosificación , Receptores de Superficie Celular/efectos de los fármacos , Receptores de Superficie Celular/metabolismo , Receptores de Péptido Intestinal Vasoactivo/efectos de los fármacos , Receptores de Péptido Intestinal Vasoactivo/metabolismo , Péptido Intestinal Vasoactivo/análogos & derivados , Animales , Señalización del Calcio/efectos de los fármacos , Citosol/metabolismo , Relación Dosis-Respuesta a Droga , Técnicas In Vitro , Insulina/metabolismo , Secreción de Insulina , Péptidos Cíclicos/farmacología , Polipéptido Hipofisario Activador de la Adenilato-Ciclasa , Ratas , Ratas Wistar , Receptores de Superficie Celular/agonistas , Receptores del Polipéptido Activador de la Adenilato-Ciclasa Hipofisaria , Receptores del Polipéptido Activador de la Adenilato-Ciclasa Hipofisaria , Receptores de Péptido Intestinal Vasoactivo/agonistas , Receptores de Tipo II del Péptido Intestinal Vasoactivo , Péptido Intestinal Vasoactivo/farmacologíaRESUMEN
Gastrointestinal (GI) smooth muscle cell activity is controlled by contractile cholinergic neurons and relaxant non-adrenergic non-cholinergic (NANC) neurons in the myenteric plexus between the circular and longitudinal muscle layer. Decreased or increased NANC relaxation might be involved in the pathophysiology of functional GI motility disorders. Vasoactive intestinal polypeptide (VIP) and nitric oxide (NO) are the primary inhibitory NANC neurotransmitters. As classic neurotransmitters, VIP is stored in vesicles in the nerve endings, while NO is synthetized on demand by the neuronal isoform of NO synthase (nNOS). The VIP/nNOS co-localization in myenteric neurons, reported for various regions of the GI tract in different species, suggests that VIP and NO are co-transmitters. At the presynaptic level, VIP and NO can induce each others release. Most clear-cut evidence for this mechanism was obtained in isolated myenteric ganglia where VIP induced NO release, and NO facilitated VIP release. At the postsynaptic level, many studies support that VIP and NO are parallel co-transmitters, acting via the adenylate cyclase/3'5' adenosine cyclic monophosphate (cAMP) and guanylate cyclase/3'5' cyclic guanosine monophosphate pathway respectively. Mainly based on results obtained in isolated GI smooth muscle cells, a serial postsynaptic VIP/NO interaction model was proposed, whereby VIP is the principle neurotransmitter, acting partially via a VPAC receptor and the adenylate cyclase/cAMP pathway but also by induction of muscular NO production. Recent results suggest that the capacity of VIP to release NO from isolated smooth muscle cells is related to the induction of inducible NOS (iNOS) in the cells during the isolation procedure. The relative contribution of NO and VIP to GI NANC relaxation differs upon tissue and nerve firing frequency, so that interference with either of them will lead to varying effects.
Asunto(s)
Tracto Gastrointestinal/fisiología , Relajación Muscular/fisiología , Músculo Liso/fisiología , Óxido Nítrico/fisiología , Péptido Intestinal Vasoactivo/farmacología , Animales , Interacciones Farmacológicas , Tracto Gastrointestinal/citología , Tracto Gastrointestinal/inervación , Humanos , Relajación Muscular/efectos de los fármacos , Músculo Liso/citología , Músculo Liso/efectos de los fármacos , Vías Nerviosas/anatomía & histología , Vías Nerviosas/química , Vías Nerviosas/fisiología , Neuronas/química , Neuronas/fisiología , Receptores de Péptido Intestinal Vasoactivo/efectos de los fármacos , Receptores de Péptido Intestinal Vasoactivo/fisiologíaRESUMEN
Receptors for vasoactive intestinal peptide (VIP) and pituitary adenylate cyclase-activating polypeptide (PACAP) in turkey cerebral cortex were characterized using two approaches: (1) in vitro radioreceptor binding of [125I]-VIP, and (2) effects of peptides from the PACAP/VIP/secretin family on cyclic AMP formation. The binding of [125I]-VIP to turkey cortical membranes was rapid, stable, and reversible. Saturation analysis resulted in a linear Scatchard plot, suggesting binding to a single class of high affinity receptor binding sites with a Kd of 0.70 nM and a Bmax of 52 fmol/mg protein. Various peptides displaced the specific binding of 0.12 nM [125I]-VIP to turkey cerebral cortical membranes in a concentration-dependent manner. The relative rank order of potency of the tested peptides to inhibit [125I]-VIP binding to turkey cerebrum was: PACAP38 approximately PACAP27 approximately chicken VIP approximately mammalian VIP >>> PHI >> secretin, chicken VIP16-28 (inactive). About 65% of specific [125I]-VIP binding sites in turkey cerebral cortex was sensitive to Gpp(NH)p, a nonhydrolysable analogue of GTP. PACAP38, PACAP27, chicken VIP and, to a lesser extent, mammalian VIP potently stimulated cyclic AMP formation in turkey cerebral cortical slices in a concentration-dependent manner, displaying EC50 values of 8.7 nM (PACAP38), 21.3 nM (PACAP27), 67.4 nM (chicken VIP), and 202 nM (mammalian VIP). On the other hand, PHI and secretin very weakly affected the nucleotide production. The obtained results indicate that cerebral cortex of turkey contains VPAC type receptors that are positively linked to cyclic AMP-generating system and are labeled with [125I]-VIP.
Asunto(s)
Corteza Cerebral/metabolismo , AMP Cíclico/biosíntesis , Receptores de la Hormona Hipofisaria/metabolismo , Receptores de Péptido Intestinal Vasoactivo/metabolismo , Pavos/metabolismo , Péptido Intestinal Vasoactivo/metabolismo , Animales , Unión Competitiva , Membrana Celular/metabolismo , Corteza Cerebral/efectos de los fármacos , Femenino , Guanilil Imidodifosfato/farmacología , Radioisótopos de Yodo , Cinética , Masculino , Neuropéptidos/metabolismo , Neuropéptidos/farmacología , Péptido PHI/farmacología , Polipéptido Hipofisario Activador de la Adenilato-Ciclasa , Receptores del Polipéptido Activador de la Adenilato-Ciclasa Hipofisaria , Receptores de la Hormona Hipofisaria/efectos de los fármacos , Receptores de Péptido Intestinal Vasoactivo/efectos de los fármacos , Secretina/farmacología , Péptido Intestinal Vasoactivo/farmacologíaRESUMEN
In this study receptors for vasoactive intestinal peptide (VIP) and pituitary adenylate cyclase-activating polypeptide (PACAP) were characterized in chick cerebral cortex by an in vitro binding technique, using 125I-labeled VIP ([125I]-VIP) as a ligand. The specific binding of [125I]-VIP to chick cerebral cortical membranes was found to be rapid, stable, saturable, reversible, and of high affinity. Saturation analysis resulted in a linear Scatchard plot, suggesting binding to a single class of receptor binding sites with high affinity (Kd = 0.21 nM) and low capacity (Bmax = 19.5 fmol/mg protein). The relative rank order of potency of the tested peptides to inhibit [125I]-VIP binding to chick cerebrum was VIP (chicken) > or = VIP (mammalian) > or = PACAP27 > or = PACAP38 >> VIP6-28 (mammalian) > PHI (porcine) >> neurotensin6-11-chicken VIP7-28 > neurotensin6-11-mammalian VIP7-28 >>> VIP16-28 (chicken; inactive) approximately secretin (inactive). About 60% of [125I]-VIP-binding sites in chick cerebral cortex were sensitive to Gpp(NH)p, a nonhydrolyzable analog of GTP. It has been concluded that the cerebral cortex of chick, in addition to PAC1 receptors, contains a population of VPAC-type receptors.
Asunto(s)
Corteza Cerebral/metabolismo , Pollos/metabolismo , Neuropéptidos/metabolismo , Receptores de la Hormona Hipofisaria/metabolismo , Receptores de Péptido Intestinal Vasoactivo/metabolismo , Péptido Intestinal Vasoactivo/metabolismo , Animales , Unión Competitiva/efectos de los fármacos , Unión Competitiva/fisiología , Membrana Celular/efectos de los fármacos , Membrana Celular/metabolismo , Corteza Cerebral/efectos de los fármacos , Guanilil Imidodifosfato/farmacología , Radioisótopos de Yodo , Masculino , Neuronas/efectos de los fármacos , Neuronas/metabolismo , Polipéptido Hipofisario Activador de la Adenilato-Ciclasa , Ensayo de Unión Radioligante , Receptores del Polipéptido Activador de la Adenilato-Ciclasa Hipofisaria , Receptores del Polipéptido Activador de la Adenilato-Ciclasa Hipofisaria , Receptores de la Hormona Hipofisaria/efectos de los fármacos , Receptores de Péptido Intestinal Vasoactivo/efectos de los fármacos , Receptores de Tipo I del Polipéptido Intestinal Vasoactivo , Fracciones SubcelularesRESUMEN
Vasoactive intestinal peptide (VIP), a neuropeptide present in the lymphoid microenvironment, acts as a potent anti-inflammatory agent that inhibits the function of activated macrophages. VIP was shown to inhibit IL-6, TNFalpha, IL-12, chemokine, and nitric oxide production in endotoxin-activated macrophages. The present study reports the effect of VIP on IL-8 production by stimulated human monocytes. VIP inhibits IL-8 production in a dose- and time-dependent manner at the mRNA level. The specific VPAC1 receptor mediates the inhibitory effect of VIP. Two transduction pathways appear to be involved, a major cAMP-independent pathway and a secondary cAMP-dependent pathway. Of obvious physiological significance is the fact that VIP, presumably through the inhibition of IL-8 production, dramatically reduces the monocyte-induced neutrophil chemotaxis, an important event in the pathogenesis of several inflammatory and autoimmune disorders. These findings support the proposed role of VIP as a key endogenous anti-inflammatory agent and describe a novel mechanism, i.e., the inhibition of the production of monocyte-derived IL-8.
Asunto(s)
Interleucina-8/biosíntesis , Monocitos/efectos de los fármacos , Monocitos/inmunología , Péptido Intestinal Vasoactivo/farmacología , Antiinflamatorios/farmacología , AMP Cíclico/metabolismo , Humanos , Técnicas In Vitro , Interleucina-8/genética , Lipopolisacáridos/farmacología , Monocitos/metabolismo , Neuropéptidos/farmacología , ARN Mensajero/genética , ARN Mensajero/metabolismo , Receptores de Péptido Intestinal Vasoactivo/efectos de los fármacos , Receptores de Péptido Intestinal Vasoactivo/metabolismo , Receptores de Tipo I del Polipéptido Intestinal Vasoactivo , Transducción de SeñalRESUMEN
Vasoactive intestinal peptide (chicken form; chVIP), peptide histidine-isoleucine (porcine and rat forms; pPHI and rPHI), D-Phe(4) derivative of porcine PHI (D-Phe(4)-pPHI), peptide histidine-methionine (PHM; human PHI), and helodermin, were tested for their ability to stimulate cAMP production in [(3)H]adenine-prelabeled slices of chick cerebral cortex (CCx) and hypothalamus (HTh). The chVIP (0.1-3 microM) concentration-dependently and potently stimulated cAMP production in HTh and CCx; the responses observed after 3 microM of chVIP were comparable to those produced by 0.1 microM PACAP38. Helodermin (5 microM) moderately but significantly stimulated cAMP formation in both HTh and CCx, whereas pPHI, rPHI, PHM at 5 microM concentration only weakly affected cAMP production in CCx, and were inactive in HTh; D-Phe(4)-pPHI was inactive in both tissues. These data demonstrate that chVIP, PACAP, and to a lesser extent helodermin were capable of potently stimulating cAMP generation in the avian central nervous system. PHI-related peptides showed only weak or no activity, depending on the tissue.
Asunto(s)
Pollos , AMP Cíclico/biosíntesis , Péptido PHI/farmacología , Isoformas de Proteínas/farmacología , Péptido Intestinal Vasoactivo/farmacología , Animales , Radioisótopos de Carbono , Células Cultivadas , Corteza Cerebral/citología , Corteza Cerebral/efectos de los fármacos , Corteza Cerebral/metabolismo , Relación Dosis-Respuesta a Droga , Humanos , Hipotálamo/citología , Hipotálamo/efectos de los fármacos , Hipotálamo/metabolismo , Péptidos y Proteínas de Señalización Intercelular , Masculino , Neuropéptidos/metabolismo , Neuropéptidos/farmacología , Fragmentos de Péptidos/metabolismo , Fragmentos de Péptidos/farmacología , Péptido PHI/metabolismo , Péptidos/metabolismo , Péptidos/farmacología , Polipéptido Hipofisario Activador de la Adenilato-Ciclasa , Receptores de Péptido Intestinal Vasoactivo/efectos de los fármacos , Receptores de Péptido Intestinal Vasoactivo/fisiología , Análisis de Secuencia de Proteína , Porcinos , Tritio , Péptido Intestinal Vasoactivo/metabolismoRESUMEN
Afferent tone is known to influence spinal opioid antinociception but the underlying neurochemical events are not well defined. This study investigates the consequence on cAMP formation of the coincident activation of signal transduction sequelae initiated by an afferent transmitter and opioid using dissociated spinal cord tissue. Afferent transmission was simulated via the addition of vasoactive intestinal polypeptide (VIP), a pelvic visceral afferent transmitter. Individually, mu, delta-, or kappa-selective opioids (1 microM each) did not alter basal spinal content of cAMP. However, VIP (1 microM) and the delta-opioid selective agonist, [D-Pen(2,5)] enkephalin (DPDPE; 1 microM), in combination, manifest a striking facilitative interaction to augment spinal levels of cAMP. Facilitative interactions between VIP and kappa- or mu-opioids were of a reduced magnitude or not observed, respectively. Blockade of delta-opioid or VIP receptors using naltrindole or VIP6-28, respectively antagonized the VIP-DPDPE facilitative interaction, as did pertussis toxin treatment. The VIP-DPDPE facilitative interaction was also eliminated by phospholipase Cbeta inhibition and inositol trisphosphate receptor blockade. This suggests that modulation of Ca(2+) trafficking by VIP and delta-opioid agonists is a point of convergence of their respective signal transduction cascades, the concomitant action at which achieves cytosolic Ca(2+) concentrations that are now sufficient for the activation of signaling molecules, e.g. Ca(2+)/calmodulin-stimulated adenylyl cyclase isoforms. These data underscore the plasticity of spinal delta-opioid neurochemical sequelae and their dependence on concomitant afferent transmitter-initiated neurochemical events.
Asunto(s)
AMP Cíclico/metabolismo , Narcóticos/farmacología , Neurotransmisores/farmacología , Transducción de Señal/efectos de los fármacos , Médula Espinal/metabolismo , Péptido Intestinal Vasoactivo/farmacología , Vías Aferentes/efectos de los fármacos , Analgésicos Opioides/farmacología , Animales , Técnicas de Cultivo de Célula , AMP Cíclico/análisis , Encefalina D-Penicilamina (2,5)/farmacología , Femenino , Plasticidad Neuronal/fisiología , Ovariectomía , Ratas , Receptores Opioides delta/agonistas , Receptores Opioides delta/antagonistas & inhibidores , Receptores de Péptido Intestinal Vasoactivo/antagonistas & inhibidores , Receptores de Péptido Intestinal Vasoactivo/efectos de los fármacos , Receptores de Péptido Intestinal Vasoactivo/metabolismo , Transducción de Señal/fisiología , Médula Espinal/químicaRESUMEN
GHRH stimulates GH secretion from somatotroph cells of the anterior pituitary via a pathway that involves GHRH receptor activation of adenylyl cyclase and increased cAMP production. The actions of GHRH to release GH can be augmented by the synthetic GH secretagogues (GHS), which bind to a distinct G protein-coupled receptor to activate phospholipase C and increase production of the second messengers calcium and diacylglycerol. The stomach peptide ghrelin represents an endogenous ligand for the GHS receptor, which does not activate the cAMP signaling pathway. This study investigates the effects of GHS and ghrelin on GHRH-induced cAMP production in a homogenous population of cells expressing the cloned GHRH and GHS receptors. Each epitope-tagged receptor was shown to be appropriately expressed and to functionally couple to its respective second messenger pathway in this heterologous cell system. Although activation of the GHS receptor alone had no effect on cAMP production, coactivation of the GHS and GHRH receptors produced a cAMP response approximately twice that observed after activation of the GHRH receptor alone. This potentiated response is dose dependent with respect to both GHRH and GHS, is dependent on the expression of both receptors, and was observed with a variety of peptide and nonpeptide GHS compounds as well as with ghrelin-(1-5). Pharmacological inhibition of signaling molecules associated with GHS receptor activation, including G protein betagamma-subunits, phospholipase C, and protein kinase C, had no effect on GHS potentiation of GHRH-induced cAMP production. Importantly, the potentiation appears to be selective for the GHRH receptor. Treatment of cells with the pharmacological agent forskolin elevated cAMP levels, but these levels were not further increased by GHS receptor activation. Similarly, activation of two receptors homologous to the GHRH receptor, the vasoactive intestinal peptide and secretin receptors, increased cAMP levels, but these levels were not further increased by GHS receptor activation. Based on these findings, we speculate that direct interactions between the GHRH and GHS receptors may explain the observed effects on signal transduction.
Asunto(s)
AMP Cíclico/biosíntesis , Hormona Liberadora de Hormona del Crecimiento/farmacología , Hormona del Crecimiento/metabolismo , Hormonas Peptídicas/farmacología , Receptores de Superficie Celular/genética , Receptores Acoplados a Proteínas G , Animales , Benzazepinas/farmacología , Colforsina/farmacología , Sinergismo Farmacológico , Inhibidores Enzimáticos/farmacología , Técnica del Anticuerpo Fluorescente , Expresión Génica , Ghrelina , Células HeLa , Hemaglutininas/genética , Proteínas de Unión al GTP Heterotriméricas/fisiología , Humanos , Técnicas de Inmunoadsorción , Proteína Quinasa C/antagonistas & inhibidores , Proteína Quinasa C/metabolismo , Ratas , Receptores de Superficie Celular/efectos de los fármacos , Receptores de Superficie Celular/fisiología , Receptores de la Hormona Gastrointestinal/efectos de los fármacos , Receptores de la Hormona Gastrointestinal/fisiología , Receptores de Ghrelina , Receptores de Neuropéptido/genética , Receptores de Hormona Reguladora de Hormona Hipofisaria/genética , Receptores de Péptido Intestinal Vasoactivo/efectos de los fármacos , Receptores de Péptido Intestinal Vasoactivo/fisiología , Proteínas Recombinantes de Fusión , Secretina/farmacología , Transducción de Señal , Tetrazoles/farmacología , Transfección , Fosfolipasas de Tipo C/antagonistas & inhibidores , Fosfolipasas de Tipo C/metabolismo , Péptido Intestinal Vasoactivo/farmacologíaRESUMEN
Vasoactive intestinal peptide (VIP) functions as a neurotransmitter involved in a number of physiological and pathological conditions. The actions of VIP are mediated through VPAC(1) and VPAC(2). In contrast to VPAC(1), which has been extensively studied, little is known about the pharmacology of VPAC(2). In this study we investigated the VIP pharmacophore for VPAC(2) by using alanine and D-amino acid scanning. We found significant species differences, and the human VPAC(2) (hVPAC(2)) expressed in Chinese hamster ovary (CHO) cells, which have been used in previous studies, differed significantly from the native hVPAC(2) in Sup T(1) cells and hVPAC(2) expressed in PANC1 cells. There was a close agreement between binding affinities and potencies for VPAC(2) activation. The amino acids whose backbone or side chain orientations were most important for high affinity potency are Asp(3), Phe(6), Thr(7), Tyr(10), Arg(12), Tyr(22), and Leu(23), whereas the side chains of Ser(2), Asp(8), Asn(9), Gln(16), Val(19), Lys(20), Lys(21), Asn(24), and Ser(25) are not essential. Comparison of the VIP pharmacophore between hVPAC(1) and hVPAC(2) demonstrated that the side chains of Thr(7), Tyr(10), Thr(11), and Tyr(22) were much more critical for high affinity for the hVPAC(2) than the hVPAC(1). In contrast, the orientation of the side chain of Asn(24) was more important for high affinity for the hVPAC(1). This study shows that in assessing the pharmacophore of VIP analogs for the VPAC(2), important species differences need to be considered as well as the expression system used. These results of our study should be useful for designing VPAC subtype-selective analogs, simplified analogs, and possibly metabolically stable analogs.
Asunto(s)
Receptores de Péptido Intestinal Vasoactivo/efectos de los fármacos , Péptido Intestinal Vasoactivo/análogos & derivados , Péptido Intestinal Vasoactivo/farmacología , Adenilil Ciclasas/biosíntesis , Adenilil Ciclasas/genética , Alanina/química , Secuencia de Aminoácidos , Sustitución de Aminoácidos , Aminoácidos/química , Animales , Células CHO , Cricetinae , Humanos , Radioisótopos de Yodo , Datos de Secuencia Molecular , Péptidos/química , Péptidos/farmacología , Péptidos Cíclicos/síntesis química , Péptidos Cíclicos/farmacología , Ratas , Receptores de Péptido Intestinal Vasoactivo/química , Receptores de Tipo II del Péptido Intestinal Vasoactivo , Receptores de Tipo I del Polipéptido Intestinal Vasoactivo , Linfocitos T/efectos de los fármacos , Linfocitos T/metabolismo , Transfección , Células Tumorales Cultivadas , Péptido Intestinal Vasoactivo/agonistas , Péptido Intestinal Vasoactivo/síntesis químicaRESUMEN
OBJECTIVE: The cellular and molecular mechanisms of hematopoietic stimulation have been studied. However, an understanding of negative effects in the hematopoietic system remains elusive. To this end, we studied the effects of vasoactive intestinal peptide (VIP) on bone marrow (BM) progenitors. MATERIALS AND METHODS: Different BM cell subsets were used to perform clonogenic assay for granulocytic (CFU-GM) or erythroid (BFU-E and CFU-E) progenitors with 10(-7)-10(-13) M VIP. The relevant receptor was verified with specific antagonists, or agonists, semi-quantitative RT-PCR, and chemical cross-linking studies with stromal membranes. RESULTS: Assays performed with unfractionated mononuclear cells and enriched CD34(+) cells showed dose-dependent inhibition on BM progenitors with significant inhibition up to 10(-10) M. Nylon wool separated cells, which depleted stroma, reversed the inhibitory effects of VIP between 10 and 20%. Combined experimental evaluation indicated that the effects of VIP on BM functions are mediated through the type 1 receptor (VPAC1). VIP induced the production of TGF-beta and TNF-alpha in BM mononuclear cells and stroma. These cytokines are partly involved in reversing the suppressive effects of VIP on CFU-GM. CONCLUSIONS: The effect of VIP on BM progenitors could be mediated through direct and indirect mechanism. Direct effects were evident by the suppressive effects of VIP on clonogenic assays with highly purified CD34(+) cells. Indirect effects were mediated through putative functions of the stromal cells and the production of TGF-beta and TNF-alpha.
Asunto(s)
Células de la Médula Ósea/efectos de los fármacos , Receptores de Péptido Intestinal Vasoactivo/efectos de los fármacos , Péptido Intestinal Vasoactivo/farmacología , Adulto , Células de la Médula Ósea/citología , División Celular/efectos de los fármacos , Línea Celular , Técnicas de Cocultivo , Ensayo de Unidades Formadoras de Colonias , Reactivos de Enlaces Cruzados/farmacología , Células Precursoras Eritroides/citología , Células Precursoras Eritroides/efectos de los fármacos , Eritropoyetina/farmacología , Factor Estimulante de Colonias de Granulocitos y Macrófagos/farmacología , Células HL-60/efectos de los fármacos , Células Madre Hematopoyéticas/efectos de los fármacos , Humanos , Interleucina-3/farmacología , Receptores de Péptido Intestinal Vasoactivo/antagonistas & inhibidores , Receptores de Péptido Intestinal Vasoactivo/biosíntesis , Receptores de Péptido Intestinal Vasoactivo/genética , Receptores de Péptido Intestinal Vasoactivo/fisiología , Receptores de Tipo II del Péptido Intestinal Vasoactivo , Receptores de Tipo I del Polipéptido Intestinal Vasoactivo , Proteínas Recombinantes/farmacología , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Células del Estroma/efectos de los fármacos , Células del Estroma/fisiología , Factor de Crecimiento Transformador beta/fisiología , Factor de Crecimiento Transformador beta1 , Factor de Necrosis Tumoral alfa/fisiologíaRESUMEN
VIP and PACAP38 are closely related peptides that are released in the adrenal gland and sympathetic ganglia and regulate catecholamine synthesis and release. We used PC12 cells as a model system to examine receptor and second messenger pathways by which each peptide stimulates transcriptional and post-transcriptional mechanisms that regulate the level of the mRNA for tyrosine hydroxylase (TH), the rate-limiting enzymatic step in catecholamine synthesis. Concentration-response studies revealed that PACAP38 had both greater efficacy and potency than VIP. The specific PAC1 receptor antagonist PACAP[6-38] blocked the effects of each peptide on TH mRNA content while the PACAP/VIP type II receptor antagonist (N-AC-Tyr(1)-D-Phe(2))-GRF-(1-29)-NH(2) was without effect. At equipotent concentrations, each peptide stimulated a transient increase in TH gene transcription lasting less than 3h. Continuous VIP treatment stimulated a transient increase in TH mRNA lasting less than 24h. In contrast, continuous exposure to PACAP38 stimulated a stable increase in TH mRNA that persisted for 2 days in the absence of elevated transcription, pointing to different post-transcriptional effects of the two peptides. PACAP38 alone had no effect on the magnitude of TH gene transcription or TH mRNA in A126-1B2 PKA-deficient PC12 cells. However, when combined with dexamethasone, PACAP38 produced a synergistic increase in TH mRNA in the absence of PACAP38-stimulated TH gene transcription. In contrast, VIP had no effect on either TH mRNA content or TH gene transcription in this model. PACAP38, but not VIP, stimulated PKC activity. Calphostin C antagonized the effect of PACAP38 on the persistent post-transcriptional elevation in TH mRNA. Thus, the results support the conclusion that VIP and PACAP38 each stimulate PAC1 receptors to increase TH gene transcription through a PKA-controlled pathway, but their divergent post-transcriptional effects result at least partly from differing abilities to stimulate PKC.
Asunto(s)
Neuropéptidos/farmacología , Células PC12/efectos de los fármacos , Procesamiento Postranscripcional del ARN/efectos de los fármacos , Transcripción Genética/efectos de los fármacos , Tirosina 3-Monooxigenasa/biosíntesis , Péptido Intestinal Vasoactivo/farmacología , Animales , Proteína Quinasa Tipo II Dependiente de AMP Cíclico , Proteínas Quinasas Dependientes de AMP Cíclico/fisiología , Dexametasona/farmacología , Sinergismo Farmacológico , Inducción Enzimática/efectos de los fármacos , Naftalenos/farmacología , Proteínas de Neoplasias/biosíntesis , Proteínas de Neoplasias/genética , Neuropéptidos/antagonistas & inhibidores , Células PC12/enzimología , Fragmentos de Péptidos/farmacología , Polipéptido Hipofisario Activador de la Adenilato-Ciclasa , Proteína Quinasa C/fisiología , ARN Mensajero/metabolismo , Ratas , Receptores del Polipéptido Activador de la Adenilato-Ciclasa Hipofisaria , Receptores del Polipéptido Activador de la Adenilato-Ciclasa Hipofisaria , Receptores de la Hormona Hipofisaria/antagonistas & inhibidores , Receptores de la Hormona Hipofisaria/efectos de los fármacos , Receptores de Péptido Intestinal Vasoactivo/efectos de los fármacos , Factores de Tiempo , Tirosina 3-Monooxigenasa/genéticaRESUMEN
Microglia react to even minor disturbances in CNS homeostasis and function as critical regulators of CNS inflammation. Activated microglia secrete inflammatory mediators such as cytokines and chemokines, which contribute to the pathophysiological changes associated with several neuroimmunologic disorders. Microglia-derived inflammatory chemokines recruit various populations of immune cells, which initiate and maintain the inflammatory response against foreign antigens. Entry and retention of activated immune cells in the CNS is a common denominator in a variety of traumatic, ischemic, and degenerative diseases. Vasoactive intestinal peptide (VIP) and pituitary adenylate cyclase-activating polypeptide (PACAP) are two structurally related neuropeptides that function as potent anti-inflammatory factors in the periphery. Here we investigated the effects of VIP and PACAP on chemokine production by activated microglia. VIP and PACAP inhibit the expression of the microglia-derived CXC chemokines MIP-2 and KC, and of the CC chemokines MIP-1alpha, -1beta, MCP-1, and RANTES. The inhibition of chemokine gene expression correlates with an inhibitory effect of VIP/PACAP on NFkB binding. The VIP/PACAP inhibition of both chemokine production and of NFkB binding is mediated through the specific receptor VPAC1 and involves a cAMP-dependent intracellular pathway. Of biological significance is the fact that the inhibition of chemokine production by VIP/PACAP leads to a significant reduction in the chemotactic activity generated by activated microglia for peripheral leukocytes, i.e., neutrophils, macrophages, and lymphocytes. Because reduction in the number and activation of infiltrating leukocytes represents an important factor in the control of inflammation in the CNS, VIP and/or PACAP released by neurons during an inflammatory response could serve as neuronal survival factors by limiting the inflammatory process.