atrial-natriuretic-factor and Neuroblastoma

To identify neural tumor cell lines that could be used as models to study growth-related natriuretic peptide actions, we determined the effects of these peptides on the proliferation of human and rodent neuroblastoma cell lines. Subnanomolar concentrations of atrial natriuretic peptide (ANP) and type C natriuretic peptide (CNP) stimulated proliferation in all four cell lines. These actions were associated with cGMP elevation and were blocked by a protein kinase G inhibitor. These data imply the involvement of guanylyl cyclase (GC)-coupled natriuretic receptors. However, higher concentrations of ANP and CNP, and low concentrations of des-[Gln(18),Ser(19),Gly(20),Leu(21),Gly(22)]-ANP(4-23)-NH(2) (desANP(4-23)) (analog for NPR-C receptor) exerted antiproliferative actions in three of the cell lines. These effects were insensitive to a protein kinase G inhibitor and to HS-142-1, suggesting that growth-inhibitory actions involved a non-GC receptor. They did not appear to involve cAMP, protein kinase A, protein kinase C, or calcium mobilization but were abolished when constitutive mitogen-activated protein kinase activity was inhibited. Radioligand binding experiments revealed the presence of a uniform class of binding sites in NG108 cells and multiple binding sites in Neuro2a cells. Northern and reverse transcriptase-polymerase chain reaction analyses revealed differential gene expression for NPR-A/B/C in NG108 and Neuro2a cells. The results indicate that natriuretic peptides stimulate neuroblastoma cell proliferation through type NPR-A/B (GC) receptors. Higher concentrations of ANP and CNP exerted a mitogen-activated protein kinase-dependent antiproliferative action mediated by a non-GC receptor that interacts with desANP(4-23) with relatively high affinity.

Topics: Atrial Natriuretic Factor; Base Sequence; Cell Division; Guanylate Cyclase; Molecular Sequence Data; Neuroblastoma; Receptors, Atrial Natriuretic Factor; Reverse Transcriptase Polymerase Chain Reaction; Tumor Cells, Cultured

The aim of this study was to evaluate plasma levels of ANF in patients with catecholamine-secreting tumors with and without hypertension and to relate ANF secretion to levels of plasma and urinary catecholamines and blood pressure. Twenty-one pheochromocytoma (15 with sustained, 6 with paroxysmal hypertension), 6 neuroblastoma (1 hypertensive) patients and 28 aged-matched controls were studied in basal conditions. Plasma and urinary norepinephrine (NE),epinephrine (E), dopamine (DA) and DOPA were determined by HPLC-ED and plasma ANF by RIA. Both neuroblastoma and pheochromocytoma patients had significantly higher plasma ANF levels than controls. Neuroblastomas showed higher ANF concentration than pheochromocytomas. No differences were found in plasma ANF between hypertensive and normotensive patients. Pheochromocytomas with ANF levels within the normal range had plasma and urinary NE and urinary DA and DOPA levels significantly higher than patients with high ANF. Plasma ANF levels were unrelated to systolic or diastolic blood pressure or heart rate. A negative correlation between plasma ANF and urinary DA was found only in the patients groups. In conclusion, plasma ANF was increased in pheochromocytoma and neuroblastoma patients. Our data suggest that the excessive catecholamine secretion is not responsible for the increased ANF secretion in these patients. The significance of the relationships among plasma ANF and urinary and plasma catecholamines requires further investigation.

Topics: Abdominal Neoplasms; Adolescent; Adrenal Gland Neoplasms; Adult; Aged; Atrial Natriuretic Factor; Biomarkers, Tumor; Blood Pressure; Catecholamines; Child; Child, Preschool; Female; Humans; Hypertension; Male; Middle Aged; Multiple Endocrine Neoplasia; Neoplasm Staging; Neuroblastoma; Pheochromocytoma; Urinary Bladder Neoplasms

Functional natriuretic peptide receptors of type A (NPR-A) were detected in the human neuroblastoma NB-OK-1, SK-N-SH and SK-N-BE, but not the SH-SY5Y, cell lines. Also, NPR-A mRNA was detected in 19 of the 25 tumor neuroblastoma samples tested in this study. Five of the eight tumor neuroblastoma samples that were assayed for atrial natriuretic peptide (ANP) binding revealed the presence of ANP-binding sites. In the human neuroblastoma NB-OK-1 cell line, [(3)H] thymidine incorporation was increased in response to ANP, decreased in response to pituitary adenylate cyclase-activating polypeptide (PACAP-27), and the stimulatory effect of ANP was inhibited by PACAP-27. Tissue transglutaminase activity was decreased by ANP and PACAP-27, and their effects were additive. However, neither cell cycle phases, cell growth, or cell apoptosis were modified by ANP or PACAP-27 treatments.

Topics: Apoptosis; Atrial Natriuretic Factor; Brain Neoplasms; Cell Division; Child; Child, Preschool; Enzyme Activation; Female; Flow Cytometry; Gene Expression Regulation, Neoplastic; Guanylate Cyclase; Humans; Infant; Male; Neuroblastoma; Neuropeptides; Neurotransmitter Agents; Pituitary Adenylate Cyclase-Activating Polypeptide; Receptors, Atrial Natriuretic Factor; RNA, Messenger; RNA, Neoplasm; Thymidine; Transglutaminases; Tritium; Tumor Cells, Cultured

Cyclic nucleotides levels and cyclic nucleotide phosphodiesterase (PDE) activities were measured in human neuroblastoma NB-OK-1 cells possessing atrial natriuretic peptide (ANP) receptors of the A type and pituitary adenylate cyclase activating polypeptide (PACAP)-preferring receptors. Adenosine 3',5'-cyclic monophosphate (cAMP) and guanosine 3',5'-cyclic monophosphate (cGMP) degradation were interrelated since the increase in cGMP, induced by ANP-(99-126), stimulated the hydrolysis of cAMP by PDE isoenzyme II. In intact NB-OK-1 cells, the levels of cAMP and cGMP attained in the presence of, respectively, 1 nM PACAP-(1-27) and 10 nM ANP-(99-126), and in the absence or presence of PDE inhibitors, strongly suggested that cAMP hydrolysis was mainly achieved by isoenzyme IV, and to a lesser extent by isoenzymes I, II, and III, while cGMP was degraded by isoenzymes I, II, III, and V. More than one-half of total cAMP- and cGMP-hydrolyzing activities was present in the membrane-bound fraction. Cyclic nucleotide PDE activities separated by anion-exchange chromatography showed that isoenzymes III and IV were mainly present in the membrane fraction, while isoenzymes I, II, and V were in the cytosolic fraction.

Topics: Atrial Natriuretic Factor; Cell Membrane; Chromatography, Ion Exchange; Cyclic AMP; Cyclic GMP; Cytosol; Exonucleases; Humans; Neuroblastoma; Peptide Fragments; Tumor Cells, Cultured

The occupancy of atrial natriuretic peptide (ANP) receptors of the ANPA type in human neuroblastoma NB-OK-1 cells elevates cGMP. In this study, ANP concentrations of 10 nM or more increased total K+ uptake. Data obtained in the presence of bumetanide and/or ouabain demonstrated that 1 microM ANP induced a primary stimulation (by 82%) of Na-K-Cl cotransport and a subsequent indirect stimulation (by 15%) of Na,K-ATPase. ANP also inhibited Na/H exchange through an amiloride-sensitive mechanism, as shown by intracellular pH measurement in cells challenged or not by an acid or alkaline load. (Bu)2cGMP mimicked all ANP effects, suggesting that ANP acted through a cGMP-dependent mechanism.

Topics: Amiloride; Atrial Natriuretic Factor; Bucladesine; Carrier Proteins; Chlorides; Humans; Hydrogen; Hydrogen-Ion Concentration; Neuroblastoma; Potassium; Sodium; Sodium-Hydrogen Exchangers; Sodium-Potassium-Chloride Symporters; Sodium-Potassium-Exchanging ATPase; Tumor Cells, Cultured

HS-142-1 is a novel non-peptide antagonist for atrial natriuretic peptide (ANP) receptor. The effect of HS-142-1 on the cyclic GMP production elicited by natriuretic peptides in neuronal cell lines, PC12 and NG108-15 was examined. Natriuretic peptides such as ANP, brain natriuretic peptide (BNP), and C-type natriuretic peptide (CNP) enhanced cyclic GMP production in a dose-dependent manner. HS-142-1 inhibited cyclic GMP accumulation elicited by natriuretic peptides in a dose-dependent fashion in both cells. The results suggest that HS-142-1 will be an important tool for identification and understanding of the mechanisms by which natriuretic peptides act in nervous systems.

Topics: Animals; Atrial Natriuretic Factor; Cell Line; Cyclic GMP; Dose-Response Relationship, Drug; Glioma; Hybrid Cells; Kinetics; Mice; Neuroblastoma; PC12 Cells; Polysaccharides; Rats

ATP dose-dependently inhibited rat 125I-ANP-(99-126) binding to membranes from the human neuroblastoma cell line NB-OK-1 by increasing the KD value for the hormone without altering the Bmax value. After a 20 min preincubation with 37.5 pM 125I-ANP-(99-126) and 0.5 mM ATP, followed by the addition of 0.3 microM unlabelled ANP-(99-126), the proportion of rapidly dissociating receptors was 4-times higher than in the absence of ATP. The other nucleotides ADP, AMP, AMP-PNP, ATP gamma S, GTP, GDP, GMP, GMP-PNP and GTP gamma S were also inhibitory but with a lower potency and/or efficacy. Binding equilibrium data were satisfactorily simulated by a computer program based on partially competitive binding of ANP-(99-126) and the nucleotides, and this, together with the data on dissociation kinetics, strongly suggests that several nucleotides, when added at concentrations up to 1 mM, form a ternary ANP-receptor-nucleotide complex.

Topics: Adenosine Monophosphate; Adenosine Triphosphate; Animals; Atrial Natriuretic Factor; Binding, Competitive; Cell Membrane; Humans; Kinetics; Neuroblastoma; Nucleotides; Peptide Fragments; Rats; Receptors, Atrial Natriuretic Factor; Receptors, Cell Surface; Tumor Cells, Cultured

The effects of seven competitive atrial natriuretic peptide (ANP) receptor antagonists were compared on cultured human neuroblastoma NB-OK-1 cells expressing exclusively ANPA receptors, by evaluating their capacity to inhibit [125I]ANP binding and to suppress ANP-stimulated cyclic GMP elevation. In ANP analogues with a shortened Cys7-Cys18 bridge, Asp13 and a hydrophobic Tic residue at position 16 expressed antagonistic activity, while Ala16 provoked lower antagonistic potency and Phe16 induced receptor activation. The binding affinity of A71915 ([Arg6, Cha8]ANP-(6-15)-D-Tic-Arg-Cys-NH2), the most potent antagonist (with a pKi of 9.18 and a pA2 of 9.48) was only 22 times less lower than that of the agonist ANP-(1-28).

Topics: Atrial Natriuretic Factor; Binding Sites; Cyclic GMP; Humans; Neuroblastoma; Neurons; Peptide Fragments; Receptors, Atrial Natriuretic Factor; Tetrahydroisoquinolines; Tumor Cells, Cultured

ANP-R1 receptors for atrial natriuretic peptide (ANP) showed the following rank order of affinity in intact human neuroblastoma cells NB-OK-1: human ANP-(99-126) approximately human ANP-(102-126) approximately rat ANP-(99-126) (K1 17-32 pM) > human ANP-(103-126) > porcine brain natriuretic peptide (BNP). Analogues truncated at the C-terminal extremity or devoid of a disulphide bridge, such as rat ANP-(103-123), rat C-ANP-(102-121), rat ANP-(111-126), rat ANP-(99-109) and rat [desCys105,Cys121]ANP-(104-126) and chicken C-type natriuretic peptide, were not recognized. The occupancy of these high affinity ANP-R1 receptors led to marked cyclic GMP accumulation in the presence of 3-isobutyl 1-methylxanthine. An ectoenzymic activity, partly shed in the incubation medium, provoked the stepwise release of Phe-Arg-[125I]Tyr, Arg-[125I]Tyr and [125I]Tyr from rat [125I]ANP-(99-126), at an optimal pH of 7.0. Its inhibition by 1,10-phenanthroline, EDTA and bacitracin but not by thiorphan suggests the contribution of at least one neutral metalloendopeptidase, distinct from EC 3.4.24.11, for which ANP showed high affinity.

Topics: Animals; Atrial Natriuretic Factor; Cell Survival; Chickens; Chromatography, High Pressure Liquid; Cyclic GMP; Dose-Response Relationship, Drug; Humans; Hydrogen-Ion Concentration; Iodine Radioisotopes; L-Lactate Dehydrogenase; Metalloendopeptidases; Neuroblastoma; Peptide Fragments; Rats; Receptors, Atrial Natriuretic Factor; Swine; Tumor Cells, Cultured

A novel metallo-endopeptidase from human neuroblastoma NB-OK-1 cells was partially purified and characterized. This enzyme activity was detected in the culture medium and could be detached from intact cells by gentle washing, suggesting a peripheral localization of the enzyme. This endopeptidase inactivated Atrial Natriuretic Peptide (ANP) by a unique and selective cleavage of the Ser123-Phe124 bond. It also produced hydrolysis at the Xaa-Phe, Xaa-Leu, or Xaa-Ile bonds of other peptide hormones such as bradykinin, somatostatin 14, litorin, substance P, neuromedin C and angiotensin II. The substrate selectivity and inhibition profile of the enzyme showed obvious similarities with the peptide hormone inactivating endopeptidase (PHIE) recently purified from Xenopus laevis skin secretions and indicated a thermolysin-like activity distinct from neutral endopeptidase (EC 3.4.24.11) and from angiotensin converting enzyme (EC 3.4.15.1).

Topics: Amino Acid Sequence; Animals; Atrial Natriuretic Factor; Cell Line; Humans; Kinetics; Metalloendopeptidases; Molecular Sequence Data; Neuroblastoma; Neuropeptides; Oligopeptides; Peptide Fragments; Phenylalanine; Protease Inhibitors; Sequence Homology, Nucleic Acid; Serine; Xenopus laevis

Characterization of the serotonin (5-HT)-induced cyclic GMP (cGMP) elevation was investigated in comparison with bradykinin- and ANP-induced elevations in NG108-15 cells. At 20 s, 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid tetraacetoxymethyl ester (BAPTA-AM, 100 microM), a membrane-permeabilized Ca2+ chelator, or N-monomethyl-L-arginine (NMMA, 300 microM), an inhibitor of L-arginine-derived nitric oxide (NO) synthesis, inhibited 5-HT-induced elevation by approximately 40%, and completely inhibited bradykinin-induced response. Neither 5-HT- nor ANP-induced cGMP elevation at 10 min was affected by BAPTA-AM or NMMA. The cGMP elevated by 5-HT as well as by ANP was effluxed to the extracellular medium. These results and our previous report suggest that 5-HT stimulates two subtypes of 5-HT receptors in NG108-15: first, 5-HT3 subtype stimulating Ca(2+)-sensitive cytosolic guanylate cyclase through NO derived from L-arginine and second, a probably novel 5-HT receptor subtype involved in activation of membrane-bound guanylate cyclase.

Topics: Animals; Atrial Natriuretic Factor; Bradykinin; Cell Line; Cell Membrane; Chelating Agents; Cyclic GMP; Egtazic Acid; Glioma; Guanylate Cyclase; Hybrid Cells; Kinetics; Neuroblastoma; Receptors, Serotonin; Serotonin

We characterized in membranes from the human neuroblastoma cell line NB-OK-1, an ANP-R1 receptor (Mr 130 kDa) for the atrial natriuretic peptide (ANP). This receptor recognized biologically active forms of ANP with high affinity but showed no affinity for truncated ANP forms. It was functional in that binding correlated with guanylate cyclase activation (a 2-fold increase in Vmax) with the following rank order of potency: rat ANP-(99-126) greater than human ANP-(99-126) greater than human ANP-(102-126) greater than porcine BNP (brain natriuretic peptide). The enzyme required free Mn2+ in addition to the Mn-GTP substrate (Km of about 0.3 mM for both basal and ANP-stimulated activity). In the presence of dithiothreitol, the dose-response curve of guanylate cyclase activation was shifted rightward by a factor of 30. ANP-R1 receptors were upregulated through protein synthesis in cells exposed to 1 mM carbamylcholine or 1 mM dibutyryl cyclic AMP for 8-24 h (ANP was ineffective).

Topics: Animals; Atrial Natriuretic Factor; Cross-Linking Reagents; Guanylate Cyclase; Humans; Iodine Radioisotopes; Kinetics; Manganese; Natriuretic Peptide, Brain; Neoplasm Proteins; Nerve Tissue Proteins; Nervous System Neoplasms; Neuroblastoma; Peptide Fragments; Rats; Receptors, Atrial Natriuretic Factor; Receptors, Cell Surface; Swine; Tumor Cells, Cultured

The content of membrane peptidases has been compared in the human astrocytoma clone D384 and the human neuroblastoma line SH-SY5Y. Endopeptidase-24.11 (neutral endopeptidase, EC 3.4.24.11) was detectable only on the astrocytoma cells whereas angiotensin-converting enzyme (EC 3.4.15.1) was selectively expressed on the neuroblastoma line. Dipeptidyl peptidase IV (EC 3.4.14.5) was also abundant on the astrocytoma line. The presence of both endopeptidase-24.11 and dipeptidyl peptidase IV on D384 cells was confirmed by immunohistochemistry. A membrane preparation from D384 cells hydrolyzed both atrial natriuretic peptide and brain natriuretic peptide and, in both cases, the pattern of metabolism was similar to that seen with purified endopeptidase-24.11. The endopeptidase-24.11 inhibitor, phosphoramidon, at 1 microM abolished natriuretic peptide metabolism. The neuroblastoma line, which lacked endopeptidase-24.11, failed to metabolise atrial natriuretic peptide and brain natriuretic peptide, emphasizing the key role of the endopeptidase in hydrolyzing these regulatory peptides at the cell surface.

Topics: Astrocytoma; Atrial Natriuretic Factor; Cell Membrane; Dipeptidyl Peptidase 4; Dipeptidyl-Peptidases and Tripeptidyl-Peptidases; Fluorescent Antibody Technique; Humans; Hydrolysis; Natriuretic Peptide, Brain; Neprilysin; Nerve Tissue Proteins; Neuroblastoma; Peptidyl-Dipeptidase A; Tumor Cells, Cultured

Topics: Astrocytoma; Atrial Natriuretic Factor; Cell Line; Cell Membrane; Clone Cells; Endopeptidases; Humans; Hydrolysis; Natriuretic Peptide, Brain; Nerve Tissue Proteins; Neuroblastoma

Although the pathology of tetanus toxin poisoning has been linked to an inhibition of neurotransmitter release, the mechanism of this inhibition is unknown. The neuroblastoma x glioma hybrid cell NG-108 is an emerging model in which to study the biochemical effect of tetanus toxin on acetylcholine secretion. In differentiated as well as undifferentiated NG-108 cells, a 4 hr tetanus toxin (10(-8) M) pretreatment had no effect on basal levels of cyclic AMP or cyclic GMP. In addition, toxin pretreatment did not affect agonist induced increases in either cyclic nucleotide. Treatment of NG-108 cells for 4 hr with 10(-10) M tetanus toxin had no effect on the subsequently measured activity of cytosolic protein kinase C. However, a 4 hr pretreatment of undifferentiated or differentiated cells with tetanus toxin (10(-8) or 10(-10) M respectively) significantly attenuated the ability of phorbol myristate acetate to mobilize cytosolic protein kinase C. Direct addition of tetanus toxin (10(-7)-10(-10) M) to isolated protein kinase C did not alter the ability of the enzyme to phosphorylate histone protein. These results suggest that one manifestation of tetanus toxin poisoning may be a disruption in protein kinase C metabolism.

Topics: Acetylcholine; Alprostadil; Atrial Natriuretic Factor; Cyclic AMP; Cyclic GMP; Cytosol; Neuroblastoma; Protein Kinase C; Tetanus Toxin; Tetradecanoylphorbol Acetate; Tumor Cells, Cultured

To clarify the presence of atrial natriuretic peptide (ANP) in neural tissue, extracts from human neuroblastoma which is considered of neural crest origin were analyzed using a specific radioimmunoassay (RIA) for ANP. High concentrations of immunoreactive ANP ranging from 2.7 to 18.4 ng per mg of protein were demonstrated in the tissue. Furthermore, high performance gel permeation chromatography (HPGPC) coupled with the RIA revealed that the immunoreactive ANP found in the tissue consisted of only one component with molecular weight of 12,000 to 13,000 daltons, corresponding to gamma-human ANP (hANP). These results could be direct evidence for generation of ANP intrinsic of human neuronal tissue, and also suggest that neuroblastoma can be used as a model for investigation of mechanism of ANP formation within the neuronal tissues.

Topics: Atrial Natriuretic Factor; Chromatography, Gel; Female; Humans; Infant; Infant, Newborn; Molecular Weight; Nervous System Neoplasms; Neuroblastoma; Radioimmunoassay

Atrial natriuretic hormones (ANHs) applied to polyploid rat glioma cells induced hyperpolarizations of about 30 s duration, followed by depolarizations lasting 1-2 min. Repeated applications of the peptide resulted in desensitization. The reversal potential of -87 mV at an extracellular K+ concentration of 5 mM and the decrease of membrane resistance during the hyperpolarization indicate that K+ channels were activated by ANH. In these cells the fluorescence signal of 2-[(2-bis[carboxymethyl]amino-5-methylphenoxy)-methyl]-6-methoxy-8-bis [carboxymethyl]aminoquinoline (quin2) was not affected by ANH suggesting that ANH did not change the cytosolic Ca2+-activity.

Topics: Animals; Atrial Natriuretic Factor; Bradykinin; Cells, Cultured; Glioma; Hybrid Cells; Ion Channels; Membrane Potentials; Nerve Tissue; Neuroblastoma; Potassium; Rats

Atriopeptin III and related atrial natriuretic peptide hormones strongly elevate the level of cyclic GMP in three neural tumor cell lines. At peptide concentrations of 1 microM clear-cut plateaus of the dose-response curves are not yet reached. Atriopeptin III increases the intracellular concentration of cyclic GMP to a maximum in the course of 30-40 min. The effect of atriopeptin III on the cellular cyclic GMP level is independent of the concentration of extracellular Ca2+ and is not affected by the Ca2+ ionophore A23187. These results suggest (1) that atrial natriuretic hormones may play an important role in the nervous system, and (2) that cultured neural cells may be useful tools in the elucidation of the mechanisms of action of these hormones.

Topics: 1-Methyl-3-isobutylxanthine; Animals; Atrial Natriuretic Factor; Calcimycin; Calcium; Cell Line; Cyclic GMP; Glioma; Hybrid Cells; Kinetics; Neuroblastoma; Rats