vasoactive-intestinal-peptide and Neuroblastoma

Topics: Amino Acid Sequence; Animals; Galanin; Humans; Immunohistochemistry; Molecular Sequence Data; Neuroblastoma; Peptide PHI; Peptides; Structure-Activity Relationship; Tumor Cells, Cultured; Vasoactive Intestinal Peptide

Topics: Adrenal Glands; Animals; Chromaffin System; Cyclic AMP; Humans; Mice; Neuroblastoma; Phorbol Esters; Tumor Cells, Cultured; Vasoactive Intestinal Peptide

As sensitive radioimmunoassays for the detection of polypeptide hormones are developed, the exciting discovery of a diffusely distributed system of interrelated endocrine cells has begun a new era of endocrinology. This system, although anatomically disassociated, is bound together by a number of common features such as its biosynthetic mechanism, histochemical and ultrastructural features, and embryologic origin (Table I). The most prominent feature, however, is their biosynthetic pathways for hormone production, from which the acronym APUD has been derived. These are the capacity for Amine Precursor Uptake such as DOPA and then subsequent Decarboxylation, resulting in the synthesis of bioactive amines or polypeptide hormones. Hyperplasias or neoplasms of these cells are defined as apudomas. In the last ten years a great deal of research has rapidly altered the original concepts of this system, especially in terms of its embryologic origin, physiologic interrelationships, classification, as well as the addition of many new APUD cell members. These will be reviewed, and the origin, diagnosis, and treatment of each recognized apudoma will be synthesized in light of its membership within the APUD system.

Topics: Adenoma, Islet Cell; APUD Cells; Apudoma; Carcinoid Tumor; Carcinoma; Endocrine Glands; Humans; Neural Crest; Neuroblastoma; Paraganglioma; Pheochromocytoma; Pituitary Neoplasms; Somatostatin; Thyroid Neoplasms; Vasoactive Intestinal Peptide

Vasoactive intestinal polypeptide (VIP) is reported to exert an autocrine control on neuroblastoma cell tumours: VIP is produced by the tumour and stimulates cell differentiation. This study tested the hypothesis that the parent peptide; the pituitary adenylate cyclase activating polypeptide (PACAP) may have a similar role. It was found that PACAP mRNA and PACAP were expressed in 12/12 tumours; it was also observed that PACAP receptor mRNA and functional PACAP receptors were expressed in 12/12 and 5/9 tumours, respectively. VIP mRNA and VIP were detected in 9/12 tumours. VIP receptor mRNA was expressed in 5/12 tumours and functional VIP receptors were never demonstrated. The tumours having the highest VIP levels also had the highest PACAP contents and were associated with a watery diarrhoea syndrome due to activation of intestinal VIP receptors. As PACAP recognizes the PACAP receptors and the VIP receptors with the same high affinity it may contribute to the syndrome and is a likely candidate for an autocrine control of neuroblastoma cell growth and differentiation.

Topics: Autocrine Communication; Binding, Competitive; Child; Child, Preschool; Female; Humans; Infant; Male; Neuroblastoma; Neuropeptides; Pituitary Adenylate Cyclase-Activating Polypeptide; Vasoactive Intestinal Peptide

We herein report a 10-month-old female infant with a 4-month history of diarrhea with electrolyte abnormalities and growth impairment. A 4-cm right adrenal tumor was detected by computed tomography. No metastasis or accumulation on I

Topics: Adrenal Gland Neoplasms; Adrenal Glands; Female; Humans; Infant; Laparoscopy; Neuroblastoma; Vasoactive Intestinal Peptide

The synaptic vesicle glycoprotein 2 (SV2) family is essential to the synaptic machinery involved in neurotransmission and vesicle recycling. The isoforms SV2A, SV2B and SV2C are implicated in neurological diseases such as epilepsy, Alzheimer's and Parkinson's disease. Suitable cell systems for studying regulation of these proteins are essential. Here we present gene expression data of SV2A, SV2B and SV2C in two human neuroblastoma cell lines after differentiation.. Human neuroblastoma cell lines SiMa and IMR-32 were treated for seven days with growth supplements (B-27 and N-2), all-trans-retinoic acid (ATRA) or vasoactive intestinal peptide (VIP) and gene expression levels of SV2 and neuronal targets were analyzed.. The two cell lines reacted differently to the treatments, and only one of the three SV2 isoforms was affected at a time. SV2B and choline O-acetyltransferase (CHAT) expression was changed in concert after growth supplement treatment, decreasing in SiMa cells while increasing in IMR-32. ATRA treatment resulted in no detected changes in SV2 expression in either cell line while VIP increased both SV2C and dopamine transporter (DAT) in IMR-32 cells.. The synergistic expression patterns between SV2B and CHAT as well as between SV2C and DAT mirror the connectivity between these targets found in disease models and knock-out animals, although here no genetic alteration was made. These cell lines and differentiation treatments could possibly be used to study SV2 regulation and function.

Topics: Binding Sites; Cell Differentiation; Cell Line, Tumor; Gene Expression Regulation, Neoplastic; Humans; Membrane Glycoproteins; Nerve Tissue Proteins; Neuroblastoma; Promoter Regions, Genetic; Transcription Factors; Transcription Initiation Site; Tretinoin; Vasoactive Intestinal Peptide

Very rarely, vasoactive intestinal peptide-related diarrhea (VIP-D) is observed in patients with high-risk neuroblastoma (HR-NB) where the associated fluid and electrolyte abnormalities can pose a major clinical challenge for administering the required aggressive multimodality treatment. Two patients with HR-NB developed VIP-D during induction and were found to have a somatic BRAF V600E mutation. Serum VIP levels and diarrhea promptly resolved in both patients after initiating treatment with BRAF and MEK inhibitors. This illustrates an association of VIP-D with BRAF V600E mutations and demonstrates a therapeutic strategy in the specific context of VIP-D and BRAF V600E mutations in HR-NB patients. The addition of BRAF and MEK inhibitors allows continued conventional tumor-directed treatment by decreasing the severity of symptoms caused by this life-threatening complication.

Topics: Diarrhea; Humans; Mitogen-Activated Protein Kinase Kinases; Mutation; N-Myc Proto-Oncogene Protein; Neuroblastoma; Protein Kinase Inhibitors; Proto-Oncogene Proteins B-raf; Vasoactive Intestinal Peptide

Pituitary adenylate cyclase-activating polypeptide (PACAP) and vasoactive intestinal peptide (VIP) are two related peptides acting as neurotransmitters/neuromodulators in central and peripheral nervous system. They are also involved in cancer showing a controversial role. Particulary, they are implicated in neuroblastoma differentiation (NB). This pediatric tumor can evolve to a malignant metastatic disease or spontaneously regress towards a benign form, known as ganglioneuroblastoma/ganglioneuroma. A negative hallmark of neoplasia progression is represented by hypoxic microenvironment. Low oxygen tension induces activation of hypoxia-inducible factors (HIFs) promoting cells proliferation and metastasis formation. Moreover, HIFs trigger vascular endothelial growth factor (VEGF) release favouring high-risk NB phenotype development. In the present work, we have investigated for the first time, if PACAP and VIP interfere with NB differentiation through modulation of hypoxic/angiogenic process. To this end, we analyzed their effect in malignant undifferentiated and all-trans retinoic acid (RA) differentiated SH-SY5Y cells, representing the benign form of this tumor. Our results have suggested tha both peptides, but predominantly VIP, induce NB differentiation into benign form by regulating HIFs, VEGF and VEGFRs expression and distribution. All these data give new insight regarding PACAP/VIP regulatory role in NB progression.

Topics: Basic Helix-Loop-Helix Transcription Factors; Cell Differentiation; Cell Line, Tumor; Humans; Hypoxia-Inducible Factor 1, alpha Subunit; Neuroblastoma; Pituitary Adenylate Cyclase-Activating Polypeptide; Receptors, Vascular Endothelial Growth Factor; Tumor Hypoxia; Vascular Endothelial Growth Factor A; Vasoactive Intestinal Peptide

Topics: Abdominal Neoplasms; Female; Humans; Infant; Neoplasm Proteins; Neuroblastoma; Steroids; Tomography, X-Ray Computed; Vasoactive Intestinal Peptide

Chronic watery diarrhoea can be a presentation of gastrointestinal disease itself or a less-evident systemic disease. A 17-month-old boy presented with intractable diarrhoea, failure to gain weight, refractory tachycardia and severe hypertension. The ability to recognise and make a quick diagnosis of secretory type of diarrhoea dictated the outcome of patients with this ailment. Catecholamine hypersecretion was considered with the additional clues of refractory tachycardia and hypertension, a well-recognised phenomenon of neuroblastic tumours. A neuroblastic tumour can lead to vasoactive intestinal peptide (VIP) overexpression, which may result in secretory diarrhoea. In this situation, measurements of plasma VIP enabled crucial diagnosis. Imaging studies were used to identify and localise a neuroblastic tumour. Subsequent removal of the tumour was curative and led to the resolution of the symptoms.

Topics: Adrenal Gland Neoplasms; Catecholamines; Diarrhea; Humans; Hypertension; Infant; Male; Neuroblastoma; Tachycardia; Treatment Outcome; Vasoactive Intestinal Peptide

Neuroblastoma (NB) is the most common extracranial solid tumor in children. Diarrheal NB is quite rare and is not easy to diagnose in the early stage. Six cases of diarrheal NB in our hospital treated from 1996 to 2006 were retrospectively analyzed, including characteristics such as electrolyte imbalance, pathologic features, vasoactive intestinal peptide (VIP) immunohistochemical staining results, treatment, and prognosis. All patients were boys with 3-8 loose or watery stools each day and routine fecal tests were normal. Abdominal tumors were identified by B-ultrasound. Drugs were ineffective. Three patients underwent surgery, and the remaining three patients received surgery and chemotherapy. Diarrhea stopped after treatment in five patients. Two patients died due to intractable hypokalemia. The tumor was located in the adrenal gland in four patients, in the upper retroperitoneum in one patient, and in the presacral area in one patient. Pathologic findings were NB and ganglioneuroblastoma. Five patients were at clinical stage I-II, and one was at stage III. Four patients survived (followed-up for 6 mo to 4 years). Immunohistochemical staining for VIP was positive. Refractory diarrhea is a paraneoplastic syndrome of NB and is rare. Patients aged 1-3 years who present with chronic intractable diarrhea should be followed closely. Intractable diarrhea, hypokalemia, and dysplasia are the initial clinical manifestations. Increased VIP is characteristic of this disease. Potassium supplementation plays a vital role in the treatment procedure, especially preoperatively. The prognosis of diarrheal NB is good following appropriate treatment.

Topics: Adrenal Gland Neoplasms; Biomarkers, Tumor; Biopsy; Child, Preschool; Diarrhea; Humans; Hypokalemia; Immunohistochemistry; Infant; Male; Neoplasm Staging; Neuroblastoma; Paraneoplastic Syndromes; Retroperitoneal Neoplasms; Retrospective Studies; Treatment Outcome; Vasoactive Intestinal Peptide

We have demonstrated previously that the Myc oncoprotein blocks cancer cell differentiation by forming a novel transcriptional repressor complex with histone deacetylase and inhibiting gene transcription of tissue transglutaminase (TG2). Moreover, induction of TG2 gene transcription and transamidase activity is essential for the differentiating effects of retinoids in cancer cells. Here, we show that two structurally distinct TG2 protein isoforms, the full-length (TG2-L) and the short form (TG2-S), exert opposing effects on cell differentiation. Repression of TG2-L with small interfering RNA, which did not affect TG2-S expression, induced dramatic neuritic differentiation in neuroblastoma cells. In contrast, overexpression of TG2-S or a GTP-binding-deficient mutant of TG2-L (R580A), both of which lack the GTP-binding Arg-580 residue, induced neuroblastoma cell differentiation, which was blocked by an inhibitor of transamidase activity. Whereas N-Myc repressed and retinoid activated both TG2 isoforms, repression of TG2-L, but not simultaneous repression of TG2-L and TG2-S, enhanced neuroblastoma cell differentiation due to N-Myc small interfering RNA or retinoid. Moreover, suppression of vasoactive intestinal peptide (VIP) expression alone induced neuroblastoma cell differentiation, and VIP was up-regulated by TG2-L, but not TG2-S. Taken together, our data indicate that TG2-L and TG2-S exert opposite effects on cell differentiation due to differences in GTP binding and modulation of VIP gene transcription. Our findings highlight the potential importance of repressing the GTP binding activity of TG2-L or activating the transamidase activity of TG2-L or TG2-S for the treatment of neuroblastoma, and possibly also other Myc-induced malignancies, and for enhancing retinoid anticancer effects.

Topics: Arginine; Binding Sites; Cell Differentiation; Cell Line, Tumor; Drug Resistance, Neoplasm; Gene Expression Regulation, Enzymologic; GTP-Binding Proteins; Guanosine Triphosphate; Humans; Immunoblotting; Isoenzymes; Neurites; Neuroblastoma; Protein Binding; Protein Glutamine gamma Glutamyltransferase 2; Proto-Oncogene Proteins c-myc; Retinoids; Reverse Transcriptase Polymerase Chain Reaction; RNA Interference; Transfection; Transglutaminases; Vasoactive Intestinal Peptide

Neuroblastic tumors (NTs) are occasionally associated with watery diarrhea, due to Vasoactive Intestinal Peptide (VIP) secretion. Most reports are single cases and suggest a great homogeny within this sub-group of NTs.. We conducted a retrospective analysis of the French experience of NTs associated with watery diarrhea due to VIP-secretion. VIP secretion was confirmed by seric dosage and/or immunohistochemistry.. Twenty-two patients met the diagnostic criteria between 1988 and 2007. Most of patients suffered from weight loss and metabolic disorders. In 16 cases, digestive symptoms preceded the diagnosis of the tumor ("Primary VIP secreting NTs"); 15 were localized and all showed a differentiated histology. Interestingly, in another 6 patients with high-risk NT, diarrhea occurred at the time of chemotherapy or retinoic acid therapy ("Secondary VIP secreting NTs"). Differentiation in response to treatment was documented in 4 cases. In all cases, only surgical excision of the tumor was able to control the digestive symptoms. Twenty children are alive and 13 are disease-free.. VIP secreting NTs are usually associated with differentiation; they can also secondarily arise from a high-risk tumor upon treatment. Primary surgery constitutes first-line treatment.

Topics: Child, Preschool; Disease-Free Survival; Female; France; Humans; Infant; Male; Neuroblastoma; Retrospective Studies; Societies, Medical; Survival Rate; Vasoactive Intestinal Peptide

Ganglia expressing the neuropeptide pituitary adenylate cyclase-activating polypeptide (PACAP) innervate vasoactive intestinal peptide (VIP) containing neurons suggesting a role of PACAP in regulating VIP expression. Human NB-1 neuroblastoma cells were applied to study PACAP regulated VIP gene expression aiming to identify the receptor and the signaling proteins involved. The PACAP receptor subtype PAC1 induced VIP gene expression as (i) PACAP and the PAC1 receptor agonist maxadilan were equally efficient and approximately 200-fold more potent than VIP, and (ii) PACAP6-38 and PG99-465, antagonists of PAC1 and VPAC2 receptors, respectively, abolished and did not affect the PACAP-induced VIP mRNA expression, respectively. A pivotal role of PKA was implicated in addition to partial involvement of PKC and ERK1/2 in PACAP-induced VIP gene expression as H-89, Bisindolylmaleimide I (BIS), Gö6976 and U0126 attenuated the VIP mRNA expression by 93%, 58%, 58% and 40%, respectively. PACAP modulated the phosphorylation of ERK1/2 (pERK1/2) and CREB/ATF-1 (pCREB/ATF-1) concomitant with a translocation of PKA to the nucleus. Inhibition of conventional PKC isoforms and MEK1/2 completely abolished pERK1/2 without affecting PACAP induced pCREB/ATF-1. In contrast, inhibiting PKA attenuated PACAP induced pCREB/ATF-1. PACAP also enhanced the FOS gene expression and individual presence of H-89, BIS, Gö6976 and U0126 partially attenuated the PACAP induced FOS mRNA expression. Combining the kinase inhibitors completely suppressed the PACAP induced FOS mRNA expression. Immunoblotting confirmed expression of FOS protein upon addition of PACAP, which was diminished by impairment of PKC, ERK1/2 and PKA activities. The resemblance of the signaling pathways involving concomitant activities of PKC, ERK1/2 and PKA in PACAP regulation of the FOS and VIP gene expressions suggest for the first time a role of FOS in PACAP-induced VIP gene expression in human NB-1 neuroblastoma cells.

Topics: Active Transport, Cell Nucleus; Animals; Cell Line, Tumor; Cyclic AMP Response Element-Binding Protein; Cyclic AMP-Dependent Protein Kinases; Extracellular Signal-Regulated MAP Kinases; Gene Expression Regulation, Neoplastic; Humans; MAP Kinase Kinase 1; MAP Kinase Kinase 2; Neuroblastoma; Pituitary Adenylate Cyclase-Activating Polypeptide; Protein Kinase C; Proto-Oncogene Proteins c-fos; Signal Transduction; Vasoactive Intestinal Peptide

To elucidate more precisely the biological characteristics of neuroblastomas, we examined four human neuroblastomas heterotransplanted into athymic nude mice NB-39 (undifferentiated type), NB-45 (poorly differentiated type with undifferentiated component), NB-52 (poorly differentiated type), and NB-726 (differentiating type) by electron microscopy, immunohistochemistry, and radioimmunoassay for the peptides in tumors. Ultrastructurally, NB-45, NB-52, and NB-726 contained more numerous and variously sized neurosecretory granules than did NB-39. Immunohistochemistry revealed neurofilament proteins, tyrosine hydroxylase, neuropeptide Y (NPY), and chromogranin A-positive cells in the four tumors in the following order of frequency: NB-726, NB-45, NB-52, and NB-39. NB-726, NB-45, and NB-52, but not NB-39, contained galanin-positive tumor cells. NB-45 and NB-726 harbored a few positive cells for calcitonin gene-related peptide. Furthermore, NB-726 exhibited positivity to leu-enkephalin, met-enkephalin, vasoactive intestinal peptide (VIP), and serotonin. Radioimmunoassay substantiated the results of immunohistochemistry, showing NPY in all tumors and either galanin or VIP in three tumors, excepting NB-39. Average doubling time of the tumor was as follows: 2 days in NB-39, 10 days in NB-45, 22 days in NB-52, and 45 days in NB-726. These results indicate that human neuroblastoma cells have different biological characteristics and reduced growth rate with differentiation in terms of ultrastructure and of peptide production abilities.

Topics: Animals; Calcitonin Gene-Related Peptide; Child; Child, Preschool; Enkephalin, Leucine; Enkephalin, Methionine; Female; Galanin; Humans; Infant; Male; Mice; Mice, Nude; Neoplasm Transplantation; Neuroblastoma; Neuropeptide Y; Secretory Vesicles; Serotonin; Transplantation, Heterologous; Tyrosine 3-Monooxygenase; Vasoactive Intestinal Peptide

Neuroblastoma is a pediatric tumor which can spontaneously regress or differentiate into a benign tumor. MYCN oncogene amplification occurs in 22% of neuroblastomas and is associated with poor prognosis. Retinoic acid (RA), a molecule able to induce differentiation and to decrease MYCN expression, is used in the therapy of neuroblastomas. The neuropeptide vasoactive intestinal peptide (VIP) is known to control proliferation or differentiation of numerous cancer cells. In vitro, VIP induces differentiation of neuroblastoma cells. To determine whether VIP could modulate MYCN expression, we carried out real-time quantitative RT-PCR and Western immunoblot analyses in human neuroblastoma SH-SY5Y and IMR-32 cells. The results indicated that VIP reduced MYCN mRNA and protein expression, especially in the MYCN-amplified IMR-32 cells, with a maximal and transient decrease by approximately 50% after few hours of treatment with VIP at 10(-6) M. This effect was compared to that of RA at 10(-5) M, which induced a diminution of MYCN mRNA expression by approximately 25% after few days of treatment. This indicated that VIP and RA display complementary kinetics. Cotreatments showed that VIP and RA had synergistic effects on regulation of expression of MYCN proteins. VIP and RA cotreatments regulated also expression of two MYCN target genes, SKP2 and TP53INP1. These results suggest that VIP, in combination with RA may have a potential therapeutic benefit in neuroblastomas with MYCN amplification, a genetic abnormality associated with poor prognosis.

Topics: Antineoplastic Agents; Carrier Proteins; Cell Line, Tumor; Cell Shape; Down-Regulation; Drug Synergism; Gene Expression Regulation, Neoplastic; Heat-Shock Proteins; Humans; Kinetics; N-Myc Proto-Oncogene Protein; Neuroblastoma; Nuclear Proteins; Oncogene Proteins; RNA, Messenger; S-Phase Kinase-Associated Proteins; Tretinoin; Vasoactive Intestinal Peptide

The intracellular signaling pathways mediating the neurotrophic actions of pituitary adenylate cyclase-activating polypeptide (PACAP) were investigated in human neuroblastoma SH-SY5Y cells. Previously, we showed that SH-SY5Y cells express the PAC(1) and VIP/PACAP receptor type 2 (VPAC(2)) receptors, and that the robust cAMP production in response to PACAP and vasoactive intestinal peptide (VIP) was mediated by PAC(1) receptors (Lutz et al. 2006). Here, we investigated the ability of PACAP-38 to differentiate SH-SY5Y cells by measuring morphological changes and the expression of neuronal markers. PACAP-38 caused a concentration-dependent increase in the number of neurite-bearing cells and an up-regulation in the expression of the neuronal proteins Bcl-2, growth-associated protein-43 (GAP-43) and choline acetyltransferase: VIP was less effective than PACAP-38 and the VPAC(2) receptor-specific agonist, Ro 25-1553, had no effect. The effects of PACAP-38 and VIP were blocked by the PAC(1) receptor antagonist, PACAP6-38. As observed with PACAP-38, the adenylyl cyclase activator, forskolin, also induced an increase in the number of neurite-bearing cells and an up-regulation in the expression of Bcl-2 and GAP-43. PACAP-induced differentiation was prevented by the adenylyl cyclase inhibitor, 2',5'-dideoxyadenosine (DDA), but not the protein kinase A (PKA) inhibitor, H89, or by siRNA-mediated knock-down of the PKA catalytic subunit. PACAP-38 and forskolin stimulated the activation of extracellular signal-regulated kinase (ERK), mitogen-activated protein kinase (MAP; p38 MAP kinase) and c-Jun N-terminal kinase (JNK). PACAP-induced neuritogenesis was blocked by the MEK1 inhibitor PD98059 and partially by the p38 MAP kinase inhibitor SB203580. Activation of exchange protein directly activated by cAMP (Epac) partially mimicked the effects of PACAP-38, and led to the phosphorylation of ERK but not p38 MAP kinase. These results provide evidence that the neurotrophic effects of PACAP-38 on human SH-SY5Y neuroblastoma cells are mediated by the PAC(1) receptor through a cAMP-dependent but PKA-independent mechanism, and furthermore suggest that this involves Epac-dependent activation of ERK as well as activation of the p38 MAP kinase signaling pathway.

Topics: Cell Size; Cyclic AMP; Dose-Response Relationship, Drug; Drug Interactions; Enzyme Activation; Enzyme Inhibitors; Extracellular Signal-Regulated MAP Kinases; Gene Expression Regulation, Neoplastic; Growth Substances; Humans; Nerve Tissue Proteins; Neuroblastoma; Neurons; p38 Mitogen-Activated Protein Kinases; Pituitary Adenylate Cyclase-Activating Polypeptide; Vasoactive Intestinal Peptide

Transduction and activation of an inducible form of STAT3 (signal transducer and activator of transcription) sufficed to increase VIP (vasoactive intestinal protein) mRNA concentrations in neuroblastoma cells. Overexpression of SOCS3 (suppressor of cytokine signaling) inhibited and mutant SOCS3 (with an inactivating point mutation in amino acid 25) enhanced the induction of VIP mRNA by CNTF (ciliary neurotrophic factor). Because mutant SOCS3 did not augment the increase in STAT transcriptional activity following CNTF stimulation, the enhancement by mutant SOCS3 of the actions of CNTF cannot be attributed to changes in STAT3 signaling. Mutant SOCS3 increased AP-1 (activator protein) transcriptional activity and JNK (c-Jun N-terminal kinase) activity and SOCS3 bound to the scaffolding protein, JNK-interacting protein-1: these observations provide a plausible explanation for the enhancement by mutant SOCS3 of the actions of CNTF. We conclude that endogenous SOCS3 inhibits AP-1 activity through blocking of JNK phosphorylation.

Topics: Adaptor Proteins, Signal Transducing; Animals; Cell Line, Tumor; Ciliary Neurotrophic Factor; Down-Regulation; Feedback, Physiological; Gene Expression Regulation; Humans; JNK Mitogen-Activated Protein Kinases; Mice; Neuroblastoma; Neurons; Phosphorylation; Rats; RNA, Messenger; STAT3 Transcription Factor; Suppressor of Cytokine Signaling 3 Protein; Suppressor of Cytokine Signaling Proteins; Transcription Factor AP-1; Transcription, Genetic; Transcriptional Activation; Vasoactive Intestinal Peptide

Parkinson's disease (PD) and 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) toxicity are both associated with dopaminergic neuron death in the substantia nigra. Although a variety of evidence has shown that degenerative cells have apoptotic features, the role of apoptosis in disease pathology remains controversial. The 1-methyl-4-phenylpyridinium ion (MPP(+)), a metabolite of MPTP, was recently shown to alter the expression of proteins involved in translational control. The initiation step of translational control is regulated by a cascade of phosphorylation affecting proteins of the antiapoptotic way controlled by mammalian target of rapamycin (mTOR) and of the proapoptotic way controlled by double-stranded RNA protein-dependent kinase (PKR). A study showed that MPP(+) induced an increase in eIF2alpha phosphorylation, leading to inhibition of protein synthesis.. (1) to assess the effects of MPP(+) toxicity on molecular factors of PKR and mTOR signaling pathways in murine neuroblastoma cells, and (2) to examine the ability of VIP and PACAP peptides to counteract the MPP(+) toxicity. Our findings showed that MPP(+) induced phosphorylation of eIF2alpha and significantly reduced the expression of phosphorylated mTOR, p70S6K, eIF4E, and 4E-BP1, suggesting its toxicity in controlling protein synthesis. Furthermore, the VIP peptide had no effect on either the PKR or the mTOR signaling pathway. On the contrary, the PACAP 27 neuropeptide prevented MPP(+)-induced eIF2alpha phosphorylation and blocked MPP(+) toxicity in molecular factors of the mTOR pathway. And last, PACAP 27 seemed to protect Neuro-2a cells from the apoptotic process as assessed by the decreased nuclear condensation after DAPI staining. These results could open new paths of research of PACAP in PD.

Topics: Animals; Blotting, Western; Brain Neoplasms; Caspase 3; Cell Line, Tumor; Fluorescent Dyes; Indoles; Mice; MPTP Poisoning; Neuroblastoma; Neuropeptides; Neuroprotective Agents; Phosphorylation; Pituitary Adenylate Cyclase-Activating Polypeptide; Protein Biosynthesis; Protein Kinases; Signal Transduction; TOR Serine-Threonine Kinases; Vasoactive Intestinal Peptide

Expression of VPAC and PAC1 receptor isoforms was determined in six neuroblastoma cell lines as well as in human embryonic and adult brain using reverse transcriptase PCR and quantitative PCR. PAC1 receptor splice variants missing a 21 amino acid sequence in the amino terminal domain were found to be the major receptor variants in the neuroblastoma cell lines and also were highly expressed in embryonic brain compared to adult brain. In four of the neuroblastoma cell lines, VIP and PACAP stimulated cyclic AMP production with different potencies and levels of maximal stimulation. High potency and greatest maximal stimulation of cyclic AMP for each peptide were recorded in SH-SY5Y cells, indicating the presence of high affinity VIP and PACAP receptors. Further characterization of specific VPAC and PAC1 receptor isoforms was carried out in the SH-SY5Y cell line, where along with known PAC1 receptor splice variants and the VPAC2 receptor, a number of novel PAC1 receptor splice variants were identified. The comparatively low level expression of the VPAC2 receptor along with the poor responsiveness of SH-SY5Y cells to the VPAC2 receptor-specific agonist Ro 25-1553 indicated that this receptor did not contribute significantly to the observed VIP responses. When the individual PAC1 receptor isoforms were expressed in COS 7 cells, the ability of VIP to activate cyclic AMP production was increased more than 50-fold at the majority of the PAC1 receptor variants lacking the 21 amino acid amino terminal domain sequence compared to those with the complete domain. Smaller changes were seen in the potency of PACAP-38. Similar trends were seen with inositol phosphate responses, where in each case agonist potencies were lower than for cyclic AMP production. The results of this study show that the combination of different amino terminal and intracellular loop 3 splicing variants in the PAC1 receptor dictates the ability of agonists, particularly VIP, to activate signaling pathways. VIP has considerably greater potency at most PAC1 receptors with the short amino terminal domain, and these therefore may mediate physiological effects of both VIP and PACAP. Furthermore, there may be a phenotypic switch in the expression of different PAC1 receptor amino terminal splice variants between embryonic and mature nervous system, indicating that regulation of this event may have an important role in VIP/PACAP function, particularly in the developing nervous system.

Topics: Adult; Alternative Splicing; Amino Acid Sequence; Animals; Base Sequence; Cell Line, Tumor; Chlorocebus aethiops; COS Cells; Cyclic AMP; Exons; Fetus; Humans; Molecular Sequence Data; Neuroblastoma; Peptide Fragments; Pituitary Adenylate Cyclase-Activating Polypeptide; Protein Isoforms; Receptors, Pituitary Adenylate Cyclase-Activating Polypeptide, Type I; Receptors, Vasoactive Intestinal Peptide; Second Messenger Systems; Sequence Alignment; Vasoactive Intestinal Peptide

The vasoactive intestinal peptide (VIP) gene has been studied extensively as a prototype neuronal gene containing multiple cis-active elements that confer responsiveness to cell lineage, neurotrophic, and activity-dependent intrinsic and extrinsic cues. However, reporter genes containing the presumptive complete regulatory region 5' to the start of transcription do not confer tissue-specific gene expression in vivo. We therefore sought cis-regulatory elements downstream of the transcriptional start that might confer additional tissue-specific and tissue-restrictive properties to the VIP transcriptional unit. We report here a repressor element, similar to the canonical restrictive element-1 (RE-1), located within the first non-coding exon of the human VIP gene. The ability of this element to regulate VIP reporter gene expression in neuroblastoma and fibroblastic cells was examined. Endogenous VIP expression is high in SH-EP neuroblastoma cells, low but inducible in SH-SY5Y cells, and absent in HeLa cells. Endogenous RE-1 silencer factor (REST) expression was highest in SH-EP and HeLa cells, and significantly lower in SH-SY5Y cells. Transient transfection of a VIP reporter gene containing a mutated RE-1 sequence revealed an RE-1-dependent regulation of VIP gene expression in all three cell types, with regulation greatest in cells (SH-EP, HeLa) with highest levels of REST expression. Serial truncation of the VIP reporter gene further revealed a specific interaction between the RE-1 and a tissue-specifier element located 5 kb upstream in the VIP gene. Thus, REST can regulate VIP gene expression in both neuroblastic and non-neuronal cells, but requires coupling to the upstream tissue specifier element.

Topics: Cell Line; Fibroblasts; Gene Expression Regulation; Genes, Reporter; HeLa Cells; Humans; Mutagenesis, Site-Directed; Neuroblastoma; Neurons; Regulatory Sequences, Nucleic Acid; Repressor Proteins; Sequence Deletion; Transcription Factors; Transfection; Vasoactive Intestinal Peptide

In children, the watery diarrhoea-hypokalemia-achlorhydria (WDHA) syndrome is uncommon and usually due to a neuroblastic tumour hypersecreting the vasoactive intestinal peptide (VIP). We report a case of WDHA syndrome secondary to hypersecretion of VIP that revealed a neuroblastoma in a 13-month-old girl. A secretory diarrhoea, characterised by the persistence of diarrhoea despite the cessation of oral feeding, led to the search of a neuroblastic tumour in the patient. The serum concentration of VIP decreased to normal values soon after the surgical excision of the tumour.

Topics: Diagnosis, Differential; Diarrhea; Female; Humans; Infant; Neuroblastoma; Vasoactive Intestinal Peptide; Vipoma

Vasoactive intestinal peptide (VIP) is known to regulate proliferation or differentiation in normal and tumoral cells. SH-SY5Y is a differentiated cell subclone derived from the SK-N-SH human neuroblastoma cell line and possess all the components for an autocrine action of VIP. In the present study, we investigated the morphological changes and intracellular signaling pathways occurring upon VIP treatment of SH-SY5Y cells. VIP induced an early remodeling of cell projections: a branched neurite network spread out and prominent varicosities developed along neurites. Although activated by VIP, the Ras/ERK pathway was not required for the remodeling process. In contrast, pull-down experiments revealed a strong Cdc42 activation by VIP while expression of a dominant-negative Cdc42 prevented the VIP-induced neurite changes, suggesting an important role for this small GTPase in the process. These data provide the first evidence for a regulation of the activity of Rho family GTPases by VIP and bring new insights in the signaling pathways implicated in neurite remodeling process induced by VIP in neuroblastoma cells.

Topics: cdc42 GTP-Binding Protein; Genes, Dominant; Humans; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; Mitogen-Activated Protein Kinases; Neurites; Neuroblastoma; Neuroprotective Agents; rac1 GTP-Binding Protein; ras Proteins; Signal Transduction; Tumor Cells, Cultured; Vasoactive Intestinal Peptide

Pituitary adenylate cyclase-activating polypeptide (PACAP), a member of the secretin/glucagon/vasoactive intestinal peptide family, induces the expression of catecholamine-synthesizing enzymes in adrenal medullary cells. In addition, PACAP and its receptor have been detected in human neuroblastoma tissues and cell lines, though it is not yet known whether PACAP enhances the expression of genes encoding catecholamine-synthesizing enzymes. To address this question, we analyzed PACAP, PACAP receptor and tyrosine hydroxylase (TH) mRNAs in neuroblastomas.. The levels of mRNA for PACAP and vasoactive intestinal peptide (VIP), as well as their receptors and the mRNA for TH were measured by RT-PCR or real-time PCR analysis.. VPAC1R mRNA was detected in all of 16 tissues and 3 cell lines that were examined, while VPAC2R mRNA was detected in 5 of 16 (31%) tissue and 2 of 3 cell lines. PAC1R mRNA was detected in 6 out of 16 (38%) tissues and none of 3 cell lines. mRNA expression of PACAP and TH were detected in many tissues (10/16 and 16/16, respectively). However, neither in tissues nor cell lines did PACAP mRNA expression correlate with TH mRNA expression.. Our findings suggest that PACAP is not involved in the regulation of expression of TH in neuroblastomas.

Topics: Base Sequence; Catecholamines; Cell Line, Tumor; Child, Preschool; DNA Primers; Female; Gene Expression Regulation, Neoplastic; Humans; Infant; Male; Nerve Growth Factors; Neuroblastoma; Neuropeptides; Neurotransmitter Agents; Pituitary Adenylate Cyclase-Activating Polypeptide; Receptors, Cell Surface; Receptors, Pituitary Adenylate Cyclase-Activating Polypeptide; Receptors, Pituitary Adenylate Cyclase-Activating Polypeptide, Type I; Receptors, Vasoactive Intestinal Peptide; Receptors, Vasoactive Intestinal Polypeptide, Type I; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; RNA, Neoplasm; Tyrosine 3-Monooxygenase; Vasoactive Intestinal Peptide

Neuroblastoma represents one of the most frequently developing malignant solid tumours in children. At the time of diagnosis, in more than half of the cases, metastatic cells are also present in the bone marrow. The present study was aimed at immunocytochemical analysis of selected neuropeptide manifestation in metastatic cells of neuroblastoma in bone marrow and at comparing the obtained results with the immunophenotype of parental neuroblastoma cells. The studies were performed on bone marrow material obtained from children treated at the Department of Paediatric Haematology and Oncology, University of Medical Sciences, Poznań, Poland, in 1998-2000. Immunocytochemical analysis of nervous tissue markers (employing the immunomax technique) involved 36 bone marrow preparations obtained from 27 children. The analysis included expression of neuron-specific enolase (NSE), PGP 9.5 protein, substance P (SP), chromogranin A (ChA), bombesin (B), galanin (G), neuropeptide Y (NPY) and vasoactive intestinal peptide (VIP). Close to 90% metastatic cells in bone marrow were found to exhibit NSE+SP+B+ phenotype and over a half of the cells manifested additionally expression of PGP 9.5+ChA+NPY+. Comparison of the obtained results with the immunophenotype of neuroblastoma cells obtained directly from the primary tumour demonstrated high correlation of NSE, SP and PGP 9.5 expression. Due to the relative ease of obtaining the bone marrow material and absence of neuromarkers in bone marrow metastatic cells in solid tumours other than neuroblastoma, determination of immunophenotype of the cells may represent a valuable supplementation in preliminary diagnosis of this tumour in children.

Topics: Adolescent; Biomarkers, Tumor; Biopsy, Needle; Bone Marrow; Child; Child, Preschool; Chromogranin A; Chromogranins; Female; Galanin; Humans; Immunohistochemistry; Immunophenotyping; Male; Neoplasm Metastasis; Neoplasm Staging; Neuroblastoma; Neuropeptide Y; Substance P; Vasoactive Intestinal Peptide

Vasoactive intestinal peptide (VIP) stimulates the neuroblastoma cell line (NMB) to proliferate. Neuropeptide activity can be inhibited by neutral endopeptidases that function intracellularly and in the extracellular milieu. NMB cells express neutral endopeptidase (NEP) activity that can be specifically inhibited by phosphoramidon (PA). Our data now show that phosphoramidon treatment increases the efficacy of VIP-stimulated neuroblastoma proliferation. These results suggest that membrane endopeptidases modulate VIP-associated cell proliferation and enhancement of endopeptidase activity may serve as a target for cancer therapy.

Topics: Cell Division; Enzyme Inhibitors; Glycopeptides; Humans; Neprilysin; Neuroblastoma; Tumor Cells, Cultured; Vasoactive Intestinal Peptide

We hypothesize that vasoactive intestinal peptide (VIP) promotes neural crest differentiation through VIP receptor type I (VPAC1). In order to test this hypothesis, SKNSH neuroblastoma cells were stably transfected with VPAC1 and receptor expression was verified by real-time RT-PCR. Overexpression of VPAC1 in SKNSH cells resulted in upregulation of endogenous retinoic acid receptor expression for both RARalpha and RXRalpha with no change in expression of RARbeta. Transfected cells demonstrated high affinity binding of VIP (K(D)=0.2 nM) and VIP-mediated stimulation of adenylate cyclase and a shift in cell cycle kinetics to a near triploid DNA index in G1. SKNSH/VPAC1 cells treated with VIP were observed to express a more differentiated phenotype compared to wild type cells as characterized by an increase in tissue transglutaminase II and a decrease in bcl-2 immunostaining. VIP-induced differentiation effects were potentiated by retinoic acid. This differentiation resulted in decreased proliferative potential in a xenograft model. Whereas, wild type SKNSH cells induced tumor growth in 100% of nude mice within 13 days post-injection, SKNSH transfected with VPAC1 demonstrated no tumor formation in xenografts followed for 6 months. Taken together, these data support the hypothesis that VIP modulation of neural crest differentiation is mediated via VPAC1 and that high expression of VPAC1 induces differentiation in and decreases tumorigenicity of neuroblastoma cells.

Topics: Cell Cycle; Cell Division; Humans; Neoplasm Transplantation; Neuroblastoma; Receptors, Retinoic Acid; Receptors, Vasoactive Intestinal Peptide; Receptors, Vasoactive Intestinal Polypeptide, Type I; RNA, Messenger; Time Factors; Transfection; Tumor Cells, Cultured; Up-Regulation; Vasoactive Intestinal Peptide

Responses to opioid agonists vary, depending on past opioid exposure and the physiological state. The intracellular signaling pathway mediated by cAMP and protein kinase A (PKA) has been linked to regulation of opioid receptor responsiveness. The role of the cAMP-PKA pathway in regulating opioid receptor gene expression is incompletely defined. Mu-opioid receptor (MuOR) and orphanin FQ/nociceptin receptor (ORL(1)) transcripts were measured after activating this pathway in human neuroblastoma cells. Human SH-SY5Y neuroblastoma cells were maintained in continuous monolayer culture. Cells were incubated with combinations of agents which activate the cAMP-PKA signal transduction pathway, including forskolin and choleratoxin (CTX). MuOR and ORL(1) transcript levels were measured by hybridization to specific probes. Activation of the cAMP-PKA signal transduction pathway with forskolin in the presence of phosphodiesterase inhibitors was associated with a time-dependent decrease in the level of MuOR mRNA; partial recovery was observed with prolonged incubations. Forskolin effects were mimicked by CTX, but not by dideoxyforskolin. The PKA inhibitor H89 blunted the actions of forskolin. However, forskolin responses persisted despite coincubation with protein synthesis inhibitors. ORL(1) transcript levels did not significantly change, but vasoactive intestinal polypeptide (VIP) transcripts exhibited substantial increases, in the presence of forskolin or CTX. These observations support a role for cAMP in regulating MuOR responsiveness through actions at the level of receptor gene expression. ORL(1) transcript levels are not effected, suggesting that the cAMP-PKA pathway has differential effects on the expression of mRNA for different, but biochemically closely related, opioid receptor subtypes.

Topics: Analgesics, Opioid; Central Nervous System; Cholera Toxin; Colforsin; Cyclic AMP; Cyclic AMP-Dependent Protein Kinases; Down-Regulation; Drug Tolerance; Enzyme Inhibitors; Gene Expression Regulation; Humans; Neuroblastoma; Nociceptin Receptor; Oligonucleotide Probes; Protein Synthesis Inhibitors; Receptors, Opioid; Receptors, Opioid, mu; RNA, Messenger; Signal Transduction; Transcription, Genetic; Tumor Cells, Cultured; Up-Regulation; Vasoactive Intestinal Peptide

SK-N-SH human neuroblastoma subclones differ widely in basal and second messenger induction of the gene encoding the neuropeptide vasoactive intestinal peptide (VIP). These differences were recapitulated by a chimeric gene which consisted of 5.2 kb of the human VIP gene 5' flanking sequence fused to a reporter. Subsequent gene deletion experiments revealed several regulatory regions on the gene, including a 645-bp sequence located approximately 4.0 upstream from the transcription start site. Here we examined this upstream region in detail. Inhibitory sequences were found to be present on each end of the 645-bp fragment. When removed, basal transcription increased more than 50-fold. Subsequent deletion/mutation analysis showed that the 213-bp fragment contained at least two enhancer elements. One of these was localized to an AT-rich 42-bp sequence shown by others to bind Oct proteins in neuroblastoma cells, while the other corresponded to a composite AP-1/ets element. In addition to these enhancers, a 28-bp sequence on the 213-bp fragment with no apparent homology to known silencers inhibited transcription. The studies provide molecular details of a complex regulatory region on the VIP gene that is likely to be used to finely tune the level of gene transcription in vivo.

Topics: 5' Untranslated Regions; Base Sequence; Consensus Sequence; DNA-Binding Proteins; Enhancer Elements, Genetic; Gene Expression Regulation, Neoplastic; Gene Silencing; Genes, Reporter; Host Cell Factor C1; Humans; Molecular Sequence Data; Mutagenesis, Site-Directed; Neuroblastoma; Octamer Transcription Factor-1; Octamer Transcription Factor-2; Regulatory Sequences, Nucleic Acid; Sequence Deletion; Transcription Factor AP-1; Transcription Factors; Transcription, Genetic; Transfection; Tumor Cells, Cultured; Vasoactive Intestinal Peptide

Brain corticotropin-releasing factor (CRF) systems integrate various responses to stress. Pathological responses to stress may result from errors in CRF receptor regulation in response to changes in synaptic CRF levels. To establish an in vitro model to study brain CRF receptors, we characterized the CRF-induced modulation of CRF(1) receptors in the human neuroblastoma cell line, IMR-32. Treatment with CRF decreased CRF(1) receptor binding and desensitized CRF-induced increases in cAMP. The decrease in binding had an EC(50) of approximately 10 nM, was maximal by 30 min, and was blocked by the CRF receptor antagonist [D-Phe(12), Nle(21,38), C(alpha)-MeLeu(37)]CRF(12-41). The desensitization was homologous as vasoactive intestinal polypeptide-induced increases in cAMP were unchanged, and elevation of cAMP did not alter CRF(1) receptor binding. Treatment with CRF for up to 24 h did not alter CRF(1) receptor mRNA levels, suggesting that a posttranscriptional mechanism maintains the decrease in receptor binding. Interestingly, recovery of CRF receptor binding and CRF-stimulated cAMP production was only partial following exposure to 100 nM CRF. In contrast, receptor binding recovered to control levels following exposure to 10 nM CRF. These data suggest that exposure to high doses of CRF result in permanent changes characterized by only partial recovery. Identifying the mechanisms underlying this partial recovery may provide insights into mechanisms underlying the acute and chronic effects of stress on CRF receptor regulation.

Topics: 1-Methyl-3-isobutylxanthine; Adenylyl Cyclases; Amphibian Proteins; Bromodeoxyuridine; Carrier Proteins; Cell Differentiation; Corticotropin-Releasing Hormone; Cyclic AMP; Down-Regulation; Nerve Tissue Proteins; Neuroblastoma; Peptide Fragments; Peptide Hormones; Peptides; Receptors, Corticotropin-Releasing Hormone; RNA, Messenger; Second Messenger Systems; Time Factors; Tumor Cells, Cultured; Vasoactive Intestinal Peptide

Activin, a member of the transforming growth factor-beta superfamily, can regulate neuropeptide gene expression in the nervous system and in neuroblastoma cells. Among the neuropeptide genes whose expression can be regulated by activin is the gene encoding the neuropeptide vasoactive intestinal peptide (VIP). To investigate the molecular mechanisms by which activin regulates neuronal gene expression, we have examined activin's regulation of VIP gene expression in NBFL neuroblastoma cells. We report here that NBFL cells respond to activin by increasing expression of VIP mRNA. Activin regulates VIP gene transcription in NBFL cells through a 180-bp element in the VIP promoter that was previously characterized to be necessary and sufficient to mediate the induction of VIP by the neuropoietic cytokines and termed the cytokine response element (CyRE). We find that the VIP CyRE is necessary and sufficient to mediate the transcriptional response to activin. In addition, ciliary neurotrophic factor (CNTF), a neuropoietic cytokine, synergizes with activin to increase VIP mRNA expression and transcription through the VIP CyRE. Mutations in either the Stat (signal transducer and activator of transcription) or AP-1 sites within the CyRE that reduce the response to CNTF, also reduce the response to activin. However, mutating both the Stat and AP-1 sites within the wild-type CyRE, while reducing the separate responses to either activin or CNTF, eliminates the synergy between them. These data suggest that activin and CNTF, two factors that appear to signal though distinct pathways, activate VIP gene transcription through a common transcriptional element, the VIP CyRE.

Topics: Activins; Animals; Binding Sites; Chickens; Ciliary Neurotrophic Factor; DNA-Binding Proteins; Drug Synergism; Gene Expression Regulation; Humans; Inhibins; Mutation; Neoplasm Proteins; Neuroblastoma; Promoter Regions, Genetic; Recombinant Fusion Proteins; Regulatory Sequences, Nucleic Acid; Signal Transduction; STAT1 Transcription Factor; Trans-Activators; Transcription Factor AP-1; Transfection; Transforming Growth Factor beta; Tumor Cells, Cultured; Vasoactive Intestinal Peptide

Vasoactive intestinal peptide (VIP) provides neuroprotection against beta-amyloid toxicity in models of Alzheimer's disease. A superactive analogue, stearyl-Nle17-VIP (SNV) is a 100-fold more potent than VIP. In primary neuronal cultures, VIP protective activity may be mediated by femtomolar-acting glial proteins such as activity-dependent neurotrophic factor (ADNF), activity-dependent neuroprotective protein (ADNP), peptide derivatives ADNF-9 (9aa) and NAP (8aa), respectively. It has been hypothesized that beta-amyloid induces oxidative stress leading to neuronal cell death. Similarly, dopamine and its oxidation products were suggested to trigger dopaminergic nigral cell death in Parkinson's disease. We now examined the possible protective effects of VIP against toxicity of dopamine, 6-hydroxydopamine (6-OHDA) and 1-methyl-4-phenylpyridinium ion (MPP+) in neuronal cultures [rat pheochromocytoma (PC12), human neuroblastoma (SH-SY5Y) and rat cerebellar granular cells]. Remarkably low concentrations of VIP (10(-16)-10(-8) M), ADNF-9 and NAP (10(-18)-10(-10) M) protected against dopamine and 6-OHDA toxicity in PC12 and neuroblastoma cells. VIP (10(-11)-10(-9) M) and SNV (10(-13)-10(-11) M), protected cerebellar granule neurons against 6-OHDA. In contrast, VIP did not rescue neurons from death associated with MPP+. Since dopamine toxicity is linked to the red/ ox state of the cellular glutathione, we investigated neuroprotection in cells depleted of reduced glutathione (GSH). Buthionine sulfoximine (BSO), a selective inhibitor of glutathione synthesis, caused a marked reduction in GSH in neuroblastoma cells and their viability decreased by 70-90%. VIP, SNV or NAP (over a wide concentration range) provided significant neuroprotection against BSO toxicity. These results show that the mechanism of neuroprotection by VIP/SNV/NAP may be mediated through raising cellular resistance against oxidative stress. Our data suggest these compounds as potential lead compounds for protective therapies against Parkinson's disease.

Topics: 1-Methyl-4-phenylpyridinium; Animals; Cell Death; Cerebellum; Dopamine; Dopamine Antagonists; Glutathione; Humans; Mice; Nerve Tissue Proteins; Neuroblastoma; Neurons; Neuroprotective Agents; Neurotoxins; Oxidopamine; Parkinson Disease; PC12 Cells; Rats; Tumor Cells, Cultured; Vasoactive Intestinal Peptide

In many ganglia, the neuropeptide pituitary adenylate cyclase-activating peptide (PACAP) innervates nerve cell bodies containing the homologous neuropeptide vasoactive intestinal polypeptide (VIP). We therefore investigated whether PACAP affected the VIP gene expression and elucidated the molecular mechanisms using the human neuroblastoma cell line NB-1. A concentration dependent induction of the VIP mRNA level was found upon PACAP stimulation. Five nM PACAP mediated transient elevation of the VIP mRNA being evident after 2 h, the maximal 65-fold induction was reached after 6-8 h and hereafter the level decreased rapidly. In cell extracts, the concentration of immunoreactive VIP was elevated four-fold upon PACAP stimulation for 8 h, and it remained elevated during the next 40 h. In conditioned medium, a stable 20-fold VIP increase was seen after 8-24 h. Experiments with the translational inhibitor cycloheximide showed a direct effect of PACAP on the VIP mRNA level, and nuclear run-on assays revealed a three- to four-fold enhancement of the VIP gene transcription rate after PACAP stimulation. The VIP mRNA induction was abolished by transcriptional inhibition with the actinomycin D, and PACAP did not seem to mediate any changes in the VIP mRNA half-life. However, the VIP mRNA level seemed very stable during the transcriptional cessation. Reporter gene constructs were used to evaluate involvement of the VIP CRE site in the PACAP mediated induction of the VIP gene transcription. Mutation of the CRE site did not abolish the induction suggesting it to be of minor if any importance for the induction. In conclusion, the PACAP mediated induction of the VIP gene expression suggests that PACAP released from nerve terminals could influence the function of VIP'ergic neurons in target tissues.

Topics: Base Sequence; Dose-Response Relationship, Drug; Exons; Gene Expression Regulation, Neoplastic; Genes, Reporter; Humans; Kinetics; Neuroblastoma; Neuropeptides; Neurotransmitter Agents; Pituitary Adenylate Cyclase-Activating Polypeptide; RNA, Messenger; Transcription, Genetic; Transfection; Tumor Cells, Cultured; Vasoactive Intestinal Peptide

Topics: Animals; Apoptosis; Brain; Cell Death; Cytokines; Humans; In Vitro Techniques; Inflammation Mediators; Intestinal Mucosa; Lung; Lung Injury; Neoplasms; Neuroblastoma; Neurons; NF-kappa B; Nitric Oxide; Peptides; Rats; Signal Transduction; Tumor Cells, Cultured; Vasoactive Intestinal Peptide

Receptors for pituitary adenylyl cyclase activating peptide (PACAP) have been identified in human SH-SY5Y neuroblastoma cells with PACAP being 1000-fold more potent than vasoactive intestinal peptide (VIP) in [(125)I]PACAP binding inhibition and stimulation of cAMP accumulation. Maxadilan, a vasodilator peptide from the salivary gland of the sand fly Lutzomyia longipalpis also specifically bound to SH-SY5Y cells, and was equipotent to PACAP in [(125)I]PACAP and [(125)I]maxadilan binding inhibition, and stimulation of cAMP accumulation. Maxadilan and PACAP also increased the cytosolic free calcium concentration. In human SK-N-MC neuroblastoma cells PACAP, VIP and maxadilan equipotently stimulated cAMP accumulation. The maximal effects of VIP and maxadilan were additive and reached those of PACAP alone. In human T47D breast carcinoma cells PACAP and VIP were also equipotent in the stimulation of cAMP accumulation, but maxadilan was inactive. The results are consistent with the interaction of maxadilan with PACAP specific PAC(1)receptors in SH-SY5Y cells, but not with VPAC receptors, not differentiating between VIP and PACAP in T47D cells. Moreover, maxadilan is a PAC(1)receptor specific agonist which allows discrimination of co-expressed PAC(1)and VPAC receptors in SK-N-MC cells.

Topics: Binding, Competitive; Calcium; Cyclic AMP; Humans; Insect Proteins; Kinetics; Neuroblastoma; Neuropeptides; Peptide Fragments; Pituitary Adenylate Cyclase-Activating Polypeptide; Receptors, Pituitary Adenylate Cyclase-Activating Polypeptide; Receptors, Pituitary Hormone; Tumor Cells, Cultured; Vasoactive Intestinal Peptide; Vasodilator Agents

The ability of cAMP-dependent protein kinase (PKA) to phosphorylate type I, II, and III inositol 1,4,5-trisphosphate (InsP3) receptors was examined. The receptors either were immunopurified from cell lines and then phosphorylated with purified PKA or were phosphorylated in intact cells after activating intracellular cAMP formation. The former studies showed that the type I receptor was a good substrate for PKA (0.65 mol Pi incorporated/mol receptor), whereas type II and III receptors were phosphorylated relatively weakly. The latter studies showed that despite these differences, each of the receptors was phosphorylated in intact cells in response to forskolin or activation of neurohormone receptors. Detailed examination of SH-SY5Y neuroblastoma cells, which express >/=99% type I receptor, revealed that minor increases in cAMP concentration were sufficient to cause maximal phosphorylation. Thus, VIP and pituitary adenylyl cyclase activating peptide (acting through Gs-coupled pituitary adenylyl cyclase activating peptide-I receptors) were potent stimuli of type I receptor phosphorylation, and remarkably, even slight increases in cAMP concentration induced by carbachol (acting through Gq-coupled muscarinic receptors) or other Ca2+ mobilizing agents were sufficient to cause phosphorylation. Finally, PKA enhanced InsP3-induced Ca2+ mobilization in a range of permeabilized cell types, irrespective of whether the type I, II, or III receptor was predominant. In summary, these data show that all InsP3 receptors are phosphorylated by PKA, albeit with marked differences in stoichiometry. The ability of both Gs- and Gq-coupled cell surface receptors to effect InsP3 receptor phosphorylation by PKA suggests that this process is widespread in mammalian cells and provides multiple routes by which the cAMP signaling pathway can influence Ca2+ mobilization.

Topics: 1-Methyl-3-isobutylxanthine; Calcium; Calcium Channels; Carbachol; Colforsin; Cyclic AMP; Cyclic AMP-Dependent Protein Kinase Type II; Cyclic AMP-Dependent Protein Kinases; Humans; Inositol 1,4,5-Trisphosphate Receptors; Neuroblastoma; Neuropeptides; Peptide Fragments; Phosphorylation; Pituitary Adenylate Cyclase-Activating Polypeptide; Receptors, Cell Surface; Receptors, Cytoplasmic and Nuclear; Tumor Cells, Cultured; Vasoactive Intestinal Peptide

The vasoactive intestinal peptide cytokine response element (VIP CyRE) is responsible for mediating the transcriptional induction of the VIP gene to the neuropoietic cytokines leukemia inhibitory factor (LIF) and ciliary neurotrophic factor (CNTF). In investigating the sequence and function of the CyRE, we found a region of DNA with homology to the distal NFAT site in the IL-2 promoter. In this paper we characterize this sequence and show that the VIP NFAT site recognizes T cell NFAT with similar affinity to the previously characterized IL-2 NFAT site. However, despite its location in the middle of the CyRE, we find no CNTF/LIF induced binding to it. Instead we show that in NBFL neuroblastoma cells, the calcium ionophore A23187 induces a protein to bind to the VIP NFAT site. This A23187-mediated induction of nuclear protein binding to an NFAT oligonucleotide is dependent on extracellular calcium but not dependent on de novo protein synthesis. Thus, this protein has the characteristics of an NFAT-like protein and is recognized by an NFAT3-specific antiserum suggesting that it is indeed an NFAT protein. The location of the NFAT site in the VIP CyRE suggests that this may be one mechanism through which different signaling pathways engage in cross talk to alter VIP gene transcription.

Topics: Animals; Base Sequence; Binding Sites; Calcimycin; Ciliary Neurotrophic Factor; Cytokines; DNA; DNA Primers; DNA-Binding Proteins; Growth Inhibitors; Humans; Interleukin-6; Leukemia Inhibitory Factor; Lymphokines; Mutagenesis, Site-Directed; Nerve Growth Factors; Nerve Tissue Proteins; Neuroblastoma; NFATC Transcription Factors; Nuclear Proteins; Recombinant Proteins; Regulatory Sequences, Nucleic Acid; Signal Transduction; Transcription Factors; Transcription, Genetic; Transfection; Tumor Cells, Cultured; Vasoactive Intestinal Peptide

The growth rate of rodent embryonic neuroblasts and human neuroblastoma cell lines is regulated in part by autocrine or paracrine actions of neuropeptides of the family that includes vasoactive intestinal peptide (VIP), peptide histidine isoleucine (PHI), and pituitary adenylate cyclase-activating peptide (PACAP). These peptides act via seven transmembrane G-protein-linked receptors coupled to cAMP elevation, phospholipase C activation, intracellular Ca2+ release, and/or of mitogen-activated protein (MAP) kinase activation. Here we investigated the action of these peptides on the mouse neuroblastoma cell line Neuro2a. PHI and VIP inhibited proliferation at concentrations as low as 10(-13) M and 10(-10) M, respectively. In contrast, PACAP action was biphasic, with stimulation occurring at subnanomolar doses and inhibition at higher doses. Peptide actions were studied further by measuring cAMP and ERK1/2 MAP kinase activity and by assessing 3H-thymidine incorporation in conjunction with a panel of signal transduction pathways inhibitors. The data obtained indicated that the PHI-inhibitory and PACAP-stimulatory activities were mediated by corresponding changes in activity of the MAP kinase pathway and independent of protein kinase A (PKA) or protein kinase C (PKC). In contrast, the inhibitory actions of VIP and PACAP were specifically blocked by antagonists of PKA. Northern blot analysis revealed gene expression for only the PACAP-preferring (PAC1) receptor. However, binding experiments using 125I-labeled PACAP27, PHI, and VIP, demonstrated the presence of PACAP-preferring sites, bivalent VIP/PACAP sites, and PHI-binding sites that did not interact with VIP. The studies demonstrate potent regulatory actions of PACAP, PHI, and VIP on neuroblastoma cell proliferation which appear to be mediated by multiple subsets of receptors which differentially couple to MAP kinase and PKA signaling pathways.

Topics: Animals; Calcium-Calmodulin-Dependent Protein Kinases; Cell Division; Cyclic AMP; Humans; Mice; Neuroblastoma; Neuropeptides; Nucleic Acid Synthesis Inhibitors; Peptide PHI; Pituitary Adenylate Cyclase-Activating Polypeptide; Receptors, Pituitary Adenylate Cyclase-Activating Polypeptide; Receptors, Pituitary Adenylate Cyclase-Activating Polypeptide, Type I; Receptors, Pituitary Hormone; RNA, Messenger; Signal Transduction; Tumor Cells, Cultured; Vasoactive Intestinal Peptide

Neuroendocrine tumors, neuroblastoma in particular, commonly express the neuropeptides vasoactive intestinal peptide (VIP) and pituitary adenylate cyclase activating peptide (PACAP) and their receptors. Retinoic acid (RA) has been shown to induce differentiation of neuroblastoma cell lines, possibly by augmenting or interfering with neuropeptide autocrine loops. We sought to determine which receptor gene subtypes are expressed in selected human neuroblastoma cell lines (SH-SY5Y, IMR-32, and LA-N-5), and the effect of RA on the VIP/PACAP ligand/receptor system. Expression of both PACAP1 and VIP1/PACAP2 receptor genes was detected by Northern analysis, which characteristically encode Type I (PACAP-preferring), and Type II (bivalent VIP/PACAP) receptors, respectively. Binding experiments carried out on IMR-32 cells, using 125I VIP and 125I PACAP-27 as tracers, corroborated that both receptor subtypes were expressed. In contrast to RA upregulation of VIP binding (confirmed here in IMR-32 cells), levels of both receptor mRNAs were reduced after RA treatment. VIP mRNA in each cell line was increased by RA, whereas PACAP mRNA, detected in IMR-32 cells only, was reduced. The studies indicate that several components of the VIP/PACAP autocrine system are regulated in neuroblastoma cell lines during RA differentiation.

Topics: Autocrine Communication; Gene Expression; Humans; Ligands; Neuroblastoma; Neuropeptides; Pituitary Adenylate Cyclase-Activating Polypeptide; Receptors, Pituitary Adenylate Cyclase-Activating Polypeptide; Receptors, Pituitary Adenylate Cyclase-Activating Polypeptide, Type I; Receptors, Pituitary Hormone; Receptors, Vasoactive Intestinal Peptide; Receptors, Vasoactive Intestinal Polypeptide, Type I; RNA, Messenger; Tretinoin; Tumor Cells, Cultured; Up-Regulation; Vasoactive Intestinal Peptide

The effects of carbamylcholine (CCh) on the gene expression of the neuropeptide vasoactive intestinal polypeptide (VIP) were studied using two human neuroblastoma cell lines. NB-1 and BE(2)M17. CCh caused a fast increase in VIP mRNA level in both cell lines which was followed by an increase in VIP immunoreactivity. The time-course of the induction of both mRNA and peptide differed, however, between the two cell lines. No morphological changes of the cells were observed during 6 days of stimulation. The effect was mediated by the muscarinic class of acetylcholine receptors, since it could be totally abolished by atropine. Since CCh caused an accumulation of inositol-1,4,5-triphosphate, it is likely that muscarinic receptor subtype M1, M3 or M5 is involved. Experiments with the translational inhibitor, cycloheximide, showed that CCh mediated a direct effect on the VIP gene expression. By combining gel permeation chromatography with radiommunoassays using antisera specific for the various VIP-precursor products, immunoreactive peaks eluting as the synthetic peptides were found in both cell lines. In addition, earlier eluting peaks which could represent partially processed or extended VIP forms were found. After CCh induction the concentration of all prepro VIP-derived products increased, and there was a tendency towards a shift to more fully processed VIP. The findings give new evidence for a direct regulation of VIP gene expression in human neuronal cells by cholinergic agents.

Topics: Brain Neoplasms; Carbachol; Cycloheximide; Gene Expression Regulation, Neoplastic; Humans; Inositol 1,4,5-Trisphosphate; Neuroblastoma; Neuropeptides; Parasympathetic Nervous System; Parasympathomimetics; Protein Synthesis Inhibitors; Radioimmunoassay; RNA, Messenger; Tumor Cells, Cultured; Vasoactive Intestinal Peptide

Neuroblastoma, a childhood tumour of the sympathetic nervous system, may in some cases differentiate to a benign ganglioneuroma or regress due to apoptosis. Somatostatin may inhibit neuroblastoma growth and induce apoptosis in vitro and was therefore investigated. Using a radioimmunoassay, we found that all ganglioneuromas contained high somatostatin concentrations (> 16 pmol/g), significantly higher than neuroblastomas (n = 117, median 2.8 pmol/g), healthy adrenals, Wilms' tumours, phaeochromocytomas and other neuroendocrine tumours (P < 0.001). Neuroblastomas contained more somatostatin than control tumours (P < 0.001-0.05). Neuroblastomas amplified for the MYCN oncogene contained less somatostatin than non-amplified tumours (1.2 pmol/g versus 4.0 pmol/g, respectively; P = 0.026). In a clinically unfavourable neuroblastoma subset (age > 12 months, stage 3 or 4) 16 children with high concentrations of somatostatin in primary tumours had a better prognosis than 23 with low somatostatin (46.7% versus 0% survival at 5 years, P < 0.005). Scintigraphy using 111In-pentetreotide identified tumours expressing high-affinity somatostatin receptors in vivo. However, no significant correlation was found between somatostatin receptor expression and peptide content in 15 tumours. Similarly, human SH-SY5Y neuroblastoma xenografts grown in nude rats showed low somatostatin concentrations, but were positive for somatostatin receptor scintigraphy. Treatment of these rats with the somatostatin analogue octreotide seemed to upregulate in vivo receptor expression of somatostatin and vasoactive intestinal peptide more effectively than 13-cis retinoic acid. In conclusion, somatostatin in neuroblastoma is associated with differentiation to benign ganglioneuromas in vivo and favourable outcome in advanced tumours. Furthermore, somatostatin receptor scintigraphy may identify tumours with high-affinity receptors in children that might benefit from targeted therapy using synthetic somatostatin analogues.

Topics: Animals; Follow-Up Studies; Ganglioneuroma; Gene Amplification; Genes, myc; Humans; Infant; Neoplasm Staging; Neuroblastoma; Octreotide; Rats; Rats, Nude; Receptors, Somatostatin; Somatostatin; Survival Rate; Transplantation, Heterologous; Tretinoin; Vasoactive Intestinal Peptide

In this study, the effects of three related peptides, pituitary adenylate cyclase-activating polypeptide 38 (PACAP38), PACAP27, and vasoactive intestinal peptide (VIP), on cyclic AMP (cAMP) accumulation and intracellular Ca2+ concentration ([Ca2+]i) were compared in N1E-115 cells. PACAP38 and PACAP27 stimulated cAMP accumulation up to 60-fold with EC50 values of 0.54 and 0.067 nM, respectively. The effect of VIP on cAMP accumulation was less potent. The binding of 125I-PACAP27 to intact cells was inhibited by PACAP38 and PACAP27 (IC50 values of 0.44 and 0.55 nM, respectively) but not by VIP. In fura-2-loaded cells, both PACAP38 and PACAP27 increased [Ca2+]i with EC50 values around 10 nM. The interactions of these three peptides with ionomycin, a Ca2+ ionophore, and 4 beta-phorbol 12-myristate 13-acetate (PMA), an activator of protein kinase C, were also determined. Ionomycin increased the cAMP accumulation caused by all three peptides. With low concentrations of PACAP38 or PACAP27, the effect of PMA was inhibitory, whereas at higher concentrations of PACAP (> 1 nM), the effect of PMA was stimulatory. Similar to other agents that elevate cAMP, PACAP38 was an effective stimulator of neurite outgrowth. These results show that (a) PACAP27 and PACAP38 stimulate cAMP accumulation and increase [Ca2+]i through the type I PACAP receptors in N1E-115 cells, (b) ionomycin enhances cAMP accumulation by all three peptides, and (c) activation of protein kinase C has a dose-dependent stimulatory or inhibitory effect on the PACAP38- or PACAP27-stimulated cAMP accumulation.

Topics: Calcium; Carcinogens; Cyclic AMP; Iodine Radioisotopes; Ionomycin; Ionophores; Neurites; Neuroblastoma; Neuropeptides; Neurotransmitter Agents; Pituitary Adenylate Cyclase-Activating Polypeptide; Protein Kinase C; Signal Transduction; Tetradecanoylphorbol Acetate; Time Factors; Tumor Cells, Cultured; Vasoactive Intestinal Peptide

The stabilities of vasoactive intestinal polypeptide (VIP) and galanin mRNAs were examined in a human neuroblastoma cell line (NBFL) treated with agents that alter second-messenger pathways. VIP and galanin mRNA stabilities were estimated by the decay of steady-state levels of transcripts following transcriptional arrest with actinomycin D or 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole (DRB). In the presence of actinomycin D, phorbol ester treatment stabilized VIP mRNA while treatment with adenylate cyclase activators, calcium ionophore, or CNTF did not. In the presence of DRB, VIP mRNA was not stabilized in phorbol ester-treated cells but instead was stabilized in cells treated with adenylate cyclase activators. With either transcriptional inhibitor, stability of galanin mRNA was not significantly altered. The difference in the behavior of VIP mRNA in the presence of actinomycin D and DRB may result from their different mechanisms of action-actinomycin D intercalates into nucleic acids while DRB is a kinase inhibitor. Using an assay for RNA stability that did not require transcriptional inhibitors, an in vitro transcribed VIP RNA fragment was relatively stable in extracts from phorbol ester-treated cells. Although treatment with phorbol ester alone resulted in stabilization of VIP mRNA, treatment with a combination of phorbol ester and adenylate cyclase activator, calcium ionophore, or CNTF did not-implying a complex interaction of these second-messenger pathways in the regulation of RNA stability.

Topics: Galanin; Humans; Neuroblastoma; Phorbol Esters; RNA, Messenger; Second Messenger Systems; Transcription, Genetic; Tumor Cells, Cultured; Vasoactive Intestinal Peptide

An upstream enhancer element [tissue specifier element (TSE)] located between 4.66 and 4.02 kb from the transcription start site is important for cell type-specific expression and phorbol ester induction of the vasoactive intestinal peptide (VIP) gene. An element located within 100 bases of the VIP promoter [the VIP cyclic AMP-responsive element (VIP-CRE)] confers cyclic AMP and phorbol ester responsiveness to heterologous promoters. The possibility that these two regions of the VIP gene function cooperatively to determine tissue-specific and second messenger-dependent expression of the VIP gene was addressed by assaying transcription from a VIP-luciferase reporter gene with progressive deletions from the 5' flanking sequence of the gene, with or without inactivation of the proximal VIP-CRE. Basal expression of the reporter gene in both SH-EP and SK-N-SH human neuroblastoma cells, which express endogenous VIP mRNA, was absolutely dependent on the presence of the upstream TSE. Full constitutive expression was also dependent on the intact VIP-CRE. Forskolin-mediated induction of the reporter gene in SH-EP and SK-N-SH cells was completely abolished by mutations in the VIP-CRE but not by deletion of the upstream sequence, indicating that the VIP-CRE alone determines cyclic AMP responsiveness. In contrast to reports that the VIP-CRE imparts 12-O-tetradecanoylphorbol 13-acetate (phorbol 12-myristate 13-acetate; PMA) responsiveness to heterologous promoters, PMA stimulation in SK-N-SH cells was independent of an intact VIP-CRE but dependent on a region between -2.5 kb and the VIP-CRE. Sequencing of the entire 5.2-kb VIP 5' flank revealed a consensus PMA-responsive element (TGACTCA) 2.25 kb upstream of the transcription start site that may represent the site imparting PMA responsiveness to the VIP gene.

Topics: Animals; Base Sequence; Cyclic AMP; DNA Primers; Gene Expression Regulation, Neoplastic; Genes, Reporter; HeLa Cells; Humans; Luciferases; Molecular Sequence Data; Neuroblastoma; Organ Specificity; PC12 Cells; Polymerase Chain Reaction; Promoter Regions, Genetic; Rats; Recombinant Fusion Proteins; Regulatory Sequences, Nucleic Acid; Restriction Mapping; Transfection; Tumor Cells, Cultured; Vasoactive Intestinal Peptide

Glia cell line-derived neurotrophic factor (GDNF), a recently cloned member of the transforming growth factor-beta (TGF-beta) superfamily, has been implicated in the survival, morphological and functional differentiation of midbrain dopaminergic neurons and motoneurons in vitro and in vivo. The factor may thus have utility in the treatment of various human neurodegenerative disorders. Mechanisms regulating expression of GDNF in normal and diseased brain as a possible means to increase the local availability of GDNF are only beginning to be explored. We have established and employed a competitive reverse transcriptase-polymerase chain reaction (RT-PCR) to study and compare levels of expression of GDNF mRNA in several cell types and to investigate its regulation. GDNF expression was clearly evident in primary cultured astrocytes, the glioma B49 and C6 cell, but less pronounced in the Schwannoma RN22 cell lines. Little or no signal could be observed in neuroblastoma cell lines (IMR32, LAN-1) or the pheochromocytoma cell line PC12, emphasizing the glial character of this factor. Using the C6 cell line we found that fibroblast growth factor-2 (FGF-2; bFGF) can increase GDNF mRNA levels, whereas FGF-1, platelet-derived growth factor (PDGF), and vasoactive intestinal polypeptide (VIP) are apparently ineffective. Several other factors (forskolin, kainic acid, triiodothyronine dexamethasone, GDNF, TGF-beta 1, and interleukin-6) appear to have slightly negative effects on GDNF mRNA levels at the concentrations tested. To further explore the relationship between FGF-2 and GDNF, we also addressed the question whether GDNF, like FGF-2, may have an effect on C6 cell proliferation. We conclude that (1) glial and glial tumor cells, rather than neuronal cell lines, express GDNF, (2) that FGF-2 has a prominent inductive effect on GDNF expression and (3) that GDNF stimulates C6 cell proliferation. Finally, these data suggest that neurotrophic actions of FGF-2 in mixed glial-neuronal cell cultures might be mediated in part by GDNF.

Topics: Animals; Astrocytes; Brain Neoplasms; Cell Division; Colforsin; Dexamethasone; Fibroblast Growth Factor 1; Fibroblast Growth Factor 2; Gene Expression Regulation, Neoplastic; Glial Cell Line-Derived Neurotrophic Factor; Glioma; Humans; Interleukin-6; Kainic Acid; Neoplasm Proteins; Nerve Growth Factors; Nerve Tissue Proteins; Neuroblastoma; Neurons; Organ Specificity; Pheochromocytoma; Platelet-Derived Growth Factor; Polymerase Chain Reaction; Rats; Recombinant Proteins; RNA, Messenger; RNA, Neoplasm; Triiodothyronine; Tumor Cells, Cultured; Vasoactive Intestinal Peptide

Clonal human neuroblastoma cells SH-IN undergo a very conspicuous phenotypic change in culture. Large substrate-adherent cells with a slow growth rate give rise to small cells emerging in focal aggregates and growing to high cell densities. This is accompanied by a dramatic switch in the expression of receptors for the structurally related neuropeptides VIP (vasoactive intestinal polypeptide) and PACAP (pituitary adenylate cyclase activating polypeptide). Large cells expressed mainly PACAP-specific receptors that triggered stimulation of intracellular cGMP production. On the other hand, polyvalent VIP/PACAP receptors positively coupled to adenylate cyclase were mostly observed in the small cells. Both neuropeptides stimulated cell proliferation in large and small cells. These data, together with the previous demonstration of autocrine/paracrine actions of VIP and PACAP in human neuroblastomas, support the idea that these neuropeptides may participate in the establishment of the apparent phenotype in these cancer cells.

Topics: Cell Division; Cyclic AMP; Cyclic GMP; Humans; Neuroblastoma; Neurons; Neuropeptides; Phenotype; Pituitary Adenylate Cyclase-Activating Polypeptide; Radioligand Assay; Receptors, Pituitary Adenylate Cyclase-Activating Polypeptide; Receptors, Pituitary Hormone; Receptors, Vasoactive Intestinal Peptide; Tumor Cells, Cultured; Vasoactive Intestinal Peptide

Topics: Animals; Calcium; Cell Line; Kinetics; Mice; Neuroblastoma; Neuropeptides; Neurotransmitter Agents; Phosphatidylinositols; Pituitary Adenylate Cyclase-Activating Polypeptide; Receptors, Pituitary Adenylate Cyclase-Activating Polypeptide; Receptors, Pituitary Hormone; Tumor Cells, Cultured; Type C Phospholipases; Vasoactive Intestinal Peptide

The potent protein kinase inhibitor staurosporine enhances cAMP-mediated responses in human neuroblastoma cells as represented by neurite extension and induction of tyrosine hydroxylase. To explore how staurosporine regulates cAMP signaling pathway, we examined transcriptional activity of the human vasoactive intestinal polypeptide (VIP) gene promoter carrying a cAMP-responsive element. In PC12 cells stably transfected with a reporter plasmid, staurosporine alone had little effect; however, in combination with forskolin or dibutyryl cAMP, staurosporine dose-dependently (1-50 nM) enhanced cAMP-mediated transcription from the VIP gene promoter, which was comparable to that maximally induced by cAMP plus 12-O-tetradecanoylphorbol-13-acetate. This is the first report of potentiation of cAMP-mediated promoter activity by staurosporine in neuroendocrine cells.

Topics: Adrenal Gland Neoplasms; Alkaloids; Animals; beta-Galactosidase; Colforsin; Cyclic AMP; Enzyme Inhibitors; Humans; Kinetics; Neurites; Neuroblastoma; PC12 Cells; Pheochromocytoma; Promoter Regions, Genetic; Protein Kinase C; Rats; Recombinant Proteins; Staurosporine; Transfection; Tumor Cells, Cultured; Tyrosine 3-Monooxygenase; Vasoactive Intestinal Peptide

Neuroblastomas and ganglioneuromas frequently produce somatostatin (SOM) and vasoactive intestinal peptide (VIP), and elevated concentrations in tumour tissue are associated with favourable outcome. Both somatostatin and VIP have been shown to have an autocrine effect on tumour growth and differentiation in vitro, and VIP may cause clinical symptoms when released systemically. Using gel-permeation chromatography and specific radioimmunoassays, we further characterised somatostatin-like immunoreactivity (SOM-LI) and VIP-like immunoreactivity (VIP-LI) in neuroblastoma and ganglioneuroma tumour tissue. The major part of SOM-LI and VIP-LI in both neuroblastoma and ganglioneuroma represents the biologically active forms SOM-28, SOM-14 and VIP-2, respectively. 21 children with neuroblastoma and ganglioneuroma were monitored with serial plasma samples during surgery. In 8 children with measurable concentrations of SOM-LI, all showed increased concentrations during tumour manipulation (P = 0.004) that subsequently decreased below preoperative levels in all but one case (P = 0.06). The only child presenting with diarrhoea showed the highest preoperative plasma VIP-LI in the study (54 pmol/l). 2 children with increased concentrations of VIP-LI preoperatively showed a rapid decrease after surgical tumour removal. These findings indicate a systemic release from the tumours. It is concluded that plasma and tumour tissue from children with neuroblastoma and ganglioneuroma contain biologically active molecular forms of somatostatin and vasoactive intestinal peptide. These peptides may bear significance both for specific symptoms in certain patients as well as influencing tumour growth and differentiation in vivo.

Topics: Child, Preschool; Chromatography, Gel; Female; Ganglioneuroma; Humans; Infant; Intraoperative Period; Male; Neoplasm Proteins; Neuroblastoma; Postoperative Period; Radioimmunoassay; Somatostatin; Vasoactive Intestinal Peptide

The presence of specific AII receptors in 6 different human neuroblastoma cell lines was investigated using binding, cAMP and [Ca2+]i studies. We found high affinity (0.1 nM), low capacity ((1-2).10(3) sites/cell) binding sites for [125I](Sar-1,Ile-8)AII in one half of the cell lines studied. In the positive cell lines mathematical modeling of multiple competition curves among AII and analogs strongly indicated the presence of a homogenous class of sites, i.e., AT1 receptors. The presence of AT1 receptors was further substantiated by AII-induced inhibition of VIP-stimulated cAMP levels and by AII-evoked [Ca2+]i transient. The density of AT1 receptors in neuroblastoma cells was not affected by treatment with pertussis toxin and retinoic acid but was significantly increased by subacute treatment with VIP. In neuroblastoma cells, AII does not stimulate DNA synthesis, suggesting other roles rather than mitogenesis. Neuroblastoma cells represents an interesting model to investigate the function of AII in neural crest derived tissues.

Topics: 1-Sarcosine-8-Isoleucine Angiotensin II; Angiotensin II; Angiotensin III; Angiotensin Receptor Antagonists; Binding, Competitive; Biphenyl Compounds; Calcium; Cyclic AMP; Dose-Response Relationship, Drug; Humans; Imidazoles; Kinetics; Losartan; Neuroblastoma; Oligopeptides; Pyridines; Receptors, Angiotensin; Tetrazoles; Tumor Cells, Cultured; Vasoactive Intestinal Peptide

Pituitary adenylate cyclase activating polypeptide (PACAP) is a newly discovered neuropeptide which exists in two biologically active forms: PACAP-38 consisting of 38 amino acids and PACAP-27, a peptide corresponding to the N-terminal 27 amino acids of PACAP-38. Both PACAPs are derived from a 176 amino acid precursor (preproPACAP) which in addition gives rise to a 29 amino acid peptide, designated PACAP-related peptide (PRP). The presence of the three preproPACAP-derived peptides (PACAP-38, PACAP-27 and PRP) in tumour tissue from nine patients with VIP-producing tumours (pancreatic carcinoma, neuroblastoma, ganglioneuroma and pheochromocytoma) and eleven patients with non-VIP-secreting tumours (gastrinoma, glucagonoma, somatostatinoma, neuroblastoma) was examined by specific radioimmunoassays. In seven out of the nine VIP-secreting tumours elevated concentrations of all the three preproPACAP-derived peptides were found compared with normal tissue, while the concentrations in the non-VIP-secreting tumours were within the normal range. PACAP-38 was in all cases the dominating peptide, the concentration ranging from 41 to 3606 pmol/g. When tumour extracts were fractionated on Sephadex G50 column, tricine gel electrophoresis or reverse-phase HPLC immunoreactive components corresponding to synthetic PACAP-38, PACAP-27 and human PRP were identified, suggesting that preproPACAP was fully processed. Immunocytochemical examination showed PACAP-immunoreactive cells in the tumour tissue which also stained for VIP. This co-localization of PACAP and VIP was confirmed by double-staining experiments on the same sections, demonstrating PHM/VIP mRNA and PACAP-immunostaining in the same cells.

Topics: Adrenal Gland Neoplasms; Ganglioneuroma; Humans; Neoplasms; Neuroblastoma; Neuropeptides; Neurotransmitter Agents; Pancreatic Neoplasms; Pheochromocytoma; Pituitary Adenylate Cyclase-Activating Polypeptide; Protein Precursors; Vasoactive Intestinal Peptide

The cytokines leukemia inhibitory factor (LIF) and ciliary neurotrophic factor (CNTF) have been implicated in determination of neuronal phenotype as well as promotion of neuronal survival. However, the intracellular mechanisms by which their signals are transduced remain poorly understood. We have previously studied the regulation of vasoactive intestinal polypeptide gene expression by LIF and CNTF in the NBFL neuroblastoma cell line. Because these cytokines induce tyrosine phosphorylation that may lead to Ras activation, we explored a possible role for Ras in LIF- and CNTF-induced signal transduction. In NBFL cells LIF increases activated Ras in a rapid, transient, and concentration-dependent manner. CNTF and a related cytokine, oncostatin M, produce similar increases. CNTF and LIF also increase activated Ras in neuron-enriched dissociated cultures of sympathetic ganglia. Moreover, these cytokines rapidly and transiently induce specific tyrosine-phosphorylated proteins, p165 and p195. The protein kinase inhibitors K252a and staurosporine block LIF-induced increases in tyrosine phosphorylation, activated Ras, and vasoactive intestinal polypeptide mRNA in NBFL cells. These data support a possible role for Ras in the cell differentiation effects of LIF and CNTF.

Topics: 1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine; Alkaloids; Animals; Animals, Newborn; Blotting, Western; Carbazoles; Cell Line; Cells, Cultured; Ciliary Neurotrophic Factor; Cytokines; Growth Inhibitors; Indole Alkaloids; Interleukin-6; Isoquinolines; Leukemia Inhibitory Factor; Lymphokines; Nerve Growth Factors; Nerve Tissue Proteins; Neuroblastoma; Neurons; Oncostatin M; Peptides; Piperazines; Protein Kinase C; Protein Kinase Inhibitors; ras Proteins; Rats; RNA; RNA, Messenger; Staurosporine; Superior Cervical Ganglion; Tumor Cells, Cultured; Vasoactive Intestinal Peptide

In this study, we investigated the vasoactive intestinal polypeptide (VIP)-stimulated cAMP production and its interaction with protein kinase C activation and elevation of intracellular Ca2+ in N1E-115 neuroblastoma cells. VIP treatment caused a 55-fold increase in cAMP accumulation. Addition of 4 beta-phorbol 12-myristate 13-acetate reduced VIP- but not forskolin-stimulated cAMP response. In comparison, ionomycin potentiated both VIP- and forskolin-induced cAMP accumulation. Our results indicate that VIP stimulates cAMP accumulation in N1E-115 cells, and that although activation of protein kinase C inhibits the VIP-stimulated cAMP response, elevation of intracellular Ca2+ potentiates this signaling pathway.

Topics: 1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine; 1-Methyl-3-isobutylxanthine; Animals; Calcium; Carcinogens; Colforsin; Cyclic AMP; Dose-Response Relationship, Drug; Drug Interactions; Drug Synergism; Enzyme Activation; Ionomycin; Isoquinolines; Kinetics; Mice; Neuroblastoma; Phorbol Esters; Piperazines; Protein Kinase C; Signal Transduction; Tetradecanoylphorbol Acetate; Tumor Cells, Cultured; Vasoactive Intestinal Peptide

Ciliary neurotrophic factor (CNTF) and leukemia inhibitory factor (LIF) are members of a family of neuropoietic cytokines that have a broad range of actions on many different neuronal populations. In cultured sympathetic neurons, CNTF and LIF induce transcription of the VIP and other neuropeptide genes as part of a program of differentiation. To gain insight into the nuclear events involved in cytokine-mediated activation of the neuropeptide genes involved in neuronal differentiation, we have investigated the mechanisms of transcriptional activation of the vasoactive intestinal peptide (VIP) gene by the CNTF family of cytokines. In the neuroblastoma cell line NBFL, CNTF, LIF, and a related cytokine, oncostatin-M, activate VIP gene transcription through a 180-base pair cytokine response element (CyRE). Deletion analysis of the VIP CyRE showed that multiple regions within the 180 base-pairs are important for cytokine-mediated transcriptional activation of the VIP gene. To one of these regions within the CyRE, cytokine treatment induces binding of a protein complex composed of members of the signal transducers and activators of transcription (STAT) transcription factor family. Mutation of this STAT-binding site attenuates cytokine-mediated transcriptional activation. LIF treatment of primary sympathetic neurons also induced binding of a STAT-containing protein complex to the VIP CyRE. Thus, activation of STAT transcription factors contributes to the induction of the VIp gene by the CNTF family of cytokines and may be involved in cytokine-mediated differentiation of sympathetic neurons.

Topics: Base Sequence; Binding Sites; Cell Differentiation; Ciliary Neurotrophic Factor; DNA; DNA-Binding Proteins; Gene Deletion; Gene Expression Regulation; Growth Inhibitors; Humans; Interleukin-6; Leukemia Inhibitory Factor; Lymphokines; Molecular Sequence Data; Nerve Tissue Proteins; Neuroblastoma; Neurons; Oncostatin M; Peptides; Phosphorylation; Signal Transduction; Transfection; Tumor Cells, Cultured; Vasoactive Intestinal Peptide

The neuropeptide vasoactive intestinal polypeptide (VIP) has a broad range of functions, and its expression has been correlated with neuronal differentiation. Here we present data on the effects of retinoic acid (RA), a known modulator of neuronal differentiation, on VIP gene expression in the human neuroblastoma cell line NB-1. Morphological data, surprisingly, indicate that these cells are not differentiated concomitant with the increase in VIP gene expression. RA was found to exert a concentration-dependent induction of peptides derived from the VIP precursor molecule, prepro-VIP. The effect at both the messenger RNA (mRNA) level, evaluated by Northern blots, and the peptide level, measured by RIAs, was found to be slow and long lasting. No changes in the processing of prepro-VIP were observed using gel chromatography and RIAs specific for various prepro-VIP sequences. Also, the expression of mRNA for the prohormone-processing enzyme PC2, present in these cells, was not altered by RA. The lag period preceding the increase in VIP mRNA led to experiments with the translational inhibitor cycloheximide showing an indirect effect of RA on VIP mRNA expression. Northern blots revealed that at least three mRNAs encoding RA receptor were expressed and rapidly induced by RA in the cells, thus making them possible candidates for the intermediate protein(s) required from the induction of VIP gene expression.

Topics: Aspartic Acid Endopeptidases; Blotting, Northern; Cycloheximide; Gene Expression Regulation, Enzymologic; Gene Expression Regulation, Neoplastic; Humans; Neuroblastoma; Proprotein Convertases; Protein Precursors; Radioimmunoassay; RNA, Messenger; Time Factors; Tretinoin; Tumor Cells, Cultured; Vasoactive Intestinal Peptide

The efficiency of ion-pair reversed-phase HPLC on a Vydac C18 column with 50 mM ammonium acetate (pH 4.75)-methanol-acetonitrile (88:9:3, v/v/v) as the mobile phase with isocratic separation and fluorescence detection for the determination of cAMP in cellular extracts was evaluated. This method was compared with a radioimmunoassay technique in terms of linearity, reproducibility and sensitivity. No interactions with other nucleotides such as AMP, ADP, ATP and cGMP were observed. Application to the measurement of cAMP modifications was studied in a neuroblastoma cell line: LA-N-2 cells stimulated by a neuropeptide, vasoactive intestinal peptide.

Topics: Acetates; Chromatography, High Pressure Liquid; Cyclic AMP; Humans; Hydrogen-Ion Concentration; Kinetics; Neuroblastoma; Radioimmunoassay; Sensitivity and Specificity; Tumor Cells, Cultured; Vasoactive Intestinal Peptide

Topics: Cell Differentiation; Cell Division; Child; Child, Preschool; Female; Ganglioneuroblastoma; Humans; Immunoenzyme Techniques; Infant; Male; Neuroblastoma; Radioligand Assay; Receptors, Vasoactive Intestinal Peptide; Survival Analysis; Vasoactive Intestinal Peptide

VIP is a widely distributed neuropeptide of 28 amino acids, whose central part is proposed to be an amphiphilic alpha-helix. In order to gain an understanding of the effect of this alpha helix on receptor binding and stimulation, a human VIP analog has been designed in which the residues 12 to 19 were replaced by a spacer of the same length, (gamma-aminobutyryl)2. This peptide altered neither the basal guinea pig tracheal smooth muscle tonus nor the VIP-induced relaxation. Conversely, the VIP analog was found to displace VIP from its binding sites on LA-N-2 human neuroblastoma cells (VIP IC50: 5.4 nM; VIP analog IC50: 52.2 nM) and to inhibit the VIP-induced cyclic AMP production of 58 +/- 15% at 1 microM and 95 +/- 2% at 10 microM. It seems that the alpha helix structure might only play the role of a spacer holding the important residues, at the N- and C-ends, respectively, at an appropriate distance. In the VIP analog structure, the (gamma-aminobutyryl)2 chain introduced in place of the alpha helix plays the role of adequate spacer to bind the LA-N-2 receptors but probably does not induce the active conformation for receptor stimulation. The lack of VIP analog effects on the tracheal receptors related to relaxation argues for a possible heterogeneity of VIP receptors on a pharmacological basis.

Topics: Amino Acid Sequence; Animals; Binding, Competitive; Cyclic AMP; Guinea Pigs; Humans; In Vitro Techniques; Male; Molecular Sequence Data; Muscle Relaxation; Neuroblastoma; Peptide Fragments; Receptors, Vasoactive Intestinal Peptide; Trachea; Tumor Cells, Cultured; Vasoactive Intestinal Peptide

Vasoactive intestinal polypeptide (VIP) has been suggested to be an inhibitory neurotransmitter in the sphincteric and nonsphincteric smooth muscles of the gut. However, the relative gene expression of VIP in these functionally diverse regions is not known.. The gastrointestinal smooth muscle sphincters of opossums were excised from the adjoining nonsphincteric smooth muscles. RNAs were isolated and subjected to blot hybridizations with VIP complementary DNA probe. Relative expression of VIP gene was quantitated using the densitometric scanning of the VIP messenger RNA (mRNA) transcripts. The cellular specificity of VIP gene expression was investigated in cultures of neuroblastoma cells and myenteric plexuses and compared with those of the smooth muscle cells.. The data showed higher levels of VIP mRNA in the sphincteric than the adjoining nonsphincteric tissues. VIP mRNA were found in significantly higher amounts in the myenteric neurons and neuroblastoma cells than in the smooth muscle cells.. VIP gene expression was significantly higher in the sphincteric smooth muscle regions than in the nonsphincteric regions of the gut. The studies provide further evidence for the role of VIP in neurotransmission of the gut.

Topics: Anal Canal; Animals; Digestive System Physiological Phenomena; Esophagogastric Junction; Female; Gene Expression; Male; Myenteric Plexus; Neuroblastoma; Neurons; Opossums; Rats; Rats, Sprague-Dawley; RNA, Messenger; Sphincter of Oddi; Tissue Distribution; Tumor Cells, Cultured; Vasoactive Intestinal Peptide

To investigate the presence of biologically active somatostatin (SS) receptors in neural crest-derived tumors, radioligand binding studies, cyclic AMP accumulation, intracellular calcium, and growth assays were performed in eight human neuroblastoma (NB) cell lines. Mathematical modeling of binding experiments strongly indicates the presence of heterogeneity of sites. The first site (SSR1) is present in 40% of the NB cell lines and binds with low capacity (0.5 pmol/mg protein) and high affinity (0.1-1 nM) SS14, SS28, and analogues. The second site (SSR2) is a high capacity site (200 pmol/mg protein), widely distributed in all of the cell lines investigated, that shows relative selectivity yet low affinity (100 nM) for SS14, SS28, and [D-Trp8]SS14 without any apparent biological activity. SSR1 is coupled to a pertussis toxin-sensitive G protein, inhibits forskolin- or VIP-stimulated adenylate cyclase activity, decreases intracellular free calcium, and mediates inhibition (30%) of both DNA synthesis and cell growth. Analysis of cell cycle distribution in aphidicolin-synchronized SSR1-positive NB cells indicated that this inhibitory effect is partially mediated by a transient accumulation in G0-G1. Our data indicate high affinity binding sites for SS14, and analogues are present and biologically active in a subset of NB cells.

Topics: Binding Sites; Calcium; Cell Cycle; Cell Division; Colforsin; Computer Simulation; Cyclic AMP; Humans; Neuroblastoma; Octreotide; Receptors, Somatostatin; Tumor Cells, Cultured; Vasoactive Intestinal Peptide

Second messenger regulation of gene expression provides a mechanism by which neurons can transduce environmental stimuli into long-term changes in the expression of molecules involved in neuronal signaling. We have investigated calcium-dependent induction of vasoactive intestinal polypeptide (VIP) mRNA and compared it with induction of VIP mRNA by cyclic AMP. Depolarization with 60 mM KCl or exposure to the calcium ionophore A23187 increases VIP mRNA levels in SH-SY5Y cells. The increase in VIP mRNA content in response to Ca2+ mobilization is slow, independent of adenylate cyclase activation, and requires de novo protein synthesis. The increase in VIP mRNA content in response to elevation of cyclic AMP levels by forskolin/isobutylmethylxanthine is independent of Ca2+ influx and does not require new protein synthesis. The mRNA for the transcription factors ATF-3, c-fos, c-jun, junB, and zif/268 is induced by A23187. Of these, ATF-3 showed the greatest relative induction by A23187 compared with induction by forskolin/isobutylmethylxanthine.

Topics: 1-Methyl-3-isobutylxanthine; Calcimycin; Calcium; Colforsin; Cyclic AMP; Gene Expression Regulation, Neoplastic; Humans; Neuroblastoma; Potassium; RNA, Messenger; Signal Transduction; Tumor Cells, Cultured; Vasoactive Intestinal Peptide

Pituitary adenylate cyclase activating peptide-38 (PACAP-38), PACAP-27 and vasoactive intestinal polypeptide (VIP) increased intracellular cAMP content in human neuroblastoma NB-OK-1 cells transiently. PACAP and VIP also arrested cell growth and induced morphological differentiation, which lasted for 24 h in spite of removal of PACAP-38 and PACAP-27. The order of potencies for the neurite outgrowth and the arrest of cell growth is PACAP-38 > PACAP-27 > VIP. The results suggest the possibility that these neuropeptides are new candidates for differentiation activity.

Topics: Cell Differentiation; Cell Division; Chromatography, High Pressure Liquid; Cyclic AMP; Humans; Neurites; Neuroblastoma; Neuropeptides; Neurotransmitter Agents; Pituitary Adenylate Cyclase-Activating Polypeptide; Tumor Cells, Cultured; Vasoactive Intestinal Peptide

Differentiation factors have been identified that influence the phenotype of sympathetic neurons by altering expression of classical neurotransmitters and neuropeptides. Investigation of the molecular mechanisms through which such factors act would be facilitated by the availability of a neuronal cell line that responds to these factors in a fashion similar to sympathetic neurons. We have identified a human neuroblastoma cell line, NBFL, that responds to the differentiation factor ciliary neurotrophic factor (CNTF) by coordinately inducing multiple neuropeptide genes as do sympathetic neurons. Treatment of NBFL cells with CNTF increases vasoactive intestinal polypeptide (VIP), somatostatin, and calcitonin gene-related peptide (CGRP) mRNAs but does not change other neurotransmitter properties. The induction of VIP mRNA by CNTF in NBFL cells is dose dependent, rapid, sustained, and independent of new protein synthesis. Genomic 5' flanking sequences located within a 1.59-kilobase region of the human VIP gene and distinct from the previously defined cAMP-responsive element subserve transcriptional activation by CNTF. Further examination of NBFL cells should permit the elucidation of the molecular mechanisms by which CNTF and other differentiation factors coordinately activate neuropeptide gene transcription to influence neuronal differentiation. Similar mechanisms may mediate the effect of CNTF on neuronal survival.

Topics: Calcitonin Gene-Related Peptide; Cell Differentiation; Ciliary Neurotrophic Factor; Gene Expression; Gene Expression Regulation; Nerve Growth Factors; Nerve Tissue Proteins; Neuroblastoma; Neurons; Neuropeptides; Regulatory Sequences, Nucleic Acid; RNA, Messenger; Somatostatin; Transcription, Genetic; Tumor Cells, Cultured; Vasoactive Intestinal Peptide

Tissue transglutaminase (tTG) activity was used to test the potent regulatory role of vasoactive intestinal peptide (VIP) on Retinoic Acid-induced effect in human neuroblastoma cell line. The comparison between both differentiation and cell death related to tissue transglutaminase was discussed in this model. VIP alone was a potent differentiating agent in SK-N-SH cells but in the presence of retinoic acid (RA), this peptide rather potentiates RA-induced tTG activity which is now considered as an apoptosis marker in neuroblastoma cell line. This paper demonstrated an additional neuromodulator role for VIP.

Topics: Apoptosis; Biomarkers; Drug Synergism; Enzyme Induction; Humans; Neoplasm Proteins; Neuroblastoma; Transglutaminases; Tretinoin; Tumor Cells, Cultured; Vasoactive Intestinal Peptide

Vasoactive intestinal peptide (VIP) is a neuromodulator, growth regulator and secretagogue for neuronal survival factors. Moreover, VIP has been suggested to be a mitogenic factor for embryonic neurons in the sympathetic nervous system. We now show that VIP had mitogenic activity in a human neuroblastoma cell line (NMB), as measured by cell number and thymidine incorporation. This mitogenic activity was dose dependent and was decreased with culture maturation. Northern blot analysis revealed VIP mRNA transcripts in this cell line suggesting an autocrine role for VIP in neurogenesis.

Topics: Blotting, Northern; Cell Division; Dose-Response Relationship, Drug; Growth Substances; Humans; Neuroblastoma; RNA, Messenger; Tumor Cells, Cultured; Vasoactive Intestinal Peptide

Direct associations between serum concentrations and immunohistochemically detectable vasoactive intestinal peptide (VIP) and maturing neuroblastoma have been documented. Furthermore, VIP has been shown to induce both the growth inhibition and morphological differentiation of cultured human neuroblastoma cell lines. As such, it is hypothesized that VIP may be operative in the autocrine regulation of neuroblastic growth and differentiation. To test this hypothesis, VIP-induced differentiation of human neuroblastoma LA-N-5 cells was performed. Significant concomitant increases in both intracellular and extracellular VIP concentrations were observed. In addition, a marked increase in VIP receptor expression was demonstrated with VIP-induced cellular differentiation. Receptor function was maintained with enhanced expression, as evidenced by an increase in the generation of intracellular cyclic adenosine monophosphate in response to exogenous VIP stimulation. Concomitant enhancement of both intracellular and extracellular VIP expression, coupled with the induction of functional specific VIP receptors during VIP-induced differentiation, provides critical evidence for the autocrine regulation of neuroblastoma maturation by this peptide.

Topics: Cell Differentiation; Cell Division; Cyclic AMP; Gene Expression; Glucose-6-Phosphate Isomerase; Humans; Iodine Radioisotopes; Neuroblastoma; Receptors, Gastrointestinal Hormone; Receptors, Vasoactive Intestinal Peptide; Tumor Cells, Cultured; Vasoactive Intestinal Peptide

Vasoactive intestinal peptide (VIP) is a potent mediator of gastrointestinal, nervous, pulmonary, vascular and immune functions. A cDNA was obtained from human HT29 intestinal epithelial cells and found to encode a 457 amino acid, 52 kDa VIP receptor. Transfection of the cDNA into COS-7 cells and 293 cells resulted in expression of specific saturable binding of VIP with a Kd of 0.8 nM, and induction of increases in intracellular cAMP by VIP with an EC50 of 1 nM. The human VIP receptor is homologous to other G protein-coupled receptors of the secretin-parathyroid hormone receptor family. A 2.8 kb transcript was detected in human lung, HT29 cells and Raji B-lymphoblasts with weaker expression in human brain, heart, kidney, liver and placenta.

Topics: Amino Acid Sequence; Animals; Base Sequence; Cell Line; Cloning, Molecular; Cyclic AMP; Gene Expression; Glioma; Humans; Hybrid Cells; Kinetics; Mice; Molecular Sequence Data; Neuroblastoma; Oligodeoxyribonucleotides; Polymerase Chain Reaction; Rats; Receptors, Gastrointestinal Hormone; Receptors, Vasoactive Intestinal Peptide; Recombinant Proteins; RNA, Messenger; Sequence Homology, Amino Acid; Transfection; Vasoactive Intestinal Peptide

The production and secretion of multiple peptide hormones and tyrosine hydroxylase by the human neuroblastoma cell line NB-1 and the effects of dibutyryl cAMP (Bt2cAMP) and phorbol esters such as 12-O-tetradecanoyl-phorbol-13-acetate (TPA) on them were investigated. The presence of messenger RNAs (mRNAs) of vasoactive intestinal peptide (VIP)/peptide histidine methionine (PHM), preprotachykinin, and tyrosine hydroxylase was detectable in the cytoplasm of cultured NB-1 cells by in situ hybridization. Treatment with Bt2cAMP and TPA markedly increased the number of cells immunoreactive to VIP, PHM, neuropeptide Y, Met-enkephalin, substance P and tyrosine hydroxylase and also the contents of VIP and Met-enkephalin in the culture medium. Bt2cAMP and TPA induced morphological changes characteristic of endocrine differentiation, such as an increase in neuroendocrine granules and the development of rough endoplasmic reticulum and Golgi apparatus. The results indicated that treatment with Bt2cAMP and TPA induces the expression of multiple genes of peptide hormone and tyrosine hydroxylase and increases hormone production and secretion through morphological changes into endocrine cells.

Topics: Bucladesine; Cytoplasmic Granules; Enkephalin, Methionine; Hormones; Humans; Immunohistochemistry; In Situ Hybridization; Microscopy, Electron; Neuroblastoma; Neuropeptide Y; Peptide PHI; Protein Precursors; Radioimmunoassay; RNA, Messenger; Substance P; Tachykinins; Tetradecanoylphorbol Acetate; Tumor Cells, Cultured; Tyrosine 3-Monooxygenase; Vasoactive Intestinal Peptide

We have used human neuroblastoma NB-OK1 cells to investigate the regulation of neurite outgrowth. Carbachol suppressed forskolin-stimulated neurite outgrowth in NB-OK1 cells although forskolin-stimulated cAMP levels were enhanced. The dose-response curve for this suppression was very similar to that for stimulation of inositol monophosphate (IP1) formation and for stimulation of the initial rise of [Ca2+]i elicited by carbachol. Carbachol-mediated changes in neurite outgrowth, IP1 formation and [Ca2+]i displayed high sensitivity for pirenzepine but low sensitivity for AF-DX116. Inhibition of intracellular calcium release with TMB-8 prevented the suppressive effect of carbachol on forskolin-stimulated neurite outgrowth. Hence we describe for the first time a relationship between neurite outgrowth and inositol triphosphate-triggered calcium release mediated by carbachol in the human neuron-derived cell line.

Topics: Atropine; Calcium; Calcium Channel Blockers; Carbachol; Cell Line; Colforsin; Cyclic AMP; Drug Interactions; Egtazic Acid; Gallic Acid; Humans; Inositol; Inositol Phosphates; Kinetics; Neurites; Neuroblastoma; Tetradecanoylphorbol Acetate; Vasoactive Intestinal Peptide

Neuroblastoma is the most common solid tumor of children less than 5 years of age; yet the biology of this tumor is poorly understood. Neuroblastoma tumors are derived from neural crest precursors; they synthesize both adrenergic and peptidergic neurotransmitters. This study determined VIP receptor expression in primary neuroblastoma tumors prior to chemotherapy. The VIP receptor was expressed in 12 of 15 neuroblastoma tumors as determined by direct binding studies (KD = 1.3-12.4 nM) and VIP-mediated stimulation of adenylate cyclase. The VIP stimulation index for adenylate cyclase in the primary tumor was inversely correlated with the VIP content of the tumor, suggesting that VIP regulates its own receptor expression. Similar observations were made in vitro by comparison of two human neuroblastoma cell lines, IMR32 and SKNSH. Both cell lines were demonstrated to express specific, high affinity VIP receptors (KD = 4 nM and 2.5 nM for IMR32 and SKNSH, respectively). IMR32 cells contained very low levels of VIP (0.6 pg VIP/10(6) cells). Exogenous VIP stimulated adenylate cyclase 22-fold over basal activity and VIP inhibited proliferation of IMR32 cells by 49% in 6-day cultures. On the other hand, SKNSH cells synthesized high levels of VIP (6.3 pg/10(6) cells), metabolized VIP rapidly and demonstrated a low level of VIP-mediated stimulation of adenylate cyclase; their proliferation rate was minimally inhibited by exogenous VIP. These observations help validate the hypothesis that VIP serves as an autocrine growth factor in neuroblastoma.

Topics: Adenylyl Cyclases; Binding, Competitive; Cell Division; Cell Survival; Humans; Neuroblastoma; Radioimmunoassay; Receptors, Gastrointestinal Hormone; Receptors, Vasoactive Intestinal Peptide; Tumor Cells, Cultured; Vasoactive Intestinal Peptide

Neuronal differentiation was induced in cultures of the human neuroblastoma cell line subclone SH-SY5Y by 14-day treatment with dibutyryl cAMP (dBcAMP), retinoic acid, and phorbol 12-myristate 13-acetate (PMA). An approximate 4-fold increase in vasoactive intestinal peptide (VIP) mRNA concentration was observed after differentiation with retinoic acid, whereas no change in VIP mRNA concentration was observed after differentiation with dBcAMP or PMA. A short-term treatment of cells with PMA did however result in a 5-fold transient increase in VIP mRNA; prior differentiation with retinoic acid or dBcAMP diminished this effect. Observed increases in VIP mRNA were in all cases accompanied by increases in VIP immunoreactivity. Remarkably, however, long-term treatment of cells with dBcAMP, which caused no change in mRNA levels, resulted in a six-fold increase in VIP immunoreactivity. Acute (36-h) treatment with carbachol also caused an increase in VIP immunoreactivity (about 2-fold, and blocked by atropine) without an increase in VIP mRNA level. Thus, a quantitative change in gene transcription or mRNA stability appears not to be a prerequisite for increased VIP expression, indicating that regulation can occur at translational or post-translational steps.

Topics: Bucladesine; Carbachol; Humans; Neuroblastoma; Peptide Biosynthesis; Radioimmunoassay; RNA, Messenger; Tetradecanoylphorbol Acetate; Tretinoin; Tumor Cells, Cultured; Vasoactive Intestinal Peptide

In this report we provide evidence for the activation of distinct differentiation pathways during treatment of the neuroblastoma cell line SMS-KCNR with 1 mM dibutyryl cyclic AMP (dbcAMP) and/or 5 microM retinoic acid (RA). Our results show that the adrenal gland specific gene pG2 is induced only during dbcAMP treatment, while RA induces a neuronal phenotype and expression of all neural related genes while decreasing the expression of many chromaffin related genes. Furthermore dbcAMP does not affect the DNA content distribution of SMS-KCNR [G1 = 61.8 +/- 4.1% (SD); S = 20.3 +/- 6.3%; G2-M = 18 +/- 5.4%] despite morphological and molecular signs of cellular differentiation. Conversely, RA arrests cell growth causing a decrease in cells in the growth fraction (S + G2 + M = 15.6 +/- 6.1%) and an increase in cells in G1 (G1 = 84.3 +/- 5%). Using cyclic AMP and RA in combination, we found that RA inhibited expression of adrenal gland specific gene pG2 and induced a neuronal phenotype. Since dbcAMP does not cause a significant G1 block in SMS-KCNR cells we propose that this agent may be able to induce SMS-KCNR only to an intermediate stage of chromaffin differentiation in which cells retain their proliferative potential.

Topics: Bucladesine; Cell Differentiation; G1 Phase; Gene Expression Regulation, Neoplastic; Genes, Retinoblastoma; Genetic Markers; Humans; Neuroblastoma; Phenotype; RNA, Messenger; RNA, Neoplasm; S Phase; Time Factors; Transcription, Genetic; Tretinoin; Tumor Cells, Cultured; Vasoactive Intestinal Peptide

Elevated serum levels of vasoactive intestinal peptide (VIP) are associated with some cases of neuroblastoma and correlate with a favorable prognosis. VIP has previously been shown in our laboratory to cause the in vitro growth inhibition and morphological differentiation of the human neuroblastoma cell line, LA-N-5. It is now shown that LA-N-5 cells express immunoreactive VIP and bear specific VIP receptors. Antagonism of endogenous VIP, either by competitive inhibition or receptor blockade, increased cell proliferation, suggesting that VIP is operative in normal growth regulation. Intracellular and extracellular levels of VIP were also shown to increase significantly during the retinoic acid-induced differentiation of these cells. Furthermore, a concomitant marked increase in VIP receptor expression was demonstrated with cellular differentiation. These receptors remain functional as evidenced by a matching increase in the level of detectable cAMP generated in response to exogenous VIP. It is concluded that VIP is a normal autoregulator of neuroblastoma cell growth and differentiation, and that retinoic acid-mediated differentiation may be, in part, due to endogenous VIP.

Topics: Cell Transformation, Neoplastic; Humans; Neuroblastoma; Receptors, Gastrointestinal Hormone; Receptors, Vasoactive Intestinal Peptide; Tretinoin; Tumor Cells, Cultured; Vasoactive Intestinal Peptide

In SK-N-SH human neuroblastoma cells, the muscarinic agonist carbachol promotes polyphosphoinositide (PPI) hydrolysis via M3 receptors and increases cyclic AMP levels through an unidentified mechanism. Activation of PPI hydrolysis by carbachol elicits a robust translocation of CaM from membranes into cytosol which was previously shown to be mimicked by the addition of the calcium ionophore ionomycin and the phorbol ester TPA28. The effect of agonist-stimulated second messenger production on CaM localization was determined by activating receptors that increase and decrease adenylyl cyclase activity on SK-N-SH cells. VIP (10 microM), prostaglandin E1 (30 microM) and forskolin (10 microM) all increased adenylyl cyclase activity 8- to 10-fold above the activity with 1 microM GTP. Carbachol (100 microM) did not stimulate adenylyl cyclase activity. The alpha 2-adrenergic agonist UK 14,304 (0.1 microM) and the delta and mu opioid DPDPE (10 microM) and DAMGO (10 microM) inhibited forskolin-stimulated cyclic AMP formation by 27-32%. CaM did not stimulate adenylyl cyclase activity. Incubation of cells with vasoactive intestinal polypeptide (VIP), dibutyryl cyclic AMP and forskolin, resulted in 30% decrease in membrane CaM and an increase in cytosolic CaM of 40-50%. The CaM translocation with the combination of an agent that elevates cyclic AMP levels and a low dose of carbachol was not different from that observed with either agent alone. UK 14,304, DPDPE and DAMGO potentiated carbachol-stimulated increases in cytosolic CaM. Upon the addition of carbachol, a 5-fold increase in intracellular calcium concentration measured with fura-2 fluorescence was observed. VIP and UK 14,304 elevated intracellular calcium concentrations 2 to 3 fold, while forskolin (10 microM) had no effect.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Adenylyl Cyclases; Adrenergic alpha-Agonists; Alprostadil; Analgesics; Brimonidine Tartrate; Bucladesine; Calcium; Calmodulin; Carbachol; Cell Line; Colforsin; Cyclic AMP; Cytosol; Edetic Acid; Egtazic Acid; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Enkephalin, D-Penicillamine (2,5)-; Enkephalins; Humans; Kinetics; Neuroblastoma; Quinoxalines; Tumor Cells, Cultured; Vasoactive Intestinal Peptide

The concentrations of immunoreactive (IR) corticotropin-releasing hormone (CRH) in 218 neuroendocrine tumors were determined by CRH radioimmunoassay. The tumors examined were 86 pancreatic endocrine tumors (PET), 22 neuroblastic tumors (NBT), 26 carcinoid tumors (CA), 24 pheochromocytomas (PHEO), 40 small cell lung carcinomas (SCLC) and 20 medullary thyroid carcinomas (MTC). IR-CRH was detectable in 21 neuroendocrine tumors (10 PET, four NBT, three CA, two PHEO and two SCLC) at levels of 10-2,700 ng/g wet weight (9.6%). The 21 patients with these CRH-producing tumors showed no clinical symptoms suggestive of Cushing's syndrome. The levels of plasma IR-CRH extracted by immunoaffinity chromatography were < 7.5 pg/ml in five normal subjects and a patient with a neuroblastic tumor containing 55 ng/g wet weight IR-CRH, but in a patient with a thymic carcinoid tumor containing 1,000 ng/g wet weight IR-CRH, the plasma level was elevated to 180 pg/ml. This patient did not have Cushing's syndrome nor an elevated plasma adrenocorticotropic hormone (ACTH) level. The concentrations of nine peptides (growth hormone-releasing hormone, somatostatin, ACTH, calcitonin, gastrin-releasing peptide, glucagon, vasoactive intestinal peptide, neuropeptide tyrosine and pancreatic polypeptide) were determined in extracts of the 21 IR-CRH-producing tumors. Some of these peptides were frequently found to be produced concomitantly with CRH. The results indicate IR-CRH to be produced by various neuroendocrine tumors, but Cushing's syndrome, due to the CRH, to be very rare. The results also show that CRH-producing tumors produce multiple hormones.

Topics: Adenoma, Islet Cell; Adrenal Gland Neoplasms; Adrenocorticotropic Hormone; Bombesin; Calcitonin; Carcinoid Tumor; Carcinoma, Small Cell; Chromatography, Gel; Corticotropin-Releasing Hormone; Gastrin-Releasing Peptide; Gastrins; Humans; Hypothalamus; Lung Neoplasms; Neoplasms; Neuroblastoma; Pancreatic Neoplasms; Peptides; Pheochromocytoma; Somatostatin; Thyroid Neoplasms; Vasoactive Intestinal Peptide

The neuropeptide contents in neuroblastomas were quantified by radioimmunoassay (RIA) to assess their possible biologic significance.. Neuroblastoma tumor tissue was obtained from the primary tumor site before therapy in 16 patients with newly diagnosed neuroblastoma and in 7 patients with central nervous system medulloblastomas or gliomas.. The tumor tissue was assayed for vasoactive intestinal peptide (VIP), somatostatin (SRIF), substance P, and neurotensin by both immunostaining and RIA techniques. Increased VIP levels of 1.2 pg/micrograms DNA or more correlated significantly with cellular differentiation (P = 0.003) and favorable disease stage (P = 0.002) in neuroblastomas. Increased SRIF contents (greater than 1.3 pg/micrograms DNA) correlated with differentiation of the tumor (P = 0.002). Increased VIP and SRIF content did not correlate with N-myc oncogene expression or ras oncogene protein immunostaining. No VIP was detectable in brain tumors, and other neuropeptides were variable in content.. RIA of VIP and SRIF levels in primary tumor tissue may offer an independent objective assay of biologic behavior in neuroblastoma biopsy specimens.

Topics: Adolescent; Adult; Aged; Aged, 80 and over; Child; Child, Preschool; Female; Ganglioneuroma; Genes, myc; Genes, ras; Humans; Immunoenzyme Techniques; Male; Neuroblastoma; Neuropeptides; Prognosis; Prospective Studies; Radioimmunoassay; Somatostatin; Substance P; Vasoactive Intestinal Peptide

Oncostatin-M (OM), a recently described glycoprotein cytokine, is structurally and functionally related to cholinergic differentiation factor/leukemia inhibitory factor (CDF/LIF) and ciliary neurotrophic factor (CNTF). To determine whether OM, like CDF/LIF and CNTF, possesses trophic or differentiative functions for neurons we examined the effects of recombinant human OM on ciliary neuron survival and neurotransmitter expression in sympathetic neurons. Like CDF/LIF, but in contrast to CNTF, OM had no effect on ciliary neuronal survival at any concentration tested. OM produced small but reproducible increases in choline acetyl transferase (ChAT) activity and vasoactive intestinal peptide (VIP) levels in rat sympathetic neuron cultures, but this effect was significantly less than that of CNTF or CDF/LIF. To determine if human OM would elicit a more robust response from human cells, we utilized a human neuroblastoma cell line, NBFL, that responds to CNTF and CDF/LIF by altering vasoactive intestinal peptide (VIP) levels. OM specifically elevated VIP and c-fos mRNA levels in NBFL cells and was as potent as CDF/LIF in this assay. Our data provides evidence that OM acts on neurons and identifies a neural cell line responsive to OM, CNTF, CDF/LIF.

Topics: Animals; Blotting, Northern; Chick Embryo; Choline O-Acetyltransferase; Growth Inhibitors; Indicators and Reagents; Interleukin-6; Leukemia Inhibitory Factor; Lymphokines; Neuroblastoma; Neurons; Neuropeptides; Oncostatin M; Peptides; Radioimmunoassay; Tumor Cells, Cultured; Vasoactive Intestinal Peptide

Eighteen commercially available antibodies were applied to formalin-fixed, paraffin-embedded neuroblastomas (NBLs, n = 20), ganglioneuroblastomas (GNBLs, n = 7), and ganglioneuromas (GNs, n = 7) to assess their reliability as markers for neuroendocrine differentiation and degree of tumor cell maturation. Incubations with a monoclonal antibody to neuron-specific enolase resulted in positive reactions in all tumors, with consistently strong staining intensities in moderate and well-differentiated NBLs, GNBLs, and GNs. Antibodies to dopamine beta-hydroxylase and protein gene product (PGP) 9.5 reacted with all tumors except two NBLs. Among the antibodies directed to chromogranins and related proteins, HISL19 was most reliable (33/34) followed by endocrine granule constituent (EGC) (30/34), chromogranin A (LK2H10) (21/34), and chromogranin A + B (CGA + B) (19/34), in proving the existence of endocrine granules in tumor cells and Neurofilament (70 + 200 kD) immunoreactivity was demonstrated in all tumors except two undifferentiated NBLs. S-100 protein-immunoreactive cells were visualized with increasing frequency in highly differentiated GNBLs and GNs, whereas Leu 7 immunoreactivity was restricted to ganglioneuromas. We conclude that antibodies directed to neuron-specific enolase, HISL19, dopamine beta-hydroxylase, neurofilaments, EGC, LK2H10, and leucocyte common antigen represent markers that might be useful in the discrimination of GNBLs from non-neuroendocrine round and small cell tumors in routinely processed tissue. Antibodies to neuron-specific enolase, PGP 9.5, different chromogranins, neurofilaments, vasoactive intestinal peptide (VIP), and S-100 protein may help to determine the grade of tumor cell maturation.

Topics: Antibodies; Antigens, Differentiation; Chromogranins; Diagnosis, Differential; Dopamine beta-Hydroxylase; Ganglioneuroma; Humans; Immunohistochemistry; Neuroblastoma; Neurofilament Proteins; Neuropeptides; Phosphopyruvate Hydratase; S100 Proteins; Ubiquitin Thiolesterase; Vasoactive Intestinal Peptide

We have investigated the modulatory action of carbachol on intracellular cAMP levels in human neuroblastoma SH-SY5Y cells. Carbachol enhanced forskolin-stimulated cAMP levels in a dose-dependent manner (EC50 = 3 microM). The enhancing effect of carbachol was completely inhibited by pirenzepine and atropine. Pertussis toxin treatment of the cells partially affected the ability of carbachol. Furthermore, carbachol also enhanced the effect of vasoactive intestinal peptide (EC50 = 3 microM)-, adenosine- and prostaglandin E1-stimulated cAMP levels. The enhancing response of carbachol was sensitive to trifluoperazine but insensitive to calphostin C. These results suggest that the mechanism for carbachol-induced cAMP levels may act, at least in part, through the activation of calmodulin system in SH-SY5Y cells. Hence we describe for the first time a synergistic interaction between calmodulin- and cAMP-dependent signal transduction pathway mediated by carbachol in neuron-derived cell line.

Topics: Adenosine; Alprostadil; Atropine; Calmodulin; Carbachol; Cell Line; Colforsin; Cyclic AMP; Drug Synergism; Humans; Kinetics; Naphthalenes; Neuroblastoma; Pertussis Toxin; Polycyclic Compounds; Protein Kinase C; Trifluoperazine; Vasoactive Intestinal Peptide; Virulence Factors, Bordetella

Topics: Cell Differentiation; Cell Division; Cell Line; DNA Replication; Drug Synergism; Glucagon; Growth Hormone-Releasing Hormone; Humans; Kinetics; Neuroblastoma; Peptide Fragments; Secretin; Sermorelin; Thymidine; Tretinoin; Vasoactive Intestinal Peptide

PACAP (pituitary adenylate-cyclase-activating peptide)-binding receptors were investigated in membranes from the rat pancreatic acinar cell line, AR 4-2J, the rat hippocampus and the human neuroblastoma cell line NB-OK, by 125I-PACAP(1-27) (amino acid residues 1-27 of N-terminal amidated PACAP) binding and adenylate cyclase activation. The relative binding of 125I-PACAP(1-27) to the receptor, and ability to activate adenylate cyclase were PACAP greater than or equal to PACAP(1-27) greater than PACAP(2-38) greater than PACAP(1-9)-VIP(10-28)(PACAP-VIP) greater than PACAP(2-27) greater than [Ser9,Tyr13]VIP greater than [Tyr13]VIP greater than or equal to [Ser9]VIP greater than or equal to VIP(1-23)-PACAP(24-27)(VIP-PACAP) greater than VIP (vasoactive intestinal peptide). The N-terminal moiety of PACAP(1-27) was more important than the three amino acids at the C-terminus for 125I-PACAP(1-27)-binding site recognition. For rat pancreatic 125I-VIP-binding sites tested with 125I-VIP, the order of binding affinity was PACAP = PACAP(1-27) greater than or equal to VIP = [Ser9]VIP = [Tyr13]VIP = [Ser9,Try13]VIP greater than or equal to PACAP-VIP greater than or equal to VIP-PACAP greater than PACAP(2-38) = PACAP(2-27). Pancreatic 125I-VIP-binding sites, when compared to 125I-PACAP(1-27)-binding sites, showed little specificity and only weak coupling, so that PACAP and VIP-PACAP acted only as partial VIP agonists on adenylate cyclase.

Topics: Adenylyl Cyclases; Amino Acid Sequence; Animals; Binding Sites; Cell Membrane; Chimera; Enzyme Activation; Hippocampus; Humans; Molecular Sequence Data; Neuroblastoma; Neurons; Neuropeptides; Pancreatic Neoplasms; Pituitary Adenylate Cyclase-Activating Polypeptide; Rats; Tumor Cells, Cultured; Vasoactive Intestinal Peptide

The human neuroblastoma clonal cell line SH-SY5Y expresses both mu- and delta-opioid receptors (ratio approximately 4.5:1). Differentiation with retinoic acid (RA) was previously shown to enhance the inhibition of adenylyl cyclase (AC) by mu-opioid agonists. We tested here the inhibition of cyclic AMP (cAMP) accumulation by morphine under a variety of conditions: after stimulation with prostaglandin E1 (PGE1), forskolin, and vasoactive intestinal peptide (VIP), both in the presence and in the absence of the phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine (IBMX). Morphine inhibition of the forskolin cAMP response (approximately 65%) was largely unaffected by the presence of IBMX. In contrast, deletion of IBMX enhanced morphine's inhibition of the PGE1 and VIP cAMP response from approximately 50 to approximately 80%. The use of highly mu- and delta-selective agents confirmed previous results that inhibition of cAMP accumulation by opioids is mostly mu, and not delta, receptor mediated in SH-SY5Y cells, regardless of the presence or absence of IBMX. Because of the large morphine inhibition and the high cAMP levels even in the absence of IBMX, PGE1-stimulated, RA-differentiated SH-SY5Y cells were subsequently used to study narcotic analgesic tolerance and dependence in vitro. Upon pretreatment with morphine over greater than or equal to 12 h, a fourfold shift of the PGE1-morphine dose-response curve was observed, whether or not IBMX was added. However, mu-opioid receptor number and affinity to the mu-selective [D-Ala2, N-Me-Phe4, Gly5-ol]enkephalin were largely unaffected, and Na(+)- and guanyl nucleotide-induced shifts of morphine-[3H]naloxone competition curves were unchanged.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: 1-Methyl-3-isobutylxanthine; Alprostadil; Cell Line; Colforsin; Cyclic AMP; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Enkephalin, D-Penicillamine (2,5)-; Enkephalins; Humans; Kinetics; Morphine; Neuroblastoma; Receptors, Opioid; Receptors, Opioid, delta; Receptors, Opioid, mu; Theophylline; Vasoactive Intestinal Peptide

We showed previously that a gene construction that consisted of 5.2 kb of 5' flanking sequence, the first exon, and part of the first intron of the human gene encoding vasoactive intestinal peptide (VIP) fused to the reporter gene chloramphenicol acetyltransferase (CAT) fully mimicked the diverse behavior of the endogenous VIP gene when transfected into subclones of the human neuroblastoma cell line SK-N-SH (Waschek et al., 1988). To determine if the same sequences were sufficient to target expression of a reporter to VIP-producing tissues in the mouse, we initiated a pilot study in which we generated four transgenic mice or mouse lines that contained the VIPCAT fusion gene. Detectable levels of CAT were found in the ileum of either founder or offspring of each of the transgenic mouse lines. In all other tissues tested, CAT activity was either below the level of detection or the transgene was not expressed, with the exception of one mouse in which ectopic expression in the cerebellum was observed. The results indicate that the VIP sequences utilized were sufficient to direct expression of the transgene to the intestine, but not necessarily to other sites of VIP expression. To investigate what specific DNA sequences might confer VIP expression in the intestine and other sites, we analyzed further the VIP gene in SK-N-SH subclones using VIP/luciferase fusion gene constructions. A 0.6 kb DNA fragment located between 4.0 kb and 4.6 kb upstream from the VIP transcriptional start site was found to impart a high level of expression in one subclone and an increased degree of phorbol ester induction in another. These and other data indicate that multiple transcriptional elements control VIP expression in neuroblastoma cells and are candidates as mediators of VIP gene expression in the intact animal.

Topics: Animals; Base Sequence; Chimera; Chloramphenicol O-Acetyltransferase; Colforsin; Gene Expression; Gene Expression Regulation, Neoplastic; Humans; Intestinal Mucosa; Luciferases; Mice; Mice, Transgenic; Molecular Sequence Data; Neuroblastoma; Tetradecanoylphorbol Acetate; Vasoactive Intestinal Peptide

We identified receptors for neuropeptide Y (NPY) on an established human neuroblastoma cell line, SK-N-MC, which are functionally coupled to adenylate cyclase through the inhibitory guanine nucleotide-binding protein of adenylate cyclase, Gi. Intact SK-N-MC cells bound radiolabeled NPY with a KD of 2 nM and contained approximately 83,000 receptors/cell. Unlabeled porcine and human NPY and structurally related porcine peptide YY (PYY) competed with labeled NPY for binding to the receptors. NPY inhibited cyclic AMP accumulation in SK-N-MC cells stimulated by isoproterenol, dopamine, vasoactive intestinal peptide, cholera toxin, and forskolin. NPY inhibited isoproterenol-stimulated cyclic AMP production in a dose-dependent manner, with half-maximal inhibition at 0.5 nM NPY. Porcine and human NPY and porcine PYY gave similar dose-response curves. NPY also inhibited basal and isoproterenol-stimulated adenylate cyclase activity in disrupted cells. Pertussis toxin treatment of the cells completely blocked the ability of NPY to inhibit cyclic AMP production and adenylate cyclase activity. The toxin catalyzed the ADP-ribosylation of a 41-kDa protein in SK-N-MC cells that corresponds to Gi. The receptors on SK-N-MC cells appeared to be specific for NPY, as other neurotransmitter drugs, such as alpha-adrenergic, dopaminergic, muscarinic, and serotonergic antagonists, did not compete for either NPY binding or NPY inhibition of adenylate cyclase. Thus, SK-N-MC cells may be a useful model for investigating NPY receptors and NPY-mediated signal transduction.

Topics: Adenosine Diphosphate Ribose; Adenylate Cyclase Toxin; Adenylyl Cyclase Inhibitors; Adenylyl Cyclases; Binding, Competitive; Cholera Toxin; Colforsin; Cyclic AMP; Dopamine; Humans; Isoproterenol; Neuroblastoma; Neuropeptide Y; Peptide YY; Peptides; Pertussis Toxin; Receptors, Neuropeptide Y; Receptors, Neurotransmitter; Tumor Cells, Cultured; Vasoactive Intestinal Peptide; Virulence Factors, Bordetella

Calcitonin gene-related peptide (CGRP) stimulated cyclic adenosine monophosphate (cAMP) levels in SK-N-MC human neuroblastoma cells in a time- and concentration-dependent manner. The efficacy order for CGRPs was human alpha-CGRP = human beta-CGRP = chick CGRP greater than rat CGRP greater than human [Tyr0]CGRP. Calcitonin (CT) failed to influence cAMP production in SK-N-MC cells. [Tyr0]CGRP27-37 which by itself did not affect cAMP levels antagonized CGRP action. Saturation analysis using [125I]CGRP showed a homogeneous population of binding sites. CGRP but not CT, vasoactive intestinal peptide (VIP) and neuropeptide Y (NPY) inhibited radioligand binding. Our results provide evidence that human neuroblastoma SK-N-MC cells contain highly specific CGRP receptors which are positively coupled to cAMP generation.

Topics: Binding Sites; Binding, Competitive; Calcitonin Gene-Related Peptide; Cyclic AMP; Humans; Neuroblastoma; Neuropeptide Y; Receptors, Cell Surface; Tumor Cells, Cultured; Vasoactive Intestinal Peptide

Release of catecholamines, a Ca2(+)-dependent process, is the most useful biochemical marker in the diagnosis of neuroblastoma. Unfortunately, its stimulus is still unknown. We found that vasoactive intestinal polypeptide (VIP), in addition to acetylcholine and muscarine (but not nicotine), causes elevation of the cytoplasmic Ca2(+)-concentration in the highly differentiated human neuroblastoma cell line SK-N-SH, with or without the presence of extracellular Ca2+. Additionally, VIP was detected in SK-N-SH cells (0.65 ng/10(6) cells). Based on these observations and the fact that neuroblastoma is not innervated in vivo, we hypothesize that in this tumor VIP is responsible for Ca2(+)-dependent release of catecholamines in an autocrine or paracrine fashion.

Topics: Acetylcholine; Calcium; Cytoplasm; Humans; Muscarine; Neuroblastoma; Nicotine; Receptors, Cholinergic; Tumor Cells, Cultured; Vasoactive Intestinal Peptide

Neuroblastoma, a tumor of the sympathetic nervous system, is the most common solid malignancy of childhood outside the central nervous system. Vasoactive intestinal peptide (VIP) is produced by some of these tumors, and elevated serum levels correlate with tumor cell differentiation and a favorable prognosis. It has previously been demonstrated that human neuroblastoma cell lines LA-N-5 and IMR-32 will differentiate in vitro when exposed to retinoic acid. It is now shown that VIP also induces in vitro differentiation of these neuroblastoma lines. LA-N-5 or IMR-32 cells were grown in the presence of different concentrations of VIP. Cell proliferation was suppressed, as measured by cell count, incorporation of [3H]thymidine, and measurement of the proliferation index. The degree of suppression correlated with the concentration of VIP, and the effect was indistinguishable, on a molar basis, from that seen when cells were treated with retinoic acid. Similarly, the morphological changes seen in the VIP-treated cells were the same as those seen in retinoic acid-treated ones. The effects of VIP on both cell lines, like those of retinoic acid, are reversible. The human neuroepithelioma line CHP-100, is much less sensitive to either agent. Vasoactive intestinal peptide is the first substance shown to cause differentiation of neuroblastoma cells in vitro which is also known clinically to have a specific association with that tumor. It is postulated that VIP may play a key role in the well-documented maturation of these tumors in vivo and in the normal development of the sympathetic nervous system. These findings may also have therapeutic implications for the management of this frustrating childhood malignancy.

Topics: Breast Neoplasms; Cell Differentiation; Cell Division; Cell Line; Dose-Response Relationship, Drug; Female; Humans; Kinetics; Leukemia, Promyelocytic, Acute; Neuroblastoma; Neuroectodermal Tumors, Primitive, Peripheral; Tumor Cells, Cultured; Vasoactive Intestinal Peptide

Three phenotypically distinct subclones (SH-SY-5Y, SH-EP, SH-IN) of the human neuroblastoma cell line SK-N-SH were found to possess vasoactive intestinal polypeptide (VIP) precursor mRNA, release immunoreactive VIP, and express high-affinity VIP receptors coupled to adenylate cyclase. The apparent molecular mass for the receptor polypeptide, as determined by covalent cross-linking of 125I-VIP, was 49 kDa. After 2 days in culture, a concentration of immunoreactive VIP equivalent to the binding affinity of VIP to its receptor was found in the medium in two of these clones (SH-IN and SH-EP). Conditioned medium from SH-IN cells competitively displaced 125I-VIP binding and increased cAMP levels in SH-EP cells, indicating that all of the necessary components for a potential autocrine action of VIP exist in SK-N-SH cells. After numerous cell passages, the SH-EP subclone converted to a distinct phenotype in which VIP precursor mRNA and VIP immunoreactivity in the cell and medium were no longer detectable. In correlation, the VIP receptor number increased, and the EC50 for VIP stimulation of cAMP production shifted to a lower concentration. This points to the possibility that the continuous presence of endogenous VIP in earlier passage SH-EP cells causes a modification in VIP receptor number and cell responsiveness to VIP.

Topics: Blotting, Northern; Cyclic AMP; Humans; Neuroblastoma; Protein Precursors; Receptors, Gastrointestinal Hormone; Receptors, Vasoactive Intestinal Peptide; RNA, Messenger; RNA, Neoplasm; Tumor Cells, Cultured; Vasoactive Intestinal Peptide

Forskolin and vasoactive intestinal polypeptide (VIP) were shown to increase cyclic AMP accumulation in a human neuroblastoma cell line, SK-N-SH cells. The alpha 2-adrenergic agonist UK 14304 decreased forskolin-stimulated cyclic AMP levels by 40 +/- 2%, with an EC50 of 83 +/- 20 nM. This response was blocked by pretreatment with pertussis toxin (PT) (EC50 = 1 ng/ml) or by the alpha 2-antagonists yohimbine, idazoxan, and phentolamine. Antagonist IC50 values were 0.3 +/- 0.1, 2.2 +/- 0.3, and 1.4 +/- 0.1 microM, respectively. This finding suggests the presence of normal inhibitory coupling of SK-N-SH cell alpha 2-adrenergic receptors to adenylate cyclase via the inhibitory GTP-binding protein species, Gi. Muscarinic receptors in many target cell types are coupled to inhibition of adenylate cyclase. However, in SK-N-SH cells, muscarinic agonists synergistically increased (67-95%) the level of cyclic AMP accumulation elicited by forskolin or VIP. EC50 values for carbamylcholine (CCh) and oxotremorine facilitation of the forskolin response were 1.2 +/- 0.2 and 0.3 +/- 0.1 microM, respectively. Pharmacological studies using the muscarinic receptor subtype-preferring antagonists 4-diphenylacetoxy-N-methylpiperidine, pirenzepine, and AF-DX 116 indicated mediation of this response by the M3 subtype. IC50 values were 14 +/- 1, 16,857 +/- 757, and 148,043 +/- 16,209 nM, respectively. CCh-elicited responses were unaffected by PT pretreatment. Muscarinic agonist binding affinity was indirectly measured by the ability of CCh to compete for [3H]quinuclidinyl benzilate binding sites on SK-N-SH cell membranes.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Adenylate Cyclase Toxin; Cell Membrane; Cyclic AMP; Humans; Neuroblastoma; Pertussis Toxin; Receptors, Adrenergic, alpha; Receptors, Gastrointestinal Hormone; Receptors, Muscarinic; Receptors, Vasoactive Intestinal Peptide; Tumor Cells, Cultured; Vasoactive Intestinal Peptide; Virulence Factors, Bordetella

Retinoic acid (RA) induces partial differentiation of neuroblastoma (NB) cells in vitro. In the human NB line, SH-SY5Y (a neuroblastic subclone of SK-N-SH), RA was previously shown to enhance the stimulatory (PGE1) and inhibitory (opioid) regulation of adenylyl cyclase. Since these cells are also sensitive to cAMP stimulation by vasoactive intestinal peptide (VIP), we have tested the effects of RA on VIP receptor expression and function. Pretreatment of SH-SY5Y cells with 10 microM RA over 6 days dramatically increased VIP receptor number from approximately 3,000 to approximately 70,000 sites per cell and enhanced threefold the cAMP accumulation after external VIP addition, while VIP immunoreactive content in the cells increased 2-3-fold. In the light of the recently proposed autocrine function of VIP in this cell lineage, the strong enhancement of the VIP system may contribute to the differentiation effects of RA.

Topics: Cell Differentiation; Cyclic AMP; Humans; Neuroblastoma; Radioimmunoassay; Receptors, Gastrointestinal Hormone; Receptors, Vasoactive Intestinal Peptide; Tretinoin; Tumor Cells, Cultured; Vasoactive Intestinal Peptide

A 18-month-old boy with stage 4 neuroblastoma needed intensive care because of prerenal acute renal failure related to an intractable watery diarrhoea syndrome occurring 10 months after the diagnosis of the primary tumor. This diarrhoea was in relation with a late hyperproduction of vasoactive intestinal peptide by the relapsing neuroblastoma itself and stopped with intravenous somatostatin administration.

Topics: Diarrhea, Infantile; Humans; Infant; Male; Neoplasm Invasiveness; Neoplasm Recurrence, Local; Neuroblastoma; Retroperitoneal Neoplasms; Vasoactive Intestinal Peptide

The expression of a transfected plasmid containing 5.2 kilobases (kb) of 5' regulatory DNA sequence of the human vasoactive intestinal peptide (VIP) gene attached to coding sequences of the reporter gene chloramphenicol acetyltransferase (CAT) was compared with endogenous VIP expression in subclones of the human neuroblastoma cell line SK-N-SH. These subclones vary widely in basal and inducible quantities of VIP and its precursor mRNA and can be interconverted under specified culture conditions. Endogenous VIP immunoreactivity, detectable in all subclones, was lowest in the neuronal subclone SH-SY-5Y, whereas 15- to 25-fold higher levels were observed in the epithelial-appearing SH-EP and intermediate SH-IN subclones. Treatment with 10 nM phorbol 12-myristate 13-acetate (PMA) stimulated VIP peptide levels approximately 5-fold in SH-SY-5Y cells but did not increase appreciably VIP levels in the other subclones. Treatment with 2.5 microM forskolin resulted in less than 50% stimulation of VIP expression in all subclones. Levels of mRNA encoding the VIP precursor generally paralleled these differences in VIP immunoreactivity. In cells transfected with the VIP/CAT fusion gene, CAT activity reflected closely these differences in basal VIP expression and the changes in response to PMA and forskolin. Deletion of 2.7 kb of the most upstream sequences resulted in an 80-90% reduction in basal CAT activity in SH-IN, but not SH-SY-5Y cells, and resulted in an 80% reduction in PMA stimulation in SH-SY-5Y cells. Deletion to within 74 nucleotides of the transcription start site resulted in CAT expression in SH-IN cells that was only 3% of that seen with the full 5.2-kb flanking sequences and further diminished the remaining PMA responsiveness in SH-SY-5Y cells. The data indicate that important cell-type-specific transcription regulatory sequences reside greater than 2.5 kb upstream from the VIP transcription start site.

Topics: Base Sequence; Cell Line; Chromosome Deletion; Colforsin; Enhancer Elements, Genetic; Humans; Molecular Sequence Data; Molecular Weight; Neuroblastoma; Plasmids; Tetradecanoylphorbol Acetate; Transfection; Vasoactive Intestinal Peptide

Neurotransmitter receptors coupling to the adenylate cyclase (AC) system were studied in the human neuroblastoma SH-SY5Y cell line. Vasoactive intestinal polypeptide (VIP) caused an up to 40-fold enhancement of the AC activity, while prostaglandin E1 (PGE1) was able to increase the cAMP accumulation 2.5-fold. Stimulation either by VIP or PGE1 was attenuated with either morphine (MOR) or oxotremorine (OXO). Prolonged exposure to MOR and OXO caused a ligand-specific, i.e. homologous desensitization of the opioid and muscarinic acetylcholine receptors, respectively. Preceding desensitization, a supersensitive response of the AC system to VIP was observed. Pretreatment of cells with PT overnight reduced the inhibitory effects of both MOR and OXO. Nevertheless, in cells pretreated with PT and then also with OXO, MOR and OXO inhibited the VIP-induced AC response. Apparently, there are both PT-sensitive and -insensitive pathways to AC inhibition in SH-SY5Y cells.

Topics: Adenylyl Cyclases; Cell Line; Humans; Morphine; Neuroblastoma; Oxotremorine; Prostaglandins E; Signal Transduction; Time Factors; Tumor Cells, Cultured; Vasoactive Intestinal Peptide

Using regional specific antisera the concentrations of vasoactive intestinal polypeptide (VIP) and the peptide with N-terminal histidine and C-terminal isoleucine (PHI) in various peripheral tissues and VIP producing tumours were compared with their immunohistochemical localization. In normal tissue the VIP levels were in general higher than the PHI levels, while the VIP/PHI ratio in tumour tissue varied considerably more than in normal tissue. By immunohistochemistry it was found that VIP and PHI immunoreactivity occurred in the same autonomic neurons. Gel chromatography revealed that VIP and PHI immunoreactivity in both normal and tumour tissue consisted of two larger molecular forms in addition to "authentic" peptides. These larger molecular forms which had overlapping elution positions probably represent VIP/PHI precursors. In tumour tissue the larger molecular forms constituted a larger proportion of the total immunoreactivity. Neurally induced relaxation of smooth muscle caused a simultaneous release of VIP and PHI which in combination with the observed relaxatory effect of the peptide suggest a role in the control of smooth muscle activity. Similarly VIP and PHI were co-secreted from tumour tissues as evidenced from elevated plasma levels in patients with VIP producing tumours. In conclusion VIP and PHI seem to co-exist and be co-secreted. Differences in posttranslational processing may create variable content and release of the two peptides.

Topics: Animals; Cats; Digestive System; Ganglioneuroma; Humans; Neuroblastoma; Pancreatic Neoplasms; Peptide PHI; Reference Values; Species Specificity; Swine; Tissue Distribution; Vasoactive Intestinal Peptide

Vasoactive intestinal peptide (VIP) and secretin are two related peptides that activate adenylate cyclase on membranes of striatal neurons and glial cells from embryonic mouse brain grown in primary culture. On the two cell types, the maximal activation that could be induced by secretin was only 40% above basal activity, which represented less than 15% of the maximal effect obtainable with VIP. From competition experiments performed on glial cells and the neuroblastoma X glioma hybrid, NG 108-15, a cell line known to possess both VIP and secretin sensitive-adenylate cyclase, we demonstrate that secretin does not activate VIP receptors. Furthermore, secretin has an apparent high affinity (EC50 10(-8) M) for its receptors on striatal neurons and NG 108-15 whereas an apparent low affinity (EC50 7 X 10(-6) M) was found on striatal glial cells. This suggests the existence of either two distinct secretin receptors or a desensitized form.

Topics: Adenylyl Cyclases; Animals; Cell Line; Corpus Striatum; Cyclic AMP; Embryo, Mammalian; Enzyme Activation; Glioma; Hybrid Cells; Mice; Neuroblastoma; Neuroglia; Neurons; Receptors, Cell Surface; Receptors, G-Protein-Coupled; Receptors, Gastrointestinal Hormone; Receptors, Vasoactive Intestinal Peptide; Secretin; Vasoactive Intestinal Peptide

The immunoreactivity of VIP and PHI standards, immobilized on a nitrocellulose membrane, was first assayed with various detection procedures. For VIP, the double bridge peroxidase-antiperoxidase (PAP) method was the most sensitive procedure, giving a detection limit of 0.1-0.3 pmol per mm2 with the 4 rabbit anti-VIP antisera tested. By contrast, the detection limit of immobilized PHI was 100 times higher with the 4 rabbit anti-PHI/PHM antisera tested presumably because major antigenic sites were masked in the immobilized peptide. With this information at hand, the VIP and PHI immunoreactivity of human neuroblastoma NB-OK-1 cells was tested after extraction, SDS-PAGE, electrotransfer, and PAP immunodetection. Two faint immunoreactive bands corresponding to two pro-VIP forms with an Mr of, respectively, 19 kDa and 18 kDa, were detected in undifferentiated cells. These distinct bands increased progressively and markedly during differentiation in the presence of dibutyryl cyclic AMP. In addition, two intermediary VIP forms of lower Mr (11 kDa and 6 kDa) and 3 kDa VIP itself were also present after 2 days of differentiation. The 19 kDa and 18 kDa pro-VIP forms were detected with a sensitivity several times higher than that of VIP and their staining was specific for VIP epitopes. By contrast, when using 4 rabbit anti-PHI/PHM antisera, we observed essentially the strong unspecific staining of a 17 kDa polypeptide. VIP immunoreactivity was also visualized by immunocytochemistry in neuroblastoma cells cultured on glass coverslips and fixed in situ. Specific VIP staining using the PAP method was present in 10 percent of the cells in the undifferentiated state.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Bucladesine; Cell Line; Golgi Apparatus; Histocytochemistry; Humans; Immunochemistry; Immunoenzyme Techniques; Neuroblastoma; Peptide PHI; Peptides; Protein Precursors; Vasoactive Intestinal Peptide

The effects of dibutyryl cAMP (Bt2cAMP) and phorbol esters such as 12-O-tetradecanoylphorbol-13-acetate (TPA) and phorbol-12,13-didecanoate (PDD) on VIP/PHM-27 gene expression in human neuroblastoma cells in culture were investigated. Bt2cAMP and phorbol esters increased the VIP/PHM-27 mRNA level by about 9- and 4-fold, respectively. In the presence of both Bt2cAMP and phorbol esters, the VIP/PHM-27 mRNA level increased by about 36-fold. The intracellular cAMP level was essentially unaffected by phorbol esters. The VIP/PHM-27 gene dosage was unchanged by Bt2cAMP and phorbol esters. The results suggest that cAMP and phorbol esters synergistically induce the VIP/PHM-27 gene expression through independent pathways.

Topics: 1-Methyl-3-isobutylxanthine; Bucladesine; Cells, Cultured; Cyclic AMP; Drug Synergism; Gene Expression Regulation; Humans; Neuroblastoma; Phorbol Esters; RNA, Messenger; Vasoactive Intestinal Peptide

The mechanism of N6,O2'-dibutyryl adenosine 3':5'-monophosphate (Bt2cAMP) induction of pro-vasoactive intestinal polypeptide (VIP)/PHM-27 biosynthesis was investigated in human neuroblastoma cells in culture. When neuroblastoma cells were grown for 48 h in the presence of 1 mM Bt2cAMP, the synthesis of pro-VIP/PHM-27 was stimulated 11-fold. The amount of prepro-VIP/PHM-27 mRNA determined both by hybridization with cloned prepro-VIP/PHM-27 cDNA and a reticulocyte cell-free translation assay was also increased 11-fold in the Bt2cAMP-induced cells. Transcription of prepro-VIP/PHM-27 mRNA in isolated nuclei was observed in induced cells, but not in uninduced cells. Blot hybridization with prepro-VIP/PHM-27 cDNA of total nuclear RNA isolated from neuroblastoma cells revealed an RNA species corresponding to mature prepro-VIP/PHM-27 mRNA, and the amount of the RNA was markedly increased in the induced cells. The quantity of VIP/PHM-27 gene in the DNA of neuroblastoma cells was analyzed after hydrolysis with a restriction endonuclease, EcoRI. However, VIP/PHM-27 gene was not amplified in the induced cells. These results indicate that Bt2cAMP-induced pro-VIP/PHM-27 synthesis is achieved by enhancing the transcription rate of prepro-VIP/PHM-27 mRNA.

Topics: Animals; Bucladesine; Cell Line; Cell Nucleus; Cyclic AMP; DNA; DNA Restriction Enzymes; DNA Transposable Elements; Humans; Kinetics; Neuroblastoma; Nucleic Acid Hybridization; Peptide PHI; Plasmids; Protein Biosynthesis; Protein Precursors; Rabbits; Reticulocytes; RNA, Messenger; Transcription, Genetic; Vasoactive Intestinal Peptide

Adenylate cyclase in plasma membranes was inhibited by micromolar concentrations of delta 8-tetrahydrocannabinol and delta 9-tetrahydrocannabinol and by levonantradol and desacetyllevonantradol. This inhibition was noncompetitive for stimulation of the enzyme at the prostanoid receptor by prostaglandin E1 or prostacyclin, or at the peptide receptor by secretin or vasoactive intestinal peptide. Forskolin-activated adenylate cyclase was also inhibited by cannabimimetic agents. Inhibition by cannabinoid compounds was neither synergistic nor additive with muscarinic or alpha-adrenergic agents when each was present at maximally inhibitory concentrations. Cannabinoid inhibition was not blocked by atropine, yohimbine, or naloxone, suggesting that muscarinic, alpha 2-adrenergic and certain opiate receptors may not be required for the response. The inhibition of adenylate cyclase was specific for psychoactive cannabinoids, since cannabinol and cannabidiol produced minimal or no response. Inhibition was also stereoselective, since dextronantradol did not produce the response. A biphasic log dose-response curve was observed for each of the cannabinoid drugs, such that reversal of the inhibition occurred at 3-10 microM. Possible mechanisms for the effects of cannabinoid drugs on adenylate cyclase activity are discussed.

Topics: Adenylyl Cyclase Inhibitors; Animals; Cannabinoids; Cell Membrane; Colforsin; Diterpenes; Dronabinol; Isomerism; Kinetics; Mice; Neuroblastoma; Phenanthridines; Prostaglandins; Secretin; Vasoactive Intestinal Peptide

Secretin, a gut-brain peptide, elicited cyclic AMP production in a clone of neuroblastoma cells derived from the C1300 mouse tumor. Adenylate cyclase (EC 4.6.1.1) in plasma membranes from these cells was stimulated by secretin greater than vasoactive intestinal peptide greater than peptide histidine isoleucine amide, but not by the related peptides glucagon, gastric inhibitory polypeptide, or human growth hormone releasing factor. Hill coefficients for stimulation approximated one and the response to submaximal peptide concentrations was additive, as expected for hormones competing for a single receptor associated with the enzyme. Binding of 125I-labeled secretin to the neuroblastoma plasma membranes was saturable, time-dependent, and reversible. The KD determined from kinetic and equilibrium binding studies approximated 1 nM. The binding site displayed marked ligand specificity that paralleled that for stimulation of adenylate cyclase. The secretin receptor was regulated by guanine nucleotides, with guanosine 5'-(beta, gamma-imino)-triphosphate being the most potent to accelerate the rate of dissociation of bound secretin. These findings demonstrate the functional association of the secretin receptor with adenylate cyclase in neuronally derived cells.

Topics: Adenylyl Cyclases; Animals; Binding, Competitive; Cell Line; Cell Membrane; Guanosine Diphosphate; Guanosine Triphosphate; Guanylyl Imidodiphosphate; Neuroblastoma; Receptors, Cell Surface; Receptors, G-Protein-Coupled; Receptors, Gastrointestinal Hormone; Secretin; Vasoactive Intestinal Peptide

Extracts of neuroblastoma X glioma hybrid cells 108CC15 and their parental lines were investigated for the presence of vasoactive intestinal polypeptide (VIP). With the aid of a radioimmunoassay and a receptor binding assay, VIP activity was found in the hybrid cells, to a lesser extent in neuroblastoma cells, but not in glioma cells. These results suggest a neuronal function of VIP. Since the hybrid cells also contain acetylcholine and opioids, they may be useful in studies of co-release of neurohormones.

Topics: Animals; Cell Line; Cells, Cultured; Gastrointestinal Hormones; Glioma; Hybrid Cells; Mice; Neoplasms, Experimental; Neuroblastoma; Rats; Vasoactive Intestinal Peptide

The concentration of vasoactive intestinal polypeptide (VIP) in plasma was measured in 22 children with neural crest tumours (NCT) during a 5-year period; the mean concentration of VIP in plasma was 22.5 pmol/l (interval 2.0-95.0 pmol/l). To establish a reference interval the plasma concentration of VIP was measured in 41 children without tumours; the mean concentration of VIP in plasma was 6.2 pmol/l (interval 0.5-19.0 pmol/l). Of the 22 children with NCT 16 had a plasma concentration of VIP within the normal range while 6 children (27%) had elevated plasma concentration of VIP between 28 and 95 pmol/l. Only one child, whose plasma concentration of VIP was 95 pmol/l had diarrhoea. Elevated plasma concentration of VIP in children with NCT but no diarrhoea has not previously been described. The urinary excretion of vanillylmandelic acid (VMA) was increased in 18 of the children with NCT (82%). In 2 of the children with normal excretion of VMA the concentration of VIP in plasma was elevated. Thus, the plasma concentration of VIP may be a supplement to VMA as a tumour marker in some cases of NCT.

Topics: Child; Child, Preschool; Diarrhea; Female; Ganglioneuroma; Gastrointestinal Hormones; Humans; Infant; Male; Neoplasms, Germ Cell and Embryonal; Neuroblastoma; Reference Values; Vasoactive Intestinal Peptide

A 1 1/2 year old child developed profuse watery diarrhoea, shown to be due to excessive plasma vasoactive intestinal peptide (VIP) levels, whilst on treatment for metastatic neuroblastoma. Because it was unresponsive to alternative treatment, an attempt was made to control the diarrhoea with a somatostatin infusion. The attempt failed despite the fact that serum VIP levels were substantially reduced. Possible reasons for failure are discussed and the importance of plasma VIP as a marker for maturation in neuroblastoma emphasised.

Topics: Diarrhea; Female; Gastrointestinal Hormones; Humans; Infant; Neoplasm Metastasis; Neuroblastoma; Somatostatin; Vasoactive Intestinal Peptide

Topics: Bucladesine; Cell Line; Cell-Free System; Chemical Precipitation; Gastrointestinal Hormones; Humans; Immunologic Techniques; Neuroblastoma; Poly A; Protein Biosynthesis; Protein Precursors; RNA; RNA, Messenger; Vasoactive Intestinal Peptide

Plasma regulatory peptide levels were studied in a group of 21 children with neurogenic tumours and in 22 control children. Plasma vasoactive intestinal polypeptide (VIP) levels were significantly higher in children with neurogenic tumours than in normal children or those with other tumours (p less than 0.05). There was no significant difference between the groups in plasma levels of gastrin, pancreatic glucagon or pancreatic polypeptide. The plasma VIP level may thus be a helpful diagnostic marker for neurogenic tumours in children.

Topics: Adolescent; Child; Child, Preschool; Gastrins; Gastrointestinal Hormones; Glucagon; Humans; Infant; Neuroblastoma; Pancreatic Polypeptide; Vasoactive Intestinal Peptide

The presence of VIP-like immunoreactivity in human neuroblastoma cell line NB-1 was demonstrated by radioimmunoassay of the crude cell extract and by immunocytochemical staining of the cells. Gel filtration profiles of VIP-like immunoreactivity in the cell extract measured by radioimmunoassays using three different region-specific antisera revealed that the immunoreactivity consists of a major molecular form corresponding to porcine VIP having 28 amino acid residues with at least two additional minor forms larger and smaller than the VIP. In addition to VIP-like immunoreactivity, the cell extract was shown to contain substance P-, neurotensin- and somatostatin-like immunoreactivity as well.

Topics: Animals; Cell Line; Female; Gastrointestinal Hormones; Humans; Neuroblastoma; Neurotensin; Somatostatin; Substance P; Swine; Vasoactive Intestinal Peptide

Immunohistochemical studies have demonstrated that immunoreactive vasoactive intestinal peptide is present in, and restricted to, the differentiating and mature ganglion cells in a variety of normal and neoplastic neural tissues. In a composite pheochromocytoma-ganglioneuroma (associated with the syndrome of watery diarrhea, hypokalemia, and hypochlorhydria), five ganglioneuroblastomas, five ganglioneuromas (two of which were associated with diarrheal syndromes), an unusual mixed neuroblastoma-ganglioneuroma, and four normal sympathetic ganglia, vasoactive intestinal peptide was present in differentiating and mature ganglion cells. The peptide was also demonstrated in isolated ganglion cells in two pheochromocytomas but was not present in pheochromocytes, Schwann cells, or undifferentiated neuroblastic cells in the neuroblastomas and ganglioneuroblastomas. These studies indicate that the presence and presumably the production of vasoactive intestinal peptide thus reflect a particular line of neuroblastic differentiation and are not merely a reflection of common derivation of these tissues. Our identification of vasoactive intestinal peptide in neurogenic tumors associated with diarrhea supports the contention that the peptide might be an important diarrheogenic factor in these tumors.

Topics: Adrenal Gland Neoplasms; Cell Differentiation; Ganglia, Autonomic; Ganglioneuroma; Gastrointestinal Hormones; Humans; Neoplasms, Nerve Tissue; Neuroblastoma; Pheochromocytoma; Vasoactive Intestinal Peptide

Topics: Cell Line; Cells, Cultured; Drug Resistance; Floxuridine; Glioma; Humans; Mercaptopurine; Morphine Dependence; Neoplasms, Experimental; Nervous System Diseases; Neuroblastoma; Neurophysiology; Thioguanine; Vasoactive Intestinal Peptide

Topics: Catecholamines; Humans; Hypertension; Neuroblastoma; Paraganglioma, Extra-Adrenal; Pheochromocytoma; Prostaglandins; Vasoactive Intestinal Peptide